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Sample records for acvr1 mutation activates

  1. Shared ACVR1 mutations in FOP and DIPG: Opportunities and challenges in extending biological and clinical implications across rare diseases.

    Science.gov (United States)

    Han, Harry J; Jain, Payal; Resnick, Adam C

    2017-08-02

    Gain-of-function mutations in the Type I Bone Morphogenic Protein (BMP) receptor ACVR1 have been identified in two diseases: Fibrodysplasia Ossificans Progressiva (FOP), a rare autosomal dominant disorder characterized by genetically driven heterotopic ossification, and in 20-25% of Diffuse Intrinsic Pontine Gliomas (DIPGs), a pediatric brain tumor with no effective therapies and dismal median survival. While the ACVR1 mutation is causal for FOP, its role in DIPG tumor biology remains under active investigation. Here, we discuss cross-fertilization between the FOP and DIPG fields, focusing on the biological mechanisms and principles gleaned from FOP that can be applied to DIPG biology. We highlight our current knowledge of ACVR1 in both diseases, and then describe the growing opportunities and barriers to effectively investigate ACVR1 in DIPG. Importantly, learning from other seemingly unrelated diseases harboring similar mutations may uncover novel mechanisms or processes for future investigation. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. RESEARCH Confirmation of the recurrent ACVR1 617G>A mutation ...

    African Journals Online (AJOL)

    mechanism remained elusive.2,3 FOP molecular investigations has revealed ... Division of Molecular Biology and Human Genetics, Faculty of Health Sciences,. Stellenbosch ... the concept of 1 specific mutation in ACVR1 is no longer tenable.

  3. Novel mutations in ACVR1 result in atypical features in two fibrodysplasia ossificans progressiva patients.

    Directory of Open Access Journals (Sweden)

    Kirsten A Petrie

    Full Text Available Fibrodysplasia Ossificans Progressiva (FOP is a rare, heritable condition typified by progression of extensive ossification within skeletal muscle, ligament and tendon together with defects in skeletal development. The condition is easily diagnosed by the presence of shortened great toes and there is severe advancement of disability with age. FOP has been shown to result from a point mutation (c.617G>A in the ACVR1 gene in almost all patients reported. Very recently two other mutations have been described in three FOP patients. We present here evidence for two further unique mutations (c.605G>T and c.983G>A in this gene in two FOP patients with some atypical digit abnormalities and other clinical features. The observation of disparate missense mutations mapped to the GS and kinase domains of the protein supports the disease model of mild kinase activation and provides a potential rationale for phenotypic variation.

  4. Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma.

    Science.gov (United States)

    Fontebasso, Adam M; Papillon-Cavanagh, Simon; Schwartzentruber, Jeremy; Nikbakht, Hamid; Gerges, Noha; Fiset, Pierre-Olivier; Bechet, Denise; Faury, Damien; De Jay, Nicolas; Ramkissoon, Lori A; Corcoran, Aoife; Jones, David T W; Sturm, Dominik; Johann, Pascal; Tomita, Tadanori; Goldman, Stewart; Nagib, Mahmoud; Bendel, Anne; Goumnerova, Liliana; Bowers, Daniel C; Leonard, Jeffrey R; Rubin, Joshua B; Alden, Tord; Browd, Samuel; Geyer, J Russell; Leary, Sarah; Jallo, George; Cohen, Kenneth; Gupta, Nalin; Prados, Michael D; Carret, Anne-Sophie; Ellezam, Benjamin; Crevier, Louis; Klekner, Almos; Bognar, Laszlo; Hauser, Peter; Garami, Miklos; Myseros, John; Dong, Zhifeng; Siegel, Peter M; Malkin, Hayley; Ligon, Azra H; Albrecht, Steffen; Pfister, Stefan M; Ligon, Keith L; Majewski, Jacek; Jabado, Nada; Kieran, Mark W

    2014-05-01

    Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

  5. 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.

  6. Fibrodysplasia ossificans progressiva: middle-age onset of heterotopic ossification from a unique missense mutation (c.974G>C, p.G325A) in ACVR1.

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    Whyte, Michael P; Wenkert, Deborah; Demertzis, Jennifer L; DiCarlo, Edward F; Westenberg, Erica; Mumm, Steven

    2012-03-01

    Fibrodysplasia ossificans progressiva (FOP) is the rare mendelian disease characterized by congenital malformation of the great toes preceding heterotopic ossification (HO) and caused by heterozygous activating mutation of the ACVR1 gene, which encodes the ALK2 receptor for bone morphogenetic proteins. Early adult life is the latest reported presentation for the HO of FOP. The patient of our report first developed HO from FOP at 47 years of age. She had congenital hallux valgus deformity but despite various traumas was previously well. HO began several months after a brief, seemingly viral, illness. Sudden and progressive pain, redness, warmth, and swelling appeared over a scapula. Computed tomography was remarkable for asymmetrical thickening of muscles and fascial planes. At first, the significance of the great toe abnormalities went unrecognized elsewhere, and biopsy for suspected inflammatory fasciitis revealed proliferating fibroblasts with scattered inflammatory cells. Prednisone improved her symptoms but, when tapered, swellings developed on her chest, posterior thorax, and flank, and FOP was diagnosed. Methylprednisolone, methotrexate, and alendronate seemed to help her symptoms, but the lesions worsened and HO appeared and rapidly progressed. Mutation analysis of the ACVR1 gene revealed heterozygosity for a unique missense defect (c.974G>C, p.G325A) that predicted a conservative (mild) amino acid change within the kinase domain of ALK2. Hence, HO in FOP can be delayed until middle-age, and perhaps provoked by a viral illness. Nevertheless, progression of HO can then be rapid despite bisphosphonate and high-dose immunosuppressive therapy. Possibly, our patient's late-onset HO reflects her mild alteration of ALK2 or some protective and therapeutically useful genetic, epigenetic, or nongenetic factor. Recognition of presymptomatic individuals or late-onset HO in FOP should have these patients avoid traumas, treatments, and maybe viral illnesses that can

  7. Palovarotene Inhibits Heterotopic Ossification and Maintains Limb Mobility and Growth in Mice With the Human ACVR1(R206H) Fibrodysplasia Ossificans Progressiva (FOP) Mutation.

    Science.gov (United States)

    Chakkalakal, Salin A; Uchibe, Kenta; Convente, Michael R; Zhang, Deyu; Economides, Aris N; Kaplan, Frederick S; Pacifici, Maurizio; Iwamoto, Masahiro; Shore, Eileen M

    2016-09-01

    Fibrodysplasia ossificans progressiva (FOP), a rare and as yet untreatable genetic disorder of progressive extraskeletal ossification, is the most disabling form of heterotopic ossification (HO) in humans and causes skeletal deformities, movement impairment, and premature death. Most FOP patients carry an activating mutation in a bone morphogenetic protein (BMP) type I receptor gene, ACVR1(R206H) , that promotes ectopic chondrogenesis and osteogenesis and, in turn, HO. We showed previously that the retinoic acid receptor γ (RARγ) agonist palovarotene effectively inhibited HO in injury-induced and genetic mouse models of the disease. Here we report that the drug additionally prevents spontaneous HO, using a novel conditional-on knock-in mouse line carrying the human ACVR1(R206H) mutation for classic FOP. In addition, palovarotene restored long bone growth, maintained growth plate function, and protected growing mutant neonates when given to lactating mothers. Importantly, palovarotene maintained joint, limb, and body motion, providing clear evidence for its encompassing therapeutic potential as a treatment for FOP. © 2016 American Society for Bone and Mineral Research.

  8. ACVR1, a Therapeutic Target of Fibrodysplasia Ossificans Progressiva, Is Negatively Regulated by miR-148a

    Directory of Open Access Journals (Sweden)

    Jun Cheng

    2012-02-01

    Full Text Available Fibrodysplasia ossificans progressiva (FOP is a rare congenital disorder of skeletal malformations and progressive extraskeletal ossification. There is still no effective treatment for FOP. All FOP individuals harbor conserved point mutations in ACVR1 gene that are thought to cause ACVR1 constitutive activation and activate BMP signal pathway. The constitutively active ACVR1 is also found to be able to cause endothelial-to-mesenchymal transition (EndMT in endothelial cells, which may cause the formation of FOP lesions. MicroRNAs (miRNAs play an essential role in regulating cell differentiation. Here, we verified that miR-148a directly targeted the 3' UTR of ACVR1 mRNA by reporter gene assays and mutational analysis at the miRNA binding sites, and inhibited ACVR1 both at the protein level and mRNA level. Further, we verified that miR-148a could inhibit the mRNA expression of the Inhibitor of DNA binding (Id gene family thereby suppressing the BMP signaling pathway. This study suggests miR-148a is an important mediator of ACVR1, thus offering a new potential target for the development of therapeutic agents against FOP.

  9. High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva.

    Science.gov (United States)

    Cappato, Serena; Tonachini, Laura; Giacopelli, Francesca; Tirone, Mario; Galietta, Luis J V; Sormani, Martina; Giovenzana, Anna; Spinelli, Antonello E; Canciani, Barbara; Brunelli, Silvia; Ravazzolo, Roberto; Bocciardi, Renata

    2016-06-01

    The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

  10. 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.

  11. 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.

  12. 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.

  13. AcvR1-mediated BMP signaling in second heart field is required for arterial pole development: implications for myocardial differentiation and regional identity.

    Science.gov (United States)

    Thomas, Penny S; Rajderkar, Sudha; Lane, Jamie; Mishina, Yuji; Kaartinen, Vesa

    2014-06-15

    BMP signaling plays an essential role in second heart field-derived heart and arterial trunk development, including myocardial differentiation, right ventricular growth, and interventricular, outflow tract and aortico-pulmonary septation. It is mediated by a number of different BMP ligands, and receptors, many of which are present simultaneously. The mechanisms by which they regulate morphogenetic events and degree of redundancy amongst them have still to be elucidated. We therefore assessed the role of BMP Type I receptor AcvR1 in anterior second heart field-derived cell development, and compared it with that of BmpR1a. By removing Acvr1 using the driver Mef2c[AHF]-Cre, we show that AcvR1 plays an essential role in arterial pole morphogenesis, identifying defects in outflow tract wall and cushion morphology that preceded a spectrum of septation defects from double outlet right ventricle to common arterial trunk in mutants. Its absence caused dysregulation in gene expression important for myocardial differentiation (Isl1, Fgf8) and regional identity (Tbx2, Tbx3, Tbx20, Tgfb2). Although these defects resemble to some degree those in the equivalent Bmpr1a mutant, a novel gene knock-in model in which Bmpr1a was expressed in the Acvr1 locus only partially restored septation in Acvr1 mutants. These data show that both BmpR1a and AcvR1 are needed for normal heart development, in which they play some non-redundant roles, and refine our understanding of the genetic and morphogenetic processes underlying Bmp-mediated heart development important in human congenital heart disease.

  14. Activating GNAS mutations in parosteal osteosarcoma.

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    Carter, Jodi M; Inwards, Carrie Y; Jin, Long; Evers, Barbara; Wenger, Doris E; Oliveira, Andre M; Fritchie, Karen J

    2014-03-01

    Parosteal osteosarcoma is a surface-based osteosarcoma that often exhibits deceptively bland cytologic features, hindering diagnosis in small biopsies or when correlative radiologic imaging is not readily available. A number of benign and malignant fibro-osseous lesions, including fibrous dysplasia (FD) and low-grade central osteosarcoma, fall within the morphologic differential diagnosis of parosteal osteosarcoma. Somatic mutations in GNAS, encoding the α-subunit of the heterotrimeric G protein complex (Gsα), occur in FD and McCune-Albright syndrome but have not been reported in parosteal osteosarcoma. We evaluated GNAS mutational status in parosteal osteosarcoma and several of its histologic mimics to determine its utility in differentiating these entities. Eleven of 14 (79%) FD cases had GNAS mutations within codon 201 (5 R201C and 6 R201H mutations). GNAS mutations were not detected in any cases of adamantinoma or osteofibrous dysplasia. Direct sequencing of 9 parosteal osteosarcomas, including 3 of low grade and 6 with dedifferentiation, revealed activating GNAS mutations in 5 cases (55%), distributed as 4 R201C-mutated tumors and 1 tumor with an R201H mutation. GNAS codon 227 mutations were not detected in any of the cases. There was no association between GNAS mutational status and patient demographics, histologic dedifferentiation, or clinical outcome. To our knowledge, we report the first series of parosteal osteosarcomas harboring activating GNAS mutations. Our data suggest that GNAS mutational status may have limited utility as an ancillary technique in differentiating benign and malignant fibro-osseous lesions of the bone.

  15. 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

  16. 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

  17. Activating FGFR2-RAS-BRAF mutations in ameloblastoma.

    Science.gov (United States)

    Brown, Noah A; Rolland, Delphine; McHugh, Jonathan B; Weigelin, Helmut C; Zhao, Lili; Lim, Megan S; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

    2014-11-01

    Ameloblastoma is an odontogenic neoplasm whose overall mutational landscape has not been well characterized. We sought to characterize pathogenic mutations in ameloblastoma and their clinical and functional significance with an emphasis on the mitogen-activated protein kinase (MAPK) pathway. A total of 84 ameloblastomas and 40 non-ameloblastoma odontogenic tumors were evaluated with a combination of BRAF V600E allele-specific PCR, VE1 immunohistochemistry, the Ion AmpliSeq Cancer Hotspot Panel, and Sanger sequencing. Efficacy of a BRAF inhibitor was evaluated in an ameloblastoma-derived cell line. Somatic, activating, and mutually exclusive RAS-BRAF and FGFR2 mutations were identified in 88% of cases. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 were also identified. BRAF V600E was the most common mutation, found in 62% of ameloblastomas and in ameloblastic fibromas/fibrodentinomas but not in other odontogenic tumors. This mutation was associated with a younger age of onset, whereas BRAF wild-type cases arose more frequently in the maxilla and showed earlier recurrences. One hundred percent concordance was observed between VE1 immunohistochemistry and molecular detection of BRAF V600E mutations. Ameloblastoma cells demonstrated constitutive MAPK pathway activation in vitro. Proliferation and MAPK activation were potently inhibited by the BRAF inhibitor vemurafenib. Our findings suggest that activating FGFR2-RAS-BRAF mutations play a critical role in the pathogenesis of most cases of ameloblastoma. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 may function as secondary mutations. BRAF V600E mutations have both diagnostic and prognostic implications. In vitro response of ameloblastoma to a BRAF inhibitor suggests a potential role for targeted therapy. ©2014 American Association for Cancer Research.

  18. Glucocerebrosidase activity in Parkinson's disease with and without GBA mutations.

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    Alcalay, Roy N; Levy, Oren A; Waters, Cheryl C; Fahn, Stanley; Ford, Blair; Kuo, Sheng-Han; Mazzoni, Pietro; Pauciulo, Michael W; Nichols, William C; Gan-Or, Ziv; Rouleau, Guy A; Chung, Wendy K; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Marder, Karen; Zhang, Xiaokui

    2015-09-01

    Glucocerebrosidase (GBA) mutations have been associated with Parkinson's disease in numerous studies. However, it is unknown whether the increased risk of Parkinson's disease in GBA carriers is due to a loss of glucocerebrosidase enzymatic activity. We measured glucocerebrosidase enzymatic activity in dried blood spots in patients with Parkinson's disease (n = 517) and controls (n = 252) with and without GBA mutations. Participants were recruited from Columbia University, New York, and fully sequenced for GBA mutations and genotyped for the LRRK2 G2019S mutation, the most common autosomal dominant mutation in the Ashkenazi Jewish population. Glucocerebrosidase enzymatic activity in dried blood spots was measured by a mass spectrometry-based assay and compared among participants categorized by GBA mutation status and Parkinson's disease diagnosis. Parkinson's disease patients were more likely than controls to carry the LRRK2 G2019S mutation (n = 39, 7.5% versus n = 2, 0.8%, P Parkinson's risk but not with Gaucher disease (E326K, P = 0.009; T369M, P Parkinson's disease were considered, they had lower mean glucocerebrosidase enzymatic activity than controls (11.14 µmol/l/h versus 11.85 µmol/l/h, P = 0.011). Difference compared to controls persisted in patients with idiopathic Parkinson's disease (after exclusion of all GBA and LRRK2 carriers; 11.53 µmol/l/h, versus 12.11 µmol/l/h, P = 0.036) and after adjustment for age and gender (P = 0.012). Interestingly, LRRK2 G2019S carriers (n = 36), most of whom had Parkinson's disease, had higher enzymatic activity than non-carriers (13.69 µmol/l/h versus 11.93 µmol/l/h, P = 0.002). In patients with idiopathic Parkinson's, higher glucocerebrosidase enzymatic activity was associated with longer disease duration (P = 0.002) in adjusted models, suggesting a milder disease course. We conclude that lower glucocerebrosidase enzymatic activity is strongly associated with GBA mutations, and modestly with idiopathic Parkinson

  19. Bi-directional SIFT predicts a subset of activating mutations.

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    Lee, William; Zhang, Yan; Mukhyala, Kiran; Lazarus, Robert A; Zhang, Zemin

    2009-12-14

    Advancements in sequencing technologies have empowered recent efforts to identify polymorphisms and mutations on a global scale. The large number of variations and mutations found in these projects requires high-throughput tools to identify those that are most likely to have an impact on function. Numerous computational tools exist for predicting which mutations are likely to be functional, but none that specifically attempt to identify mutations that result in hyperactivation or gain-of-function. Here we present a modified version of the SIFT (Sorting Intolerant from Tolerant) algorithm that utilizes protein sequence alignments with homologous sequences to identify functional mutations based on evolutionary fitness. We show that this bi-directional SIFT (B-SIFT) is capable of identifying experimentally verified activating mutants from multiple datasets. B-SIFT analysis of large-scale cancer genotyping data identified potential activating mutations, some of which we have provided detailed structural evidence to support. B-SIFT could prove to be a valuable tool for efforts in protein engineering as well as in identification of functional mutations in cancer.

  20. TERT promoter mutations and monoallelic activation of TERT in cancer.

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    Huang, F W; Bielski, C M; Rinne, M L; Hahn, W C; Sellers, W R; Stegmeier, F; Garraway, L A; Kryukov, G V

    2015-12-14

    Here we report that promoter mutations in telomerase (TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer.

  1. 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.

  2. Whole-exome sequencing identified mutational profiles of high-grade colon adenomas

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    Kim, Tae-Min; Rhee, Je-Keun; Park, Hyeon-Chun; Sung, Min Kim; Kim, Sung Soo; Hyeok, Chang An; Lee Hyung, Sug; Chung, Yeun-Jun

    2017-01-01

    Although gene-to-gene analyses identified genetic alterations such as APC, KRAS and TP53 mutations in colon adenomas, it is largely unknown whether there are any others in them. Mutational profiling of high-grade colon adenoma (HGCA) that just precedes colon carcinoma might identify not only novel adenoma-specific genes but also critical genes for its progression to carcinoma. For this, we performed whole-exome sequencing (WES) of 12 HGCAs and identified 11 non-hypermutated and one hypermutated (POLE-mutated) cases. We identified 22 genes including APC, KRAS, TP53, GNAS, NRAS, SMAD4, ARID2, and PIK3CA with non-silent mutations in the cancer Census Genes. Bi-allelic and mono-allelic APC alterations were found in nine and one HGCAs, respectively, while the other two harbored wild-type APC. Five HGCAs harbored either mono-allelic (four HGCAs) or bi-allelic (one HGCA) SMAD4 mutation or 18q loss that had been known as early carcinoma-specific changes. We identified MTOR, ACVR1B, GNAQ, ATM, CNOT1, EP300, ARID2, RET and MAP2K4 mutations for the first time in colon adenomas. Our WES data is largely matched with the earlier ‘adenoma-carcinoma model’ (APC, KRAS, NRAS and GNAS mutations), but there are newly identified SMAD4, MTOR, ACVR1B, GNAQ, ATM, CNOT1, EP300, ARID2, RET and MAP2K4 mutations in this study. Our findings provide resource for understanding colon premalignant lesions and for identifying genomic clues for differential diagnosis and therapy options for colon adenomas and carcinomas. PMID:28179590

  3. The impact of point mutations in the human androgen receptor: classification of mutations on the basis of transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Colin W Hay

    Full Text Available Androgen receptor mediated signaling drives prostate cancer cell growth and survival. Mutations within the receptor occur infrequently in prostate cancer prior to hormonal therapy but become prevalent in incurable androgen independent and metastatic tumors. Despite the determining role played by the androgen receptor in all stages of prostate cancer progression, there is a conspicuous dearth of comparable data on the consequences of mutations. In order to remedy this omission, we have combined an expansive study of forty five mutations which are predominantly associated with high Gleason scores and metastatic tumors, and span the entire length of the receptor, with a literature review of the mutations under investigation. We report the discovery of a novel prevalent class of androgen receptor mutation that possesses loss of function at low levels of androgen yet transforms to a gain of function at physiological levels. Importantly, mutations introducing constitutive gain of function are uncommon, with the majority of mutations leading to either loss of function or no significant change from wild-type activity. Therefore, the widely accepted supposition that androgen receptor mutations in prostate cancer result in gain of function is appealing, but mistaken. In addition, the transcriptional outcome of some mutations is dependent upon the androgen receptor responsive element. We discuss the consequences of these findings and the role of androgen receptor mutations for prostate cancer progression and current treatment options.

  4. Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis

    Science.gov (United States)

    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

  5. Activin receptor-like kinase 7 suppresses lipolysis to accumulate fat in obesity through downregulation of peroxisome proliferator-activated receptor γ and C/EBPα.

    Science.gov (United States)

    Yogosawa, Satomi; Mizutani, Shin; Ogawa, Yoshihiro; Izumi, Tetsuro

    2013-01-01

    We previously identified a quantitative trait locus for adiposity, non-insulin-dependent diabetes 5 (Nidd5), on mouse chromosome 2. In the current study, we identified the actual genetic alteration at Nidd5 as a nonsense mutation of the Acvr1c gene encoding activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-β receptors, which results in a COOH-terminal deletion of the kinase domain. We further showed that the ALK7 dysfunction causes increased lipolysis in adipocytes and leads to decreased fat accumulation. Conversely, ALK7 activation inhibits lipolysis by suppressing the expression of adipose lipases. ALK7 and activated Smads repress those lipases by downregulating peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBP) α. Although PPARγ and C/EBPα act as adipogenic transcription factors during adipocyte differentiation, they are lipolytic in sum in differentiated adipocytes and are downregulated by ALK7 in obesity to accumulate fat. Under the obese state, ALK7 deficiency improves glucose tolerance and insulin sensitivity by preferentially increasing fat combustion in mice. These findings have uncovered a net lipolytic function of PPARγ and C/EBPα in differentiated adipocytes and point to the ALK7-signaling pathway that is activated in obesity as a potential target of medical intervention.

  6. Activin Receptor-Like Kinase 7 Suppresses Lipolysis to Accumulate Fat in Obesity Through Downregulation of Peroxisome Proliferator–Activated Receptor γ and C/EBPα

    Science.gov (United States)

    Yogosawa, Satomi; Mizutani, Shin; Ogawa, Yoshihiro; Izumi, Tetsuro

    2013-01-01

    We previously identified a quantitative trait locus for adiposity, non-insulin-dependent diabetes 5 (Nidd5), on mouse chromosome 2. In the current study, we identified the actual genetic alteration at Nidd5 as a nonsense mutation of the Acvr1c gene encoding activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-β receptors, which results in a COOH-terminal deletion of the kinase domain. We further showed that the ALK7 dysfunction causes increased lipolysis in adipocytes and leads to decreased fat accumulation. Conversely, ALK7 activation inhibits lipolysis by suppressing the expression of adipose lipases. ALK7 and activated Smads repress those lipases by downregulating peroxisome proliferator–activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBP) α. Although PPARγ and C/EBPα act as adipogenic transcription factors during adipocyte differentiation, they are lipolytic in sum in differentiated adipocytes and are downregulated by ALK7 in obesity to accumulate fat. Under the obese state, ALK7 deficiency improves glucose tolerance and insulin sensitivity by preferentially increasing fat combustion in mice. These findings have uncovered a net lipolytic function of PPARγ and C/EBPα in differentiated adipocytes and point to the ALK7-signaling pathway that is activated in obesity as a potential target of medical intervention. PMID:22933117

  7. Three faces of recombination activating gene 1 (RAG1) mutations.

    Science.gov (United States)

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

  8. 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

  9. 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 ident...

  10. 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.

  11. 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.

  12. Targetable activating mutations are very frequent in GCB and ABC diffuse large B-cell lymphoma.

    Science.gov (United States)

    Bohers, Elodie; Mareschal, Sylvain; Bouzelfen, Abdelilah; Marchand, Vinciane; Ruminy, Philippe; Maingonnat, Catherine; Ménard, Anne-Lise; Etancelin, Pascaline; Bertrand, Philippe; Dubois, Sydney; Alcantara, Marion; Bastard, Christian; Tilly, Hervé; Jardin, Fabrice

    2014-02-01

    Diffuse large B cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy that can be divided in two major subgroups, germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Activating mutations of genes involved in the BCR and NF-κB pathways (CD79A, CD79B, MYD88, and CARD11) or in epigenetic regulation (EZH2) have been recently reported, preferentially in one of the two DLBCL subtypes. We analyzed the mutational status of these five recurrently mutated genes in a cohort of 161 untreated de novo DLBCL. Overall, 93 mutations were detected, in 61 (38%) of the patients. The L265P MYD88 mutation was the most frequent MYD88 variant (n = 18), observed exclusively in the ABC subtype. CD79A/CD79B ITAM domains were targeted in ABC DLBCL (12/77; 16%), whereas CARD11 mutations were equally distributed in the two subtypes. The EZH2 Y641 substitution was found almost exclusively in the GCB subgroup (15/62; 24%). Twenty cases (12%) displayed two activating mutations, including the most frequent CD79/MYD88 variants combination (n = 8) which is observed exclusively in the ABC subtype. When considering only ABC DLBCL patients treated by rituximab plus chemotherapy, the presence of an activating NF-κB mutation was associated with an unfavorable outcome (3-years OS 26% for mutated cases versus 67% for the cases without mutations, P = 0.0337). Our study demonstrates that activating and targetable mutations are observed at a very high frequency in DLBCL at the time of diagnosis, indicating that sequencing of a limited number of genes could help tailor an optimal treatment strategy in DLBCL. Copyright © 2013 Wiley Periodicals, Inc.

  13. Mutated human androgen receptor gene detected in a prostatic cancer patient is also activated by estradiol

    Energy Technology Data Exchange (ETDEWEB)

    Elo, J.P.; Kvist, L.; Leinonen, K.; Isomaa, V. [Univ. of Oulu (Finland)] [and others

    1995-12-01

    Androgens are necessary for the development of prostatic cancer. The mechanisms by which the originally androgen-dependent prostatic cancer cells are relieved of the requirement to use androgen for their growth are largely unknown. The human prostatic cancer cell line LNCaP has been shown to contain a point mutation in the human androgen receptor gene (hAR), suggesting that changes in the hAR may contribute to the abnormal hormone response of prostatic cells. To search for point mutations in the hAR, we used single strand conformation polymorphism analysis and a polymerase chain reaction direct sequencing method to screen 23 prostatic cancer specimens from untreated patients, 6 prostatic cancer specimens from treated patients, and 11 benign prostatic hyperplasia specimens. One mutation was identified in DNA isolated from prostatic cancer tissue, and the mutation was also detected in the leukocyte DNA of the patient and his offspring. The mutation changed codon 726 in exon E from arginine to leucine and was a germ line mutation. The mutation we found in exon E of the hAR gene does not alter the ligand binding specificity of the AR, but the mutated receptor was activated by estradiol to a significantly greater extent than the wild-type receptor. The AR gene mutation described in this study might be one explanation for the altered biological activity of prostatic cancer. 36 refs., 4 figs.

  14. Inhibition of mutated, activated BRAF in metastatic melanoma.

    Science.gov (United States)

    Flaherty, Keith T; Puzanov, Igor; Kim, Kevin B; Ribas, Antoni; McArthur, Grant A; Sosman, Jeffrey A; O'Dwyer, Peter J; Lee, Richard J; Grippo, Joseph F; Nolop, Keith; Chapman, Paul B

    2010-08-26

    The identification of somatic mutations in the gene encoding the serine-threonine protein kinase B-RAF (BRAF) in the majority of melanomas offers an opportunity to test oncogene-targeted therapy for this disease. We conducted a multicenter, phase 1, dose-escalation trial of PLX4032 (also known as RG7204), an orally available inhibitor of mutated BRAF, followed by an extension phase involving the maximum dose that could be administered without adverse effects (the recommended phase 2 dose). Patients received PLX4032 twice daily until they had disease progression. Pharmacokinetic analysis and tumor-response assessments were conducted in all patients. In selected patients, tumor biopsy was performed before and during treatment to validate BRAF inhibition. A total of 55 patients (49 of whom had melanoma) were enrolled in the dose-escalation phase, and 32 additional patients with metastatic melanoma who had BRAF with the V600E mutation were enrolled in the extension phase. The recommended phase 2 dose was 960 mg twice daily, with increases in the dose limited by grade 2 or 3 rash, fatigue, and arthralgia. In the dose-escalation cohort, among the 16 patients with melanoma whose tumors carried the V600E BRAF mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response and 1 had a complete response. Among the 32 patients in the extension cohort, 24 had a partial response and 2 had a complete response. The estimated median progression-free survival among all patients was more than 7 months. Treatment of metastatic melanoma with PLX4032 in patients with tumors that carry the V600E BRAF mutation resulted in complete or partial tumor regression in the majority of patients. (Funded by Plexxikon and Roche Pharmaceuticals.)

  15. Activating mutation in MET oncogene in familial colorectal cancer

    Directory of Open Access Journals (Sweden)

    Schildkraut Joellen M

    2011-10-01

    Full Text Available Abstract Background In developed countries, the lifetime risk of developing colorectal cancer (CRC is 5%, and it is the second leading cause of death from cancer. The presence of family history is a well established risk factor with 25-35% of CRCs attributable to inherited and/or familial factors. The highly penetrant inherited colon cancer syndromes account for approximately 5%, leaving greater than 20% without clear genetic definition. Familial colorectal cancer has been linked to chromosome 7q31 by multiple affected relative pair studies. The MET proto-oncogene which resides in this chromosomal region is considered a candidate for genetic susceptibility. Methods MET exons were amplified by PCR from germline DNA of 148 affected sibling pairs with colorectal cancer. Amplicons with altered sequence were detected with high-resolution melt-curve analysis using a LightScanner (Idaho Technologies. Samples demonstrating alternative melt curves were sequenced. A TaqMan assay for the specific c.2975C >T change was used to confirm this mutation in a cohort of 299 colorectal cancer cases and to look for allelic amplification in tumors. Results Here we report a germline non-synonymous change in the MET proto-oncogene at amino acid position T992I (also reported as MET p.T1010I in 5.2% of a cohort of sibling pairs affected with CRC. This genetic variant was then confirmed in a second cohort of individuals diagnosed with CRC and having a first degree relative with CRC at prevalence of 4.1%. This mutation has been reported in cancer cells of multiple origins, including 2.5% of colon cancers, and in Conclusions Although the MET p.T992I genetic mutation is commonly found in somatic colorectal cancer tissues, this is the first report also implicating this MET genetic mutation as a germline inherited risk factor for familial colorectal cancer. Future studies on the cancer risks associated with this mutation and the prevalence in different at-risk populations will

  16. 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

  17. Mutations induced by dacarbazine activated with cytochrome P-450.

    Science.gov (United States)

    Mudipalli, A; Nadadur, S S; Maccubbin, A E; Gurtoo, H L

    1995-03-01

    The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to alkylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system. In the present study, we used an in vitro shuttle vector assay to study the base and sequence specificity of mutagenesis by DTIC. The shuttle vector plasmid pSP189 was treated with DTIC (1-2.5 mM) in vitro in a reconstituted cytochrome P-450 system at 37 degrees C for either 30 or 60 min. SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplification in indicator bacteria. Mutagenesis of DTIC in this system was dependent upon the presence of the cytochrome P-450 reconstituted system and NADPH. Mutations induced by DTIC included single base substitutions (35%), single base deletions (30.5%), single base insertions (19.4%) and large deletions (13.8%). Among the substitutions, transversions and transitions were in the ratio of 1:0.7. Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots.

  18. 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.

  19. Activating GNAS and KRAS mutations in gastric foveolar metaplasia, gastric heterotopia, and adenocarcinoma of the duodenum

    OpenAIRE

    Matsubara, A.; Ogawa, R; Suzuki, H; Oda, I.; Taniguchi, H; Kanai, Y.; Kushima, R; Sekine, S

    2015-01-01

    Background: Heterotopic gastric-type epithelium, including gastric foveolar metaplasia (GFM) and gastric heterotopia (GH), is a common finding in duodenal biopsy specimens; however, there is still controversy regarding their histogenetic backgrounds. Methods: We analysed a total of 177 duodenal lesions, including 66 GFM lesions, 81 GH lesions, and 30 adenocarcinomas, for the presence of GNAS, KRAS, and BRAF mutations. Results: Activating GNAS mutations were identified in 27 GFM lesions (41%) ...

  20. Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.

    Science.gov (United States)

    Flanagan, Sarah E; Haapaniemi, Emma; Russell, Mark A; Caswell, Richard; Lango Allen, Hana; De Franco, Elisa; McDonald, Timothy J; Rajala, Hanna; Ramelius, Anita; Barton, John; Heiskanen, Kaarina; Heiskanen-Kosma, Tarja; Kajosaari, Merja; Murphy, Nuala P; Milenkovic, Tatjana; Seppänen, Mikko; Lernmark, Åke; Mustjoki, Satu; Otonkoski, Timo; Kere, Juha; Morgan, Noel G; Ellard, Sian; Hattersley, Andrew T

    2014-08-01

    Monogenic causes of autoimmunity provide key insights into the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in five individuals with a spectrum of early-onset autoimmune disease, including type 1 diabetes. These findings emphasize the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in hyper IgE syndrome.

