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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

    2018-04-01

    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 The Authors. Published by Elsevier Inc. All rights reserved.

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

    African Journals Online (AJOL)

    Objective. Fibrodysplasia ossificans progressiva (FOP) is a rare genetic condition in which progressive ossification of fibrous tissue, tendons and ligaments leads to severe physical handicap. Most affected individuals who have been studied have a recurrent 617G>A mutation in the ACVR1/ALK2 gene that codes for activin ...

  3. 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.

  4. 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.

  5. 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.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  7. ACVR1B rs2854464 Is Associated with Sprint/Power Athletic Status in a Large Cohort of Europeans but Not Brazilians.

    Science.gov (United States)

    Voisin, Sarah; Guilherme, João Paulo F L; Yan, Xu; Pushkarev, Vladimir P; Cieszczyk, Pawel; Massidda, Myosotis; Calò, Carla M; Dyatlov, Dmitry A; Kolupaev, Vitaliy A; Pushkareva, Yuliya E; Maciejewska, Agnieszka; Sawczuk, Marek; Lancha, Antonio H; Artioli, Guilherme G; Eynon, Nir

    2016-01-01

    Skeletal muscle strength and mass, major contributors to sprint/power athletic performance, are influenced by genetics. However, to date, only a handful of genetic variants have been associated with sprint/power performance. The ACVR1B A allele (rs rs2854464) has previously been associated with increased muscle-strength in non-athletic cohort. However, no follow-up and/or replications studies have since been conducted. Therefore, the aim of the present study was to compare the genotype distribution of ACVR1B rs2854464 between endurance athletes (E), sprint/power (S/P) athletes, mixed athletes (M), and non-athletic control participants in 1672 athletes (endurance athletes, n = 482; sprint/power athletes, n = 578; mixed athletes, n = 498) and 1089 controls (C) of both European Caucasians (Italian, Polish and Russians) and Brazilians. We have also compared the genotype distribution according to the athlete's level of competition (elite vs. sub-elite). DNA extraction and genotyping were performed using various methods. Fisher's exact test (adjusted for multiple comparisons) was used to test whether the genotype distribution of rs2854464 (AA, AG and GG) differs between groups. The A allele was overrepresented in S/P athletes compared with C in the Caucasian sample (adjusted p = 0.048), whereas there were no differences in genotype distribution between E athletes and C, in neither the Brazilian nor the Caucasian samples (adjusted p > 0.05). When comparing all Caucasian athletes regardless of their sporting discipline to C, we found that the A allele was overrepresented in athletes compared to C (adjusted p = 0.024). This association was even more pronounced when only elite-level athletes were considered (adjusted p = 0.00017). In conclusion, in a relatively large cohort of athletes from Europe and South America we have shown that the ACVR1B rs2854464 A allele is associated with sprint/power performance in Caucasians but not in Brazilian athletes. This reinforces the

  8. p53 mutations promote proteasomal activity.

    Science.gov (United States)

    Oren, Moshe; Kotler, Eran

    2016-07-27

    p53 mutations occur very frequently in human cancer. Besides abrogating the tumour suppressive functions of wild-type p53, many of those mutations also acquire oncogenic gain-of-function activities. Augmentation of proteasome activity is now reported as a common gain-of-function mechanism shared by different p53 mutants, which promotes cancer resistance to proteasome inhibitors.

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

    NARCIS (Netherlands)

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

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

  10. 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

  11. Clustered Mutation Signatures Reveal that Error-Prone DNA Repair Targets Mutations to Active Genes.

    Science.gov (United States)

    Supek, Fran; Lehner, Ben

    2017-07-27

    Many processes can cause the same nucleotide change in a genome, making the identification of the mechanisms causing mutations a difficult challenge. Here, we show that clustered mutations provide a more precise fingerprint of mutagenic processes. Of nine clustered mutation signatures identified from >1,000 tumor genomes, three relate to variable APOBEC activity and three are associated with tobacco smoking. An additional signature matches the spectrum of translesion DNA polymerase eta (POLH). In lymphoid cells, these mutations target promoters, consistent with AID-initiated somatic hypermutation. In solid tumors, however, they are associated with UV exposure and alcohol consumption and target the H3K36me3 chromatin of active genes in a mismatch repair (MMR)-dependent manner. These regions normally have a low mutation rate because error-free MMR also targets H3K36me3 chromatin. Carcinogens and error-prone repair therefore redistribute mutations to the more important regions of the genome, contributing a substantial mutation load in many tumors, including driver mutations. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    Rafael Herrera-Esparza

    2013-01-01

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

  14. Oncogenically active MYD88 mutations in human lymphoma

    Science.gov (United States)

    Ngo, Vu N.; Young, Ryan M.; Schmitz, Roland; Jhavar, Sameer; Xiao, Wenming; Lim, Kian-Huat; Kohlhammer, Holger; Xu, Weihong; Yang, Yandan; Zhao, Hong; Shaffer, Arthur L.; Romesser, Paul; Wright, George; Powell, John; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Ott, German; Gascoyne, Randy D.; Connors, Joseph M.; Rimsza, Lisa M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Fisher, Richard I.; Braziel, Rita M.; Tubbs, Raymond R.; Cook, J. R.; Weisenburger, Denny D.; Chan, Wing C.; Staudt, Louis M.

    2016-01-01

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy1. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling2,3, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt’s lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, theMYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations

  15. 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.

  16. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    Science.gov (United States)

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease.

  17. GCM2-Activating Mutations in Familial Isolated Hyperparathyroidism.

    Science.gov (United States)

    Guan, Bin; Welch, James M; Sapp, Julie C; Ling, Hua; Li, Yulong; Johnston, Jennifer J; Kebebew, Electron; Biesecker, Leslie G; Simonds, William F; Marx, Stephen J; Agarwal, Sunita K

    2016-11-03

    Primary hyperparathyroidism (PHPT) is a common endocrine disease characterized by parathyroid hormone excess and hypercalcemia and caused by hypersecreting parathyroid glands. Familial PHPT occurs in an isolated nonsyndromal form, termed familial isolated hyperparathyroidism (FIHP), or as part of a syndrome, such as multiple endocrine neoplasia type 1 or hyperparathyroidism-jaw tumor syndrome. The specific genetic or other cause(s) of FIHP are unknown. We performed exome sequencing on germline DNA of eight index-case individuals from eight unrelated kindreds with FIHP. Selected rare variants were assessed for co-segregation in affected family members and screened for in an additional 32 kindreds with FIHP. In eight kindreds with FIHP, we identified three rare missense variants in GCM2, a gene encoding a transcription factor required for parathyroid development. Functional characterization of the GCM2 variants and deletion analyses revealed a small C-terminal conserved inhibitory domain (CCID) in GCM2. Two of the three rare variants were recurrent, located in the GCM2 CCID, and found in seven of the 40 (18%) kindreds with FIHP. These two rare variants acted as gain-of-function mutations that increased the transcriptional activity of GCM2, suggesting that GCM2 is a parathyroid proto-oncogene. Our results demonstrate that germline-activating mutations affecting the CCID of GCM2 can cause FIHP. Published by Elsevier Inc.

  18. R and D activities on radiation induced mutation breeding

    International