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

    Science.gov (United States)

    Chen, Chen; Han, Sheng; Meng, Lingxuan; Li, Zhonghua; Zhang, Xue; Wu, Anhua

    2014-01-01

    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.

  2. 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

    cells that show a paternal age effect. Screening of 30 spermatocytic seminomas for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G, encoding K650E, which causes thanatophoric dysplasia in the germline) and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA...... showed that levels of the FGFR3 mutation increase with paternal age and that the mutation spectrum at the Lys650 codon is similar to that observed in bladder cancer. Most spermatocytic seminomas show increased immunoreactivity for FGFR3 and/or HRAS. We propose that paternal age-effect mutations activate...

  3. 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.

  4. 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.

  5. Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.

    Science.gov (United States)

    Van Gorp, Hanne; Saavedra, Pedro H V; de Vasconcelos, Nathalia M; Van Opdenbosch, Nina; Vande Walle, Lieselotte; Matusiak, Magdalena; Prencipe, Giusi; Insalaco, Antonella; Van Hauwermeiren, Filip; Demon, Dieter; Bogaert, Delfien J; Dullaers, Melissa; De Baere, Elfride; Hochepied, Tino; Dehoorne, Joke; Vermaelen, Karim Y; Haerynck, Filomeen; De Benedetti, Fabrizio; Lamkanfi, Mohamed

    2016-12-13

    Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.

  6. 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.

  7. mTOR activation in medullary thyroid carcinoma with RAS mutation.

    Science.gov (United States)

    Lyra, Joana; Vinagre, João; Batista, Rui; Pinto, Vasco; Prazeres, Hugo; Rodrigues, Fernando; Eloy, Catarina; Sobrinho-Simões, Manuel; Soares, Paula

    2014-11-01

    Rearranged during transfection (RET) mutations are well-known genetic events in sporadic and familial medullary thyroid carcinoma (FMTC). The presence of RAS mutations in sporadic cases, challenging the RET paradigm in these tumors, has been recently reported. We intend to evaluate mTOR pathway activation in RET- and RAS-mutated MTC. In this study, we analysed the presence of RET, H-RAS, and K-RAS mutations in a series of 87 MTCs (82 apparently sporadic and five FMTCs; five apparently sporadic MTCs were eventually found to be familial). We also evaluated mTOR activation--using the expression of its downstream effector phospho-S6 ribosomal protein (p-S6) and the expression of the mTOR inhibitor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN)--by immunohistochemistry. Our results revealed that RET mutations were present in 52.9% of the cases (46/87) and RAS mutations in 12.6% (11/87) of the whole series of MTCs and 14.3% of the 77 sporadic MTCs. The presence of RET and RAS mutations was mutually exclusive. RAS mutations were significantly associated with higher intensity of p-S6 expression (P=0.007), suggesting that the mTOR pathway is activated in such MTCs. We observed also an increased expression of p-S6 in invasive tumors (P=0.042) and in MTCs with lymph node metastases (P=0.046). Cytoplasmic PTEN expression was detected in 58.8% of the cases; cases WT for RAS showed a significantly lower expression of PTEN (P=0.045). We confirmed the presence of RAS mutation in 14.3% of sporadic MTCs and report, for the first time, an association between such mutations and the activation of the mTOR pathway. The evaluation of the mTOR activation by pS6 expression may serve as an indicator of invasive MTC. © 2014 European Society of Endocrinology.

  8. 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.

  9. 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.

  10. Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Jeffrey C Lee

    2006-12-01

    Full Text Available BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132 of glioblastomas and 12.5% (1/8 of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

  11. 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.

  12. Contribution of arylsulfatase A mutations located on the same allele to enzyme activity reduction and metachromatic leukodystrophy severity.

    Science.gov (United States)

    Regis, Stefano; Corsolini, Fabio; Stroppiano, Marina; Cusano, Roberto; Filocamo, Mirella

    2002-04-01

    The occurrence of different mutations on the same arylsulfatase A allele is not uncommon, due to the high frequency of several variants, among which the pseudodeficiency mutations are particularly important. We identified a late infantile metachromatic leukodystrophy patient carrying on one allele the new E253K mutation and the known T391S polymorphism, and on the other allele the common P426L mutation, usually associated with the adult or juvenile form of the disease, and the N350S and *96A>G pseudodeficiency mutations. To analyze the contribution of mutations located on the same allele to enzyme activity reduction, as well as the possible phenotype implications, we performed transient expression experiments using arylsulfatase A cDNAs carrying the identified mutations separately and in combination. Our results indicate that mutants containing multiple mutations cause a greater reduction of ARSA activity than do the corresponding single mutants, the total deficiency likely corresponding to the sum of the reductions attributed to each mutation. Consequently, each mutation may contribute to ARSA activity reduction, and, therefore, to the degree of disease severity. This is particularly important for the alleles containing a disease-causing mutation and the pseudodeficiency mutations: in these alleles pseudodeficiency could play a role in affecting the clinical phenotype.

  13. Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay.

    Science.gov (United States)

    Valdez-Flores, Marco A; Vargas-Poussou, Rosa; Verkaart, Sjoerd; Tutakhel, Omar A Z; Valdez-Ortiz, Angel; Blanchard, Anne; Treard, Cyrielle; Hoenderop, Joost G J; Bindels, René J M; Jeleń, Sabina

    2016-12-01

    Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations. Copyright © 2016 the American Physiological Society.

  14. Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C.

    Science.gov (United States)

    Maccubbin, A E; Mudipalli, A; Nadadur, S S; Ersing, N; Gurtoo, H L

    1997-01-01

    Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P-postlabeling and was characterized as a monoadduct of guanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations at G:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations.

  15. Mechanisms of Non-canonical Activation of Ataxia Telangiectasia Mutated.

    Science.gov (United States)

    Khoronenkova, S V

    2016-12-01

    ATM is a master regulator of the cellular response to DNA damage. The classical mechanism of ATM activation involves its monomerization in response to DNA double-strand breaks, resulting in ATM-dependent phosphorylation of more than a thousand substrates required for cell cycle progression, DNA repair, and apoptosis. Here, new experimental evidence for non-canonical mechanisms of ATM activation in response to stimuli distinct from DNA double-strand breaks is discussed. It includes cytoskeletal changes, chromatin modifications, RNA-DNA hybrids, and DNA single-strand breaks. Noncanonical ATM activation may be important for the pathology of the multisystemic disease Ataxia Telangiectasia.

  16. 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.

  17. 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.

  18. Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA

    OpenAIRE

    Miner, Maurine D.; Port, Gary C.; Freitag, Nancy E.

    2008-01-01

    The transcriptional activator PrfA is required for the expression of virulence factors necessary for Listeria monocytogenes pathogenesis. PrfA is believed to become activated following L. monocytogenes entry into the cytosol of infected host cells resulting in the induction of target genes whose products are required for bacterial intracellular growth and cell-to-cell spread. Several mutations have been identified that appear to lock PrfA into its highly activated cytosolic form (known as prf...

  19. Additional prognostic role of EGFR activating mutations in lung adenocarcinoma patients with brain metastasis: integrating with lung specific GPA score.

    Science.gov (United States)

    Lee, Dae-Won; Shin, Dong-Yeop; Kim, Jin Wook; Keam, Bhumsuk; Kim, Tae Min; Kim, Hak Jae; Kim, Dong-Wan; Wu, Hong-Gyun; Paek, Sun Ha; Kim, Young Whan; Heo, Dae Seog; Kim, Dong Gyu; Lee, Se-Hoon

    2014-12-01

    While several prognostic models have been presented in NSCLC patients with brain metastasis, none of these models have included molecular markers as an index. The aim of our study was to evaluate the prognostic value of EGFR mutations and to integrate these EGFR mutations into the prognostic index in NSCLC patients with brain metastasis. We analyzed retrospectively 292 lung adenocarcinoma patients with brain metastasis. Clinico-pathological features and overall survival (OS) were compared between patients with EGFR mutations and patients with EGFR wild type. EGFR mutation status was integrated with lung specific graded prognostic assessment (GPA) score. Among 292 patients, EGFR mutation status was tested in 183 patients. One hundred and five patients (57.4%) had EGFR activating mutations, 14 (7.7%) had EGFR non-activating mutations and 64 (35.0%) had EGFR wild type. OS was significantly longer in patients with EGFR activating mutations than in those with EGFR wild type patients (20.4 vs. 10.1 months, p = 0.002). However, patients with EGFR non-activating mutations did not show superior OS compared with EGFR wild type patients (14.6 vs. 10.1 months, p = 0.83). Multivariate analysis revealed that the presence of EGFR activating mutation is an independent positive prognostic factor for OS (adjusted hazard ratio 0.56, p = 0.002). EGFR activating mutations have a prognostic role in lung adenocarcinoma patients with brain metastasis that is independent of other known prognostic factors. The frequency of EGFR mutation was higher than expected. The presence of EGFR activating mutations should be included as an index in the prognostic models for lung adenocarcinoma patients with brain metastasis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  20. 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.

  1. 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.

  2. Troponin activator augments muscle force in nemaline myopathy patients with nebulin mutations.

    Science.gov (United States)

    de Winter, Josine Marieke; Buck, Danielle; Hidalgo, Carlos; Jasper, Jeffrey R; Malik, Fady I; Clarke, Nigel F; Stienen, Ger J M; Lawlor, Michael W; Beggs, Alan H; Ottenheijm, Coen A C; Granzier, Henk

    2013-06-01

    Nemaline myopathy-the most common non-dystrophic congenital myopathy-is caused by mutations in thin filament genes, of which the nebulin gene is the most frequently affected one. The nebulin gene codes for the giant sarcomeric protein nebulin, which plays a crucial role in skeletal muscle contractile performance. Muscle weakness is a hallmark feature of nemaline myopathy patients with nebulin mutations, and is caused by changes in contractile protein function, including a lower calcium-sensitivity of force generation. To date no therapy exists to treat muscle weakness in nemaline myopathy. Here, we studied the ability of the novel fast skeletal muscle troponin activator, CK-2066260, to augment force generation at submaximal calcium levels in muscle cells from nemaline myopathy patients with nebulin mutations. Contractile protein function was determined in permeabilised muscle cells isolated from frozen patient biopsies. The effect of 5 μM CK-2066260 on force production was assessed. Nebulin protein concentrations were severely reduced in muscle cells from these patients compared to controls, while myofibrillar ultrastructure was largely preserved. Both maximal active tension and the calcium-sensitivity of force generation were lower in patients compared to controls. Importantly, CK-2066260 greatly increased the calcium-sensitivity of force generation-without affecting the cooperativity of activation-in patients to levels that exceed those observed in untreated control muscle. Fast skeletal troponin activation is a therapeutic mechanism to augment contractile protein function in nemaline myopathy patients with nebulin mutations and with other neuromuscular diseases.

  3. TERT Promoter Mutations Lead to High Transcriptional Activity under Hypoxia and Temozolomide Treatment and Predict Poor Prognosis in Gliomas

    OpenAIRE

    Chen Chen; Sheng Han; Lingxuan Meng; Zhonghua Li; Xue Zhang; Anhua Wu

    2014-01-01

    Objective 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. Methods 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 promo...

  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

    The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different...... surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3...

  5. Promiscuous Mutations Activate the Non-Canonical NF-kB Pathway in Multiple Myeloma

    OpenAIRE

    Keats, Jonathan J.; Fonseca, Rafael; Chesi, Marta; Schop, Roelandt; Baker, Angela; Chng, Wee-Joo; Van Wier, Scott; Tiedemann, Rodger; Shi, Chang-Xin; Sebag, Michael; Braggio, Esteban; Henry, Travis; Zhu, Yuan-Xiao; Fogle, Homer; Price-Troska, Tammy

    2007-01-01

    Activation of NF-kB has been noted in many tumor types, however only rarely has this been linked to an underlying genetic mutation. An integrated analysis of high-density oligonucleotide array CGH and gene expression profiling data from 155 multiple myeloma samples identified a promiscuous array of abnormalities contributing to the dysregulation of NF-kB in approximately 20% of patients. We report mutations in ten genes causing the inactivation of TRAF2, TRAF3, CYLD, cIAP1/cIAP2, and activati...

  6. The second activating glucokinase mutation (A456V)

    DEFF Research Database (Denmark)

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

    2002-01-01

    In this study, a second case of hyperinsulinemic hypoglycemia due to activation of glucokinase is reported. The 14-year-old proband had a history of neonatal hypoglycemia, treated with diazoxide. He was admitted with coma and convulsions due to nonketotic hypoglycemia. His BMI was 34 kg/m(2......), and his fasting blood glucose ranged from 2.1 to 2.7 mmol/l, associated with inappropriately high serum levels of insulin, C-peptide, and proinsulin. An oral glucose tolerance test (OGTT) showed exaggerated responses of these peptides followed by profound hypoglycemia. Treatment with diazoxide...... and chlorothiazide was effective. His mother never had clinical hypoglycemic symptoms, even though her fasting blood glucose ranged from 2.9 to 3.5 mmol/l. Increases in serum insulin, C-peptide, and proinsulin in response to an OGTT suggested a lower threshold for glucose-stimulated insulin release (GSIR). Screening...

  7. 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

    2016-09-07

    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.

  8. 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.

  9. 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.

  10. Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3.

    Science.gov (United States)

    Haapaniemi, Emma M; Kaustio, Meri; Rajala, Hanna L M; van Adrichem, Arjan J; Kainulainen, Leena; Glumoff, Virpi; Doffinger, Rainer; Kuusanmäki, Heikki; Heiskanen-Kosma, Tarja; Trotta, Luca; Chiang, Samuel; Kulmala, Petri; Eldfors, Samuli; Katainen, Riku; Siitonen, Sanna; Karjalainen-Lindsberg, Marja-Liisa; Kovanen, Panu E; Otonkoski, Timo; Porkka, Kimmo; Heiskanen, Kaarina; Hänninen, Arno; Bryceson, Yenan T; Uusitalo-Seppälä, Raija; Saarela, Janna; Seppänen, Mikko; Mustjoki, Satu; Kere, Juha

    2015-01-22

    The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Activating ESR1 mutations in hormone-resistant metastatic breast cancer.

    Science.gov (United States)

    Robinson, Dan R; Wu, Yi-Mi; Vats, Pankaj; Su, Fengyun; Lonigro, Robert J; Cao, Xuhong; Kalyana-Sundaram, Shanker; Wang, Rui; Ning, Yu; Hodges, Lynda; Gursky, Amy; Siddiqui, Javed; Tomlins, Scott A; Roychowdhury, Sameek; Pienta, Kenneth J; Kim, Scott Y; Roberts, J Scott; Rae, James M; Van Poznak, Catherine H; Hayes, Daniel F; Chugh, Rashmi; Kunju, Lakshmi P; Talpaz, Moshe; Schott, Anne F; Chinnaiyan, Arul M

    2013-12-01

    Breast cancer is the most prevalent cancer in women, and over two-thirds of cases express estrogen receptor-α (ER-α, encoded by ESR1). Through a prospective clinical sequencing program for advanced cancers, we enrolled 11 patients with ER-positive metastatic breast cancer. Whole-exome and transcriptome analysis showed that six cases harbored mutations of ESR1 affecting its ligand-binding domain (LBD), all of whom had been treated with anti-estrogens and estrogen deprivation therapies. A survey of The Cancer Genome Atlas (TCGA) identified four endometrial cancers with similar mutations of ESR1. The five new LBD-localized ESR1 mutations identified here (encoding p.Leu536Gln, p.Tyr537Ser, p.Tyr537Cys, p.Tyr537Asn and p.Asp538Gly) were shown to result in constitutive activity and continued responsiveness to anti-estrogen therapies in vitro. Taken together, these studies suggest that activating mutations in ESR1 are a key mechanism in acquired endocrine resistance in breast cancer therapy.

  17. Effects of amino-terminal extensions and specific mutations on the activity of restrictocin.

    Science.gov (United States)

    Yang, R; Kenealy, W R

    1992-08-25

    The cytotoxic activities of restrictocin with aminoterminal extensions and specific mutations were investigated using in vivo and in vitro systems. Genes were constructed from the cDNA clone of restrictocin which encode: the native form of restrictocin (including the leader sequence); Met-prorestrictocin, in which a codon for methionine was placed before a putative pro region; Met-mature restrictocin, with a methionine codon prior to the mature form of restrictocin; and three mutated forms of Met-mature restrictocin, E95G, E115G/H136L, and H136L. These constructions were placed under the control of the GAL1 promoter and were transformed into Saccharomyces cerevisiae. Transformants were killed, and a new RNA band formed when any of these genes except those containing the H136L mutation were expressed. Restrictocin protein was detected by immunoblot only in cells expressing the native form of restrictocin and the forms containing the H136L mutation. Native restrictocin, Met-prorestrictocin, and Met-mature restrictocin mRNA were translated in an in vitro system resulting in proteins of the expected molecular weight and inactivation of the translation system. Restrictocin was not inactivated by the presence of the leader sequence and the putative prosequence. Amino acid His136 is putatively in the active site of restrictocin by analogy to ribonuclease U2 and the elimination of toxic effects in the S. cerevisiae expression and in vitro translation systems.

  18. An activating Pik3ca mutation coupled with Pten loss is sufficient to initiate ovarian tumorigenesis in mice.

    Science.gov (United States)

    Kinross, Kathryn M; Montgomery, Karen G; Kleinschmidt, Margarete; Waring, Paul; Ivetac, Ivan; Tikoo, Anjali; Saad, Mirette; Hare, Lauren; Roh, Vincent; Mantamadiotis, Theo; Sheppard, Karen E; Ryland, Georgina L; Campbell, Ian G; Gorringe, Kylie L; Christensen, James G; Cullinane, Carleen; Hicks, Rodney J; Pearson, Richard B; Johnstone, Ricky W; McArthur, Grant A; Phillips, Wayne A

    2012-02-01

    Mutations in the gene encoding the p110α subunit of PI3K (PIK3CA) that result in enhanced PI3K activity are frequently observed in human cancers. To better understand the role of mutant PIK3CA in the initiation or progression of tumorigenesis, we generated mice in which a PIK3CA mutation commonly detected in human cancers (the H1047R mutation) could be conditionally knocked into the endogenous Pik3ca locus. Activation of this mutation in the mouse ovary revealed that alone, Pik3caH1047R induced premalignant hyperplasia of the ovarian surface epithelium but no tumors. Concomitantly, we analyzed several human ovarian cancers and found PIK3CA mutations coexistent with KRAS and/or PTEN mutations, raising the possibility that a secondary defect in a co-regulator of PI3K activity may be required for mutant PIK3CA to promote transformation. Consistent with this notion, we found that Pik3caH1047R mutation plus Pten deletion in the mouse ovary led to the development of ovarian serous adenocarcinomas and granulosa cell tumors. Both mutational events were required for early, robust Akt activation. Pharmacological inhibition of PI3K/mTOR in these mice delayed tumor growth and prolonged survival. These results demonstrate that the Pik3caH1047R mutation with loss of Pten is enough to promote ovarian cell transformation and that we have developed a model system for studying possible therapies.

  19. Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits.

    Science.gov (United States)

    Pavlov, Michael Y; Zorzet, Anna; Andersson, Dan I; Ehrenberg, Måns

    2011-01-19

    We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.

  20. Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits

    Science.gov (United States)

    Pavlov, Michael Y; Zorzet, Anna; Andersson, Dan I; Ehrenberg, Måns

    2011-01-01

    We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNAi). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNAi, implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNAi and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10−4 for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases. PMID:21151095

  1. Improving Polymerase Activity with Unnatural Substrates by Sampling Mutations in Homologous Protein Architectures.

    Science.gov (United States)

    Dunn, Matthew R; Otto, Carine; Fenton, Kathryn E; Chaput, John C

    2016-05-20

    The ability to synthesize and propagate genetic information encoded in the framework of xeno-nucleic acid (XNA) polymers would inform a wide range of topics from the origins of life to synthetic biology. While directed evolution has produced examples of engineered polymerases that can accept XNA substrates, these enzymes function with reduced activity relative to their natural counterparts. Here, we describe a biochemical strategy that enables the discovery of engineered polymerases with improved activity for a given unnatural polymerase function. Our approach involves identifying specificity determining residues (SDRs) that control polymerase activity, screening mutations at SDR positions in a model polymerase scaffold, and assaying key gain-of-function mutations in orthologous protein architectures. By transferring beneficial mutations between homologous protein structures, we show that new polymerases can be identified that function with superior activity relative to their starting donor scaffold. This concept, which we call scaffold sampling, was used to generate engineered DNA polymerases that can faithfully synthesize RNA and TNA (threose nucleic acid), respectively, on a DNA template with high primer-extension efficiency and low template sequence bias. We suggest that the ability to combine phenotypes from different donor and recipient scaffolds provides a new paradigm in polymerase engineering where natural structural diversity can be used to refine the catalytic activity of synthetic enzymes.

  2. 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.

  3. 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.

  4. Activating glucokinase (GCK) mutations as a cause of medically responsive congenital hyperinsulinism: prevalence in children and characterisation of a novel GCK mutation

    DEFF Research Database (Denmark)

    Christesen, Henrik B T; Tribble, Nicholas D; Molven, Anders;

    2008-01-01

    OBJECTIVE: Activating glucokinase (GCK) mutations are a rarely reported cause of congenital hyperinsulinism (CHI), but the prevalence of GCK mutations is not known. METHODS: From a pooled cohort of 201 non-syndromic children with CHI from three European referral centres (Denmark, n=141; Norway, n......=26; UK, n=34), 108 children had no K(ATP)-channel (ABCC8/KCNJ11) gene abnormalities and were screened for GCK mutations. Novel GCK mutations were kinetically characterised. RESULTS: In five patients, four heterozygous GCK mutations (S64Y, T65I, W99R and A456V) were identified, out of which S64Y...... was novel. Two of the mutations arose de novo, three were dominantly inherited. All the five patients were medically responsive. In the combined Danish and Norwegian cohort, the prevalence of GCK-CHI was estimated to be 1.2% (2/167, 95% confidence interval (CI) 0-2.8%) of all the CHI patients. In the three...

  5. Catalytic activity of tripeptidase from Lactococcus lactis to which amino acid substitution was introduced according to natural mutation.

    Science.gov (United States)

    Mori, Sumiko; Kaneko, Satoshi; Kasumi, Takafumi

    2004-05-01

    Four mutations observed between tripeptidases from Lactococcus lactis subsp. lactis and subsp. cremoris were introduced one by one to the corresponding points in wild-type tripeptidase from L. lactis subsp. lactis. The k(cat) values of four resultant mutants were analyzed and discussed in stereographical terms. Change in catalytic activity appeared to be related to the sequential and steric location of mutation point within the enzyme protein, even though no drastic change was observed with one point mutation.

  6. Oculocutaneous Albinism Type 1: Link between Mutations, Tyrosinase Conformational Stability, and Enzymatic Activity

    Science.gov (United States)

    Dolinska, Monika B.; Kus, Nicole; Farney, Katie; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2017-01-01

    Summary Oculocutaneous albinism Type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intra-melanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities- tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants show very low protein expression, protein yield, and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactive tyrosinase and results in OCA1B albinism. PMID:27775880

  7. 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.

  8. 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.

  9. Sperm enzyme activity analysis of individual sperm for detection of heritable mutations in mammals.

    Science.gov (United States)

    Panda, B B; Ficsor, G; Ginsberg, L C

    1988-03-15

    Male mice were injected i.p. with 2.5 mg/kg mitomycin C, 100 mg/kg ethyl nitrosourea or saline and mated with untreated virgin females five weeks later. Sperm from 64 of the F1 male progeny were analyzed histochemically for acrosin, succinic dehydrogenase and alpha-glycerophosphate dehydrogenase activity. The frequency of F1 males with sub-normal sperm enzyme activity was significantly higher among progeny from treated males than in controls. These results show that analysis of sperm enzyme activity in F1 males is a practical method for detection of transmitted mutations induced in a treated parent.

  10. 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.

  11. 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

  12. Improvement of Bacillus circulans beta-amylase activity attained using the ancestral mutation method.

    Science.gov (United States)

    Yamashiro, Kan; Yokobori, Shin-ichi; Koikeda, Satoshi; Yamagishi, Akihiko

    2010-07-01

    Thermostabilization of enzymes is one of the greatest challenges of protein engineering. The ancestral mutation method, which introduces ancestral residues into a target enzyme, has previously been developed and used to improve the thermostabilities of thermophilic enzymes. Herein, we report a study that used the ancestral mutation method to improve the thermostability of Bacillus circulans beta-amylase, a mesophilic enzyme. A bacterial, common-ancestral beta-amylase sequence was inferred using a phylogenetic tree composed of higher plant and bacterial amylase sequences. Eighteen mutants containing ancestral residues were designed, expressed in Escherichia coli and purified. Several of these mutants were more thermostable than that of the wild-type amylase. Notably, one mutant had both greater activity and greater thermostability. The relationship between the extent to which the amino acid residues within 5 A of the mutation site were evolutionarily conserved and the extent to which thermostability was improved was examined. Apparently, it is necessary to conserve the residues surrounding an ancestral residue if thermostability is to be improved by the ancestral mutation method.

  13. A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

    Science.gov (United States)

    Katz, W S; Lesa, G M; Yannoukakos, D; Clandinin, T R; Schlessinger, J; Sternberg, P W

    1996-01-01

    The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates. PMID:8552080

  14. 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.

  15. The effects of expression of an activated rasG mutation on the differentiation of Dictyostelium.

    Science.gov (United States)

    Thiery, R; Robbins, S; Khosla, M; Spiegelman, G B; Weeks, G

    1992-01-01

    Dictyostelium discoideum contains two ras genes, rasG and rasD, that are expressed during growth and differentiation, respectively. It was shown previously that Dictyostelium transformants expressing an activated rasD gene (a mutation producing a change in amino acid 12 from glycine to threonine) developed abnormally. When developed on filters these transformants formed multitipped aggregates, which did not go on to produce final fruiting bodies, but in a submerged culture assay on a plastic surface they either formed small aggregates or did not aggregate. In this study we transformed cells with the rasG gene, mutated to change amino acid 12 from glycine to threonine. The resulting transformants developed normally on filters, but aggregation under other conditions was impaired. In particular, in submerged culture on a plastic surface they either produced very small aggregates or did not aggregate, one of the phenotypes exhibited by the activated rasD transformants. Molecular analysis of the transformants revealed the presence of high copy numbers of the mutated rasG gene, but the level of expression of the mutant gene never exceeded the level of expression of the endogenous gene. These results indicate a powerful dominant effect of a relatively small amount of the activated RasG protein in Dictyostelium.

  16. Probing the effect of MODY mutations near the co-activator-binding pocket of HNF4α.

    Science.gov (United States)

    Rha, Geun Bae; Wu, Guangteng; Chi, Young-In

    2011-10-01

    HNF4α (hepatocyte nuclear factor 4α) is a culprit gene product for a monogenic and dominantly inherited form of diabetes, referred to as MODY (maturity onset diabetes of the young). As a member of the NR (nuclear receptor) superfamily, HNF4α recruits transcriptional co-activators such as SRC-1α (steroid receptor co-activator-1α) and PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) through the LXXLL-binding motifs for its transactivation, and our recent crystal structures of the complex provided the molecular details and the mechanistic insights into these co-activator recruitments. Several mutations have been identified from the MODY patients and, among these, point mutations can be very instructive site-specific measures of protein function and structure. Thus, in the present study, we probed the functional effects of the two MODY point mutations (D206Y and M364R) found directly near the LXXLL motif-binding site by conducting a series of experiments on their structural integrity and specific functional roles such as overall transcription, ligand selectivity, target gene recognition and co-activator recruitment. While the D206Y mutation has a subtle effect, the M364R mutation significantly impaired the overall transactivation by HNF4α. These functional disruptions are mainly due to their reduced ability to recruit co-activators and lowered protein stability (only with M364R mutation), while their DNA-binding activities and ligand selectivities are preserved. These results confirmed our structural predictions and proved that MODY mutations are loss-of-function mutations leading to impaired β-cell function. These findings should help target selective residues for correcting mutational defects or modulating the overall activity of HNF4α as a means of therapeutic intervention.

  17. KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes.

    Science.gov (United States)

    Massa, Ornella; Iafusco, Dario; D'Amato, Elena; Gloyn, Anna L; Hattersley, Andrew T; Pasquino, Bruno; Tonini, Giorgio; Dammacco, Francesco; Zanette, Giorgio; Meschi, Franco; Porzio, Ottavia; Bottazzo, Gianfranco; Crinó, Antonino; Lorini, Renata; Cerutti, Franco; Vanelli, Maurizio; Barbetti, Fabrizio

    2005-01-01

    Permanent neonatal diabetes mellitus (PNDM) is a rare condition characterized by severe hyperglycemia constantly requiring insulin treatment from its onset. Complete deficiency of glucokinase (GCK) can cause PNDM; however, the genetic etiology is unknown in most PNDM patients. Recently, heterozygous activating mutations of KCNJ11, encoding Kir6.2, the pore forming subunit of the ATP-dependent potassium (K(ATP)) channel of the pancreatic beta-cell, were found in patients with PNDM. Closure of the K(ATP) channel exerts a pivotal role in insulin secretion by modifying the resting membrane potential that leads to insulin exocytosis. We screened the KCNJ11 gene in 12 Italian patients with PNDM (onset within 3 months from birth) and in six patients with non-autoimmune, insulin-requiring diabetes diagnosed during the first year of life. Five different heterozygous mutations were identified: c.149G>C (p.R50P), c.175G>A (p.V59M), c.509A>G (p.K170R), c.510G>C (p.K170N), and c.601C>T (p.R201C) in eight patients with diabetes diagnosed between day 3 and 182. Mutations at Arg50 and Lys170 residues are novel. Four patients also presented with motor and/or developmental delay as previously reported. We conclude that KCNJ11 mutations are a common cause of PNDM either in isolation or associated with developmental delay. Permanent diabetes of non autoimmune origin can present up to 6 months from birth in individuals with KCNJ11 and EIF2AK3 mutations. Therefore, we suggest that the acronym PNDM be replaced with the more comprehensive permanent diabetes mellitus of infancy (PDMI), linking it to the gene product (e.g., GCK-PDMI, KCNJ11-PDMI) to avoid confusion between patients with early-onset, autoimmune type 1 diabetes.

  18. 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.

  19. LYN-activating mutations mediate antiestrogen resistance in estrogen receptor–positive breast cancer

    Science.gov (United States)

    Schwarz, Luis J.; Fox, Emily M.; Balko, Justin M.; Garrett, Joan T.; Kuba, María Gabriela; Estrada, Mónica Valeria; González-Angulo, Ana María; Mills, Gordon B.; Red-Brewer, Monica; Mayer, Ingrid A.; Abramson, Vandana; Rizzo, Monica; Kelley, Mark C.; Meszoely, Ingrid M.; Arteaga, Carlos L.

    2014-01-01

    Estrogen receptor–positive (ER+) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER+ tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER+ breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER+ LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYND189Y has higher catalytic activity than WT protein. Further, LYND189Y exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYNWT. Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYND189Y overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER+ breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER+ xenografts but not LYND189Y-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER+ breast cancers. PMID:25401474

  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...... mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning...

  1. Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models.

    Science.gov (United States)

    Taylor, James G; Cheuk, Adam T; Tsang, Patricia S; Chung, Joon-Yong; Song, Young K; Desai, Krupa; Yu, Yanlin; Chen, Qing-Rong; Shah, Kushal; Youngblood, Victoria; Fang, Jun; Kim, Su Young; Yeung, Choh; Helman, Lee J; Mendoza, Arnulfo; Ngo, Vu; Staudt, Louis M; Wei, Jun S; Khanna, Chand; Catchpoole, Daniel; Qualman, Stephen J; Hewitt, Stephen M; Merlino, Glenn; Chanock, Stephen J; Khan, Javed

    2009-11-01

    Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS.

  2. 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.

  3. 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

    % 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......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......'s skeletal muscle, suggesting that virtually no truncated receptor was expressed. Receptor kinase activity was, however, reduced by 95 and 91% in the compound heterozygous brothers. This suggests that the mother's mutated allele contributes little to the generation of functional receptor protein...

  4. Matriptase-2 mutations in iron-refractory iron deficiency anemia patients provide new insights into protease activation mechanisms.

    Science.gov (United States)

    Ramsay, Andrew J; Quesada, Victor; Sanchez, Mayka; Garabaya, Cecilia; Sardà, María P; Baiget, Montserrat; Remacha, Angel; Velasco, Gloria; López-Otín, Carlos

    2009-10-01

    Mutations leading to abrogation of matriptase-2 proteolytic activity in humans are associated with an iron-refractory iron deficiency anemia (IRIDA) due to elevated hepcidin levels. Here we describe two novel heterozygous mutations within the matriptase-2 (TMPRSS6) gene of monozygotic twin girls exhibiting an IRIDA phenotype. The first is the frameshift mutation (P686fs) caused by the insertion of the four nucleotides CCCC in exon 16 (2172_2173insCCCC) that is predicted to terminate translation before the catalytic serine. The second mutation is the di-nucleotide substitution c.467C>A and c.468C>T in exon 3 that causes the missense mutation A118D in the SEA domain of the extracellular stem region of matriptase-2. Functional analysis of both variant matriptase-2 proteases has revealed that they lead to ineffective suppression of hepcidin transcription. We also demonstrate that the A118D SEA domain mutation causes an intra-molecular structural imbalance that impairs matriptase-2 activation. Collectively, these results extend the pattern of TMPRSS6 mutations associated with IRIDA and functionally demonstrate that mutations affecting protease regions other than the catalytic domain may have a profound impact in the regulatory role of matriptase-2 during iron deficiency.

  5. Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations.

    Science.gov (United States)

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir; Wang, KeWei; Zheng, Jie

    2012-04-01

    The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate.

  6. Mutations that extend the specificity of the endonuclease activity of lambda terminase.

    Science.gov (United States)

    Arens, J S; Hang, Q; Hwang, Y; Tuma, B; Max, S; Feiss, M

    1999-01-01

    Terminase, an enzyme encoded by the Nu1 and A genes of bacteriophage lambda, is crucial for packaging concatemeric DNA into virions. cosN, a 22-bp segment, is the site on the virus chromosome where terminase introduces staggered nicks to cut the concatemer to generate unit-length virion chromosomes. Although cosN is rotationally symmetric, mutations in cosN have asymmetric effects. The cosN G2C mutation (a G-to-C change at position 2) in the left half of cosN reduces the phage yield 10-fold, whereas the symmetric mutation cosN C11G, in the right half of cosN, does not affect the burst size. The reduction in phage yield caused by cosN G2C is correlated with a defect in cos cleavage. Three suppressors of the cosN G2C mutation, A-E515G, A-N509K, and A-R504C, have been isolated that restore the yield of lambda cosN G2C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. lambda A-E515G, A-N509K, and A-R504C phages, which are cosN+, also had wild-type burst sizes. In vitro cos cleavage experiments on cosN G2C C11G DNA showed that the rate of cleavage for A-E515G terminase is three- to fourfold higher than for wild-type terminase. The A-E515G mutation changes residue 515 of gpA from glutamic acid to glycine. Uncharged polar and hydrophobic residues at position 515 suppressed the growth defect of lambda cosN G2C C11G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of lambda cosN G2C C11G. In a lambda cosN+ background, all amino acids tested at position 515 were functional. These results suggest that A-E515G plays an indirect role in extending the specificity of the endonuclease activity of lambda terminase.

  7. Pore mutations in ammonium transporter AMT1 with increased electrogenic ammonium transport activity.

    Science.gov (United States)

    Loqué, Dominique; Mora, Silvia I; Andrade, Susana L A; Pantoja, Omar; Frommer, Wolf B

    2009-09-11

    AMT/Mep ammonium transporters mediate high affinity ammonium/ammonia uptake in bacteria, fungi, and plants. The Arabidopsis AMT1 proteins mediate uptake of the ionic form of ammonium. AMT transport activity is controlled allosterically via a highly conserved cytosolic C terminus that interacts with neighboring subunits in a trimer. The C terminus is thus capable of modulating the conductivity of the pore. To gain insight into the underlying mechanism, pore mutants suppressing the inhibitory effect of mutations in the C-terminal trans-activation domain were characterized. AMT1;1 carrying the mutation Q57H in transmembrane helix I (TMH I) showed increased ammonium uptake but reduced capacity to take up methylammonium. To explore whether the transport mechanism was altered, the AMT1;1-Q57H mutant was expressed in Xenopus oocytes and analyzed electrophysiologically. AMT1;1-Q57H was characterized by increased ammonium-induced and reduced methylammonium-induced currents. AMT1;1-Q57H possesses a 100x lower affinity for ammonium (K(m)) and a 10-fold higher V(max) as compared with the wild type form. To test whether the trans-regulatory mechanism is conserved in archaeal homologs, AfAmt-2 from Archaeoglobus fulgidus was expressed in yeast. The transport function of AfAmt-2 also depends on trans-activation by the C terminus, and mutations in pore-residues corresponding to Q57H of AMT1;1 suppress nonfunctional AfAmt-2 mutants lacking the activating C terminus. Altogether, our data suggest that bacterial and plant AMTs use a conserved allosteric mechanism to control ammonium flux, potentially using a gating mechanism that limits flux to protect against ammonium toxicity.

  8. Activating Mutations in ERBB2 and Their Impact on Diagnostics and Treatment

    Science.gov (United States)

    Herter-Sprie, Grit S.; Greulich, Heidi; Wong, Kwok-Kin

    2013-01-01

    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/HER2, 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. PMID:23630663

  9. 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.

  10. The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.

    Science.gov (United States)

    Ando, Maya; Fiesel, Fabienne C; Hudec, Roman; Caulfield, Thomas R; Ogaki, Kotaro; Górka-Skoczylas, Paulina; Koziorowski, Dariusz; Friedman, Andrzej; Chen, Li; Dawson, Valina L; Dawson, Ted M; Bu, Guojun; Ross, Owen A; Wszolek, Zbigniew K; Springer, Wolfdieter

    2017-04-24

    Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays. Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon

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

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    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

  12. 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

  13. Modeling tumor predisposing FH mutations in yeast: effects on fumarase activity, growth phenotype and gene expression profile.

    Science.gov (United States)

    Kokko, Antti; Ylisaukko-Oja, Sanna S K; Kiuru, Maija; Takatalo, Maarit S; Salmikangas, Paula; Tuimala, Jarno; Arango, Diego; Karhu, Auli; Aaltonen, Lauri A; Jäntti, Jussi

    2006-03-15

    Heterozygous mutations in the fumarase (FH) gene cause the tumor predisposition syndrome hereditary leiomyomatosis and renal cell cancer (MIM 605839). While most families segregate a benign phenotype of multiple leiomyomas, others display a phenotype with early-onset renal cancer and leiomyosarcoma. Modifier genes may play a role in this, but an alternative explanation is simple genotype-phenotype association. FH mutations predisposing to cancer appear to be truncating or in fully conserved amino acids, suggesting that mutations severely affecting FH activity might predispose to malignancy. In the present study, we analyzed 2 conserved fumarase mutations in yeast. H153R has been described in 3 cancer predisposition families; whereas all 3 reported K187R families have displayed the benign phenotype. Examining H153R and K187R should clarify whether cancer-related FH mutations differ from their benign phenotype-associated counterparts. Yeast strains containing the 2 mutations, and knockout and wild type (WT) references, were created and the growth phenotypes studied on selected carbon sources to assess mitochondrial function. Additionally, Fum1 protein production and activity were measured, and the strains were subjected to transcriptional profiling. On nonfermentable lactate medium, the fumarase knockout strains did not grow, whereas the mutants showed no differences, as compared to WT yeast. Although both mutant strains produced fumarase, a considerable decrease in enzyme activity was seen in mutants with respect to WT. Transcription of the majority of Krebs cycle enzymes was downregulated in response to mutations in fumarase. In conclusion, both mutants displayed some, albeit greatly reduced, fumarase activity. This activity was sufficient to support normal growth on nonfermentable carbon source, unlike the deletion phenotype, demonstrating the significance of the residual activity. The findings support the hypothesis that modifier gene(s), rather than phenotype

  14. Mutation analysis of mitogen activated protein kinase 1 gene in Indian cases of 46,XY disorder of sex development

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    Dhanjit Kumar Das

    2013-01-01

    Full Text Available Background: Determination of sex is the result of cascade of molecular events that cause undifferentiated bipotential gonad to develop as a testis or an ovary. A series of genes such as SRY, steroidogenic factor-1 (SF1, AR, SRD5 α, Desert hedgehog (DHH etc., have been reported to have a significant role in development of sex in the fetus and secondary sexual characteristics at the time of puberty. Recently, mitogen activated protein kinase kinase kinase 1 (MAP3K1 gene was found to be associated with 46, XY disorders of sex development (DSD. Aim: The present study is focused to identify mutations in MAP3K1 gene in the cohort of 10 Indian patients with 46,XY DSD including one family with two affected sisters. These patients were already screened for SRY, SF1 and DHH gene, but no mutation was observed in any of these genes. Materials and Methods: The entire coding regions of MAP3K1 were amplified and sequenced using the gene specific primers. Results and Discussions: Sequence analysis of MAP3K1 gene has revealed four variants including one missense, two silent and one deletion mutation. The missense mutation p.D806N was observed in four patients with hypospadias. Two patients showed the presence of silent mutation p.Q1028Q present in exon 14. Another silent mutation p.T428T was observed in a patient with gonadal dysgenesis. We have also observed one deletion mutation p. 942insT present in two patients. The pathogenicity of the missense mutation p.D806N was carried out using in-silico approach. Sequence homology analysis has revealed that the aspartate at 806 was found to be well-conserved across species, indicated the importance of this residue. The score for polyphen analysis of this mutation was found to be 0.999 indicating to be pathogenic mutation. Since, p.D806N mutation was found to be important residue; it might contribute to sexual development. We have reported the presence of mutations/polymorphism in MAP3K1 gene. All the mutations were

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

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    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.

  16. LYN-activating mutations mediate antiestrogen resistance in estrogen receptor-positive breast cancer.

    Science.gov (United States)

    Schwarz, Luis J; Fox, Emily M; Balko, Justin M; Garrett, Joan T; Kuba, María Gabriela; Estrada, Mónica Valeria; González-Angulo, Ana María; Mills, Gordon B; Red-Brewer, Monica; Mayer, Ingrid A; Abramson, Vandana; Rizzo, Monica; Kelley, Mark C; Meszoely, Ingrid M; Arteaga, Carlos L

    2014-12-01

    Estrogen receptor-positive (ER(+)) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER(+) tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER(+) breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER(+) LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYN(D189Y) has higher catalytic activity than WT protein. Further, LYN(D189Y) exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYN(WT). Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYN(D189Y) overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER(+) breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER(+) xenografts but not LYN(D189Y)-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER(+) breast cancers.

  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. Prognostic quality of activating TERT promoter mutations in glioblastoma: interaction with the rs2853669 polymorphism and patient age at diagnosis.

    Science.gov (United States)

    Spiegl-Kreinecker, Sabine; Lötsch, Daniela; Ghanim, Bahil; Pirker, Christine; Mohr, Thomas; Laaber, Magdalena; Weis, Serge; Olschowski, Alfred; Webersinke, Gerald; Pichler, Josef; Berger, Walter

    2015-09-01

    Expression of the telomerase reverse transcriptase (TERT) might be altered by activating mutations of the rs2853669 polymorphism within the promoter region. Here we investigate the impact of these genomic alterations on telomerase activation and dissect their prognostic potential in glioblastoma (GBM). The respective TERT promoter region was sequenced in 126 GBM tissues and compared with clinical parameters and glioma biomarkers MGMT promoter methylation and IDH1 mutation. TERT mRNA expression, telomerase activity, and telomere lengths were determined by reverse transcriptase PCR, TRAP assay, and real-time PCR, respectively. Seventy-three percent of GBM patients harbored TERT promoter mutations associated with enhanced telomerase activity and TERT mRNA expression but reduced telomere lengths (P < .001 for all). Patients with mutated tumors exhibited significantly shorter overall survival in the entire cohort (11.5 vs 23.1 months; P < .0001) and in the primary GBM patient subgroup lacking IDH1 mutations (n = 120; P = .0084). This prognostic impact was confined to younger patients (aged <65 years), while the negative prognostic power of enhanced age at diagnosis was limited to those patients lacking TERT promoter mutations. Presence of the common single nucleotide polymorphism rs2853669, disrupting an endogenous Ets2 transcription factor-binding site, was associated with improved survival exclusively in patients with a wild-type TERT promoter. On the contrary, the shortest mean overall survival was detected in those patients harboring both an activating TERT promoter mutation and homozygous rs2853669 alleles. In summary, TERT promoter mutations are powerful prognosticators for worse course of disease in human GBM patients but their prognostic value is influenced by the rs2853669 polymorphism and age at diagnosis. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e

  19. Hotspot mutations in KIT receptor differentially modulate its allosterically coupled conformational dynamics: impact on activation and drug sensitivity.

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    Isaure Chauvot de Beauchêne

    2014-07-01

    Full Text Available Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D localized in crucial regulatory segments, the juxtamembrane region (JMR and the activation (A- loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts.

  20. Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

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    Miriam Kaltenbach

    2016-10-01

    Full Text Available The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1 from phosphotriesterase to arylesterase, and (2 from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak

  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. Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel.

    Science.gov (United States)

    Rojas, C V; Neely, A; Velasco-Loyden, G; Palma, V; Kukuljan, M

    1999-01-01

    Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant alpha-subunits with the beta1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.

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

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    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. The circadian Clock mutation increases exploratory activity and escape-seeking behavior.

    Science.gov (United States)

    Easton, A; Arbuzova, J; Turek, F W

    2003-02-01

    Disturbances of circadian rhythms are associated with many types of mood disorders; however, it is unknown whether a dysfunctional circadian pacemaker can be the primary cause of altered emotional behavior. To test this hypothesis, male and female mice carrying a mutation of the circadian gene, Clock, were compared to wild-type mice in an array of behavioral tests used to measure exploratory activity, anxiety, and behavioral despair. Female Clock mutant mice exhibited significantly greater activity and rearing in an open field and a greater number of total arm entries in the elevated plus maze. In addition, female Clock mutant mice spent significantly more time swimming in the forced swim test than wild-type mice on both days of a 2-day test. Male Clock mutant mice also exhibited increased exploration of the open field and increased swimming in the forced swim test; however, behavioral changes were less robust in Clock mutant males compared to Clock mutant females. These changes in behavior were not dependent on the expression of a lengthened free-running period but were more or less striking depending on the testing conditions. These data indicate that the Clock mutation leads to increased exploratory behavior and increased escape-seeking behavior, and, conversely, does not result in increased anxiety or depressive-like behavior. These results suggest that the Clock gene is involved in regulating behavioral arousal, and that Clock may interact with sex hormones to produce these behavioral changes.

  5. 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.

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

    Science.gov (United States)

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

    2015-10-01

    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 pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-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.

  7. Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction.

    Science.gov (United States)

    Kiss, Márton; Kiss, András A; Radics, Monika; Popovics, Nikoletta; Hermesz, Edit; Csiszár, Katalin; Mink, Mátyás

    2016-01-01

    The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that

  8. Activating mutations of the TRPML1 channel revealed by proline-scanning mutagenesis.

    Science.gov (United States)

    Dong, Xian-ping; Wang, Xiang; Shen, Dongbiao; Chen, Su; Liu, Meiling; Wang, Yanbin; Mills, Eric; Cheng, Xiping; Delling, Markus; Xu, Haoxing

    2009-11-13

    The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1(V432P)) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1(V432P)-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca(2+)-dependent, constitutive Ca(2+) permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca(2+) release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca(2+) and Fe(2+)/Mn(2+) dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the

  9. 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.

  10. A study of KIT activating mutations in acute myeloid leukemia M0 ...

    African Journals Online (AJOL)

    Syed Rizwan Hussain

    2012-03-04

    Mar 4, 2012 ... tify and determine the frequency and prevalence of mutations in North Indian patients suffering ..... Table 2 Amino acid sequences of KIT gene point mutations at exon .... primary adenoid cystic carcinoma of the salivary gland.

  11. 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.

  12. 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...

  13. 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.

  14. Psoriasis mutations disrupt CARD14 autoinhibition promoting BCL10-MALT1-dependent NF-κB activation.

    Science.gov (United States)

    Howes, Ashleigh; O'Sullivan, Paul A; Breyer, Felix; Ghose, Ashavari; Cao, Li; Krappmann, Daniel; Bowcock, Anne M; Ley, Steven C

    2016-06-15

    Inherited and de novo mutations in the CARD14 gene promote the development of psoriasis, an inflammatory disease of the skin. Caspase recruitment domain-containing protein 14 (CARD14) is a member of the CARMA protein family that includes the structurally related CARD11 adaptor that mediates NF-κB activation by antigen receptors. We investigated the mechanism by which CARD14 mutation in psoriasis activates NF-κB. In contrast with wild-type CARD14, CARD14(E138A) and CARD14(G117S) psoriasis mutants interacted constitutively with BCL10 and MALT1, and triggered BCL10- and MALT1-dependent activation of NF-κB in keratinocytes. These alterations disrupted the inhibitory effect of the CARD14 linker region (LR) on NF-κB activation by facilitating BCL10 binding. Therefore, psoriasis mutations activated CARD14 by a mechanism analogous to oncogenic CARD11 mutations in non-Hodgkin B cell lymphomas. CARD14(E138A) also stimulated MALT1 paracaspase activity and activated both ERK1/2 and p38α MAP kinases. Inhibition of MALT1 with mepazine reduced CARD14(E138A)-induced expression of specific psoriasis-associated transcripts in keratinocytes. Our results establish the mechanism whereby gain-of-function CARD14 variants, which induce psoriatic disease in affected individuals, activate pro-inflammatory signalling.

  15. Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks.

    Science.gov (United States)

    Sordet, Olivier; Redon, Christophe E; Guirouilh-Barbat, Josée; Smith, Susan; Solier, Stéphanie; Douarre, Céline; Conti, Chiara; Nakamura, Asako J; Das, Benu B; Nicolas, Estelle; Kohn, Kurt W; Bonner, William M; Pommier, Yves

    2009-08-01

    Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

  16. Mdm2 RING mutation enhances p53 transcriptional activity and p53-p300 interaction.

    Directory of Open Access Journals (Sweden)

    Hilary V Clegg

    Full Text Available The p53 transcription factor and tumor suppressor is regulated primarily by the E3 ubiquitin ligase Mdm2, which ubiquitinates p53 to target it for proteasomal degradation. Aside from its ubiquitin ligase function, Mdm2 has been believed to be capable of suppressing p53's transcriptional activity by binding with and masking the transactivation domain of p53. The ability of Mdm2 to restrain p53 activity by binding alone, without ubiquitination, was challenged by a 2007 study using a knockin mouse harboring a single cysteine-to-alanine point mutation (C462A in Mdm2's RING domain. Mouse embryonic fibroblasts with this mutation, which abrogates Mdm2's E3 ubiquitin ligase activity without affecting its ability to bind with p53, were unable to suppress p53 activity. In this study, we utilized the Mdm2(C462A mouse model to characterize in further detail the role of Mdm2's RING domain in the control of p53. Here, we show in vivo that the Mdm2(C462A protein not only fails to suppress p53, but compared to the complete absence of Mdm2, Mdm2(C462A actually enhances p53 transcriptional activity toward p53 target genes p21/CDKN1A, MDM2, BAX, NOXA, and 14-3-3σ. In addition, we found that Mdm2(C462A facilitates the interaction between p53 and the acetyltransferase CBP/p300, and it fails to heterodimerize with its homolog and sister regulator of p53, Mdmx, suggesting that a fully intact RING domain is required for Mdm2's inhibition of the p300-p53 interaction and for its interaction with Mdmx. These findings help us to better understand the complex regulation of the Mdm2-p53 pathway and have important implications for chemotherapeutic agents targeting Mdm2, as they suggest that inhibition of Mdm2's E3 ubiquitin ligase activity may be sufficient for increasing p53 activity in vivo, without the need to block Mdm2-p53 binding.

  17. Mutational activation of the AmgRS two-component system in aminoglycoside-resistant Pseudomonas aeruginosa.

    Science.gov (United States)

    Lau, Calvin Ho-Fung; Fraud, Sebastien; Jones, Marcus; Peterson, Scott N; Poole, Keith

    2013-05-01

    The amgRS operon encodes a presumed membrane stress-responsive two-component system linked to intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Genome sequencing of a lab isolate showing modest pan-aminoglycoside resistance, strain K2979, revealed a number of mutations, including a substitution in amgS that produced an R182C change in the AmgS sensor kinase product of this gene. Introduction of this mutation into an otherwise wild-type strain recapitulated the resistance phenotype, while correcting the mutation in the resistant mutant abrogated the resistant phenotype, confirming that the amgS mutation is responsible for the aminoglycoside resistance of strain K2979. The amgSR182 mutation promoted an AmgR-dependent, 2- to 3-fold increase in expression of the AmgRS target genes htpX and PA5528, mirroring the impact of aminoglycoside exposure of wild-type cells on htpX and PA5528 expression. This suggests that amgSR182 is a gain-of-function mutation that activates AmgS and the AmgRS two-component system in promoting modest resistance to aminoglycosides. Screening of several pan-aminoglycoside-resistant clinical isolates of P. aeruginosa revealed three that showed elevated htpX and PA5528 expression and harbored single amino acid-altering mutations in amgS (V121G or D106N) and no mutations in amgR. Introduction of the amgSV121G mutation into wild-type P. aeruginosa generated a resistance phenotype reminiscent of the amgSR182 mutant and produced a 2- to 3-fold increase in htpX and PA5528 expression, confirming that it, too, is a gain-of-function aminoglycoside resistance-promoting mutation. These results highlight the contribution of amgS mutations and activation of the AmgRS two-component system to acquired aminoglycoside resistance in lab and clinical isolates of P. aeruginosa.

  18. 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.

  19. Increased activation of blood coagulation in pregnant women with the Factor V Leiden mutation.

    Science.gov (United States)

    Kjellberg, Ulla; van Rooijen, Marianne; Bremme, Katarina; Hellgren, Margareta

    2014-10-01

    The risk of venous thromboembolism is enhanced in pregnant carriers of the Factor V Leiden mutation. The primary aim of the study was to compare prothrombin fragments 1+2, soluble fibrin and D-dimer levels in pregnant Factor V Leiden mutation carriers with those in non-carriers. Secondary aims were to evaluate whether these biomarkers could predict placenta-mediated complications or venous thromboembolism, and to study blood coagulation after caesarean section with thromboprophylaxis and after vaginal delivery without thromboprophylaxis. Prothrombin fragments 1+2, soluble fibrin and D-dimer levels were studied longitudinally in 476 carriers with singleton pregnancies from gestational weeks 23-25 until 8-10 weeks postpartum. Prothrombin fragments 1+2 and D-dimer levels gradually increased during pregnancy. D-dimer levels were higher in carriers, both during pregnancy and puerperium, compared to non-carriers. D-dimer levels above 0.5mg/l were found in about 30% and 20% of the heterozygous carriers at 4-5 and 8-10 weeks postpartum, respectively. Soluble fibrin levels were mainly unchanged during pregnancy, with no difference between carriers and non-carriers. Biomarker levels were similar in carriers with uncomplicated and complicated pregnancies. Higher D-dimer levels indicate increased blood coagulation and fibrinolysis activity in carriers. The high proportion of carriers with D-dimer levels exceeding 0.5mg/l postpartum must be considered when assessing the probability of venous thromboembolism. Large overlaps in biomarker levels in normal and complicated pregnancies suggest that these biomarkers cannot be used as predictors. Thromboprophylaxis following caesarean section may prevent increased activation of blood coagulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. 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

  1. Mutations in Recombination Activating Gene 1 and 2 in patients with severe combined immunodeficiency disorders in Egypt.

    Science.gov (United States)

    Meshaal, Safa; El Hawary, Rabab; Elsharkawy, Marwa; Mousa, Reem K; Farid, Reem J; Abd Elaziz, Dalia; Alkady, Radwa; Galal, Nermeen; Massaad, Michel J; Boutros, Jeannette; Elmarsafy, Aisha

    2015-06-01

    The Recombination Activating Genes (RAG) 1/2 are important for the development and function of T and B cells. Loss of RAG1/2 function results in severe combined immunodeficiency (SCID), which could lead to early death. We studied the prevalence of RAG1/2 mutations in ten SCID patients in Egypt. We identified two novel homozygous nonsense mutations in RAG1, a novel homozygous deletion, and a previously reported homozygous missense mutation from four patients, as well as two homozygous mutations in RAG2 from the same patient. Prenatal diagnosis performed in the mother of a patient with RAG1 deficiency determined that the fetus was heterozygous for the same mutation. This represents the first report on RAG1/2 mutations in SCID patients in Egypt. The early diagnosis dramatically affects the outcome of the disease by allowing bone marrow transplantation at an early age, and providing prenatal diagnosis and genetic counseling for families with a history of SCID.

  2. Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity

    Science.gov (United States)

    Li, Linhua; Ye, Yujia; Sang, Peng; Yin, Yirui; Hu, Wei; Wang, Jing; Zhang, Chao; Li, Deyun; Wan, Wen; Li, Rui; Li, Longjun; Ma, Linling; Xie, Yuehui

    2017-01-01

    Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+. The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL. PMID:28194412

  3. Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Linhua Li

    2017-01-01

    Full Text Available Pyrroline-5-carboxylate reductase (P5CR1 is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C to proline with concomitant oxidation of NAD(PH to NAD(P+. The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL.

  4. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

    Science.gov (United States)

    Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

    2017-01-01

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

  5. Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes.

    Science.gov (United States)

    Sanguinetti, M C; Xu, Q P

    1999-02-01

    1. The structural basis for the activation gate of voltage-dependent K+ channels is not known, but indirect evidence has implicated the S4-S5 linker, the cytoplasmic region between the fourth and fifth transmembrane domains of the channel subunit. We have studied the effects of mutations in the S4-S5 linker of HERG (human ether-á-go-go-related gene), a human delayed rectifier K+ channel, in Xenopus oocytes. 2. Mutation of acidic residues (D540, E544) in the S4-S5 linker of HERG channels to neutral (Ala) or basic (Lys) residues accelerated the rate of channel deactivation. Most mutations greatly accelerated the rate of activation. However, E544K HERG channels activated more slowly than wild-type HERG channels. 3. Mutation of residues in the S4-S5 linker had little or no effect on fast inactivation, consistent with independence of HERG channel activation and inactivation 4. In response to large hyperpolarizations, D540K HERG channels can reopen into a state that is distinct from the normal depolarization-induced open state. It is proposed that substitution of a negatively charged Asp with the positively charged Lys disrupts a subunit interaction that normally stabilizes the channel in a closed state at negative transmembrane potentials. 5. The results indicate that the S4-S5 linker is a crucial component of the activation gate of HERG channels.

  6. 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)

  7. 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.

  8. Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Gabriel Lima Lopes

    2015-08-01

    Full Text Available AbstractLung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21, first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs. Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC.

  9. 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...

  10. 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

  11. Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer *

    Science.gov (United States)

    Lopes, Gabriel Lima; Vattimo, Edoardo Filippo de Queiroz; de Castro, Gilberto

    2015-01-01

    Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21), first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC) patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs). Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC. PMID:26398757

  12. FUNCTIONOMICS OF NCC MUTATIONS IN GITELMAN SYNDROME USING A NOVEL MAMMALIAN CELL-BASED ACTIVITY ASSAY.

    NARCIS (Netherlands)

    Valdez-Flores, M.A.; Vargas-Poussou, R.; Verkaart, S.; Tutakhel, O.A.Z.; Valdez-Ortiz, A.; Blanchard, A.; Treard, C.; Hoenderop, J.G.J.; Bindels, R.J.M.; Jelen, S.

    2016-01-01

    Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was,

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

    OpenAIRE

    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...

  14. 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.

  15. RUNX1 and CBFβ Mutations and Activities of Their Wild-Type Alleles in AML.

    Science.gov (United States)

    Hyde, R Katherine; Liu, Paul; Friedman, Alan D

    2017-01-01

    Mutations in RUNX1 and CBFB have long been recognized as important in hematological malignancies. Point mutations and deletions of RUNX1 are frequently found in myelodysplastic syndrome, myeloproliferative disease, and acute myeloid leukemia. Germline mutations in RUNX1 are associated with familial platelet disorder with predisposition to AML. In addition, as will be discussed in other chapters, both RUNX1 and CBFB are involved in recurrent chromosomal rearrangements in leukemia. More recently, roles for the non-mutated RUNX1 and CBFB genes have been identified in multiple leukemia subtypes. This chapter will discuss the roles of RUNX1 and CBFB, both in diseases caused by their mutations or deletions, as well as in the context of chromosomal rearrangements.

  16. Evidence in Latin America of recurrence of V388M, a phenylketonuria mutation with high in vitro residual activity

    Energy Technology Data Exchange (ETDEWEB)

    Desviat, L.R.; Perez, B.; De Lucca, M. [Universidad Autonoma de Madrid, (Spain)] [and others

    1995-08-01

    Phenylketonuria mutation V388M is frequent in the Iberian Peninsula. In vitro, the V388M mutant enzyme has similar immunoreactive protein and phenylalanine hydroxylase mRNA and had 43% residual activity, which correlates well with the mild phenotype exhibited by the homozygous patients. In Spain it has been detected in 5.7% of the mutant alleles and is always associated with haplotype 1.7. This mutation is also present in high frequency in some Latin American countries (Brazil, 9% Chile, 13%). It is interesting that in Chile most of the alleles bearing this mutation carry haplotype 4.3, although in Brazil it is found only on the background of haplotype 1.7. The origin of V388M in Spain on haplotype 1.7 and in Chile on haplotype 4.3 is clearly different. Recurrence is the most plausible explanation, because the mutation involves a CpG dinucleotide, and a recombination event transferring the mutation from haplotype 1 to 4 is unlikely. 29 refs., 2 figs., 3 tabs.

  17. Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+),K(+)-ATPase activity and ouabain binding.

    Science.gov (United States)

    Weigand, Karl M; Messchaert, Muriël; Swarts, Herman G P; Russel, Frans G M; Koenderink, Jan B

    2014-07-01

    De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na(+),K(+)-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K(+)/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na(+),K(+)-ATPase. Functional impairment of Na(+),K(+)-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.

  18. Subclinical Nonautoimmune Hyperthyroidism in a Family Segregates with a Thyrotropin Receptor Mutation with Weakly Increased Constitutive Activity

    Science.gov (United States)

    Chen, Chun-Rong; Higashiyama, Takuya; Mizutori-Sasai, Yumiko; Ito, Mitsuru; Kubota, Sumihisa; Amino, Nobuyuki; Miyauchi, Akira; Rapoport, Basil

    2010-01-01

    Background Subclinical hyperthyroidism is usually associated with Graves' disease or toxic nodular goiter. Here we report a family with hereditary subclinical hyperthyroidism caused by a constitutively activating germline mutation of the thyrotropin receptor (TSHR) gene. Methods The proband was a 64-year-old Japanese woman who presented with a thyroid nodule and was found to be euthyroid with a suppressed serum TSH. The nodule was not hot. Although antibodies to thyroid peroxidase and thyroglobulin antibodies were present, TSHR antibodies were not detected by TSH-binding inhibition or by bioassay. Two of her middle-aged sons, but not her daughter, also had subclinical hyperthyroidism without TSHR antibodies. Without therapy, the clinical condition of the affected individuals remained unchanged over 3 years without development of overt hyperthyroidism. Results A novel heterozygous TSHR point mutation causing a glutamic acid to lysine substitution at codon 575 (E575K) in the second extracellular loop was detected in the three family members with subclinical hyperthyroidism, but was absent in her one daughter with normal thyroid function. In vitro functional studies of the E575K TSHR mutation demonstrated a weak, but significant, increase in constitutive activation of the cAMP pathway. Conclusion Although hereditary nonautoimmune overt hyperthyroidism is very rare, TSHR activating mutations as a cause of subclinical hyperthyroidism may be more common and should be considered in the differential diagnosis, especially if familial. PMID:20929407

  19. The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.

    Science.gov (United States)

    Bertrand, Carol A; Mitra, Shalini; Mishra, Sanjay K; Wang, Xiaohui; Zhao, Yu; Pilewski, Joseph M; Madden, Dean R; Frizzell, Raymond A

    2017-03-30

    Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. This association is thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it's unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 co-expressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared to cells co-expressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when co-expressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with co-expression of wt CFTR. However, co-expression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is co-expressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

  20. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  1. Male mutation bias and possible long-term effects of human activities.

    Science.gov (United States)

    Cotton, Samuel; Wedekind, Claus

    2010-10-01

    The ability of a population to adapt to changing environments depends critically on the amount and kind of genetic variability it possesses. Mutations are an important source of new genetic variability and may lead to new adaptations, especially if the population size is large. Mutation rates are extremely variable between and within species, and males usually have higher mutation rates as a result of elevated rates of male germ cell division. This male bias affects the overall mutation rate. We examined the factors that influence male mutation bias, and focused on the effects of classical life-history parameters, such as the average age at reproduction and elevated rates of sperm production in response to sexual selection and sperm competition. We argue that human-induced changes in age at reproduction or in sexual selection will affect male mutation biases and hence overall mutation rates. Depending on the effective population size, these changes are likely to influence the long-term persistence of a population.

  2. Activating mutations of the Gs alpha-gene in nonfunctioning pituitary tumors.

    Science.gov (United States)

    Tordjman, K; Stern, N; Ouaknine, G; Yossiphov, Y; Razon, N; Nordenskjöld, M; Friedman, E

    1993-09-01

    The majority of pituitary tumors are of monoclonal origin; however, the molecular basis for their formation is poorly understood. Somatic mutations in the alpha-subunit of the GTP-binding protein, Gs alpha (gsp oncogene) have been found in about one third of GH-secreting tumors. Mutations in another alpha-subunit of a GTP-binding protein, Gi2 alpha (gip mutations) have been described in other endocrine tumors. In this study, we examined 21 nonfunctioning pituitary tumors and 4 macroprolactinomas for gsp mutations and 27 nonfunctioning tumors and 4 macroprolactinomas for gip mutations. Using the polymerase chain reaction and denaturing gradient gel electrophoresis, 2 nonfunctioning pituitary tumors displayed migration abnormalities when the Gs alpha-gene was analyzed. Sequence analysis of these abnormally migrating polymerase chain reaction products revealed two previously known gsp mutations: arginine at codon 201 altered to cysteine, and glutamine at codon 227 changed to leucine. No gip mutations could be demonstrated. These findings emphasize the monoclonal origin of nonfunctioning pituitary tumors and suggest that cAMP may play a role in tumorigenesis of nonfunctioning pituitary tumors.

  3. 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.

  4. Human NR5A1/SF-1 mutations show decreased activity on BDNF (brain-derived neurotrophic factor), an important regulator of energy balance: testing impact of novel SF-1 mutations beyond steroidogenesis.

    Science.gov (United States)

    Malikova, Jana; Camats, Núria; Fernández-Cancio, Mónica; Heath, Karen; González, Isabel; Caimarí, María; del Campo, Miguel; Albisu, Marian; Kolouskova, Stanislava; Audí, Laura; Flück, Christa E

    2014-01-01

    Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations.

  5. Human NR5A1/SF-1 mutations show decreased activity on BDNF (brain-derived neurotrophic factor, an important regulator of energy balance: testing impact of novel SF-1 mutations beyond steroidogenesis.

    Directory of Open Access Journals (Sweden)

    Jana Malikova

    Full Text Available Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF, an important regulator of energy balance in the ventromedial hypothalamus.To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance.5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency.SF-1 mutations were studied in cell systems (HEK293, JEG3 for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2 and in energy balance (BDNF. BDNF regulation by SF-1 was studied by promoter assays (JEG3.Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159 were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters. However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI.Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop. In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations.

  6. UNTAGGED MUTATION IN RICE GAL4/VP16 TRANSCRIPTIONAL ACTIVATOR FACILITATED-ENHANCER TRAP LINES

    Directory of Open Access Journals (Sweden)

    Sri Koerniati

    2013-04-01

    Full Text Available An enhancer trap system is an insertional mutagenesis based upon gene expression, instead of gene knock-out, so its insertion in genome is  expected not linked to any dramatic changes in plant phenotypes. Gene  knock-out, leading to lossof- function (LoF mutation, is a dominant  approach for rice functional genomic studies. The objective of this study was to find out whether Transcriptional Activator-Facilitated Enhancer Trap (TAFET T-DNA insertion inducing mutant phenotypes in rice TAFET population. Materials used in this experiment were T1 generation of 270 rice TAFET lines. Eight plants of each were grown in the greenhouse and observed for any mutant phenotypes. Phenotypic, histochemical, Southernblot analyses were carried out to define a mutant of pSKC66.1- 8e. Result showed that about 10% of the 270 lines produced chlorophyll-deficient  leaves, ranged from yellowish green (viridis, white stripe green zebra-like stripe to completely white (albino. Albino plants died after two weeks,  whilst white stripe or viridis mutants became normal in the next generation(T2. Another mutant was pSKC66.1-8e line which had floral dramatic phenotype change with various spikelet shapes and number of organs, and had a single twisted culm. The flower of mutant also had gus gene expression. Plants with wild type did not express gus gene and had six or more straight culms. Molecular, histochemical and phenotypic analyses of this particular line for three generations indicated that mutant phenotype was not due to the T-DNA insertion. Since there was approved that Tos17 is activated during tissue culture and induced mutant phenotype, this line might relate to Tos17 insertion, but it needs further investigation to gain such conclusion.

  7. A novel activation-induced cytidine deaminase (AID) mutation in Brazilian patients with hyper-IgM type 2 syndrome.

    Science.gov (United States)

    Caratão, Nadine; Cortesão, Catarina S; Reis, Pedro H; Freitas, Raquel F; Jacob, Cristina M A; Pastorino, Antonio C; Carneiro-Sampaio, Magda; Barreto, Vasco M

    2013-08-01

    Activation-induced cytidine deaminase (AID) is a DNA editing protein that plays an essential role in three major events of immunoglobulin (Ig) diversification: somatic hypermutation, class switch recombination and Ig gene conversion. Mutations in the AID gene (AICDA) have been found in patients with autosomal recessive Hyper-IgM (HIGM) syndrome type 2. Here, two 9- and 14-year-old Brazilian sisters, from a consanguineous family, were diagnosed with HIGM2 syndrome. Sequencing analysis of the exons from AICDA revealed that both patients are homozygous for a single C to G transversion in the third position of codon 15, which replaces a conserved Phenylalanine with a Leucine. To our knowledge, this is a new AICDA mutation found in HIGM2 patients. Functional studies confirm that the homologous murine mutation leads to a dysfunctional protein with diminished intrinsic cytidine deaminase activity and is unable to rescue CSR when introduced in Aicda(-/-)stimulated murine B cells. We briefly discuss the relevance of AICDA mutations found in patients for the biology of this molecule.

  8. Pathologic Active mTOR Mutation in Brain Malformation with Intractable Epilepsy Leads to Cell-Autonomous Migration Delay.

    Science.gov (United States)

    Hanai, Sae; Sukigara, Sayuri; Dai, Hongmei; Owa, Tomoo; Horike, Shin-Ichi; Otsuki, Taisuke; Saito, Takashi; Nakagawa, Eiji; Ikegaya, Naoki; Kaido, Takanobu; Sato, Noriko; Takahashi, Akio; Sugai, Kenji; Saito, Yuko; Sasaki, Masayuki; Hoshino, Mikio; Goto, Yu-Ichi; Koizumi, Schuichi; Itoh, Masayuki

    2017-05-01

    The activation of phosphatidylinositol 3-kinase-AKTs-mammalian target of rapamycin cell signaling pathway leads to cell overgrowth and abnormal migration and results in various types of cortical malformations, such as hemimegalencephaly (HME), focal cortical dysplasia, and tuberous sclerosis complex. However, the pathomechanism underlying abnormal cell migration remains unknown. With the use of fetal mouse brain, we performed causative gene analysis of the resected brain tissues from a patient with HME and investigated the pathogenesis. We obtained a novel somatic mutation of the MTOR gene, having approximately 11% and 7% mutation frequency in the resected brain tissues. Moreover, we revealed that the MTOR mutation resulted in hyperphosphorylation of its downstream molecules, S6 and 4E-binding protein 1, and delayed cell migration on the radial glial fiber and did not affect other cells. We suspect cell-autonomous migration arrest on the radial glial foot by the active MTOR mutation and offer potential explanations for why this may lead to cortical malformations such as HME. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. 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.

  10. Impact of active smoking on survival of patients with metastatic lung adenocarcinoma harboring an epidermal growth factor receptor (EGFR) mutation.

    Science.gov (United States)

    Erdogan, Bulent; Kodaz, Hilmi; Karabulut, Senem; Cinkaya, Ahmet; Tozkir, Hilmi; Tanriverdi, Ozgur; Cabuk, Devrim; Hacioglu, Muhammed Bekir; Turkmen, Esma; Hacibekiroglu, Ilhan; Uzunoglu, Sernaz; Cicin, Irfan

    2016-11-10

    Lung cancer in smokers and non-smokers demonstrates distinct genetic profiles, and cigarette smoking affects epidermal growth factor receptor (EGFR) function and causes secondary EGFR tyrosine kinase resistance. We evaluated the effect of active smoking in patients with metastatic lung adenocarcinoma. A total of 132 metastatic lung adenocarcinoma patients, diagnosed between 2008 and 2013, with known EGFR mutation status, were evaluated retrospectively. Among these patients, 40 had an activating EGFR mutation. Patients who continued smoking during the treatment were defined as active smokers. Former smokers and never smokers were together defined as non-smokers. The outcomes of the treatment in relation to the EGFR mutation and smoking status were evaluated. The median follow-up time was 10.5 months. The overall response rate for the first-line therapy was significantly higher among the EGFR-mutant patients (p = 0.01), however, smoking status had no impact on the response rate (p = 0.1). The EGFR-mutant active smokers progressed earlier than the non-smokers (p Smoking status did not affect the OS in EGFR wild type tumors (p = 0.49) but EGFR-mutant non-smokers had a longer OS than the active smokers (p = 0.01).The active smokers treated with erlotinib had poorer survival than the non-smokers (p = 0.03). Multivariate analysis of EGFR-mutant patients showed that erlotinib treatment at any line and non-smoking were independent prognostic factors for the OS (p = 0.04 and p = 0.01, respectively). Smoking during treatment is a negative prognostic factor in metastatic lung adenocarcinoma with an EGFR mutation.

  11. A missense mutation P136L in the arylsulfatase A gene causes instability and loss of activity of the mutant enzyme.

    Science.gov (United States)

    Kafert, S; Heinisch, U; Zlotogora, J; Gieselmann, V

    1995-02-01

    Metachromatic leukodystrophy is a lysosomal storage disease caused by deficiency of arylsulfatase A. Sequencing of the arylsulfatase A genes of an Ashkenazi Jewish patient suffering from the severe late infantile form of the disease revealed a point mutation in exon 2 causing proline 136 to be substituted by leucine. The patient was homozygous for this mutation. Studies on Ltk- cells stably expressing the mutant enzyme show that the mutation causes complete loss of enzyme activity and rapid degradation in an early biosynthetic compartment.

  12. 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

  13. 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.

  14. Effect of KRAS exon 2 mutations on antitumor activity of afatinib and gefitinib.

    Science.gov (United States)

    Gamba, Sebastian; Camaj, Peter; Heinemann, Volker; Laubender, Rüdiger P; Wang, Yan; Zhao, Yue; Stintzing, Sebastian; Giessen, Clemens; Boeck, Stefan; Haertl, Christoph; Bruns, Christiane J; Modest, Dominik P

    2015-04-01

    The aim of this study was to investigate the impact of different KRAS mutations on the inhibitory potential of afatinib and gefitinib in SW48 colorectal cancer cells. The influence of afatinib/gefitinib on cell viability and cell cycle was evaluated in isogenic SW48 KRAS wild-type/mutant cells. Protein levels of phosphorylated/total EGFR, HER-2, HER-3, ERK, and AKT were compared between treated/untreated samples using western blotting. The activity of both afatinib and gefitinib was the lowest in KRAS G12C/G12S/G12D and the highest in G13D/G12A mutant subtypes. A 50% decrease in cell viability was achieved at concentrations of 3.0-7.7 μmol/l for afatinib and 5.4-19.5 μmol/l for gefitinib. The effect of both drugs on apoptosis appeared to be stronger than their influence on proliferation and was generally less pronounced in mutant cells than in wild-type cells. The average number of apoptotic cells after treatment with afatinib was 2.6 times as high as the corresponding value following treatment with gefitinib (Pafatinib than by gefitinib (Pafatinib and gefitinib whereas others seem to increase sensitivity to treatment (G13D/G12A) compared with the parental clone (KRAS wild-type). In SW48 colorectal cancer cells, afatinib seems to be more potent than gefitinib because of its superior efficacy in inhibiting both EGFR and HER-2, suppressing signaling along both MEK/ERK and PI3K/AKT pathways to a greater extent.

  15. Targeting wild-type and mutationally activated FGFR4 in rhabdomyosarcoma with the inhibitor ponatinib (AP24534).

    Science.gov (United States)

    Li, Samuel Q; Cheuk, Adam T; Shern, Jack F; Song, Young K; Hurd, Laura; Liao, Hongling; Wei, Jun S; Khan, Javed

    2013-01-01

    Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

  16. Targeting wild-type and mutationally activated FGFR4 in rhabdomyosarcoma with the inhibitor ponatinib (AP24534.

    Directory of Open Access Journals (Sweden)

    Samuel Q Li

    Full Text Available Rhabdomyosarcoma (RMS is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4 is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534 to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

  17. The Pro115Gln Missense Mutation of Peroxisome Proliferator Activated Receptor γ (PPARγ Gene in Diabetes in the Pakistani Population

    Directory of Open Access Journals (Sweden)

    Sumbal

    2016-05-01

    Full Text Available Background Diabetes is an alarmingly increasing public health concern. According to a World Health Organization (WHO report, diabetes is highly prevalent in almost all regions of Pakistan, with an overall prevalence of 22.04% in urban and 17.15% in rural areas. In addition to lifestyle factors, genetics plays an important part in the progression to diabetes, of which peroxisome proliferator activated receptors (PPARs, a super-family of transcriptional regulators, are considered to be very critical. Objectives The aim of the current study was to investigate the prevalence of a missense mutation Pro115Gln in the PPARγ; gene in Pakistan and search for its effects on certain serum biochemical parameters. Materials and Methods A total of 1015 subjects (490 diabetics and 525 healthy controls were genotyped by PCR-RFLP and serum profiling was performed. Results We could not detect the mutation in the study subjects, but the anthropometric and biochemical parameters were significantly different between the cases and controls. Conclusions In conclusion, as this mutation is present in a highly conserved region and any change in this region would adversely affect the protein structure and function, so the prevalence of this mutation is very low.

  18. 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......M in the presence of RyR2 CaMBD. The D95V, N97S and D129G mutations lowered the affinity of Ca2+ binding of the C-lobe of CaM, to apparent KDs of ~ 140, 150, and 4000 nM, respectively, consistent with the critical role of these residues in Ca2+ binding to the C-lobe. Thus, we suggest that these mutations may shift...... to an apo-CaM binding state during diastole, leading to dysregulation of RyR2 mediated Ca2+ release. Despite the pronounced impact on RyR2 mediated Ca2+ release, the N-lobe N53I mutation only imposed a small lowering of the N-lobe Ca2+ affinity (KD ~1200 nM). Thus, the RyR2 mediated Ca2+ release is either...

  19. The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors.

    OpenAIRE

    Chatzidaki, A.; D Oyley, J. M.; Gill-Thind, J. K.; Sheppard, T. D.; Millar, N S

    2015-01-01

    Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have divers...

  20. The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors

    OpenAIRE

    Chatzidaki, Anna; D'Oyley, Jarryl M; Gill-Thind, JasKiran K.; Sheppard, Tom D; Millar, Neil S.

    2015-01-01

    Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have divers...

  1. The nemaline myopathy-causing E117K mutation in β-tropomyosin reduces thin filament activation.

    Science.gov (United States)

    Karpicheva, Olga E; Robinson, Paul; Piers, Adam; Borovikov, Yurii S; Redwood, Charles S

    2013-08-01

    The effect of the nemaline myopathy-causing E117K mutation in β-tropomyosin (TM) on the structure and function of this regulatory protein was studied. The E117K mutant was found to have indistinguishable actin affinity compared with wild-type (WT) and similar secondary structure as measured by circular dichroism. However the E117K mutation significantly lowered maximum activation of actomyosin ATPase. To explain the molecular mechanism of impaired ATPase activation, WT and E117K TMs were covalently labeled at Cys-36 with 5-iodoacetimido-fluorescein and incorporated into ghost muscle fibers. The changes in the position and flexibility of tropomyosin strands on the thin filaments were observed at simulation of weak and strong binding states of actomyosin at high or low Ca(2+) by polarized fluorescence techniques. The E117K mutation was found to shift the tropomyosin strands towards the closed position and restrict the tropomyosin displacement during the transformation of actomyosin from weak to strong binding state thus leading to a reduction in thin filament activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis.

    Science.gov (United States)

    Sermet-Gaudelus, Isabelle; Boeck, Kris De; Casimir, Georges J; Vermeulen, François; Leal, Teresinha; Mogenet, Agnès; Roussel, Delphine; Fritsch, Janine; Hanssens, Laurence; Hirawat, Samit; Miller, Nilsen L; Constantine, Scott; Reha, Allen; Ajayi, Temitayo; Elfring, Gary L; Miller, Langdon L

    2010-11-15

    Nonsense (premature stop codon) mutations in mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) in approximately 10% of patients. Ataluren (PTC124) is an oral drug that permits ribosomes to readthrough premature stop codons in mRNA to produce functional protein. To evaluate ataluren activity, safety, and pharmacokinetics in children with nonsense mutation CF. Patients were assessed in two 28-day cycles, comprising 14 days on and 14 days off ataluren. Patients took ataluren three times per day (morning, midday, and evening) with randomization to the order of receiving a lower dose (4, 4, and 8 mg/kg) and a higher dose (10, 10, and 20 mg/kg) in the two cycles. The study enrolled 30 patients (16 male and 14 female, ages 6 through 18 yr) with a nonsense mutation in at least one allele of the CFTR gene, a classical CF phenotype, and abnormal baseline nasal epithelial chloride transport. Ataluren induced a nasal chloride transport response (at least a -5-mV improvement) or hyperpolarization (value more electrically negative than -5 mV) in 50% and 47% of patients, respectively, with more hyperpolarizations at the higher dose. Improvements were seen in seven of nine nonsense mutation genotypes represented. Ataluren significantly increased the proportion of nasal epithelial cells expressing apical full-length CFTR protein. Adverse events and laboratory abnormalities were infrequent and usually mild. Ataluren pharmacokinetics were similar to those in adults. In children with nonsense mutation CF, ataluren can induce functional CFTR production and is well tolerated.

  7. Missense mutations in SLC26A8, encoding a sperm-specific activator of CFTR, are associated with human asthenozoospermia.

    Science.gov (United States)

    Dirami, Thassadite; Rode, Baptiste; Jollivet, Mathilde; Da Silva, Nathalie; Escalier, Denise; Gaitch, Natacha; Norez, Caroline; Tuffery, Pierre; Wolf, Jean-Philippe; Becq, Frédéric; Ray, Pierre F; Dulioust, Emmanuel; Gacon, Gérard; Bienvenu, Thierry; Touré, Aminata

    2013-05-02

    The cystic fibrosis transmembrane conductance regulator (CFTR) is present in mature sperm and is required for sperm motility and capacitation. Both these processes are controlled by ions fluxes and are essential for fertilization. We have shown that SLC26A8, a sperm-specific member of the SLC26 family of anion exchangers, associates with the CFTR channel and strongly stimulates its activity. This suggests that the two proteins cooperate to regulate the anion fluxes required for correct sperm motility and capacitation. Here, we report on three heterozygous SLC26A8 missense mutations identified in a cohort of 146 men presenting with asthenozoospermia: c.260G>A (p.Arg87Gln), c.2434G>A (p.Glu812Lys), and c.2860C>T (p.Arg954Cys). These mutations were not present in 121 controls matched for ethnicity, and statistical analysis on a control population of 8,600 individuals (from dbSNP and 1000 Genomes) showed them to be associated with asthenozoospermia with a power > 95%. By cotransfecting Chinese hamster ovary (CHO)-K1 cells with SLC26A8 variants and CFTR, we showed that the physical interaction between the two proteins was partly conserved but that the capacity to activate CFTR-dependent anion transport was completely abolished for all mutants. Biochemical studies revealed the presence of much smaller amounts of protein for all variants, but these amounts were restored to wild-type levels upon treatment with the proteasome inhibitor MG132. Immunocytochemistry also showed the amounts of SLC26A8 in sperm to be abnormally small in individuals carrying the mutations. These mutations might therefore impair formation of the SLC26A8-CFTR complex, principally by affecting SLC26A8 stability, consistent with an impairment of CFTR-dependent sperm-activation events in affected individuals.

  8. CACNA1D de novo mutations in autism spectrum disorders activate Cav1.3 L-type calcium channels.

    Science.gov (United States)

    Pinggera, Alexandra; Lieb, Andreas; Benedetti, Bruno; Lampert, Michaela; Monteleone, Stefania; Liedl, Klaus R; Tuluc, Petronel; Striessnig, Jörg

    2015-05-01

    Cav1.3 voltage-gated L-type calcium channels (LTCCs) are part of postsynaptic neuronal signaling networks. They play a key role in brain function, including fear memory and emotional and drug-taking behaviors. A whole-exome sequencing study identified a de novo mutation, p.A749G, in Cav1.3 α1-subunits (CACNA1D), the second main LTCC in the brain, as 1 of 62 high risk-conferring mutations in a cohort of patients with autism and intellectual disability. We screened all published genetic information available from whole-exome sequencing studies and identified a second de novo CACNA1D mutation, p.G407R. Both mutations are present only in the probands and not in their unaffected parents or siblings. We functionally expressed both mutations in tsA-201 cells to study their functional consequences using whole-cell patch-clamp. The mutations p.A749G and p.G407R caused dramatic changes in channel gating by shifting (~15 mV) the voltage dependence for steady-state activation and inactivation to more negative voltages (p.A749G) or by pronounced slowing of current inactivation during depolarizing stimuli (p.G407R). In both cases, these changes are compatible with a gain-of-function phenotype. Our data, together with the discovery that Cav1.3 gain-of-function causes primary aldosteronism with seizures, neurologic abnormalities, and intellectual disability, suggest that Cav1.3 gain-of-function mutations confer a major part of the risk for autism in the two probands and may even cause the disease. Our findings have immediate clinical relevance because blockers of LTCCs are available for therapeutic attempts in affected individuals. Patients should also be explored for other symptoms likely resulting from Cav1.3 hyperactivity, in particular, primary aldosteronism. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  9. 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.

  10. A Mosaic Activating Mutation in AKT1 Associated with the Proteus Syndrome

    NARCIS (Netherlands)

    Lindhurst, Marjorie J.; Sapp, Julie C.; Teer, Jamie K.; Johnston, Jennifer J.; Finn, Erin M.; Peters, Kathryn; Turner, Joyce; Cannons, Jennifer L.; Bick, David; Blakemore, Laurel; Blumhorst, Catherine; Brockmann, Knut; Calder, Peter; Cherman, Natasha; Deardorff, Matthew A.; Everman, David B.; Golas, Gretchen; Greenstein, Robert M.; Kato, B. Maya; Keppler-Noreuil, Kim M.; Kuznetsov, Sergei A.; Miyamoto, Richard T.; Newman, Kurt; Ng, David; O'Brien, Kevin; Rothenberg, Steven; Schwartzentruber, Douglas J.; Singhal, Virender; Tirabosco, Roberto; Upton, Joseph; Wientroub, Shlomo; Zackai, Elaine H.; Hoag, Kimberly; Whitewood-Neal, Tracey; Robey, Pamela G.; Schwartzberg, Pamela L.; Darling, Thomas N.; Tosi, Laura L.; Mullikin, James C.; Biesecker, Leslie G.

    2011-01-01

    BACKGROUND The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS We performed exome sequencing of DNA from

  11. Identification of an AR mutation-negative class of androgen insensitivity by determining endogenous AR activity

    NARCIS (Netherlands)

    Hornig, N.C.; Ukat, M.; H.U. Schweikert (H.); O. Hiort (Olaf); Werner, R.; S.L.S. Drop (Stenvert); M.L. Cools (Martine); I.A. Hughes (Ieuan A.); L. Audí (Laura); S.F. Ahmed (S. Faisal); Demiri, J.; Rodens, P.; Worch, L.; Wehner, G.; Kulle, A.E.; Dunstheimer, D.; Müller-Roßberg, E.; T. Reinehr (Thomas); Hadidi, A.T.; Eckstein, A.K.; Van Der Horst, C.; Seif, C.; R. Siebert (Reiner); O. Ammerpohl (Ole); P-M. Holterhus (Paul-Martin)

    2016-01-01

    textabstractContext: Only approximately 85%of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30%with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. Objective: The objective of the study

  12. A Mosaic Activating Mutation in AKT1 Associated with the Proteus Syndrome

    NARCIS (Netherlands)

    Lindhurst, Marjorie J.; Sapp, Julie C.; Teer, Jamie K.; Johnston, Jennifer J.; Finn, Erin M.; Peters, Kathryn; Turner, Joyce; Cannons, Jennifer L.; Bick, David; Blakemore, Laurel; Blumhorst, Catherine; Brockmann, Knut; Calder, Peter; Cherman, Natasha; Deardorff, Matthew A.; Everman, David B.; Golas, Gretchen; Greenstein, Robert M.; Kato, B. Maya; Keppler-Noreuil, Kim M.; Kuznetsov, Sergei A.; Miyamoto, Richard T.; Newman, Kurt; Ng, David; O'Brien, Kevin; Rothenberg, Steven; Schwartzentruber, Douglas J.; Singhal, Virender; Tirabosco, Roberto; Upton, Joseph; Wientroub, Shlomo; Zackai, Elaine H.; Hoag, Kimberly; Whitewood-Neal, Tracey; Robey, Pamela G.; Schwartzberg, Pamela L.; Darling, Thomas N.; Tosi, Laura L.; Mullikin, James C.; Biesecker, Leslie G.

    2011-01-01

    BACKGROUND The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS We performed exome sequencing of DNA from b

  13. Insights into the biochemical and genetic basis of glucokinase activation from naturally occurring hypoglycemia mutations.

    NARCIS (Netherlands)

    Gloyn, A.L.; Noordam, C.; Willemsen, M.A.A.P.; Ellard, S.; Lam, W.W.; Campbell, I.W.; Midgley, P.; Shiota, C.; Buettger, C.; Magnuson, M.A.; Matschinsky, F.M.; Hattersley, A.T.

    2003-01-01

    Glucokinase (GCK) is a key regulatory enzyme in the pancreatic beta-cell and catalyzes the rate-limiting step for beta-cell glucose metabolism. We report two novel GCK mutations (T65I and W99R) that have arisen de novo in two families with familial hypoglycemia. Insulin levels, although inappropriat

  14. An active hAT transposable element causing bud mutation of carnation by insertion into the flavonoid 3'-hydroxylase gene.

    Science.gov (United States)

    Momose, Masaki; Nakayama, Masayoshi; Itoh, Yoshio; Umemoto, Naoyuki; Toguri, Toshihiro; Ozeki, Yoshihiro

    2013-04-01

    The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3'H, the gene for flavonoid 3'-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.

  15. FAD-mutation of APP is associated with a loss of its synaptotrophic activity.

    Science.gov (United States)

    Seeger, Gudrun; Gärtner, Ulrich; Ueberham, Uwe; Rohn, Susanne; Arendt, Thomas

    2009-08-01

    Alzheimer's disease (AD) is a chronic neurodegenerative disorder associated with extracellular accumulation of Abeta peptide that derives from the amyloid precursor protein (APP). While amyloidogenic processing of APP has received most attention, the physiological function of APP and the sequelae of potentially impaired APP function are less understood. APP is a transmembrane glycoprotein being widely expressed in neurons in both central and peripheral nervous system. Its physiological function has been associated with neuronal survival, neurite outgrowth and neuronal plasticity. The aim of the present study was to determine whether FAD-linked mutations of APP, known to be associated with early onset of the disease, might impair its synaptotrophic function, potentially contributing to synaptic deficiencies seen in AD. We performed a quantitative electron microscopy study on synapses in well characterized expression-matched transgenic mice lines expressing either wildtype or FAD-mutated hAPP. Using serial electron microscopic sections, we comparatively analyzed by stereological methods the number and sizes of synaptic contacts and the number of synaptic vesicles in the neocortex. We could clearly show a synaptotrophic effect in mice overexpressing wildtype hAPP evidenced by a significant increase in the number of synapses and the number of vesicles per synapse. This effect was abolished when FAD-mutated APP(Sw,Ind) was expressed instead of wildtype APP. The present study demonstrates a synaptotrophic effect of APP which is lost in the presence of a FAD-mutation. This failure could either be due to a synaptotoxic effect of Abeta potentially counteracting the synaptotrophic effect of APP. Alternatively, the FAD-mutation might impair the physiological function of the extracellular domain of APP and its fragments which might be required for the synaptotrophic effect. This suggests that not only "too much Abeta" but also "too less functional intact APP" might be relevant

  16. Massively parallel sequencing reveals an accumulation of de novo mutations and an activating mutation of LPAR1 in a patient with metastatic neuroblastoma.

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    Jun S Wei

    Full Text Available Neuroblastoma is one of the most genomically heterogeneous childhood malignances studied to date, and the molecular events that occur during the course of the disease are not fully understood. Genomic studies in neuroblastoma have showed only a few recurrent mutations and a low somatic mutation burden. However, none of these studies has examined the mutations arising during the course of disease, nor have they systemically examined the expression of mutant genes. Here we performed genomic analyses on tumors taken during a 3.5 years disease course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal primary tumor taken at surgical resection, and a liver metastasis at autopsy. Whole genome sequencing of the index liver metastasis identified 44 non-synonymous somatic mutations in 42 genes (0.85 mutation/MB and a large hemizygous deletion in the ATRX gene which has been recently reported in neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the primary tumor and bone marrow biopsy, while the other 30 were unique to the index tumor, indicating accumulation of de novo mutations during therapy. Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1 had high level of expression of the mutant alleles, suggesting potential oncogenic driver roles of these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1 was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant demonstrated a significantly increased motility through elevated Rho signaling, but had no effect on growth. Therefore, this study highlights the need for multiple biopsies and sequencing during progression of a cancer and combinatorial DNA and RNA sequencing approach for systematic identification of expressed driver mutations.

  17. 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.

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

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    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.

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

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    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.

  20. Activating Mutations of the TRPML1 Channel Revealed by Proline-scanning Mutagenesis*

    OpenAIRE

    2009-01-01

    The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1V432P) results in a...

  1. A Presenilin-1 Mutation Identified in Familial Alzheimer Disease with Cotton Wool Plaques Causes a Nearly Complete Loss of γ-Secretase Activity*

    Science.gov (United States)

    Heilig, Elizabeth A.; Xia, Weiming; Shen, Jie; Kelleher, Raymond J.

    2010-01-01

    Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit γ-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by γ-secretase produces β-amyloid peptides (Aβ40 and Aβ42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on γ-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of γ-secretase activity, including >90% reductions in the generation of Aβ40, Aβ42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of γ-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Aβ40, Aβ42, and the APP and Notch intracellular domains. These results argue against overproduction of Aβ42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function. PMID:20460383

  2. Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry

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    Michael J. Greig

    2015-01-01

    Full Text Available Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug’s binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR. Wild-type (WT EGFR, EGFR with a single activating mutant (Del 746–750 or L858R, and the drug-resistant double mutant (L858R/T790M EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT to 7.5 hours in the PC-9 cells (Del 746–750 mutant. The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10–20% reduction in rates.

  3. Lessons from McCune-Albright syndrome-associated intraductal papillary mucinous neoplasms: : GNAS-activating mutations in pancreatic carcinogenesis.

    Science.gov (United States)

    Parvanescu, Alina; Cros, Jérôme; Ronot, Maxime; Hentic, Olivia; Grybek, Virginie; Couvelard, Anne; Levy, Philippe; Chanson, Philippe; Ruszniewski, Philippe; Sauvanet, Alain; Gaujoux, Sebastien

    2014-08-01

    GNAS-activating mutations are reported in intraductal papillary mucinous neoplasms (IPMNs) and in McCune-Albright syndrome, characterized by fibrous dysplasia, precocious puberty, and café au lait spots. Recently, IPMNs have been described as a McCune-Albright syndrome-associated tumor, present in about 15% of patients. The aim of the present work was to assess the prevalence of polyostotic fibrous dysplasia and McCune-Albright syndrome among patients operated on for presumptive sporadic IPMNs. All patients operated on for IPMNs between January 1, 2007, and December 31, 2012, with available imaging were retrospectively screened for polyostotic fibrous dysplasia based on their preoperative abdominal or thoracoabdominal spiral computed tomography images. Systematic screening of 272 patients operated on for IPMNs revealed 1 patient with axial and peripheral polyostotic fibrous dysplasia and café au lait spots on clinical examination suggestive of McCune-Albright syndrome. This patient had been operated on for an unusually large invasive colloid adenocarcinoma (pT3N0M0 R0) derived from an intestinal subtype GNAS-mutated IPMN. The patient underwent adjuvant chemotherapy with gemcitabine for 6 months and was alive without recurrence 6 years later. Besides providing additional evidence of a syndromic IPMN as a feature of McCune-Albright syndrome, this observation is further evidence of the functional oncogenic consequences of GNAS mutations in the pancreas.

  4. Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator).

    Science.gov (United States)

    Ramjeesingh, M; Li, C; Garami, E; Huan, L J; Galley, K; Wang, Y; Bear, C E

    1999-02-02

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basis of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the present study, to determine the structural basis for the ATPase activity of CFTR, we assessed the effect of mutations within the "Walker A" consensus motifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysine residue in the "Walker A" motif of either the first nucleotide binding fold (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibited the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding folds of CFTR are functioning cooperatively in catalysis. However, the rate of channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly coupled to channel gating as previously hypothesized.

  5. 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.

  6. P04.24MUTATIONS OF THE TERT PROMOTER CORRELATE WITH ENHANCED TELOMERASE ACTIVATION AND PREDICT A WORSE PROGNOSIS IN PRIMARY GLIOBLASTOMA PATIENTS

    Science.gov (United States)

    Spiegl-Kreinecker, S.; Loetsch, D.; Laaber, M.; Pichler, J.; Weis, S.; Ghanim, B.; Webersinke, G.; Olschowski, A.; Berger, W.

    2014-01-01

    The stabilization of telomeres by upregulation of telomerase is compulsive for indefinite proliferation and cell immortalization therefore representing a main feature of malignant solid tumors, including gliomas. However, the mechanisms responsible for cancer-associated telomerase activation are not completely understood. Recently, defined mutations in the TERT promoter were identified in a variety of tumors, most frequent in primary glioblastomas (GBM). Presence of the mutations was associated with increased TERT expression. In the present study GBM derived tumor tissue from 126 patients operated at the Wagner Jauregg Hospital were screened for TERT promoter mutations. Subsequently the collected data were correlated with telomere associated parameters (telomerase activity, TERT mRNA expression, telomere lengths), the glioma biomarkers MGMT promoter methylation and IDH1 mutation as well as clinical parameters including patient survival. Using direct sequencing, the TERT promoter mutations (C228T, C250T) were found in 73% of tumors with predominance of C228T (72% of the mutated cases). Thirty-four (27%) samples contained none of the investigated TERT promoter mutations while mutations at both sites occurred in none of the investigated GBM cases. TERT promoter mutations were accompanied by a significant upregulation of telomerase activity (p = 0.0005) and TERT mRNA expression (p = 0.0004). Accordingly, telomere lengths of TERT promoter mutated tumors were significantly shorter compared to the TERT promoter wild-type subgroup (p = 0.001). Moreover, Kaplan-Meier survival analyses revealed a significantly shorter overall survival for GBM patients harbouring mutant tumors (p < 0.0001). In the multivariate Cox regression analysis TERT promoter mutation was found to have independent prognostic power (p = 0.049) but reached elevated significance in the interaction with age (p = 0.007). Accordingly, the prognostic quality of TERT promoter mutations was confined to the

  7. Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110α (PIK3CA)

    Science.gov (United States)

    Burke, John E.; Perisic, Olga; Masson, Glenn R.; Vadas, Oscar; Williams, Roger L.

    2012-01-01

    The p110α catalytic subunit (PIK3CA) is one of the most frequently mutated genes in cancer. We have examined the activation of the wild-type p110α/p85α and a spectrum of oncogenic mutants using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We find that for the wild-type enzyme, the natural transition from an inactive cytosolic conformation to an activated form on membranes entails four distinct events. Analysis of oncogenic mutations shows that all up-regulate the enzyme by enhancing one or more of these dynamic events. We provide the first insight into the activation mechanism by mutations in the linker between the adapter-binding domain (ABD) and the Ras-binding domain (RBD) (G106V and G118D). These mutations, which are common in endometrial cancers, enhance two of the natural activation events: movement of the ABD and ABD–RBD linker relative to the rest of the catalytic subunit and breaking the C2–iSH2 interface on binding membranes. C2 domain mutants (N345K and C420R) also mimic these events, even in the absence of membranes. A third event is breaking the nSH2–helical domain contact caused by phosphotyrosine-containing peptides binding to the enzyme, which is mimicked by a helical domain mutation (E545K). Interaction of the C lobe of the kinase domain with membranes is the fourth activation event, and is potentiated by kinase domain mutations (e.g., H1047R). All mutations increased lipid binding and basal activity, even mutants distant from the membrane surface. Our results elucidate a unifying mechanism in which diverse PIK3CA mutations stimulate lipid kinase activity by facilitating allosteric motions required for catalysis on membranes. PMID:22949682

  8. 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

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

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    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.

  10. 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.

  11. The G1613A mutation in the HBV genome affects HBeAg expression and viral replication through altered core promoter activity.

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    Man-Shan Li

    Full Text Available Infection of hepatitis B virus (HBV causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC. Previously, we demonstrated that the G1613A mutation in the HBV negative regulatory element (NRE is a hotspot mutation in HCC patients. In this study, we further investigated the functional consequences of this mutation in the context of the full length HBV genome and its replication. We showed that the G1613A mutation significantly suppresses the secretion of e antigen (HBeAg and enhances the synthesis of viral DNA, which is in consistence to our clinical result that the G1613A mutation associates with high viral load in chronic HBV carriers. To further investigate the molecular mechanism of the mutation, we performed the electrophoretic mobility shift assay with the recombinant RFX1 protein, a trans-activator that was shown to interact with the NRE of HBV. Intriguingly, RFX1 binds to the G1613A mutant with higher affinity than the wild-type sequence, indicating that the mutation possesses the trans-activating effect to the core promoter via NRE. The trans-activating effect was further validated by the enhancement of the core promoter activity after overexpression of RFX1 in liver cell line. In summary, our results suggest the functional consequences of the hotspot G1613A mutation found in HBV. We also provide a possible molecular mechanism of this hotspot mutation to the increased viral load of HBV carriers, which increases the risk to HCC.

  12. Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity

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    Shirou Tsuchida

    2015-03-01

    Full Text Available d-bifunctional protein (d-BP deficiency is thought to lead to severe lipid metabolism disorders. To investigate the effect of naturally occurring missense mutations in the hydratase domain in d-BP, we constructed several d-BP hydratase variants and measured their activities. Missense mutations at sites whose conservation rates among 30 eukaryotes were < 70% did not affect hydratase activity. We predicted that missense mutations of highly conserved amino acids would markedly reduce activity. However, R562H and R562L, naturally occurring missense mutations of highly conserved amino acids, did not reduce activity. This result suggests that a missense mutation in a highly conserved amino acid does not always lead to severe lipid metabolism disorders. We also investigated the effect of G525V, which had been found in a mildly symptomatic patient with d-BP deficiency who was heterozygous for G525 and G658X. G525V markedly reduced hydratase activity. We had predicted that heterozygous G525V and G658X would lead to severely disordered lipid metabolism. However, the symptoms were inconsistent with this prediction. Characterizing mutations in the d-BP gene and the symptoms of d-BP deficiency may require pleiotropy, not only in vitro, studies.

  13. 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.

  14. 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.

  15. Mutations in the c-Kit Gene Disrupt Mitogen-Activated Protein Kinase Signaling during Tumor Development in Adenoid Cystic Carcinoma of the Salivary Glands

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    Osamu Tetsu

    2010-09-01

    Full Text Available The Ras/mitogen-activated protein kinase (MAPK pathway is considered to be a positive regulator of tumor initiation, progression, and maintenance. This study reports an opposite finding: we have found strong evidence that the MAPK pathway is inhibited in a subset of adenoid cystic carcinomas (ACCs of the salivary glands. ACC tumors consistently overexpress the receptor tyrosine kinase (RTK c-Kit, which has been considered a therapeutic target. We performed mutational analysis of the c-Kit gene (KIT in 17 cases of ACC and found that 2 cases of ACC had distinct missense mutations in KIT at both the genomic DNA and messenger RNA levels. These mutations caused G664R and R796G amino acid substitutions in the kinase domains. Surprisingly, the mutations were functionally inactive in cultured cells. We observed a significant reduction of MAPK (ERK1/2 activity in tumor cells, as assessed by immunohistochemistry. We performed further mutational analysis of the downstream effectors in the c-Kit pathway in the genes HRAS, KRAS, NRAS, BRAF, PIK3CA, and PTEN. This analysis revealed that two ACC tumors without KIT mutations had missense mutations in either KRAS or BRAF, causing S17N K-Ras and V590I B-Raf mutants, respectively. Our functional analysis showed that proteins with these mutations were also inactive in cultured cells. This is the first time that MAPK activity from the RTK signaling has been shown to be inhibited by gene mutations during tumor development. Because ACC seems to proliferate despite inactivation of the c-Kit signaling pathway, we suggest that selective inhibition of c-Kit is probably not a suitable treatment strategy for ACC.

  16. Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity.

    Science.gov (United States)

    De Samblanx, G W; Goderis, I J; Thevissen, K; Raemaekers, R; Fant, F; Borremans, F; Acland, D P; Osborn, R W; Patel, S; Broekaert, W F

    1997-01-10

    Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.

  17. Mutation of a diacidic motif in SIV-PBj Nef impairs T-cell activation and enteropathic disease

    Directory of Open Access Journals (Sweden)

    Prüfer Steffen

    2011-03-01

    Full Text Available Abstract Background The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM. Results Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. Conclusions In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.

  18. 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

  19. 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

    and ammonia production. Peak oxidative capacity (VO2max) and cardiac output were determined, using cycle ergometry as the exercise modality. The two patients with atypical McArdle disease carried common mutations on one allele (R50X and G205S), and novel splice mutations in introns 3 [IVS3-26A>G (c.425-26A...... features of two patients with a variant form of McArdle disease, associated with unusually high exercise capacity. Physiologic findings were compared to those in 47 patients with typical McArdle disease, and 17 healthy subjects. Subjects performed an ischaemic forearm exercise test to assess lactate......>G)] and 5 [IVS5-601G>A (c.856-601G>A)] on the other allele. Plasma lactate after ischaemic exercise decreased in all typical McArdle patients, but increased in the two atypical McArdle patients (10% of that in healthy subjects). Peak workload and oxidative capacity were 2-fold higher in patients...

  20. Decreased gene expression activity as a result of a mutation in the calreticulin gene promoter in a family case of schizoaffective disorder.

    Science.gov (United States)

    Farashi, S; Ohadi, M; Hosseinkhani, S; Darvish, H; Mirabzadeh, A

    2016-06-01

    Accumulating evidence of population association studies support the hypothesis that the high heritability of major psychiatric disorders is a combination of relatively common alleles of modest effect, and rare alleles some with relatively larger effects. We have previously reported low frequency mutations in the proximal promoter of the human calreticulin (CALR) gene that co-occur with the spectrum of major psychiatric disorders. One of those mutations at -205C>T (rs556992558) was detected in an isolate case of schizoaffective disorder. In the current study, the functional implication of mutation -205T is studied in the human neuronal cell lines LAN-5, BE(2)-C and HEK-293. In contrast with other mutations in the promoter region which increase gene expression activity, the -205T mutation significantly decreased gene expression in those cell lines in comparison with the wild-type -205C nucleotide (p expression activity in the mutant -205T versus the wild-type -205C construct. VPA increased gene expression activity in both constructs, while a significantly higher expression activity was observed in the mutant construct (p < 0.01), indicative of the creation of a positive effector binding site for VPA as a result of the -205T mutation. We conclude that deviation from normalcy in the level of CALR in either direction is associated with major psychiatric disorders.

  1. 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.

  2. Dexamethasone partially rescues ataxia telangiectasia-mutated (ATM) deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity.

    Science.gov (United States)

    Menotta, Michele; Biagiotti, Sara; Bianchi, Marzia; Chessa, Luciana; Magnani, Mauro

    2012-11-30

    Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of nucleotide residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.

  3. The Ag-195 (C®G mutation in hereditary persistence of fetal hemoglobin is not associated with activation of a reporter gene in vitro

    Directory of Open Access Journals (Sweden)

    Schreiber R.

    2001-01-01

    Full Text Available Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G mutation. Furthermore, this is the first time that the -195 (C->G mutation of the Agamma-globin gene has been evaluated by in vitro gene expression.

  4. Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2

    DEFF Research Database (Denmark)

    Seemann, Petra; Schwappacher, Raphaela; Kjær, Klaus Wilbrandt

    2005-01-01

    Here we describe 2 mutations in growth and differentiation factor 5 (GDF5) that alter receptor-binding affinities. They cause brachydactyly type A2 (L441P) and symphalangism (R438L), conditions previously associated with mutations in the GDF5 receptor bone morphogenetic protein receptor type 1b...... activity when compared with WT GDF5. Biosensor interaction analyses revealed loss of binding to BMPR1A and BMPR1B ectodomains for the L441P mutant, whereas the R438L mutant showed normal binding to BMPR1B but increased binding to BMPR1A, the receptor normally activated by BMP2. The binding to NOGGIN...... was normal for both mutants. Thus, the brachydactyly type A2 phenotype (L441P) is caused by inhibition of the ligand-receptor interaction, whereas the symphalangism phenotype (R438L) is caused by a loss of receptor-binding specificity, resulting in a gain of function by the acquisition of BMP2-like...

  5. 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

  6. 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.

  7. P04.24MUTATIONS OF THE TERT PROMOTER CORRELATE WITH ENHANCED TELOMERASE ACTIVATION AND PREDICT A WORSE PROGNOSIS IN PRIMARY GLIOBLASTOMA PATIENTS

    OpenAIRE

    Spiegl-Kreinecker, S.; Loetsch, D.; Laaber, M.; Pichler, J; Weis, S.; Ghanim, B; Webersinke, G; Olschowski, A; Berger, W

    2014-01-01

    The stabilization of telomeres by upregulation of telomerase is compulsive for indefinite proliferation and cell immortalization therefore representing a main feature of malignant solid tumors, including gliomas. However, the mechanisms responsible for cancer-associated telomerase activation are not completely understood. Recently, defined mutations in the TERT promoter were identified in a variety of tumors, most frequent in primary glioblastomas (GBM). Presence of the mutations was associat...

  8. 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.

  9. 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

  10. Computational Identification of Amino-Acid Mutations that Further Improve the Activity of a Chalcone-Flavonone Isomerase from Glycine max.

    Science.gov (United States)

    Yuan, Hui; Wu, Jiaqi; Wang, Xiaoqiang; Chen, Jiakuan; Zhong, Yang; Huang, Qiang; Nan, Peng

    2017-01-01

    Protein design for improving enzymatic activity remains a challenge in biochemistry, especially to identify target amino-acid sites for mutagenesis and to design beneficial mutations for those sites. Here, we employ a computational approach that combines multiple sequence alignment, positive selection detection, and molecular docking to identify and design beneficial amino-acid mutations that further improve the intramolecular-cyclization activity of a chalcone-flavonone isomerase from Glycine max (GmCHI). By this approach, two GmCHI mutants with higher activities were predicted and verified. The results demonstrate that this approach could determine the beneficial amino-acid mutations for improving the enzymatic activity, and may find more applications in engineering of enzymes.

  11. Computational Identification of Amino-Acid Mutations that Further Improve the Activity of a Chalcone–Flavonone Isomerase from Glycine max

    Science.gov (United States)

    Yuan, Hui; Wu, Jiaqi; Wang, Xiaoqiang; Chen, Jiakuan; Zhong, Yang; Huang, Qiang; Nan, Peng

    2017-01-01

    Protein design for improving enzymatic activity remains a challenge in biochemistry, especially to identify target amino-acid sites for mutagenesis and to design beneficial mutations for those sites. Here, we employ a computational approach that combines multiple sequence alignment, positive selection detection, and molecular docking to identify and design beneficial amino-acid mutations that further improve the intramolecular-cyclization activity of a chalcone–flavonone isomerase from Glycine max (GmCHI). By this approach, two GmCHI mutants with higher activities were predicted and verified. The results demonstrate that this approach could determine the beneficial amino-acid mutations for improving the enzymatic activity, and may find more applications in engineering of enzymes. PMID:28286513

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

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    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.

  13. 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.

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

    Science.gov (United States)

    Turner, Claire E; Sommerlad, Mary; McGregor, Karen; Davies, Frances J; Pichon, Bruno; Chong, Deborah L W; Farzaneh, Leili; Holden, Matthew T G; Spratt, Brian G; Efstratiou, Androulla; Sriskandan, Shiranee

    2012-01-01

    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.

  15. Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains

    Science.gov (United States)

    Palma, Eleonora; Fucile, Sergio; Barabino, Benedetta; Miledi, Ricardo; Eusebi, Fabrizio

    1999-01-01

    Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric α7 receptors, and that mutating Leu-247 of the α7 nicotinic AcCho receptor-channel domain (L247Tα7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type α7 or L247Tα7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Tα7 receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189–190Sα7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker α-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic α7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical α7 subunits may be sufficient to determine the functional properties of α7 receptors. PMID:10557336

  16. An activating G{sub s}{alpha} mutation is present in fibrous dysplasia of bone in the McCune-Albright syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Shenker, A.; Weinstein, L.S.; Spiegel, A.M. [National Institutes of Health, Bethesda, MD (United States); Sweet, D.E. [Armed Forces Institute of Pathology, Washington, DC (United States)

    1994-09-01

    McCune-Albright syndrome (MAS) is a sporadic disease characterized by polyostotic fibrous dysplasia, cafe-au-lait spots, and multiple endocrinopathies. The etiology of fibrous dysplasia is unknown. Activating mutations of codon 201 in the gene encoding the {alpha}-subunit of G{sub s}, the G-protein that stimulates adenylyl cyclase, have been found in all affected MAS tissues that have been studied. Initial attempts to amplify DNA from decalcified paraffin-embedded bone specimens from frozen surgical bone specimens from five MAS patients using polymerase chain reaction and allele-specific oligonucleotide hybridization. Most of the cells in four specimens of dysplastic bone contained a heterozygous mutation encoding substitution of Arg{sup 201} of G{sub s}{alpha} with His, but the mutation was barely detectable in peripheral blood specimens from the patients. Only a small amount of mutant allele was detected in a specimen of normal cortical bone from the fifth patient, although this patients had a high proportion of mutation in other, affected tissues. The mosaic distribution of mutant alleles is consistent with an embryological somatic cell mutation of the G{sub s}{alpha} gene in MAS. The presence of an activating mutation of G{sub s}{alpha} in osteoblastic progenitor cells may cause them to exhibit increased proliferation and abnormal differentiation, thereby producing the lesions of fibrous dysplasia. 43 refs., 2 figs.

  17. Disrupted coupling of gating charge displacement to Na+ current activation for DIIS4 mutations in hypokalemic periodic paralysis.

    Science.gov (United States)

    Mi, Wentao; Rybalchenko, Volodymyr; Cannon, Stephen C

    2014-08-01

    Missense mutations at arginine residues in the S4 voltage-sensor domains of NaV1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K(+). Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K(+) during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K(+), yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na(+) current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type NaV1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na(+) was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼ 10 µS/cm(2) or 1% of the total resting conductance. Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na(+) current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na(+) current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced

  18. CACNA1H missense mutations associated with amyotrophic lateral sclerosis alter Cav3.2 T-type calcium channel activity and reticular thalamic neuron firing.

    Science.gov (United States)

    Rzhepetskyy, Yuriy; Lazniewska, Joanna; Blesneac, Iulia; Pamphlett, Roger; Weiss, Norbert

    2016-11-01

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. In a recent study by Steinberg and colleagues, 2 recessive missense mutations were identified in the Cav3.2 T-type calcium channel gene (CACNA1H), in a family with an affected proband (early onset, long duration ALS) and 2 unaffected parents. We have introduced and functionally characterized these mutations using transiently expressed human Cav3.2 channels in tsA-201 cells. Both of these mutations produced mild but significant changes on T-type channel activity that are consistent with a loss of channel function. Computer modeling in thalamic reticular neurons suggested that these mutations result in decreased neuronal excitability of thalamic structures. Taken together, these findings implicate CACNA1H as a susceptibility gene in amyotrophic lateral sclerosis.

  19. 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.

  20. 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.

  1. The helicase and ATPase activities of RECQL4 are compromised by mutations reported in three human patients

    DEFF Research Database (Denmark)

    Jensen, Martin Borch; Dunn, Christopher A; Keijzers, Guido

    2012-01-01

    RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations....... These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase...... a consistent pattern of functional deficiency and provide further support for a helicase-dependent cellular function of RECQL4 in addition to its N-terminus-dependent role in initiation of replication, a function that may underlie the phenotype of RECQL4-linked disease....

  2. 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.

  3. 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.

  4. Scale up and pharmacokinetic study of a novel mutated chimeric tissue plasminogen activator (mt-PA) in rats

    Science.gov (United States)

    Raigani, Mozhgan; Rouini, Mohammad-Reza; Golabchifar, Ali-Akbar; Mirabzadeh, Esmat; Vaziri, Behrouz; Barkhordari, Farzaneh; Davami, Fatemeh; Mahboudi, Fereidoun

    2017-01-01

    Because of high mortality caused by cardiovascular diseases, various fibrinolytic agents with diverse pharmacokinetic and pharmacodynamic properties have been developed. A novel mutated chimeric tissue plasminogen activator (mt-PA) was developed by the removal of first three domains of t-PA, insertion of GHRP sequence and mutation towards resistance to plasminogen activator inhibitor-1 (PAI-1). Mt-PA protein was expressed in Expi293F cells. The expression level of mt-PA was found to be 5000 IU/mL. Following purification, the pharmacokinetic properties of mt-PA were evaluated in three doses in rats. Data related to mt-PA were best fitted to two compartment model. With the increase in dose, the Area Under the plasma concentration-time Curve (AUC0→∞) increased. The elimination half-life (t1/2) of mt-PA was in the range of 19.1–26.1 min in three doses while that of Alteplase was 8.3 min. The plasma clearance (CLp) of mt-PA ranged from 3.8 to 5.9 mL/min in three doses, which was several times lower than that of Alteplase (142.6 mL/min). The mean residence time (MRT) of mt-PA ranged from 23.3–31.8 min in three doses, which was 4–5 times greater than that of Alteplase (6 min). Mt-PA showed extended half-life and mean residence time and is a good candidate for further clinical studies. PMID:28223717

  5. An X-linked channelopathy with cardiomegaly due to a CLIC2 mutation enhancing ryanodine receptor channel activity.

    Science.gov (United States)

    Takano, Kyoko; Liu, Dan; Tarpey, Patrick; Gallant, Esther; Lam, Alex; Witham, Shawn; Alexov, Emil; Chaubey, Alka; Stevenson, Roger E; Schwartz, Charles E; Board, Philip G; Dulhunty, Angela F

    2012-10-15

    Chloride intracellular channel 2 (CLIC2) protein is a member of the glutathione transferase class of proteins. Its' only known function is the regulation of ryanodine receptor (RyR) intracellular Ca(2+) release channels. These RyR proteins play a major role in the regulation of Ca(2+) signaling in many cells. Utilizing exome capture and deep sequencing of genes on the X-chromosome, we have identified a mutation in CLIC2 (c.303C>G, p.H101Q) which is associated with X-linked intellectual disability (ID), atrial fibrillation, cardiomegaly, congestive heart failure (CHF), some somatic features and seizures. Functional studies of the H101Q variant indicated that it stimulated rather than inhibited the action of RyR channels, with channels remaining open for longer times and potentially amplifying Ca(2+) signals dependent on RyR channel activity. The overly active RyRs in cardiac and skeletal muscle cells and neuronal cells would result in abnormal cardiac function and trigger post-synaptic pathways and neurotransmitter release. The presence of both cardiomegaly and CHF in the two affected males and atrial fibrillation in one are consistent with abnormal RyR2 channel function. Since the dysfunction of RyR2 channels in the brain via 'leaky mutations' can result in mild developmental delay and seizures, our data also suggest a vital role for the CLIC2 protein in maintaining normal cognitive function via its interaction with RyRs in the brain. Therefore, our patients appear to suffer from a new channelopathy comprised of ID, seizures and cardiac problems because of enhanced Ca(2+) release through RyRs in neuronal cells and cardiac muscle cells.

  6. Pacemaker Activity of the Human Sinoatrial Node: An Update on the Effects of Mutations in HCN4 on the Hyperpolarization-Activated Current

    Directory of Open Access Journals (Sweden)

    Arie O. Verkerk

    2015-01-01

    Full Text Available Since 2003, several loss-of-function mutations in the HCN4 gene, which encodes the HCN4 protein, have been associated with sinus node dysfunction. In human sinoatrial node (SAN, HCN4 is the most abundant of the four isoforms of the HCN family. Tetramers of HCN subunits constitute the ion channels that conduct the hyperpolarization-activated “funny” current (If, which plays an important modulating role in SAN pacemaker activity. Voltage-clamp experiments on HCN4 channels expressed in COS-7, CHO and HEK-293 cells, as well as in Xenopus oocytes have revealed changes in the expression and kinetics of mutant channels, but the extent to which especially the kinetic changes would affect If flowing during a human SAN action potential often remains unresolved. In our contribution to the Topical Collection on Human Single Nucleotide Polymorphisms and Disease Diagnostics, we provide an updated review of the mutation-induced changes in the expression and kinetics of HCN4 channels and provide an overview of their effects on If during the time course of a human SAN action potential, as assessed in simulated action potential clamp experiments. Future research may solve apparent inconsistencies between data from clinical studies and data from in vitro and in silico experiments.

  7. 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.

  8. mTOR signaling is activated by FLT3 kinase and promotes survival of FLT3-mutated acute myeloid leukemia cells

    Directory of Open Access Journals (Sweden)

    Panayiotidis Panayiotis

    2010-11-01

    Full Text Available Abstract Activating mutations of the FLT3 gene mediate leukemogenesis, at least in part, through activation of PI3K/AKT. The mammalian target of rapamycin (mTOR-Raptor signaling pathway is known to act downstream of AKT. Here we show that the mTOR effectors, 4EBP1, p70S6K and rpS6, are highly activated in cultured and primary FLT3-mutated acute myeloid leukemia (AML cells. Introduction of FLT3-ITD expressing constitutively activated FLT3 kinase further activates mTOR and its downstream effectors in BaF3 cells. We also found that mTOR signaling contributes to tumor cell survival, as demonstrated by pharmacologic inhibition of PI3K/AKT/mTOR, or total silencing of the mTOR gene. Furthermore, inhibition of FLT3 kinase results in downregulation of mTOR signaling associated with decreased survival of FLT3-mutated AML cells. These findings suggest that mTOR signaling operates downstream of activated FLT3 kinase thus contributing to tumor cell survival, and may represent a promising therapeutic target for AML patients with mutated-FLT3.

  9. Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity

    DEFF Research Database (Denmark)

    Nørgaard, P; Winther, Jakob R.

    2001-01-01

    Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during the folding of secretory proteins. PDI is composed of domains with structural homology...... to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight...... into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild...

  10. DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN.

    Science.gov (United States)

    Ito, Yasuhiro; Kitagawa, Mamiko; Ihashi, Nao; Yabe, Kimiko; Kimbara, Junji; Yasuda, Junichi; Ito, Hirotaka; Inakuma, Takahiro; Hiroi, Seiji; Kasumi, Takafumi

    2008-07-01

    The RIN gene encodes a putative MADS box transcription factor that controls tomato fruit ripening, and its ripening inhibitor (rin) mutation yields non-ripening fruit. In this study, the molecular properties of RIN and the rin mutant protein were clarified. The results revealed that the RIN protein accumulates in ripening fruit specifically and is localized in the nucleus of the cell. In vitro studies revealed that RIN forms a stable homodimer that binds to MADS domain-specific DNA sites. Analysis of binding site selection experiments revealed that the consensus binding sites of RIN highly resemble those of the SEPALLATA (SEP) proteins, which are Arabidopsis MADS box proteins that control the identity of floral organs. RIN exhibited a transcription-activating function similar to that exhibited by the SEP proteins. These results indicate that RIN exhibits similar molecular functions to SEP proteins although they play distinctly different biological roles. In vivo assays revealed that RIN binds to the cis-element of LeACS2. Our results also revealed that the rin mutant protein accumulates in the mutant fruit and exhibits a DNA-binding activity similar to that exhibited by the wild-type protein, but has lost its transcription-activating function, which in turn would inhibit ripening in mutant fruit.

  11. An Activating Mutation in sos-1 Identifies Its Dbl Domain as a Critical Inhibitor of the Epidermal Growth Factor Receptor Pathway during Caenorhabditis elegans Vulval Development▿

    Science.gov (United States)

    Modzelewska, Katarzyna; Elgort, Marc G.; Huang, Jingyu; Jongeward, Gregg; Lauritzen, Amara; Yoon, Charles H.; Sternberg, Paul W.; Moghal, Nadeem

    2007-01-01

    Proper regulation of receptor tyrosine kinase (RTK)-Ras-mitogen-activated protein kinase (MAPK) signaling pathways is critical for normal development and the prevention of cancer. SOS is a dual-function guanine nucleotide exchange factor (GEF) that catalyzes exchange on Ras and Rac. Although the physiologic role of SOS and its CDC25 domain in RTK-mediated Ras activation is well established, the in vivo function of its Dbl Rac GEF domain is less clear. We have identified a novel gain-of-function missense mutation in the Dbl domain of Caenorhabditis elegans SOS-1 that promotes epidermal growth factor receptor (EGFR) signaling in vivo. Our data indicate that a major developmental function of the Dbl domain is to inhibit EGF-dependent MAPK activation. The amount of inhibition conferred by the Dbl domain is equal to that of established trans-acting inhibitors of the EGFR pathway, including c-Cbl and RasGAP, and more than that of MAPK phosphatase. In conjunction with molecular modeling, our data suggest that the C. elegans mutation, as well as an equivalent mutation in human SOS1, activates the MAPK pathway by disrupting an autoinhibitory function of the Dbl domain on Ras activation. Our work suggests that functionally similar point mutations in humans could directly contribute to disease. PMID:17339331

  12. An activating mutation in sos-1 identifies its Dbl domain as a critical inhibitor of the epidermal growth factor receptor pathway during Caenorhabditis elegans vulval development.

    Science.gov (United States)

    Modzelewska, Katarzyna; Elgort, Marc G; Huang, Jingyu; Jongeward, Gregg; Lauritzen, Amara; Yoon, Charles H; Sternberg, Paul W; Moghal, Nadeem

    2007-05-01

    Proper regulation of receptor tyrosine kinase (RTK)-Ras-mitogen-activated protein kinase (MAPK) signaling pathways is critical for normal development and the prevention of cancer. SOS is a dual-function guanine nucleotide exchange factor (GEF) that catalyzes exchange on Ras and Rac. Although the physiologic role of SOS and its CDC25 domain in RTK-mediated Ras activation is well established, the in vivo function of its Dbl Rac GEF domain is less clear. We have identified a novel gain-of-function missense mutation in the Dbl domain of Caenorhabditis elegans SOS-1 that promotes epidermal growth factor receptor (EGFR) signaling in vivo. Our data indicate that a major developmental function of the Dbl domain is to inhibit EGF-dependent MAPK activation. The amount of inhibition conferred by the Dbl domain is equal to that of established trans-acting inhibitors of the EGFR pathway, including c-Cbl and RasGAP, and more than that of MAPK phosphatase. In conjunction with molecular modeling, our data suggest that the C. elegans mutation, as well as an equivalent mutation in human SOS1, activates the MAPK pathway by disrupting an autoinhibitory function of the Dbl domain on Ras activation. Our work suggests that functionally similar point mutations in humans could directly contribute to disease.

  13. Specific effects of BCL10 Serine mutations on phosphorylations in canonical and noncanonical pathways of NF-κB activation following carrageenan

    Science.gov (United States)

    Bhattacharyya, Sumit; Borthakur, Alip; Anbazhagan, Arivarasu N.; Katyal, Shivani; Dudeja, Pradeep K.

    2011-01-01

    To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-κB-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-κB nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-κB-inducing kinase (NIK) at Thr559, transforming growth factor-β-activating kinase (TAK) 1 at Thr184, Thr187, and Ser192, and inhibitory factor κBα (IκBα) at Ser32, were examined following mutation of BCL10 at Ser138 and at Ser218. Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser32)-IκBα to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser138 mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr559 phosphorylation. Hence, the phosphorylation of BCL10 Ser138, but not Ser218, emerged as a critical event in activation of the noncanonical pathway of NF-κB activation. Either BCL10 Ser138 or Ser218 mutation inhibited the phosphorylation of TAK1 at Thr184 and at Thr187, but not at Ser192. These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and IκBα and differentially affect the canonical and noncanonical pathways of NF-κB activation. PMID:21700900

  14. 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.

  15. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

    Science.gov (United States)

    Young, Christian D; Zimmerman, Lisa J; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B; Gatza, Michael L; Morrison, Meghan M; Moore, Preston D; Whitwell, Corbin A; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E; Silva, Grace O; Patel, Premal; Brantley-Sieders, Dana M; Levin, Maren; Horiates, Marina; Palma, Norma A; Wang, Kai; Stephens, Philip J; Perou, Charles M; Weaver, Alissa M; O'Shaughnessy, Joyce A; Chang, Jenny C; Park, Ben Ho; Liebler, Daniel C; Cook, Rebecca S; Arteaga, Carlos L

    2015-07-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer*

    Science.gov (United States)

    Young, Christian D.; Zimmerman, Lisa J.; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B.; Gatza, Michael L.; Morrison, Meghan M.; Moore, Preston D.; Whitwell, Corbin A.; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E.; Silva, Grace O.; Patel, Premal; Brantley-Sieders, Dana M.; Levin, Maren; Horiates, Marina; Palma, Norma A.; Wang, Kai; Stephens, Philip J.; Perou, Charles M.; Weaver, Alissa M.; O'Shaughnessy, Joyce A.; Chang, Jenny C.; Park, Ben Ho; Liebler, Daniel C.; Cook, Rebecca S.; Arteaga, Carlos L.

    2015-01-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR. PMID:25953087

  17. Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase.

    Science.gov (United States)

    Tamura, H; Tameishi, K; Yamada, A; Tomita, M; Matsuo, Y; Nishikawa, K; Ikezawa, H

    1995-01-01

    Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126-->Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80%, whereas that of D233G was not so profoundly affected. Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine(HNP), lysophosphatidylcholine (lysoPC) and p-nitro-phenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases. Images Figure 2 Figure 3 Figure 4 Figure 6 PMID:7639690

  18. 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.

  19. 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.

  20. 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

    detectable increases in phosphorylation of ERK (pERK), we stained biopsies from 36 CRC patients with activating mutations in the BRAF gene (BRAFV600E: BRAF(m)), the KRAS gene (KRAS(m)) or in neither (BRAF/KRAS(n)) with this optimized method. Staining was scored in blind-coded specimens by two observers....... Staining of stromal cells was used as a positive control. BRAF(m) or KRAS(m) tumors did not show higher staining scores than BRAF/KRAS(n) tumors. Although BRAFV600E staining occurred in over 90% of cancer cells in all 9 BRAF(m) tumors, 3 only showed staining for pERK in less than 10% of cancer cell nuclei....... 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...

  1. Effects of GSH1 and GSH2 Gene Mutation on Glutathione Synthetases Activity of Saccharomyces cerevisiae.

    Science.gov (United States)

    Xu, Wen; Jia, Haiyan; Zhang, Longmei; Wang, Haiyan; Tang, Hui; Zhang, Liping

    2017-08-01

    In this paper, three mutants from wild Saccharomyces cerevisiae HBU2.558, called U2.558, UN2.558, and UNA2.558, were screened by UV, sodium nitrite, Atmospheric and room temperature plasma, respectively. Glutathione production of the three mutants increased by 41.86, 72.09 and 56.76%, respectively. We detected the activity of glutathione synthetases and found that its activity was improved. Amino acid sequences of three mutant colonies were compared with HBU2.558. Four mutants: Leu51→Pro51 (L51P), Glu62→Val62 (E62V), Ala332→Glu332 (A332E) and Ser653→Gly653 (S653G) were found in the analysis of γ-glutamylcysteine ligase. L51 is located adjacently to the two active sites of GCL/E/Mg(2+)/ADP complex in the overall GCL structure. L51P mutant spread distortion on the β-sheet due to the fact that the φ was changed from -50.4° to -40.2°. A mutant Leu54→Pro54 (L54P) was found in the analysis of glutathione synthetase, and L54 was an amino acid located between an α-helix and a β-sheet. The results confirm that introduction of proline located at the middle of the β-sheet or at the N- or C-terminal between α-helix and β-sheet or, i.e., L51P and L54P, changed the φ, rigidity, hydrophobicity and conformational entropy, thus increased protein stability and improved the enzyme activity.

  2. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    Science.gov (United States)

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

  3. 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...

  4. Exploring the mechanism of DNA polymerases by analyzing the effect of mutations of active site acidic groups in Polymerase β.

    Science.gov (United States)

    Matute, Ricardo A; Yoon, Hanwool; Warshel, Arieh

    2016-11-01

    Elucidating the catalytic mechanism of DNA polymerase is crucial for a progress in the understanding of the control of replication fidelity. This work tries to advance the mechanistic understanding by analyzing the observed effect of mutations of the acidic groups in the active site of Polymerase β as well as the pH effect on the rate constant. The analysis involves both empirical valence bond (EVB) free energy calculations and considerations of the observed pH dependence of the reaction. The combined analysis indicates that the proton transfer (PT) from the nucleophilic O3' has two possible pathways, one to D256 and the second to the bulk. We concluded based on calculations and the experimental pH profile that the most likely path for the wild-type (WT) and the D256E and D256A mutants is a PT to the bulk, although the WT may also use a PT to Asp 256. Our analysis highlights the need for very extensive sampling in the calculations of the activation barrier and also clearly shows that ab initio QM/MM calculations that do not involve extensive sampling are unlikely to give a clear quantitative picture of the reaction mechanism. Proteins 2016; 84:1644-1657. © 2016 Wiley Periodicals, Inc.

  5. Ataxia Telangiectasia-Mutated (ATM) kinase activity is regulated by ATP-driven conformational changes in the Mre11/Rad50/Nbs1 (MRN) complex

    NARCIS (Netherlands)

    J.-H. Lee (Ji-Hoon); M.R. Mand (Michael); R.A. Deshpande (Rajashree); E. Kinoshita (Eri); S.-H. Yang (Soo-Hyun); C. Wyman (Claire); T.T. Paull

    2013-01-01

    textabstractThe Ataxia Telangiectasia-Mutated (ATM) protein kinase is recruited to sites of double-strand DNA breaks by the Mre11/Rad50/Nbs1 (MRN) complex, which also facilitates ATM monomerization and activation. MRN exists in at least two distinct conformational states, dependent on ATP binding an

  6. Activating E17K mutation in the gene encoding the protein kinase AKT1 in a subset of squamous cell carcinoma of the lung.

    Science.gov (United States)

    Malanga, Donatella; Scrima, Marianna; De Marco, Carmela; Fabiani, Fernanda; De Rosa, Nicla; De Gisi, Silvia; Malara, Natalia; Savino, Rocco; Rocco, Gaetano; Chiappetta, Gennaro; Franco, Renato; Tirino, Virginia; Pirozzi, Giuseppe; Viglietto, Giuseppe

    2008-03-01

    Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G-->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).

  7. Effects of mutations in active site heme ligands on the spectroscopic and catalytic properties of SoxAX cytochromes.

    Science.gov (United States)

    Kilmartin, James R; Bernhardt, Paul V; Dhouib, Rabeb; Hanson, Graeme R; Riley, Mark J; Kappler, Ulrike

    2016-09-01

    By attaching a sulfur substrate to a conserved cysteine of the SoxYZ carrier protein SoxAX cytochromes initiate the reaction cycle of the Sox (sulfur oxidation) multienzyme complex, which is the major pathway for microbial reoxidation of sulfur compounds in the environment. Despite their important role in this process, the reaction mechanism of the SoxAX cytochromes has not been fully elucidated. Here we report the effects of several active site mutations on the spectroscopic and enzymatic properties of the type II SoxAX protein from Starkeya novella, which in addition to two heme groups also contains a Cu redox centre. All substituted proteins contained these redox centres except for His231Ala which was unable to bind Cu(II). Substitution of the SoxA active site heme cysteine ligand with histidine resulted in increased microheterogeneity around the SoxA heme as determined by CW-EPR, while a SnSoxAX(C236A) substituted protein revealed a completely new, nitrogenous SoxA heme ligand. The same novel ligand was present in SnSoxAX(H231A) CW-EPR spectra, the first time that a ligand switch of the SoxA heme involving a nearby amino acid has been demonstrated. Kinetically, SnSoxAX(C236A) and SnSoxAX(C236H) showed reduced turnover, and in assays containing SoxYZ these mutants retained only ~25% of the wildtype activity. Together, these data indicate that the Cu redox centre can mediate a low level of activity, and that a possible ligand switch can occur during catalysis. It also appears that the SoxA heme cysteine ligand (and possibly the low redox potential) is important for an efficient reaction with SnSoxYZ/thiosulfate. Copyright © 2016 Elsevier Inc. All rights reserved.

  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. The prognostic IDH1( R132 ) mutation is associated with reduced NADP+-dependent IDH activity in glioblastoma

    NARCIS (Netherlands)

    Bleeker, F.E.; Atai, N.A.; Lamba, S.; Jonker, A.; Rijkeboer, D.; Bosch, K.S.; Tigchelaar, W.; Troost, D.; Vandertop, W.P.; Bardelli, A.; van Noorden, C.J.F.

    2010-01-01

    Somatic mutations in the isocitrate dehydrogenase 1 gene (IDH1) occur at high frequency in gliomas and seem to be a prognostic factor for survival in glioblastoma patients. In our set of 98 glioblastoma patients, IDH1 ( R132 ) mutations were associated with improved survival of 1 year on average, af

  10. 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.

  11. Single point mutations of aromatic residues in transmembrane helices 5 and -6 differentially affect TRPV4 activation by 4α-PDD and hypotonicity

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjær; Janssens, Annelies; Prenen, Jean

    2014-01-01

    The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due....... Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants - F617L and Y621L - showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli...

  12. 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.

  13. Impacts of the G145R Mutation on the Structure and Immunogenic Activity of the Hepatitis B Surface Antigen: A Computational Analysis

    Directory of Open Access Journals (Sweden)

    Rezaee

    2016-06-01

    Full Text Available Background Vaccine-escaped hepatitis B virus (HBV mutations occur within the “a” determinant area, which is located in the major hydrophilic region (MHR of the hepatitis B surface antigen (HBsAg protein. It is now well established that the common G145R mutation is highly capable of escaping from HBsAg immune recognition. However, the impacts of this mutation on the structure and immunogenic activity of HBsAg have been poorly investigated. Objectives The present study analyzed the effects of the G145R mutation on the structure and immunogenic activity of the HBsAg. Materials and Methods Three-dimensional (3D structure of HBsAg for both the wild-type and G145R mutant were predicted and refined using several web tools. After quantitative evaluations, the effects of the G145R mutation on the secondary and 3D structures of the HBsAg were investigated. In parallel, the immunogenic activity of the wild-type and mutant HBsAg was also analyzed using a ClusPro docking server as well as the IEDB web tool. Further analyses were performed via molecular dynamics (MD simulations using the GROMACS v5.0.2 simulation package. Results The G145R mutation causes a considerable reduction in the immunogenic activity of the HBsAg through a conformational change in the HBsAg antigenic loops. This mutation inserts a new β-strand in the “a” determinant region of the HBsAg, leading to a reduced binding affinity to its monoclonal antibody, MAb12. The G145R mutation also increased the compactness and stability of the HBsAg by enhancing the rigidity of the “a” determinant. Conclusions These data will be beneficial for designing more advanced antibodies for the recognition of the HBsAg in diagnostics. In addition, the results of this study may assist in the design or development of more effective hepatitis B vaccines.

  14. 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...... patients: two patients with OHS and one patient with classical Menkes disease. The pseudo-exons were inserted between exons 10 and 11, between exons 16 and 17 and between exons 14 and 15 in the three patients, as a result of deep intronic mutations. This is the first time the activation of pseudo...... mechanism, which has hitherto been overlooked.European Journal of Human Genetics advance online publication, 4 September 2013; doi:10.1038/ejhg.2013.191....

  15. Association of resistance to activated protein with the presence of Leiden and Cambridge Factor V mutations in Mexican patients with primary thrombophilia.

    Science.gov (United States)

    Zavala Hernández, César; Hernández Zamora, Edgar; Martínez Murillo, Carlos; Arenas Sordo, María de la Luz; González Orozco, Ana Elena; Reyes Maldonado, Elba

    2010-01-01

    Leiden and Cambridge factor V coagulation mutations and activated protein C resistance (RaPC) are alterations related with vein and artery thrombosis. In this study we aimed to determine whether RaPC is associated with the presence of Leiden and Cambridge mutation and the frequency of these mutations in the racially mestizo Mexican population. We included 150 Mexican patients with primary thrombophilia and 100 healthy subjects in this study. RaPC was determined using commercial methods and genotypes FV Leiden and FV Cambridge with PCR-RFLPs. RaPC was positive in four patients and in one control individual; however, there was no presence of Leiden or Cambridge mutation in the studied group; thus, RaPC was not correlated with the presence of any of the studied mutations. These results indicate that there are other primary or secondary causes different from those studied, which condition the presence of RaPC. Furthermore, the frequency obtained for RaPC in our thrombophilic population of racially mixed Mexicans is lower compared to that obtained in the Caucasian population, most probably because they are genetically different populations.

  16. 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.

  17. 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

  18. Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.

    Science.gov (United States)

    Maruyama, K; MacLennan, D H

    1988-01-01

    Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis. Images PMID:2966962

  19. Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects

    Directory of Open Access Journals (Sweden)

    Yang Ruifeng

    2012-04-01

    Full Text Available Abstract Background Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection. Results To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A or hyperstable (E45A capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5α. Conclusions The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the

  20. 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

  1. X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Yuki; Kawata, Masakado; Komura, Jun-ichiro; Ono, Tetsuya; Yamamoto, Kazuo

    2003-07-25

    Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T{yields}T:A transversion and G:C{yields}T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T{yields}T:A transversion and G:C{yields}T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis.

  2. 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

  3. Mutation of residues 423 (Met/Ile), 444 (Thr/Met), and 506 (Asn/Ser) confer cholesteryl esterase activity on rat lung carboxylesterase. Ser-506 is required for activation by cAMP-dependent protein kinase.

    Science.gov (United States)

    Wallace, T J; Kodsi, E M; Langston, T B; Gergis, M R; Grogan, W M

    2001-08-31

    Site-directed mutagenesis is used to identify amino acid residues that dictate reported differences in substrate specificity between rat hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) and rat lung carboxylesterase (LCE), proteins differing by only 4 residues in their primary sequences. Beginning with LCE, the substitution Met(423) --> Ile(423) alone or in combination with other mutations increased activity with p-nitrophenylcaprylate (PNPC) relative to more hydrophilic p-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr(444) --> Met(444) was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn(506) --> Ser(506), creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase, a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase activity by the mutated LCE. The substitution Gln(186) --> Arg(186) selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that provide insight into the effects of these mutations on substrate specificity.

  4. 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.

  5. Single exon mutation in arylsulfatase A gene has two effects: loss of enzyme activity and aberrant splicing.

    Science.gov (United States)

    Hasegawa, Y; Kawame, H; Ida, H; Ohashi, T; Eto, Y

    1994-04-01

    The arylsulfatase A gene of a Japanese patient who has the juvenile form of metachromatic leukodystrophy, and who has been previously reported as a heterozygote of the 1070A mutation, was investigated. Nucleotide sequence analysis revealed the presence of a previously unreported C-to-T substitution (designated 2330T), 22 nucleotides downstream from the exon 8 splice acceptor site. Although the 2330T mutation itself results in a single amino acid substitution of Thr409 by Ile, the analysis of the patient's cDNA fragments amplified by the reverse transcription-polymerase chain reaction revealed that transcripts of the 2330T allele were spliced both normally and aberrantly. The aberrant splicing produced a 27-nucleotide deletion from the usual exon 8 splice acceptor site. These results indicate that the new mutation is a rare case of an exon mutation affecting splice site selection. The mechanism of this aberrant pre-mRNA splicing is discussed.

  6. Small-fiber neuropathy Nav1.8 mutation shifts activation to hyperpolarized potentials and increases excitability of dorsal root ganglion neurons.

    Science.gov (United States)

    Huang, Jianying; Yang, Yang; Zhao, Peng; Gerrits, Monique M; Hoeijmakers, Janneke G J; Bekelaar, Kim; Merkies, Ingemar S J; Faber, Catharina G; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2013-08-28

    Idiopathic small-fiber neuropathy (I-SFN), clinically characterized by burning pain in distal extremities and autonomic dysfunction, is a disorder of small-caliber nerve fibers of unknown etiology with limited treatment options. Functional variants of voltage-gated sodium channel Nav1.7, encoded by SCN9A, have been identified in approximately one-third of I-SFN patients. These variants render dorsal root ganglion (DRG) neurons hyperexcitable. Sodium channel Nav1.8, encoded by SCN10A, is preferentially expressed in small-diameter DRG neurons, and produces most of the current underlying the upstroke of action potentials in these neurons. We previously demonstrated two functional variants of Nav1.8 that either enhance ramp current or shift activation in a hyperpolarizing direction, and render DRG neurons hyperexcitable, in I-SFN patients with no mutations of SCN9A. We have now evaluated additional I-SFN patients with no mutations in SCN9A, and report a novel I-SFN-related Nav1.8 mutation I1706V in a patient with painful I-SFN. Whole-cell voltage-clamp recordings in small DRG neurons demonstrate that the mutation hyperpolarizes activation and the response to slow ramp depolarizations. However, it decreases fractional channels resistant to fast inactivation and reduces persistent currents. Current-clamp studies reveal that mutant channels decrease current threshold and increase the firing frequency of evoked action potentials within small DRG neurons. These observations suggest that the effects of this mutation on activation and ramp current are dominant over the reduced persistent current, and show that these pro-excitatory gating changes confer hyperexcitability on peripheral sensory neurons, which may contribute to pain in this individual with I-SFN.

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

    Science.gov (United States)

    Mondini, Anna; Sassone, Francesca; Civello, Davide Antonio; Garavaglia, Maria Lisa; Bazzini, Claudia; Rodighiero, Simona; Vezzoli, Valeria; Conti, Fabio; Torielli, Lucia; Capasso, Giovanbattista; Paulmichl, Markus; Meyer, Giuliano

    2012-01-01

    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.

  8. 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.

  9. 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.

  10. 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......-associated segmental overgrowth syndrome overlaps with CLOVES syndrome and fibroadipose hyperplasia but is distinct from each of these entities....

  11. Next-Generation Sequencing Reveals Deep Intronic Cryptic ABCC8 and HADH Splicing Founder Mutations Causing Hyperinsulinism by Pseudoexon Activation

    Science.gov (United States)

    Flanagan, Sarah E.; Xie, Weijia; Caswell, Richard; Damhuis, Annet; Vianey-Saban, Christine; Akcay, Teoman; Darendeliler, Feyza; Bas, Firdevs; Guven, Ayla; Siklar, Zeynep; Ocal, Gonul; Berberoglu, Merih; Murphy, Nuala; O’Sullivan, Maureen; Green, Andrew; Clayton, Peter E.; Banerjee, Indraneel; Clayton, Peter T.; Hussain, Khalid; Weedon, Michael N.; Ellard, Sian

    2013-01-01

    Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ∼10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals’ fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease. PMID:23273570

  12. Molecular Effects of cTnC DCM Mutations on Calcium Sensitivity and Myofilament Activation-An Integrated Multiscale Modeling Study.

    Science.gov (United States)

    Dewan, Sukriti; McCabe, Kimberly J; Regnier, Michael; McCulloch, Andrew D; Lindert, Steffen

    2016-08-25

    Mutations in cardiac troponin C (D75Y, E59D, and G159D), a key regulatory protein of myofilament contraction, have been associated with dilated cardiomyopathy (DCM). Despite reports of altered myofilament function in these mutants, the underlying molecular alterations caused by these mutations remain elusive. Here we investigate in silico the intramolecular mechanisms by which these mutations affect myofilament contraction. On the basis of the location of cardiac troponin C (cTnC) mutations, we tested the hypothesis that intramolecular effects can explain the altered myofilament calcium sensitivity of force development for D75Y and E59D cTnC, whereas altered cardiac troponin C-troponin I (cTnC-cTnI) interaction contributes to the reported contractile effects of the G159D mutation. We employed a multiscale approach combining molecular dynamics (MD) and Brownian dynamics (BD) simulations to estimate cTnC calcium association and hydrophobic patch opening. We then integrated these parameters into a Markov model of myofilament activation to compute the steady-state force-pCa relationship. The analysis showed that myofilament calcium sensitivity with D75Y and E59D can be explained by changes in calcium binding affinity of cTnC and the rate of hydrophobic patch opening, if a partial cTnC interhelical opening angle (110°) is sufficient for cTnI switch peptide association to cTnC. In contrast, interactions between cTnC and cTnI within the cardiac troponin complex must also be accounted for to explain contractile alterations due to G159D. In conclusion, this is the first multiscale in silico study to elucidate how direct molecular effects of genetic mutations in cTnC translate to altered myofilament contractile function.

  13. Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza.

    Science.gov (United States)

    Schulert, Grant S; Zhang, Mingce; Fall, Ndate; Husami, Ammar; Kissell, Diane; Hanosh, Andrew; Zhang, Kejian; Davis, Kristina; Jentzen, Jeffrey M; Napolitano, Lena; Siddiqui, Javed; Smith, Lauren B; Harms, Paul W; Grom, Alexei A; Cron, Randy Q

    2016-04-01

    Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza. Sixteen cases of fatal influenza A(H1N1) infection, 81% with histopathologic hemophagocytosis, were identified and analyzed for clinical and laboratory features of HLH, using modified HLH-2004 and macrophage activation syndrome (MAS) criteria. Fourteen specimens were subject to whole-exome sequencing. Sequence alignment and variant filtering detected HLH gene mutations and potential disease-causing variants. Cytolytic function of the PRF1 p.A91V mutation was tested in lentiviral-transduced NK-92 natural killer (NK) cells. Despite several lacking variables, cases of influenza A(H1N1) infection met 44% and 81% of modified HLH-2004 and MAS criteria, respectively. Five subjects (36%) carried one of 3 heterozygous LYST mutations, 2 of whom also possessed the p.A91V PRF1 mutation, which was shown to decrease NK cell cytolytic function. Several patients also carried rare variants in other genes previously observed in MAS. This cohort of fatal influenza A(H1N1) infections confirms the presence of hemophagocytosis and HLH pathology. Moreover, the high percentage of HLH gene mutations suggests they are risk factors for mortality among individuals with influenza A(H1N1) infection. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  14. 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.

  15. 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

  16. 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.

  17. Plasminogen activator inhibitor-1 4G/5G polymorphism, factor V Leiden, prothrombin mutations and the risk of VTE recurrence.

    Science.gov (United States)

    Sundquist, Kristina; Wang, Xiao; Svensson, Peter J; Sundquist, Jan; Hedelius, Anna; Larsson Lönn, Sara; Zöller, Bengt; Memon, Ashfaque A

    2015-11-25

    Plasminogen-activator inhibitor (PAI)-1 is an important inhibitor of the plasminogen/plasmin system. PAI-1 levels are influenced by the 4G/5G polymorphism in the PAI-1 promoter. We investigated the relationship between the PAI-1 polymorphism and VTE recurrence, and its possible modification by factor V Leiden (FVL) and prothrombin (PTM) mutations. Patients (n=1,069) from the Malmö Thrombophilia Study were followed from discontinuation of anticoagulant treatment until diagnosis of VTE recurrence or the end of the study (maximum follow-up 9.8 years). One hundred twenty-seven patients (11.9 %) had VTE recurrence. PAI-1 was genotyped by TaqMan PCR. Cox regression analysis adjusted for age, sex and acquired risk factors of VTE showed no evidence of an association between PAI-1 genotype and risk of VTE recurrence in the study population as a whole. However, by including an interaction term in the analysis we showed that FVL but not PTM modified the effect of PAI-1 genotype: patients with the 4G allele plus FVL had a higher risk of VTE recurrence [hazard ratio (HR) =2.3, 95 % confidence interval (CI) =1.5-3.3] compared to patients with the 4G allele but no FVL (reference group) or FVL irrespective of PAI-1 genotype (HR=1.8, 95 % CI=1.3-2.5). Compared to reference group, 5G allele irrespective of FVL was associated with lower risk of VTE recurrence only when compared with 4G allele together with FVL. In conclusion, FVL has a modifying effect on PAI-1 polymorphism in relation to risk of VTE recurrence. The role of PAI-1 polymorphism as a risk factor of recurrent VTE may be FVL dependent.

  18. 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.

  19. Dysregulation of large-conductance Ca2+-activated K+ channel expression in nonsyndromal mental retardation due to a cereblon p.R419X mutation.

    Science.gov (United States)

    Higgins, Joseph J; Hao, Jin; Kosofsky, Barry E; Rajadhyaksha, Anjali M

    2008-07-01

    A nonsense mutation (R419X) in the human cereblon gene [mutation (mut) CRBN] causes a mild type of autosomal recessive nonsyndromal mental retardation (ARNSMR). CRBN, a cytosolic protein, regulates the assembly and neuronal surface expression of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) in brain regions involved in memory and learning. Using the real-time quantitative polymerase chain reaction, we show that mut CRBN disturbs the development of adult brain BK(Ca) isoforms. These changes are predicted to result in BK(Ca) channels with a higher intracellular Ca(2+) sensitivity, faster activation, and slower deactivation kinetics. Such alterations may contribute to cognitive impairments in patients with mild ARNSMR.

  20. Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Tachibana, C; Winther, Jakob R.

    1997-01-01

    Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS....... Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC...... active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important...

  1. Complete androgen insensitivity syndrome caused by a novel splice donor site mutation and activation of a cryptic splice donor site in the androgen receptor gene.

    Science.gov (United States)

    Infante, Joana B; Alvelos, Maria I; Bastos, Margarida; Carrilho, Francisco; Lemos, Manuel C

    2016-01-01

    The androgen insensitivity syndrome is an X-linked recessive genetic disorder characterized by resistance to the actions of androgens in an individual with a male karyotype. We evaluated a 34-year-old female with primary amenorrhea and a 46,XY karyotype, with normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the androgen receptor (AR) gene revealed a novel splice donor site mutation in intron 4 (c.2173+2T>C). RT-PCR analysis showed that this mutation resulted in the activation of a cryptic splice donor site located in the second half of exon 4 and in the synthesis of a shorter mRNA transcript and an in-frame deletion of 41 amino acids. This novel mutation associated with a rare mechanism of abnormal splicing further expands the spectrum of mutations associated with the androgen insensitivity syndrome and may contribute to the understanding of the molecular mechanisms involved in splicing defects.

  2. The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants.

    Science.gov (United States)

    Medová, Michaela; Pochon, Benoît; Streit, Bruno; Blank-Liss, Wieslawa; Francica, Paola; Stroka, Deborah; Keogh, Adrian; Aebersold, Daniel M; Blaukat, Andree; Bladt, Friedhelm; Zimmer, Yitzhak

    2013-11-01

    The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results show a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five of the eight cell lines (IC50 2-43 nmol/L). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell-cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biologic functions, such as cellular morphology, MET-dependent cell motility, and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET-mutated variants. Animals were randomized for the treatment with EMD1214063 (50 mg/kg/d) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, whereas tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small-molecule inhibitor with selective activity towards mutated MET variants. ©2013 AACR.

  3. Thrombocytosis and STAT5 activation in chronic myelogenous leukaemia are not associated with JAK2 V617F or calreticulin mutations.

    Science.gov (United States)

    Turakhia, Samir K; Murugesan, Gurunathan; Cotta, Claudiu V; Theil, Karl S

    2016-08-01

    Marked thrombocytosis is uncommon in chronic myelogenous leukaemia (CML) but may be associated with mutation of JAK2 V617F, calreticulin (CALR) and/or phospho-STAT5 (p-STAT5) activation in other myeloproliferative neoplasms (MPNs), particularly essential thrombocythaemia (ET). We investigated the JAK2 V617F, CALR and STAT5 activation status in patients with CML and thrombocytosis (CML-T) that mimicked ET, trying to identify a common mechanism for thrombocytosis in MPN. Blood and bone marrow morphological findings were reviewed from seven CML-T, four otherwise typical CML and one CML in blast phase. All cases were analysed for BCR-ABL1, JAK2 V617F and CALR exon 9 mutation and p-STAT5 expression. Four of seven cases of CML-T had marked thrombocytosis (>1000×10(9)/L). Eleven of 12 cases had megakaryocyte morphology typical for CML. All cases were BCR-ABL1 positive. Eleven of 12 cases were negative for JAK2 V617F, while STAT5 was activated in six of seven CML-T and in four of five CML cases. No case had a detectable CALR exon 9 mutation. One case of CML developed ET-like morphology and had JAK2 V617F detected while in molecular remission for CML. Detection of BCR-ABL1 is critical in the distinction of ET from CML. Thrombocytosis and STAT5 activation in CML-T are not consistently associated with CALR exon 9 or JAK2 V617F mutation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  4. 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.

  5. 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.

  6. Whacking a mole-cule: clinical activity and mechanisms of resistance to third generation EGFR inhibitors in EGFR mutated lung cancers with EGFR-T790M.

    Science.gov (United States)

    Costa, Daniel B; Kobayashi, Susumu S

    2015-12-01

    Epidermal growth factor receptor (EGFR) mutations, especially EGFR-exon 19 deletions and EGFR-L858R, are the most frequent actionable genomic events in lung adenocarcinomas. Tumors arise due to constitutively activated EGFR signaling and are susceptible to EGFR tyrosine kinase inhibitors (TKIs). First generation EGFR TKIs (gefitinib and erlotinib) and the second generation EGFR TKI afatinib are approved worldwide. Although targeted therapies against EGFR mutants induce dramatic initial responses, acquired resistance (through multiple biological mechanisms) to erlotinib, gefitinib and afatinib emerges within the first 1-2 years of continued monotherapy. EGFR-T790M accounts for more than half of acquired resistance to first or second generation EGFR TKIs by modifying ATP affinity and drug binding kinetics. Two new studies have shown that two covalent pyrimidine inhibitors-AZD9291 and rociletinib of EGFR-T790M (i.e., third generation EGFR TKIs) shown remarkable clinical activity in patients with acquired resistance to erlotinib, gefitinib and afatinib when the tumor carries EGFR-T790M in conjunction with an activating mutation. However, and regrettably, acquired resistance to these third generation EGFR TKIs has already been reported in preclinical models and clinical specimens; such as a tertiary mutation at EGFR-C797S that prevents covalent binding of EGFR TKIs. The experience with sequential EGFR TKI monotherapy highlights tumor heterogeneity and adaptability (i.e., relentless game of whack-a-mole played between TKIs and cancer), and will help shape future clinical development of novel combinatory approaches to manage EGFR mutated lung adenocarcinomas.

  7. Oncogenic mutations in adenomatous polyposis coli (Apc activate mechanistic target of rapamycin complex 1 (mTORC1 in mice and zebrafish

    Directory of Open Access Journals (Sweden)

    Alexander J. Valvezan

    2014-01-01

    Full Text Available Truncating mutations in adenomatous polyposis coli (APC are strongly linked to colorectal cancers. APC is a negative regulator of the Wnt pathway and constitutive Wnt activation mediated by enhanced Wnt–β-catenin target gene activation is believed to be the predominant mechanism responsible for APC mutant phenotypes. However, recent evidence suggests that additional downstream effectors contribute to APC mutant phenotypes. We previously identified a mechanism in cultured human cells by which APC, acting through glycogen synthase kinase-3 (GSK-3, suppresses mTORC1, a nutrient sensor that regulates cell growth and proliferation. We hypothesized that truncating Apc mutations should activate mTORC1 in vivo and that mTORC1 plays an important role in Apc mutant phenotypes. We find that mTORC1 is strongly activated in apc mutant zebrafish and in intestinal polyps in Apc mutant mice. Furthermore, mTORC1 activation is essential downstream of APC as mTORC1 inhibition partially rescues Apc mutant phenotypes including early lethality, reduced circulation and liver hyperplasia. Importantly, combining mTORC1 and Wnt inhibition rescues defects in morphogenesis of the anterior-posterior axis that are not rescued by inhibition of either pathway alone. These data establish mTORC1 as a crucial, β-catenin independent effector of oncogenic Apc mutations and highlight the importance of mTORC1 regulation by APC during embryonic development. Our findings also suggest a new model of colorectal cancer pathogenesis in which mTORC1 is activated in parallel with Wnt/β-catenin signaling.

  8. Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice.

    Science.gov (United States)

    Xu, Haoxing; Delling, Markus; Li, Linyu; Dong, Xianping; Clapham, David E

    2007-11-13

    Transient receptor potential (TRP) genes of the mucolipin subfamily (TRPML1-3 and MCOLN1-3) are presumed to encode ion channel proteins of intracellular endosomes and lysosomes. Mutations in human TRPML1 (mucolipin 1/MCOLN1) result in mucolipidosis type IV, a severe inherited neurodegenerative disease associated with defective lysosomal biogenesis and trafficking. A mutation in mouse TRPML3 (A419P; TRPML3(Va)) results in the varitint-waddler (Va) phenotype. Va mice are deaf, exhibit circling behavior due to vestibular defects, and have variegated/dilute coat color as a result of pigmentation defects. Prior electrophysiological studies of presumed TRPML plasma membrane channels are contradictory and inconsistent with known TRP channel properties. Here, we report that the Va mutation produces a gain-of-function that allows TRPML1 and TRPML3 to be measured and identified as inwardly rectifying, proton-impermeant, Ca(2+)-permeant cation channels. TRPML3 is highly expressed in normal melanocytes. Melanocyte markers are lost in the Va mouse, suggesting that their variegated and hypopigmented fur is caused by severe alteration of melanocyte function or cell death. TRPML3(Va) expression in melanocyte cell lines results in high resting Ca(2+) levels, rounded, poorly adherent cells, and loss of membrane integrity. We conclude that the Va phenotype is caused by mutation-induced TRPML3 gain-of-function, resulting in cell death.

  9. Concerted action of target-site mutations and high EPSPS activity in glyphosate-resistant junglerice (Echinochloa colona) from California

    Science.gov (United States)

    Glyphosate is the most widely used non-selective herbicide and Echinochloa colona is an annual weed affecting field crops and orchards in California. A population carrying a glyphosate-resistance-endowing mutation in the EPSPS gene was found in the Northern Sacramento Valley. We used selfed lines ...

  10. 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.

  11. Septin mutations in human cancers

    Directory of Open Access Journals (Sweden)

    Elias T Spiliotis

    2016-11-01

    Full Text Available Septins are GTP-binding proteins that are evolutionarily and structurally related to the RAS oncogenes. Septin expression levels are altered in many cancers and new advances point to how abnormal septin expression may contribute to the progression of cancer. In contrast to the RAS GTPases, which are frequently mutated and actively promote tumorigenesis, little is known about the occurrence and role of septin mutations in human cancers. Here, we review septin missense mutations that are currently in the Catalog of Somatic Mutations in Cancer (COSMIC database. The majority of septin mutations occur in tumors of the large intestine, skin, endometrium and stomach. Over 25% of the annotated mutations in SEPT2, SEPT4 and SEPT9 belong to large intestine tumors. From all septins, SEPT9 and SEPT14 exhibit the highest mutation frequencies in skin, stomach and large intestine cancers. While septin mutations occur with frequencies lower than 3%, recurring mutations in several invariant and highly conserved amino acids are found across different septin paralogs and tumor types. Interestingly, a significant number of these mutations occur in the GTP-binding pocket and septin dimerization interfaces. Future studies may determine how these somatic mutations affect septin structure and function, whether they contribute to the progression of specific cancers and if they could serve as tumor-specific biomarkers.

  12. 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.

  13. Friend or foe: can activating mutations in NOTCH1 contribute to a favorable treatment outcome in patients with T-ALL?

    Science.gov (United States)

    Goldshtein, Aviya; Berger, Michael

    2014-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Despite significant improvement in the treatment of T-ALL, approximately 20% of children and most adults succumb to resistant or relapsed disease. Transformation events occur during crucial steps of thymocyte development and have been related to the expression of certain oncogenes such as TAL2, TLX1, LYL1, LMO1, and NOTCH1. Mutations that lead to constitutive activation of NOTCH1 are most commonly found in human patients with T-ALL. Moreover, overexpression of the intracellular portion of NOTCH1 can lead to the initiation of T-ALL in mouse models. These findings suggest that NOTCH1 may promote tumorigenesis through the regulation of differentiation of leukemic cells, and, potentially, of leukemia-initiating cell identity and function. Multiple studies and clinical trials aimed at targeting NOTCH1 in T-ALL or using NOTCH1 mutations as a prognostic tool are currently underway. Recent studies unexpectedly found that activating mutations in NOTCH1 are correlated with better treatment outcome. Here we review these studies and discuss possible explanations for these findings.

  14. 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...... 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...

  15. 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.

  16. 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.

  17. Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system.

    Science.gov (United States)

    Ulbrich, Lisa; Favaloro, Flores Lietta; Trobiani, Laura; Marchetti, Valentina; Patel, Vruti; Pascucci, Tiziana; Comoletti, Davide; Marciniak, Stefan J; De Jaco, Antonella

    2016-02-15

    Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations.

  18. [Effect of α -1,2 fucosyltransferase gene 682A> G and 547_552delAG mutations on the activity of fucosyltransferase].

    Science.gov (United States)

    Tao, Sudan; He, Yanmin; Xu, Xianguo; Hong, Xiaozhen; He, Ji; Zhu, Faming; Lyu, Hangjun

    2014-10-01

    To explore the effect of α -1,2 fucosyltransferase (FUT1) gene 682A> G and 547_552delAG mutations on the expression of FUT1 mRNA and activity of α -1,2 fucosyltransferase. Recombinant expression vectors of FUT1 682A> G and FUT1 547_552delAG were constructed and transfected into COS-7 cells for stable expression screening. Expression of FUT1 mRNA was determined using real-time quantitative PCR. The activity of FUT1 was measured with high-performance liquid chromatography. Stably transfected COS-7 cells with wild type FUT1, FUT1 682A> G and FUT1 547_552delAG were respectively obtained. The FUT1 mRNA level of transfected cells with 682A> G and 547_552delAG recombination vectors have measured 101.69% and 102.79% compared with that of wild type FUT1 transfected cells. A specific protein band with about 46 kD was confirmed in the 682A> G transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6× His Tag antibody. Similar protein was not identified in the 547_552delAG cells lysates. Enzymes activity of FUT1 682A> G has measured 61.01% compared with wild type FUT1 protein, whilst the activity of FUT1 547_557delAG was completely abolished. FUT1 682A> G and 547_552delAG mutations do not affect the transcript efficiency, although various mutations have different impact on the enzyme's activity.

  19. 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.

  20. 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.

  1. 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.

  2. Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia.

    Science.gov (United States)

    Dawson, M A; Gudgin, E J; Horton, S J; Giotopoulos, G; Meduri, E; Robson, S; Cannizzaro, E; Osaki, H; Wiese, M; Putwain, S; Fong, C Y; Grove, C; Craig, J; Dittmann, A; Lugo, D; Jeffrey, P; Drewes, G; Lee, K; Bullinger, L; Prinjha, R K; Kouzarides, T; Vassiliou, G S; Huntly, B J P

    2014-02-01

    Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common 'core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain 'super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.

  3. 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.

  4. Activating somatic mutations in diffuse large B-cell lymphomas: lessons from next generation sequencing and key elements in the precision medicine era.

    Science.gov (United States)

    Bohers, Elodie; Mareschal, Sylvain; Bertrand, Philippe; Viailly, Pierre Julien; Dubois, Sydney; Maingonnat, Catherine; Ruminy, Philippe; Tilly, Hervé; Jardin, Fabrice

    2015-05-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30-40% of newly diagnosed non-Hodgkin lymphomas. Historically, DLBCL has been thought to involve recurrent translocations of the immunoglobulin heavy (IGH) locus and the deregulation of rearranged oncogenes. Whole exome sequencing (WES) of more than 200 DLBCLs has completely redefined the genetic landscape of the disease by identifying recurrent single nucleotide variants and providing new therapeutic opportunities in DLBCL molecular subtypes. Some of these somatic mutations target genes that play a crucial role in B-cell function (B cell receptor [BCR] signaling, nuclear factor κB [NF-κB] pathway, Toll-like receptor [TLR] signaling and phosphatidylinositol 3-kinase [PI3K] pathway), immunity, cell cycle/apoptosis or chromatin modification. In this review, following an overview of the somatic mutations reported in DLBCL, we focus on activating and clustered mutations targeting genes including MYD88, CD79A/B, EZH2 and CARD11 and discuss their clinical and therapeutic relevance in the precision medicine era.

  5. Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Herraiz, Cecilia; Jiménez-Cervantes, Celia; Zanna, Paola; García-Borrón, José C

    2009-10-06

    Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogen-activated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose-response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.

  6. The glucose-dependent insulinotropic polypeptide receptor is overexpressed amongst GNAS1 mutation-negative somatotropinomas and drives growth hormone (GH)-promoter activity in GH3 cells.

    Science.gov (United States)

    Occhi, G; Losa, M; Albiger, N; Trivellin, G; Regazzo, D; Scanarini, M; Monteserin-Garcia, J L; Fröhlich, B; Ferasin, S; Terreni, M R; Fassina, A; Vitiello, L; Stalla, G; Mantero, F; Scaroni, C

    2011-07-01

    Somatic mutations in the GNAS1 gene, encoding the α-subunit of the heterotrimeric stimulatory G protein (Gαs), occur in approximately 40% of growth hormone (GH)-secreting pituitary tumours. By altering the adenylate cyclase-cAMP-protein kinase A pathway, they unequivocally give somatotroph cells a growth advantage. Hence, the pathogenesis of somatotropinomas could be linked to anomalies in receptors coupled to the cAMP second-messenger cascade. Among them, the glucose-dependent insulinotropic polypeptide receptor (GIPR) is already known to play a primary role in the impaired cAMP-dependent cortisol secretion in patients affected by food-dependent Cushing's syndrome. In the present study, 43 somatotropinomas and 12 normal pituitary glands were investigated for GIPR expression by quantitative reverse transcriptase-polymerase chain reaction, western blotting and immunohistochemistry. Tumoural specimens were also evaluated for GNAS1 mutational status. The effect of GIPR overexpression on cAMP levels and GH transcription was evaluated in an in vitro model of somatotropinomas, the GH-secreting pituitary cell line GH3. GIPR was expressed at higher levels compared to normal pituitaries in 13 GNAS1 mutation-negative somatotropinomas. GIP stimulated adenylyl cyclase and GH-promoter activity in GIPR-transfected GH3 cells, confirming a correct coupling of GIPR to Gαs. In a proportion of acromegalic patients, GIPR overexpression appeared to be associated with a paradoxical increase in GH after an oral glucose tolerance test. Whether GIPR overexpression in acromegalic patients may be associated with this paradoxical response or more generally involved in the pathogenesis of acromegaly, as suggested by the mutually exclusive high GIPR levels and GNAS1 mutations, remains an open question.

  7. 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.

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

    Science.gov (United States)

    Fong, Keith S K; Hufnagel, Robert B; Khadka, Vedbar S; Corley, Michael J; Maunakea, Alika K; Fogelgren, Ben; Ahmed, Zubair M; Lozanoff, Scott

    2016-05-01

    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.

  9. 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 cDNA...

  10. UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

    Energy Technology Data Exchange (ETDEWEB)

    Arimoto-Kobayashi, Sakae, E-mail: arimoto@cc.okayama-u.ac.jp [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan); Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan)

    2010-09-10

    We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in {phi}X174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

  11. Point Mutations in Transmembrane Helices 2 and 3 of ExbB and TolQ Affect Their Activities in Escherichia coli K-12

    OpenAIRE

    Braun, Volkmar; Herrmann, Christina

    2004-01-01

    Replacement of glutamate 176, the only charged amino acid in the third transmembrane helix of ExbB, with alanine (E176A) abolished ExbB activity in all determined ExbB-dependent functions of Escherichia coli. Combination of the mutations T148A in the second transmembrane helix and T181A in the third transmembrane helix, proposed to form part of a proton pathway through ExbB, also resulted in inactive ExbB. E176 and T148 are strictly conserved in ExbB and TolQ proteins, and T181 is almost stri...

  12. 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.

  13. Characterization of a novel BCHE "silent" allele: point mutation (p.Val204Asp causes loss of activity and prolonged apnea with suxamethonium.

    Directory of Open Access Journals (Sweden)

    Herve Delacour

    Full Text Available Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp. Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 µM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.

  14. 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.

  15. Amplification of a novel c-Kit activating mutation Asn(822)-Lys in the Kasumi-1 cell line: a t(8;21)-Kit mutant model for acute myeloid leukemia.

    Science.gov (United States)

    Beghini, Alessandro; Magnani, Ivana; Ripamonti, Carla B; Larizza, Lidia

    2002-01-01

    A subset of AML-M2/M4Eo patients has been shown to carry c-kit mutations suggesting that myelomonoblastic leukemia cells, disrupting core binding factor through t(8;21) or inv(16) chromosomal rearrangements, have a common differentiation stage suitable to c-kit mutation. In rare core binding factor leukemia patients an increased dosage of a mutated Asp816(Tyr/Val) kit allele is achieved through nonrandom duplication of chromosome 4 where the c-kit gene is located. The c-kit gene was studied in the core binding factor leukemia cell line Kasumi-1 with t(8;21) by fluorescence in situ hybridization and mutation analysis. The dosage of Asn822(Lys) mutated allele was evaluated by fluorescence semiquantitative PCR. The correct membrane homing of KIT receptor and its activating status was analysed by immunofluorescence and Western blotting respectively. We identified in the Kasumi-1 cell line a novel Asn822(Lys) ligand-independent c-kit activating mutation and demonstrated by semiquantitative PCR that the mutated allele is about fivefold amplified compared to the normal allele. Fluorescence In Situ Hybridization analysis revealed that c-kit amplification maps to minute 4cen-q11 derived marker chromosome, often carrying duplicated signals, which are unequally distributed in the cell population. The Asn822(Lys) mutation affects a highly conserved codon within the tyrosine kinase activation loop leading, likewise the Asp(816) mutants, to constitutive ligand-independent activation of the KIT receptor. Results obtained point to the Kasumi-1 cell line as powerful in-vitro model for further investigation of altered KIT signal transduction pathways in acute myeloid leukemia with core binding factor rearrangements and a useful tool for pharmacological therapeutic targeting.

  16. Cardiomyopathy-related mutation (A30V) in mouse cardiac troponin T divergently alters the magnitude of stretch activation in α- and β-myosin heavy chain fibers.

    Science.gov (United States)

    Mickelson, Alexis V; Gollapudi, Sampath K; Chandra, Murali

    2017-01-01

    The present study investigated the functional consequences of the human hypertrophic cardiomyopathy (HCM) mutation A28V in cardiac troponin T (TnT). The A28V mutation is located within the NH2 terminus of TnT, a region known to be important for full activation of cardiac thin filaments. The functional consequences of the A28V mutation in TnT remain unknown. Given how α- and β-myosin heavy chain (MHC) isoforms differently alter the functional effect of the NH2 terminus of TnT, we hypothesized that the A28V-induced effects would be differently modulated by α- and β-MHC isoforms. Recombinant wild-type mouse TnT (TnTWT) and the mouse equivalent of the human A28V mutation (TnTA30V) were reconstituted into detergent-skinned cardiac muscle fibers extracted from normal (α-MHC) and transgenic (β-MHC) mice. Dynamic and steady-state contractile parameters were measured in reconstituted muscle fibers. Step-like length perturbation experiments demonstrated that TnTA30V decreased the magnitude of the muscle length-mediated recruitment of new force-bearing cross bridges (ER) by 30% in α-MHC fibers. In sharp contrast, TnTA30V increased ER by 55% in β-MHC fibers. Inferences drawn from other dynamic contractile parameters suggest that directional changes in ER in TnTA30V + α-MHC and TnTA30V + β-MHC fibers result from a divergent impact on cross bridge-regulatory unit (troponin-tropomyosin complex) cooperativity. TnTA30V-mediated effects on Ca(2+)-activated maximal tension and instantaneous muscle fiber stiffness (ED) were also divergently affected by α- and β-MHC. Our study demonstrates that TnTA30V + α-MHC and TnTA30V + β-MHC fibers show contrasting contractile phenotypes; however, only the observations from β-MHC fibers are consistent with the clinical data for A28V in humans.

  17. 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......AIMS: Exercise increases risk of ventricular arrhythmia in subjects with arrhythmogenic right ventricular cardiomyopathy (ARVC). We aimed to investigate the impact of exercise on myocardial function in ARVC subjects. METHODS AND RESULTS: We included 110 subjects (age 42 ± 17 years), 65 ARVC...... family members (47 ± 3% vs. 52 ± 6%, P 

  18. Anaerobically Grown Escherichia coli Has an Enhanced Mutation Rate and Distinct Mutational Spectra

    Science.gov (United States)

    Shewaramani, Sonal; Finn, Thomas J.; Kassen, Rees; Rainey, Paul B.

    2017-01-01

    Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in Escherichia coli populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes. PMID:28103245

  19. Msx1 Mutations

    Science.gov (United States)

    Wang, Y.; Kong, H.; Mues, G.; D’Souza, R.

    2011-01-01

    Mutations in the transcription factors PAX9 and MSX1 cause selective tooth agenesis in humans. In tooth bud mesenchyme of mice, both proteins are required for the expression of Bmp4, which is the key signaling factor for progression to the next step of tooth development. We have previously shown that Pax9 can transactivate a 2.4-kb Bmp4 promoter construct, and that most tooth-agenesis-causing PAX9 mutations impair DNA binding and Bmp4 promoter activation. We also found that Msx1 by itself represses transcription from this proximal Bmp4 promoter, and that, in combination with Pax9, it acts as a potentiator of Pax9-induced Bmp4 transactivation. This synergism of Msx1 with Pax9 is significant, because it is currently the only documented mechanism for Msx1-mediated activation of Bmp4. In this study, we investigated whether the 5 known tooth-agenesis-causing MSX1 missense mutations disrupt this Pax9-potentiation effect, or if they lead to deficiencies in protein stability, protein-protein interactions, nuclear translocation, and DNA-binding. We found that none of the studied molecular mechanisms yielded a satisfactory explanation for the pathogenic effects of the Msx1 mutations, calling for an entirely different approach to the investigation of this step of odontogenesis on the molecular level. PMID:21297014

  20. 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

  1. 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.

  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. 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

  4. Traceless splicing enabled by substrate-induced activation of the Nostoc punctiforme Npu DnaE intein after mutation of a catalytic cysteine to serine.

    Science.gov (United States)

    Cheriyan, Manoj; Chan, Siu-Hong; Perler, Francine

    2014-12-12

    Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices. Copyright © 2014. Published by Elsevier Ltd.

  5. Analysis of non-typeable Haemophilous influenzae VapC1 mutations reveals structural features required for toxicity and flexibility in the active site.

    Directory of Open Access Journals (Sweden)

    Brooke Hamilton

    Full Text Available Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.

  6. Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase.

    Science.gov (United States)

    Hwang, Y; Feiss, M

    1996-08-30

    Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimulated endonuclease activity of terminase is located in gpA, the large terminase subunit. There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490. Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase. In the present work, mutations causing the gpA changes K497A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo. Purified K497A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in cos cleavage are sufficient to explain the lethality of the K497A and K497D defects. In in vitro packaging studies using mature (cleaved) phage DNA, the K497A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 microM ATP, indicating that the K497D change does not affect DNA translocation. In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase.

  7. An adaptive mutation in adenylate kinase that increases organismal fitness is linked to stability-activity trade-offs

    National Research Council Canada - National Science Library

    Couñago, Rafael; Wilson, Corey J; Peña, Matthew I; Wittung-Stafshede, Pernilla; Shamoo, Yousif

    Protein function is a balance between activity and stability. However, the relevance of stability-activity trade-offs for protein evolution and their impact on organismal fitness have been difficult to determine...

  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. IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced

    Science.gov (United States)

    Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin

    2017-01-01

    Aim of study Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Materials and methods Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. Results We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Conclusion Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment. PMID:28052098

  10. Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16.

    Science.gov (United States)

    Sheu, Der-Shyan; Chen, Wen-Ming; Lai, Yung-Wei; Chang, Rey-Chang

    2012-05-01

    The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.

  11. Altered CYP19A1 and CYP3A4 Activities Due to Mutations A115V, T142A, Q153R and P284L in the Human P450 Oxidoreductase

    Directory of Open Access Journals (Sweden)

    Sameer S. Udhane

    2017-08-01

    Full Text Available All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein–protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

  12. 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...

  13. 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...

  14. 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

  15. 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.

  16. 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...

  17. Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface*

    Science.gov (United States)

    Chin, Stephanie; Yang, Donghe; Miles, Andrew J.; Eckford, Paul D. W.; Molinski, Steven; Wallace, B. A.; Bear, Christine E.

    2017-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. PMID:28003367

  18. 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.

  19. Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface.

    Science.gov (United States)

    Chin, Stephanie; Yang, Donghe; Miles, Andrew J; Eckford, Paul D W; Molinski, Steven; Wallace, B A; Bear, Christine E

    2017-02-03

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation.

  20. 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.

  1. Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine.

    Science.gov (United States)

    Montioli, Riccardo; Oppici, Elisa; Dindo, Mirco; Roncador, Alessandro; Gotte, Giovanni; Cellini, Barbara; Borri Voltattorni, Carla

    2015-10-01

    Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.

  2. 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.

  3. 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.

  4. IDH mutations in acute myeloid leukemia.

    Science.gov (United States)

    Rakheja, Dinesh; Konoplev, Sergej; Medeiros, L Jeffrey; Chen, Weina

    2012-10-01

    Acute myeloid leukemia is a heterogeneous group of diseases. Mutations of the isocitrate dehydrogenase (IDH) genes represent a novel class of point mutations in acute myeloid leukemia. These mutations prevent oxidative decarboxylation of isocitrate to α-ketoglutarate and confer novel enzymatic activity, facilitating the reduction of α-ketoglutarate to d-2-hydroxyglutarate, a putative oncometabolite. IDH1/IDH2 mutations are heterozygous, and their combined frequency is approximately 17% in unselected acute myeloid leukemia cases, 27% in cytogenetically normal acute myeloid leukemia cases, and up to 67% in acute myeloid leukemia cases with cuplike nuclei. These mutations are largely mutually exclusive. Despite many similarities of IDH1 and IDH2 mutations, it is possible that they represent distinct molecular or clinical subgroups of acute myeloid leukemia. All known mutations involve arginine (R), in codon 132 of IDH1 or codon 140 or 172 of IDH2. IDH1(R132) and IDH2(R140) mutations are frequently accompanied by normal cytogenetics and NPM1 mutation, whereas IDH2(R172) is frequently the only mutation detected in acute myeloid leukemia. There is increasing evidence that the prognostic impact of IDH1/2 mutations varies according to the specific mutation and also depends on the context of concurrent mutations of other genes. IDH1(R132) mutation may predict poor outcome in a subset of patients with molecular low-risk acute myeloid leukemia, whereas IDH2(R172) mutations confer a poor prognosis in patients with acute myeloid leukemia. Expression of IDH1/2 mutants induces an increase in global DNA hypermethylation and inhibits TET2-induced cytosine 5-hydroxymethylation, DNA demethylation. These data suggest that IDH1/2 mutations constitute a distinct mutational class in acute myeloid leukemia, which affects the epigenetic state, an important consideration for the development of therapeutic agents.

  5. Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses

    Science.gov (United States)

    Madhuprakash, Jogi; Bobbili, Kishore Babu; Moerschbacher, Bruno M.; Singh, Tej Pal; Swamy, Musti J.; Podile, Appa Rao

    2015-01-01

    Serratia proteamaculans chitinase-D (SpChiD) has a unique combination of hydrolytic and transglycosylation (TG) activities. The TG activity of SpChiD can be used for large-scale production of chito-oligosaccharides (CHOS). The multiple activities (hydrolytic and/or chitobiase activities and TG) of SpChiD appear to be strongly influenced by the substrate-binding cleft. Here, we report the unique property of SpChiD substrate-binding cleft, wherein, the residues Tyr28, Val35 and Thr36 control chitobiase activity and the residues Trp160 and Trp290 are crucial for TG activity. Mutants with reduced (V35G and T36G/F) or no (SpChiDΔ30–42 and Y28A) chitobiase activity produced higher amounts of the quantifiable even-chain TG product with degree of polymerization (DP)-6, indicating that the chitobiase and TG activities are inversely related. In addition to its unprecedented catalytic properties, unlike other chitinases, the single modular SpChiD showed dual unfolding transitions. Ligand-induced thermal stability studies with the catalytically inactive mutant of SpChiD (E153A) showed that the transition temperature increased upon binding of CHOS with DP2–6. Isothermal titration calorimetry experiments revealed the exceptionally high binding affinities for E153A to CHOS with DP2–6. These observations strongly support that the architecture of SpChiD substrate-binding cleft adopted to control chitobiase and TG activities, in addition to usual chitinase-mediated hydrolysis. PMID:26493546

  6. Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements.

    Science.gov (United States)

    Ochi, Kozo; Tanaka, Yukinori; Tojo, Shigeo

    2014-02-01

    Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often "silent" under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed.

  7. Mutations in exons 10 and 11 of human glucokinase result in conformational variations in the active site of the structure contributing to poor substrate binding - explains hyperglycemia in type 2 diabetic patients.

    Science.gov (United States)

    Yellapu, Nandakumar; Mahto, Manoj Kumar; Valasani, Koteswara Rao; Sarma, P V G K; Matcha, Bhaskar

    2015-01-01

    Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of β-α-β units, hairpins, β-bulges, strands, helices, helix-helix interactions, β-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.

  8. 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.

  9. Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity.

    Science.gov (United States)

    Jablonski, S A; Morrow, C D

    1995-01-01

    The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the

  10. Inhibition of Transforming Growth Factor-Beta1 SignalingAttenuates Ataxia Telangiectasia Mutated Activity in Response toGenotoxic Stress

    Energy Technology Data Exchange (ETDEWEB)

    Kirshner, Julia; Jobling, Michael F.; Pajares, Maria Jose; Ravani, Shraddha A.; Glick, Adam; Lavin, Martin F.; Koslov, Sergei; Shiloh, Yosef; Barcellos-Hoff, Mary Helen

    2006-01-01

    Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor {beta} (TGF{beta})-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF{beta} inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf{beta}I null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF{beta} type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced H2AX radiation-induced foci; and increased radiosensitivity compared with TGF{beta} competent cells. We determined that loss of TGF{beta} signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF{beta} restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf{beta}I, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF{beta} may be used to advantage in cancer therapy.

  11. Structure-activity relationships of diverse oxazolidinones for linezolid-resistant Staphylococcus aureus strains possessing the cfr methyltransferase gene or ribosomal mutations.

    Science.gov (United States)

    Locke, Jeffrey B; Finn, John; Hilgers, Mark; Morales, Gracia; Rahawi, Shahad; G C, Kedar; Picazo, Juan José; Im, Weonbin; Shaw, Karen Joy; Stein, Jeffrey L

    2010-12-01

    Staphylococcal resistance to linezolid (LZD) is mediated through ribosomal mutations (23S rRNA or ribosomal proteins L3 and L4) or through methylation of 23S rRNA by the horizontally transferred Cfr methyltransferase. To investigate the structural basis for oxazolidinone activity against LZD-resistant (LZD(r)) strains, we compared structurally diverse, clinically relevant oxazolidinones, including LZD, radezolid (RX-1741), TR-700 (torezolid), and a set of TR-700 analogs (including novel CD-rings and various A-ring C-5 substituents), against a panel of laboratory-derived and clinical LZD(r) Staphylococcus aureus strains possessing a variety of resistance mechanisms. Potency against all strains was correlated with optimization of C- and D-rings, which interact with more highly conserved regions of the peptidyl transferase center binding site. Activity against cfr strains was retained with either hydroxymethyl or 1,2,3-triazole C-5 groups but was reduced by 2- to 8-fold in compounds with acetamide substituents. LZD, which possesses a C-5 acetamide group and lacks a D-ring substituent, demonstrated the lowest potency against all strains tested, particularly against cfr strains. These data reveal key features contributing to oxazolidinone activity and highlight structural tradeoffs between potency against susceptible strains and potency against strains with various resistance mechanisms.

  12. 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.

  13. Genetic analysis of the Kirsten-ras-revertant 1 gene: Potentiation of its tumor suppressor activity by specific point mutations

    Energy Technology Data Exchange (ETDEWEB)

    Kitayama, Hitoshi (Institute of Physical and Chemical Research, Ibaraki (Japan) Univ. of Tsukuba, Ibaraki (Japan)); Matsuzaki, Tomoko; Ikawa, Yoji; Noda, Makoto (Institute of Physical and Chemical Research, Ibaraki (Japan))

    1990-06-01

    Kirsten-ras-revertant 1 (Krev-1) cDNA encodes a ras-related protein and exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when introduced into a v-K-ras-transformed mouse NIH 3T3 cell line, DT. Toward understanding the mechanism of action of Krev-1 protein, the authors constructed a series of point mutants of Krev-1 cDNA and tested their biological activities in DT cells and HT1080 human fibrosarcoma cells harboring the activated N-ras gene. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp{sup 17} and Asn{sup 116}), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys{sup 181}), and at the unique Thr{sup 61} all decreased the transformation suppressor activity. On the other hand, substitutions such as Gly{sup 12} to Val{sup 12} and Gln{sup 63} to Glu{sup 63} were found to significantly increase the transformation suppressor/tumor suppressor activity of Krev-1. These findings are consistent with the idea that Krev-1 protein is regulated like many other G proteins by the guanine triphosphate/guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.

  14. Ponatinib (AP24534), a multitargeted pan-FGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models.

    Science.gov (United States)

    Gozgit, Joseph M; Wong, Matthew J; Moran, Lauren; Wardwell, Scott; Mohemmad, Qurish K; Narasimhan, Narayana I; Shakespeare, William C; Wang, Frank; Clackson, Tim; Rivera, Victor M

    2012-03-01

    Members of the fibroblast growth factor receptor family of kinases (FGFR1-4) are dysregulated in multiple cancers. Ponatinib (AP24534) is an oral multitargeted tyrosine kinase inhibitor being explored in a pivotal phase II trial in patients with chronic myelogenous leukemia due to its potent activity against BCR-ABL. Ponatinib has also been shown to inhibit the in vitro kinase activity of all four FGFRs, prompting us to examine its potential as an FGFR inhibitor. In Ba/F3 cells engineered to express activated FGFR1-4, ponatinib potently inhibited FGFR-mediated signaling and viability with IC(50) values FGFR-mediated signaling with IC(50) values FGFR1-4 inhibition can be sustained in patients. These results show that ponatinib is a potent pan-FGFR inhibitor and provide strong rationale for its evaluation in patients with FGFR-driven cancers.

  15. CF Mutation Panel

    Science.gov (United States)

    ... Testing; Cystic Fibrosis Transmembrane Conductance Regulator Mutation Analysis; CFTR Mutation Analysis Formal name: Cystic Fibrosis Gene Mutation ... an elevated immunoreactive trypsinogen (IRT) or positive sweat chloride test , to confirm the diagnosis of cystic fibrosis. ...

  16. A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

    Directory of Open Access Journals (Sweden)

    Thomas J Lampert

    Full Text Available Although G-protein coupled receptors (GPCRs are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490. Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba²⁺ and K⁺, suggesting a decrease in basal excitability (decrease in Ca²⁺ channel activity. The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA and proteose peptone (PP, two known chemoattractants in Tetrahymena. Using microsomal [³⁵S]GTPγS binding assays, we found that wild-type (CU427 have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor, addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.

  17. The Arabidopsis thaliana rlp mutations revert the ectopic leaf blade formation conferred by activation tagging of the LEP gene

    DEFF Research Database (Denmark)

    van der Graaff, Eric; Nussbaumer, C; Keller, Bente

    2003-01-01

    Activation tagging of the gene LEAFY PETIOLE (LEP) with a T-DNA construct induces ectopic leaf blade formation in Arabidopsis, which results in a leafy petiole phenotype. In addition, the number of rosette leaves produced prior to the onset of bolting is reduced, and the rate of leaf initiation...... is retarded by the activation tagged LEP gene. The ectopic leaf blade results from an invasion of the petiole region by the wild-type leaf blade. In order to isolate mutants that are specifically disturbed in the outgrowth of the leaf blade, second site mutagenesis was performed using ethane methanesulphonate...

  18. Novel marker for the onset of frontotemporal dementia: early increase in activity-dependent neuroprotective protein (ADNP in the face of Tau mutation.

    Directory of Open Access Journals (Sweden)

    Yulie Schirer

    Full Text Available Tauopathy, a major pathology in Alzheimer's disease, is also found in ~50% of frontotemporal dementias (FTDs. Tau transcript, a product of a single gene, undergoes alternative splicing to yield 6 protein species, each with either 3 or 4 microtubule binding repeat domains (tau 3R or 4R, associated with dynamic and stable microtubules, respectively. While the healthy human brain shows a 1/1 ratio of tau 3R/4R, this ratio may be dramatically changed in the FTD brain. We have previously discovered that activity-dependent neuroprotective protein (ADNP is essential for brain formation in the mouse, with ADNP+/- mice exhibiting tauopathy, age-driven neurodegeneration and behavioral deficits. Here, in transgenic mice overexpressing a mutated tau 4R species, in the cerebral cortex but not in the cerebellum, we showed significantly increased ADNP expression (~3-fold transcripts in the cerebral cortex of young transgenic mice (~disease onset, but not in the cerebellum, as compared to control littermates. The transgene-age-related increased ADNP expression paralleled augmented dynamic tau 3R transcript level compared to control littermates. Blocking mutated tau 4R transgene expression resulted in normalization of ADNP and tau 3R expression. ADNP was previously shown to be a member of the SWItch/Sucrose NonFermentable (SWI/SNF chromatin remodeling complex. Here, Brahma (Brm, a component of the SWI/SNF complex regulating alternative splicing, showed a similar developmental expression pattern to ADNP. Immunoprecipitations further suggested Brm-ADNP interaction coupled to ADNP - polypyrimidine tract-binding protein (PTB-associated splicing factor (PSF-binding, with PSF being a direct regulator of tau transcript splicing. It should be noted that although we have shown a correlation between levels of ADNP and tau isoform expression three months of age, we are not presenting evidence of a direct link between the two. Future research into ADNP/tau relations is

  19. Effects of familial hemiplegic migraine type 1 mutations on neuronal P/Q-type Ca2+ channel activity and inhibitory synaptic transmission.

    Science.gov (United States)

    Cao, Yu-Qing; Tsien, Richard W

    2005-02-15

    Inhibitory synapses play key roles in the modulatory circuitry that regulates pain signaling and generation of migraine headache. A rare, dominant form of this common disease, familial hemiplegic migraine type 1 (FHM1), arises from missense mutations in the pore-forming alpha1A subunit of P/Q-type Ca2+ channels. These channels are normally vital for presynaptic Ca2+ entry and neurotransmitter release at many central synapses, raising questions about effects of FHM1 mutations on neuronal Ca2+ influx and inhibitory and excitatory neurotransmission. We have expressed the four original FHM1 mutant channels in hippocampal neurons from alpha1A knockout mice. Whole-cell recordings indicated that FHM1 mutant channels were less effective than wild-type channels in their ability to conduct P/Q-type current, but not generally different from wild type in voltage-dependent channel gating. Ca2+ influx triggered by action potential waveforms was also diminished. In keeping with decreased channel activity, FHM1 mutant channels were correspondingly impaired in supporting the P/Q-type component of inhibitory neurotransmission. When expressed in wild-type inhibitory neurons, FHM1 mutant channels reduced the contribution of P/Q-type channels to GABAergic synaptic currents, consistent with a competition of mutant and endogenous channels for P/Q-specific slots. In all cases, N-type channels took up the burden of supporting transmission and homeostatic mechanisms maintained overall synaptic strength. The shift to reliance on N-type channels greatly increased the susceptibility to G protein-coupled modulation of neurotransmission, studied with the GABAB agonist baclofen. Thus, mutant-expressing synapses might be weakened in a heightened state of neuromodulation like that provoked by triggers of migraine such as stress.

  20. 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.

  1. Mutational profiling reveals PIK3CA mutations in gallbladder carcinoma

    Directory of Open Access Journals (Sweden)

    Bardeesy Nabeel

    2011-02-01

    Full Text Available Abstract Background The genetics of advanced biliary tract cancers (BTC, which encompass intra- and extra-hepatic cholangiocarcinomas as well as gallbladder carcinomas, are heterogeneous and remain to be fully defined. Methods To better characterize mutations in established known oncogenes and tumor suppressor genes we tested a mass spectrometric based platform to interrogate common cancer associated mutations across a panel of 77 formalin fixed paraffin embedded archived BTC cases. Results Mutations among three genes, KRAS, NRAS and PIK3CA were confirmed in this cohort. Activating mutations in PIK3CA were identified exclusively in GBC (4/32, 12.5%. KRAS mutations were identified in 3 (13% intra-hepatic cholangiocarcinomas and 1 (33% perihillar cholangiocarcinoma but were not identified in gallbladder carcinomas and extra-hepatic cholangiocarcinoma. Conclusions The presence of activating mutations in PIK3CA specifically in GBC has clinical implications in both the diagnosis of this cancer type, as well as the potential utility of targeted therapies such as PI3 kinase inhibitors.

  2. Enhancement of Cry19Aa Mosquitocidal Activity against Aedes aegypti by Mutations in the Putative Loop Regions of Domain II

    OpenAIRE

    Abdullah, Mohd Amir F.; Donald H Dean

    2004-01-01

    Improvements in the mosquitocidal activity of Bacillus thuringiensis Cry19Aa were achieved by protein engineering of putative surface loop residues in domain II through rational design. The improvement of Aedes toxicity in Cry19Aa was 42,000-fold and did not affect its toxicity against Anopheles or Culex.

  3. 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.

  4. Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi.

    Science.gov (United States)

    Hwang, Y; Catalano, C E; Feiss, M

    1996-02-27

    Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively. Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine. By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3[32P] ATP in the presence of UV light. Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively. ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM). The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase. ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA. In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA.

  5. Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification.

    Science.gov (United States)

    Cano, Veridiana S P; Jeon, Geoung A; Johansson, Hans E; Henderson, C Allen; Park, Jong-Hwan; Valentini, Sandro R; Hershey, John W B; Park, Myung Hee

    2008-01-01

    The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.

  6. 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

  7. 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

  8. Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity.

    Science.gov (United States)

    Tanabe, Maiko; Ishino, Sonoko; Ishino, Yoshizumi; Nishida, Hirokazu

    2014-01-21

    The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.

  9. Vav1 Oncogenic Mutation Inhibits T Cell Receptor-induced Calcium Mobilization through Inhibition of Phospholipase Cγ1 Activation*

    Science.gov (United States)

    Knyazhitsky, Mira; Moas, Etay; Shaginov, Ekaterina; Luria, Anna; Braiman, Alex

    2012-01-01

    Robust elevation of the cytosolic calcium concentration is a crucial early step for T cell activation triggered by the T cell antigen receptor. Vav1 is a proto-oncogene expressed in hematopoietic cells that is indispensable for transducing the calcium-mobilizing signal. Following T cell receptor stimulation, Vav1 facilitates formation of signaling microclusters through multiple interactions with other proteins participating in the signaling cascade. Truncation of the N terminus of Vav1 produces its oncogenic version, which is unable to support normal calcium flux following T cell activation. We show here that truncation of the N-terminal region of Vav1 alters the fine structure of protein complexes in the signaling clusters, affecting the interaction of Vav1 with phospholipase Cγ1 (PLCγ1). This alteration is accompanied by a decrease in PLCγ1 phosphorylation and inhibition of inositol 1,4,5-trisphosphate production. We suggest that the structural integrity of the N-terminal region of Vav1 is important for the proper formation of the Vav1-associated signaling complexes. The oncogenic truncation of this region elicits conformational changes that interfere with the Vav1-mediated activation of PLCγ1 and that inhibit calcium mobilization. PMID:22474331

  10. 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.

  11. The truncate mutation of Notch2 enhances cell proliferation through activating the NF-κB signal pathway in the diffuse large B-cell lymphomas.

    Directory of Open Access Journals (Sweden)

    Xinxia Zhang

    Full Text Available The Notch2 is a critical membrane receptor for B-cell functions, and also displays various biological roles in lymphoma pathogenesis. In this article, we reported that 3 of 69 (4.3% diffuse large B-cell lymphomas (DLBCLs exhibited a truncate NOTCH2 mutation at the nucleotide 7605 (G/A in the cDNA sequence, which led to partial deletion of the C-terminal of PEST (proline-, glutamic acid-, serine- and threonine-rich domain. The truncate Notch2 activated both the Notch2 and the NF-κB signals and promoted the proliferation of B-cell lymphoma cell lines, including DLBCL and Burkitt's lymphoma cell lines. Moreover, the ectopic proliferation was completely inhibited by ammonium pyrrolidinedithiocarbamate (PDTC, an NF-κB inhibitor. Simultaneously, PDTC also reduced the expression level of Notch2. Based on these results, we conclude that the Notch2 receptor with PEST domain truncation enhances cell proliferation which may be associated with the activation of the Notch2 and the NF-κB signaling. Our results are expected to provide a possible target for new DLBCL therapies by suppressing the Notch2 and the NF-κB signaling.

  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. 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

  14. Functional effects of diphosphomimetic mutations at cAbl-mediated phosphorylation sites on Rad51 recombinase activity.

    Science.gov (United States)

    Alligand, Brendan; Le Breton, Magali; Marquis, Damien; Vallette, François; Fleury, Fabrice

    2017-08-01

    Homologous Recombination enables faithful repair of the deleterious double strand breaks of DNA. This pathway relies on Rad51 to catalyze homologous DNA strand exchange. Rad51 is known to be phosphorylated in a sequential manner on Y315 and then on Y54, but the effect of such phosphorylation on Rad51 function remains poorly understood. We have developed a phosphomimetic model in order to study all the phosphorylation states. With the purified phosphomimetic proteins we performed in vitro assays to determine the activity of Rad51. Here we demonstrate the inhibitory effect of the double phosphomimetic mutant and suggest that it may be due to a defect in nucleofilament formation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Coat protein mutations in an attenuated Cucumber mosaic virus encoding mutant 2b protein that lacks RNA silencing suppressor activity induces chlorosis with photosynthesis gene repression and chloroplast abnormalities in infected tobacco plants.

    Science.gov (United States)

    Mochizuki, Tomofumi; Yamazaki, Ryota; Wada, Tomoya; Ohki, Satoshi T

    2014-05-01

    In tobacco plants, the Cucumber mosaic virus (CMV) pepo strain induces mosaic symptoms, including pale green chlorosis and malformed tissues. Here, we characterized the involvement of 2b protein and coat protein (CP) in the development of mosaic symptoms. A 2b mutant (R46C) that lacks viral suppressor of RNA silencing (VSR) activity showed an asymptomatic phenotype with low levels of virus accumulation. Tomato spotted wilt virus NSs protein did not complement the virulence of the R46C, although it did restore high-level virus accumulation. However, R46C mutants expressing mutated CP in which the amino acid P129 was mutated to A, E, C, Q, or S induced chlorosis that was associated with reduced expression of chloroplast and photosynthesis related genes (CPRGs) and abnormal chloroplasts with fewer thylakoid membranes. These results suggest that the CP of the CMV pepo strain acquires virulence by amino acid mutations, which causes CPRG repression and chloroplast abnormalities.

  16. [TP53 mutations and molecular epidemiology].

    Science.gov (United States)

    Otsuka, Kazunori; Ishioka, Chikashi

    2007-05-01

    Tumor suppressor p53 protein is activated by a variety of cellular stresses through several pathways and transactivates its downstream genes, including regulators of cell cycle, apoptosis and DNA repair. The loss of p53 function by TP53 gene mutations therefore fails to activate these genes and is thought to be a critical cause of carcinogenesis and/or tumor progression. TP53 is one of the most frequently mutated genes in human cancer. TP53 mutations are found in about 50% of human cancers, although the frequency of TP53 mutations differs among tumor types. However, the degree of functional disorder of mutant p53 varies according to the type of TP53 mutation. And the effects of p53 on cancer formation and/or progression are influenced by the degree of p53 dysfunction. So it is important to analyze the effects of TP53 mutations carefully according to the oncogenicity of each mutation from the molecular epidemiological point of view. Here, together with some cautions needed for analyzing and interpreting the significance of TP53 gene mutations, we present some examples of the identified specific mutation spectrum and the correlation between the prognosis and TP53 mutation in some cancers.

  17. Nutritional status in the era of target therapy: poor nutrition is a prognostic factor in non-small cell lung cancer with activating epidermal growth factor receptor mutations.

    Science.gov (United States)

    Park, Sehhoon; Park, Seongyeol; Lee, Se-Hoon; Suh, Beomseok; Keam, Bhumsuk; Kim, Tae Min; Kim, Dong-Wan; Kim, Young Whan; Heo, Dae Seog

    2016-11-01

    Pretreatment nutritional status is an important prognostic factor in patients treated with conventional cytotoxic chemotherapy. In the era of target therapies, its value is overlooked and has not been investigated. The aim of our study is to evaluate the value of nutritional status in targeted therapy. A total of 2012 patients with non-small cell lung cancer (NSCLC) were reviewed and 630 patients with activating epidermal growth factor receptor (EGFR) mutation treated with EGFR tyrosine kinase inhibitor (TKI) were enrolled for the final analysis. Anemia, body mass index (BMI), and prognostic nutritional index (PNI) were considered as nutritional factors. Hazard ratio (HR), progression-free survival (PFS) and overall survival (OS) for each group were calculated by Cox proportional analysis. In addition, scores were applied for each category and the sum of scores was used for survival analysis. In univariable analysis, anemia (HR, 1.29; p = 0.015), BMI lower than 18.5 (HR, 1.98; p = 0.002), and PNI lower than 45 (HR, 1.57; p nutritional status is a prognostic marker in NSCLC patients treated with EGFR TKI. Hence, baseline nutritional status should be more carefully evaluated and adequate nutrition should be supplied to these patients.

  18. 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.

  19. 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

  20. 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

  1. A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB*

    Science.gov (United States)

    Biedermannová, Lada; Prokop, Zbyněk; Gora, Artur; Chovancová, Eva; Kovács, Mihály; Damborský, Jiří; Wade, Rebecca C.

    2012-01-01

    Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels. PMID:22745119

  2. A mutation in MYD88 (L265P) supports the survival of lymphoplasmacytic cells by activation of Bruton tyrosine kinase in Waldenström macroglobulinemia.

    Science.gov (United States)

    Yang, Guang; Zhou, Yangsheng; Liu, Xia; Xu, Lian; Cao, Yang; Manning, Robert J; Patterson, Christopher J; Buhrlage, Sara J; Gray, Nathanael; Tai, Yu-Tzu; Anderson, Kenneth C; Hunter, Zachary R; Treon, Steven P

    2013-08-15

    Myeloid differentiation factor 88 (MYD88) L265P somatic mutation is highly prevalent in Waldenström macroglobulinemia (WM) and supports malignant growth through nuclear factor κB (NF-κB). The signaling cascade(s) by which MYD88 L265P promotes NF-κB activation in WM remain unclear. By lentiviral knockdown or use of a MYD88 inhibitor, decreased phosphorylation of the NF-κB gatekeeper IκBα and survival occurred in MYD88 L265P-expressing WM cells. Conversely, WM cells engineered to overexpress MYD88 L265P showed enhanced survival. Coimmunoprecipitation studies identified Bruton tyrosine kinase (BTK) complexed to MYD88 in L265P-expressing WM cells, with preferential binding of MYD88 to phosphorylated BTK (pBTK). Increased pBTK was also observed in WM cells transduced to overexpress L265P vs wild-type MYD88. Importantly, MYD88 binding to BTK was abrogated following treatment of MYD88 L265P-expressing cells with a BTK kinase inhibitor. Inhibition of BTK or interleukin-1 receptor-associated kinase 1 and 4 (IRAK-1 and -4) kinase activity induced apoptosis of WM cells, and their combination resulted in more robust inhibition of NF-κB signaling and synergistic WM cell killing. The results establish BTK as a downstream target of MYD88 L265P signaling, and provide a framework for the study of BTK inhibitors alone, and in combination with IRAK inhibitors for the treatment of WM.

  3. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang, E-mail: gux2002@suda.edu.cn

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.

  4. 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 (Shin); 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

  5. Telomerase reverse transcriptase promoter mutations in bladder cancer

    DEFF Research Database (Denmark)

    Allory, Yves; Beukers, Willemien; Sagrera, Ana

    2014-01-01

    BACKGROUND: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. OBJECTIVES: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential ...

  6. 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 (Shin); 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

  7. Somatic mutations of calreticulin in myeloproliferative neoplasms.

    Science.gov (United States)

    Klampfl, Thorsten; Gisslinger, Heinz; Harutyunyan, Ashot S; Nivarthi, Harini; Rumi, Elisa; Milosevic, Jelena D; Them, Nicole C C; Berg, Tiina; Gisslinger, Bettina; Pietra, Daniela; Chen, Doris; Vladimer, Gregory I; Bagienski, Klaudia; Milanesi, Chiara; Casetti, Ilaria Carola; Sant'Antonio, Emanuela; Ferretti, Virginia; Elena, Chiara; Schischlik, Fiorella; Cleary, Ciara; Six, Melanie; Schalling, Martin; Schönegger, Andreas; Bock, Christoph; Malcovati, Luca; Pascutto, Cristiana; Superti-Furga, Giulio; Cazzola, Mario; Kralovics, Robert

    2013-12-19

    Approximately 50 to 60% of patients with essential thrombocythemia or primary myelofibrosis carry a mutation in the Janus kinase 2 gene (JAK2), and an additional 5 to 10% have activating mutations in the thrombopoietin receptor gene (MPL). So far, no specific molecular marker has been identified in the remaining 30 to 45% of patients. We performed whole-exome sequencing to identify somatically acquired mutations in six patients who had primary myelofibrosis without mutations in JAK2 or MPL. Resequencing of CALR, encoding calreticulin, was then performed in cohorts of patients with myeloid neoplasms. Somatic insertions or deletions in exon 9 of CALR were detected in all patients who underwent whole-exome sequencing. Resequencing in 1107 samples from patients with myeloproliferative neoplasms showed that CALR mutations were absent in polycythemia vera. In essential thrombocythemia and primary myelofibrosis, CALR mutations and JAK2 and MPL mutations were mutually exclusive. Among patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 or MPL, CALR mutations were detected in 67% of those with essential thrombocythemia and 88% of those with primary myelofibrosis. A total of 36 types of insertions or deletions were identified that all cause a frameshift to the same alternative reading frame and generate a novel C-terminal peptide in the mutant calreticulin. Overexpression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with mutated CALR had a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F

  8. Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease.

    Science.gov (United States)

    Fourrier, Mickael; Lester, Katherine; Markussen, Turhan; Falk, Knut; Secombes, Christopher J; McBeath, Alastair; Collet, Bertrand

    2015-01-01

    In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.

  9. 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.

  10. Identification of recurrent SMO and BRAF mutations in ameloblastomas

    OpenAIRE

    2014-01-01

    Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation...

  11. Copper(II) complexes of alloferon 1 with point mutations (H1A) and (H9A) stability structure and biological activity.

    Science.gov (United States)

    Matusiak, Agnieszka; Kuczer, Mariola; Czarniewska, Elżbieta; Rosiński, Grzegorz; Kowalik-Jankowska, Teresa

    2014-09-01

    Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations (H1A) A(1)GVSGH(6)GQH(9)GVH(12)G (Allo1A) and (H9A) H(1)GVSGH(6)GQA(9)GVH(12)G (Allo9A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied. The presence of the His residue in first position of the peptide chain changes the coordination abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal amino group. Furthermore, the presence of two additional histidine residues in the Allo9A peptide (H(6),H(12)) leads to the formation of the CuL complex with 4N {NH2,NIm-H(1),NIm-H(6),NIm-H(12)} binding site in wide pH range (5-8). For the Cu(II)-Allo1A system, the results demonstrated that at physiological pH7.4 the predominant complex the CuH-1L consists of the 3N {NH2,N(-),CO,NIm} coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed the lowest hemocytotoxic activity while the most active was the Cu(II)-Allo9A complex formed at pH7.4. The results may suggest that the N-terminal-His(1) and His(6) residues may be more important for their proapoptotic properties in insects than those at positions 9 and 12 in the peptide chain.

  12. [Jaridonin, a new diterpenoid from Isodon rubescens, induces cell cycle arrest in gastric cancer cells through activating ataxia telangiectasia mutated kinase].

    Science.gov (United States)

    Ma, Y C; Su, N; Zhao, N M; Li, Q Y; Zhang, M; Zhao, H W; Liu, H M; Qin, Y H

    2016-04-01

    To study the effects of Jaridonin, a novel diterpenoid from isodon rubescens, on the cell cycle of human gastric cancer cells and its molecular mechanism of action. Flow cytometry was used to analyze the cell cycle distribution and expression of ataxia telangiectasia mutated kinase (ATM) after Jaridonin treatment. Western blot was performed to detect the expression of cell cycle-related proteins. The results of flow cytometry showed that the percentages of MGC-803 cells in G(2)/M phase at 6 hours after 0, 10, 20 μmol/L Jaridonin-treatment were (10.8±2.2)%, (18.2±2.5)%, (27.3±3.2)%, respectively; those at 12 hours after Jaridonin-treatment were (12.0±1.5)%, (24.1±2.0)% and (39.7±5.2)%, respectively, indicating a G2/M phase arrest of MGC-803 cells was resulted in a time- and dose-dependent manner. The expressions of ATM, Chk1, Chk2, phosphorylated Cdc2 and CDK2 were up-regulated in the MGC-803 cells after Jaridonin treatment, while the levels of Cdc2 and CDK2 were decreased. KU-55933, an inhibitor of ATM, reversed the expression of relevant proteins and G(2)/M phase arrest induced by Jaridonin. Jaridonin can significantly induce G(2)/M arrest in gastric cancer MGC-803 cells. Its mechanism may be related to the activation of ATM and Chk1/2, and inactivation of Cdc2 and CDK2 phosphorylation.

  13. Grape seed extract induces anoikis and caspase-mediated apoptosis in human prostate carcinoma LNCaP cells: possible role of ataxia telangiectasia mutated-p53 activation.

    Science.gov (United States)

    Kaur, Manjinder; Agarwal, Rajesh; Agarwal, Chapla

    2006-05-01

    Prostate cancer is the second leading cancer diagnosed in elderly males in the Western world. Epidemiologic studies suggest that dietary modifications could be an effective approach in reducing various cancers, including prostate cancer, and accordingly cancer-preventive efficacy of dietary nutrients has gained increased attention in recent years. We have recently shown that grape seed extract (GSE) inhibits growth and induces apoptotic death of advanced human prostate cancer DU145 cells in culture and xenograft. Because prostate cancer is initially an androgen-dependent malignancy, here we used LNCaP human prostate cancer cells as a model to assess GSE efficacy and associated mechanisms. GSE treatment of cells led to their detachment within 12 hours, as occurs in anoikis, and caused a significant decrease in live cells mostly due to their apoptotic death. GSE-induced anoikis and apoptosis were accompanied by a strong decrease in focal adhesion kinase levels, but an increase in caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage; however, GSE caused both caspase-dependent and caspase-independent apoptosis as evidenced by cytochrome c and apoptosis-inducing factor release into cytosol. Additional studies revealed that GSE causes DNA damage-induced activation of ataxia telangiectasia mutated kinase and Chk2, as well as p53 Ser(15) phosphorylation and its translocation to mitochondria, suggesting this to be an additional mechanism for apoptosis induction. GSE-induced apoptosis, cell growth inhibition, and cell death were attenuated by pretreatment with N-acetylcysteine and involved reactive oxygen species generation. Together, these results show GSE effects in LNCaP cells and suggest additional in vivo efficacy studies in prostate cancer animal models.

  14. Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity in E. coli via Mutation of Free Cysteine Residues

    Science.gov (United States)

    2017-01-01

    Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1) can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields in E. coli. In this study, a wild-type hSOD1 (wtSOD1) and three mutant SOD1s (mhSOD1s), in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S) increased the expression of soluble hSOD1 in E. coli whereas substitution of the internal Cys6 (mhSOD1/C6S) decreased it. Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

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

    Science.gov (United States)

    Hirano, Ken-ichi; Tanaka, Tatsuya; Ikeda, Yoshihiko; Yamaguchi, Satoshi; Zaima, Nobuhiro; Kobayashi, Kazuhiro; Suzuki, Akira; Sakata, Yasuhiko; Sakata, Yasushi; Kobayashi, Kunihisa; Toda, Tatsushi; Fukushima, Norihide; Ishibashi-Ueda, Hatsue; Tavian, Daniela; Nagasaka, Hironori; Hui, Shu-Ping; Chiba, Hitoshi; Sawa, Yoshiki; Hori, Masatsugu

    2014-01-10

    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 is defective, the marked up-regulation of PPARγ and related genes may lead to increased uptake of LCFAs, the substrates for TG synthesis. This potentially vicious cycle of LCFAs could explain the massive accumulation of TG and severe clinical course for this rare

  16. 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.

  17. Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity in E. coli via Mutation of Free Cysteine Residues

    Directory of Open Access Journals (Sweden)

    Kun Zhang

    2017-01-01

    Full Text Available Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1 can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields in E. coli. In this study, a wild-type hSOD1 (wtSOD1 and three mutant SOD1s (mhSOD1s, in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S increased the expression of soluble hSOD1 in E. coli whereas substitution of the internal Cys6 (mhSOD1/C6S decreased it. Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

  18. 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.

  19. Substitution of isoleucine for methionine at position 1153 in the beta-subunit of the human insulin receptor. A mutation that impairs receptor tyrosine kinase activity, receptor endocytosis, and insulin action.

    Science.gov (United States)

    Cama, A; Quon, M J; de la Luz Sierra, M; Taylor, S I

    1992-04-25

    The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase. The receptor tyrosine kinase is stimulated by insulin binding to the extracellular domain of the receptor. Previously, we have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Ile for Met1153 in the tyrosine kinase domain of the receptor near the cluster of the three major autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163). In this investigation, the Ile1153 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs receptor tyrosine kinase activity and also inhibits the ability of insulin to stimulate 2-deoxyglucose uptake and thymidine incorporation. These data support the hypothesis that the receptor tyrosine activity plays a necessary role in the ability of the receptor to mediate insulin action in vivo. Furthermore, expression of the Ile1153 mutant receptor exerted a dominant negative effect to inhibit the ability of endogenous murine receptors for insulin and insulin-like growth factor I to mediate their actions upon the cell. This observation is consistent with previous suggestions that mutant receptors dimerize with wild type receptors, thereby creating hybrid molecules which lack biological activity. The dominant negative effect of the mutant receptor may explain the dominant mode of inheritance of insulin resistance caused by the Ile1153 mutation. Finally, the mutation inhibits the ability of insulin to stimulate receptor endocytosis. This may explain the normal number of insulin receptors on the surface of the patient's cells in vivo. Despite the presence of markedly elevated levels of insulin in the patient's plasma, the receptors were resistant to down-regulation.

  20. SQSTM1 Mutations and Glaucoma.

    Directory of Open Access Journals (Sweden)

    Todd E Scheetz

    Full Text Available Glaucoma is the most common cause of irreversible blindness worldwide. One subset of glaucoma, normal tension glaucoma (NTG occurs in the absence of high intraocular pressure. Mutations in two genes, optineurin (OPTN and TANK binding kinase 1 (TBK1, cause familial NTG and have known roles in the catabolic cellular process autophagy. TKB1 encodes a kinase that phosphorylates OPTN, an autophagy receptor, which ultimately activates autophagy. The sequestosome (SQSTM1 gene also encodes an autophagy receptor and also is a target of TBK1 phosphorylation. Consequently, we hypothesized that mutations in SQSTM1 may also cause NTG. We tested this hypothesis by searching for glaucoma-causing mutations in a cohort of NTG patients (n = 308 and matched controls (n = 157 using Sanger sequencing. An additional 1098 population control samples were also analyzed using whole exome sequencing. A total of 17 non-synonymous mutations were detected which were not significantly skewed between cases and controls when analyzed separately, or as a group (p > 0.05. These data suggest that SQSTM1 mutations are not a common cause of NTG.

  1. Science Letters: Screen p53 mutations in hepatocellular carcinoma by FASAY: A novel splicing mutation

    Institute of Scientific and Technical Information of China (English)

    WU Xiao-mo; FU Jing-geng; GE Wang-zhong; ZHU Jiang-yan; WANG Jun-yong; ZHANG Wei; QIAN Wei; HUO Ke-ke

    2007-01-01

    Objective: To establish a routine procedure for the detection of p53 mutations in hepatocellular carcinoma (HCC)surgical resections using the FASAY (functional analysis of separated alleles of p53 on yeast) procedure. Methods: p53 status was analyzed by FASAY and cDNA sequencing in 50 cases of HCC. After the extraction of RNA from the frozen tumor and corresponding normal tissues, reverse transcription RT-PCR was carried out using these samples. The assay can detect mutations of p53mRNA between codons 67 and 347 by the DNA-binding activity of the protein and reveal them as red colonies. Results: Of the 50specimens, 29 (58%) were positive (mutant) by FASAY. Sequencing analysis confirmed that all 29 FASAY positive tumors harbored mutations, and that no mutations were detectable in any FASAY negative tumors. In 29 p53 mutations, 22 mutations were point missense mutation, 5 were deletions and 2 were splicing mutations. A novel splice mutation on splice donor of intron 6was reported, which could produce two different mRNAs, respectively using the nearest upstream and downstream recessive splice donor sites. Conclusion: FASAY is a sensitive method for detecting the various types of p53 mutations in HCC, suggesting that the yeast functional assay for the detection of p53 mutations may be essential for elucidating their clinical significance.

  2. Mutations in ANKH cause chondrocalcinosis.

    Science.gov (United States)

    Pendleton, Adrian; Johnson, Michelle D; Hughes, Anne; Gurley, Kyle A; Ho, Andrew M; Doherty, Michael; Dixey, Josh; Gillet, Pierre; Loeuille, Damien; McGrath, Rodney; Reginato, Antonio; Shiang, Rita; Wright, Gary; Netter, Patrick; Williams, Charlene; Kingsley, David M

    2002-10-01

    Chondrocalcinosis (CC) is a common cause of joint pain and arthritis that is caused by the deposition of calcium-containing crystals within articular cartilage. Although most cases are sporadic, rare familial forms have been linked to human chromosomes 8 (CCAL1) or 5p (CCAL2) (Baldwin et al. 1995; Hughes et al. 1995; Andrew et al. 1999). Here, we show that two previously described families with CCAL2 have mutations in the human homolog of the mouse progressive ankylosis gene (ANKH). One of the human mutations results in the substitution of a highly conserved amino acid residue within a predicted transmembrane segment. The other creates a new ATG start site that adds four additional residues to the ANKH protein. Both mutations segregate completely with disease status and are not found in control subjects. In addition, 1 of 95 U.K. patients with sporadic CC showed a deletion of a single codon in the ANKH gene. The same change was found in a sister who had bilateral knee replacement for osteoarthritis. Each of the three human mutations was reconstructed in a full-length ANK expression construct previously shown to regulate pyrophosphate levels in cultured cells in vitro. All three of the human mutations showed significantly more activity than a previously described nonsense mutation that causes severe hydroxyapatite mineral deposition and widespread joint ankylosis in mice. These results suggest that small sequence changes in ANKH are one cause of CC and joint disease in humans. Increased ANK activity may explain the different types of crystals commonly deposited in human CCAL2 families and mutant mice and may provide a useful pharmacological target for treating some forms of human CC.

  3. The Startle Disease Mutation E103K Impairs Activation of Human Homomeric α1 Glycine Receptors by Disrupting an Intersubunit Salt Bridge across the Agonist Binding Site*

    Science.gov (United States)

    Safar, Fatemah; Hurdiss, Elliot; Erotocritou, Marios; Greiner, Timo; Irvine, Mark W.; Fang, Guangyu; Jane, David; Yu, Rilei; Dämgen, Marc A.

    2017-01-01

    Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine (N-methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules. PMID:28174298

  4. 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.

    Science.gov (United States)

    Mayfield, Jeffrey A; Liang, Zhong; Agrahari, Garima; Lee, Shaun W; Donahue, Deborah L; Ploplis, Victoria A; Castellino, Francis J

    2014-01-01

    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.

  5. 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.

  6. 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

    2012-09-07

    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.

  7. 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.

  8. Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza

    OpenAIRE

    Schulert, Grant S; Zhang, Mingce; Fall, Ndate; Husami, Ammar; Kissell, Diane; Hanosh, Andrew; Zhang, Kejian; Davis, Kristina; Jentzen, Jeffrey M.; Napolitano, Lena; Siddiqui, Javed; Smith, Lauren B.; Harms, Paul W.; Grom, Alexei A.; Cron, Randy Q

    2015-01-01

    Background.?Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza.

  9. 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.

  10. 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.

  11. 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

  12. Depolarized Inactivation Overcomes Impaired Activation to Produce DRG Neuron Hyperexcitability in a Nav1.7 Mutation in a Patient with Distal Limb Pain

    NARCIS (Netherlands)

    Huang, J.; Yang, Y; Dib-Hajj, S.D.; Es, M. van; Zhao, P.; Salomon, J.; Drenth, J.P.; Waxman, S.G.

    2014-01-01

    Sodium channel Nav1.7, encoded by SCN9A, is expressed in DRG neurons and regulates their excitability. Genetic and functional studies have established a critical contribution of Nav1.7 to human pain disorders. We have now characterized a novel Nav1.7 mutation (R1279P) from a female human subject wit

  13. Afatinib versus cisplatin plus pemetrexed in Japanese patients with advanced non-small cell lung cancer harboring activating EGFR mutations: Subgroup analysis of LUX-Lung 3.

    Science.gov (United States)

    Kato, Terufumi; Yoshioka, Hiroshige; Okamoto, Isamu; Yokoyama, Akira; Hida, Toyoaki; Seto, Takashi; Kiura, Katsuyuki; Massey, Dan; Seki, Yoko; Yamamoto, Nobuyuki

    2015-09-01

    In LUX-Lung 3, afatinib significantly improved progression-free survival (PFS) versus cisplatin/pemetrexed in EGFR mutation-positive lung adenocarcinoma patients and overall survival (OS) in Del19 patients. Preplanned analyses in Japanese patients from LUX-Lung 3 were performed. Patients were randomized 2:1 to afatinib or cisplatin/pemetrexed, stratified by mutation type (Del19/L858R/Other). Primary endpoint was PFS (independent review). Secondary endpoints included OS, objective response, and safety. Median PFS (data cut-off: February 2012) for afatinib versus cisplatin/pemetrexed was 13.8 vs 6.9 months (hazard ratio [HR], 0.38; 95% confidence interval [CI], 0.20-0.70; P = 0.0014) in all Japanese patients (N = 83), with more pronounced improvements in those with common mutations (Del19/L858R; HR, 0.28; 95% CI, 0.15-0.52; P < 0.0001) and Del19 mutations (HR, 0.16; 95% CI, 0.06-0.39; P < 0.0001). PFS was also improved in L858R patients (HR, 0.50; 95% CI, 0.20-1.25; P = 0.1309). Median OS (data cut-off: November 2013) with afatinib versus cisplatin/pemetrexed was 46.9 vs 35.8 months (HR, 0.75; 95% CI, 0.40-1.43; P = 0.3791) in all Japanese patients, with greater benefit in patients with common mutations (HR, 0.57; 95% CI, 0.29-1.12; P = 0.0966) and Del19 mutations (HR, 0.34; 95% CI, 0.13-0.87; P = 0.0181); OS was not significantly different in L858R patients (HR, 1.13; 95% CI, 0.40-3.21; P = 0.8212). Following study treatment discontinuation, most patients (93.5%) received subsequent anticancer therapy. The most common treatment-related adverse events were diarrhea, rash/acne, nail effects and stomatitis with afatinib and nausea, decreased appetite, neutropenia, and leukopenia with cisplatin/pemetrexed. Afatinib significantly improved PFS versus cisplatin/pemetrexed in Japanese EGFR mutation-positive lung adenocarcinoma patients and OS in Del19 but not L858R patients (www.clinicaltrials.gov; NCT00949650).

  14. Whole Exome Sequencing, Familial Genomic Triangulation, and Systems Biology Converge to Identify a Novel Nonsense Mutation in TAB2-encoded TGF-beta Activated Kinase 1 in a Child with Polyvalvular Syndrome.

    Science.gov (United States)

    Ackerman, Jaeger P; Smestad, John A; Tester, David J; Qureshi, Muhammad Y; Crabb, Beau A; Mendelsohn, Nancy J; Ackerman, Michael J

    2016-09-01

    To use whole exome sequencing (WES) of a family trio to identify a genetic cause for polyvalvular syndrome. A male child was born with mild pulmonary valve stenosis and mild aortic root dilatation, and an atrial septal defect, ventricular septal defect, and patent ductus arteriosus that were closed surgically. Subsequently, the phenotype of polyvalvular syndrome with involvement of both semilunar and both atrioventricular valves emerged. His family history was negative for congenital heart disease. Because of hypotonia, myopia, soft pale skin, joint hypermobility, and mild facial dysmorphism, either Noonan syndrome- or William syndrome-spectrum disorders were suspected clinically. However, chromosomal analysis was normal and commercially available Noonan syndrome and William syndrome genetic tests were negative. Whole exome sequencing of the patient and both parents was performed. Variants were analyzed by sporadic and autosomal recessive inheritance models. A sporadic mutation, annotated as c.1491 T > A, in TAB2, resulting in a nonsense mutation, p.Y497X, in the TAB2-encoded TGF-beta activated kinase 1 (TAK1) was identified as the most likely disease-susceptibility gene. This mutation results in elimination of the terminal 197 amino acids, including the C-terminal binding motif critical for interactions with TRAF6 and TAK1. The combination of WES, genomic triangulation, and systems biology has uncovered perturbations in TGF-beta activated kinase 1 signaling as a novel pathogenic substrate for polyvalvular syndrome. © 2016 Wiley Periodicals, Inc.

  15. 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

  16. Familial hemiplegic migraine CaV2.1 channel mutation R192Q enhances ATP-gated P2X3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain

    Directory of Open Access Journals (Sweden)

    Nair Asha

    2010-08-01

    Full Text Available Abstract Background The R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Cav2.1, is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X3 receptors that are thought to be important contributors to migraine pain. Results Using in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca2+ imaging that showed how these knockin ganglion neurons generated P2X3 receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Cav2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X3 receptors of knockin neurons. In such cells we observed strong activation of CaMKII reversed by ω-agatoxin treatment. The CaMKII inhibitor KN-93 blocked CaMKII phosphorylation and the hyperesponsive P2X3 phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X3 receptors was constitutively decreased and restored by KN-93. No change in threonine or tyrosine phosphorylation was detected. Finally, pharmacological inhibitors of the phosphatase calcineurin normalized the enhanced P2X3 receptor responses of knockin neurons and increased their serine phosphorylation. Conclusions The present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X3 receptors of trigeminal ganglion neurons via CaMKII-dependent activation of calcineurin that selectively impaired the serine phosphorylation state of such receptors, thus potentiating their effects in transducing trigeminal nociception.

  17. A frequent splicing mutation and novel missense mutations color the updated mutational spectrum of classic galactosemia in Portugal.

    Science.gov (United States)

    Coelho, Ana I; Ramos, Ruben; Gaspar, Ana; Costa, Cláudia; Oliveira, Anabela; Diogo, Luísa; Garcia, Paula; Paiva, Sandra; Martins, Esmeralda; Teles, Elisa Leão; Rodrigues, Esmeralda; Cardoso, M Teresa; Ferreira, Elena; Sequeira, Sílvia; Leite, Margarida; Silva, Maria João; de Almeida, Isabel Tavares; Vicente, João B; Rivera, Isabel

    2014-01-01

    Classic galactosemia is an autosomal recessive disorder caused by deficient galactose-1-phosphate uridylyltransferase (GALT) activity. Patients develop symptoms in the neonatal period, which can be ameliorated by dietary restriction of galactose. Many patients develop long-term complications, with a broad range of clinical symptoms whose pathophysiology is poorly understood. The high allelic heterogeneity of GALT gene that characterizes this disorder is thought to play a determinant role in biochemical and clinical phenotypes. We aimed to characterize the mutational spectrum of GALT deficiency in Portugal and to assess potential genotype-phenotype correlations. Direct sequencing of the GALT gene and in silico analyses were employed to evaluate the impact of uncharacterized mutations upon GALT functionality. Molecular characterization of 42 galactosemic Portuguese patients revealed a mutational spectrum comprising 14 nucleotide substitutions: ten missense, two nonsense and two putative splicing mutations. Sixteen different genotypic combinations were detected, half of the patients being p.Q188R homozygotes. Notably, the second most frequent variation is a splicing mutation. In silico predictions complemented by a close-up on the mutations in the protein structure suggest that uncharacterized missense mutations have cumulative point effects on protein stability, oligomeric state, or substrate binding. One splicing mutation is predicted to cause an alternative splicing event. This study reinforces the difficulty in establishing a genotype-phenotype correlation in classic galactosemia, a monogenic disease whose complex pathogenesis and clinical features emphasize the need to expand the knowledge on this "cloudy" disorder.

  18. 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

  19. Mutations affecting gyrase in Haemophilus influenzae

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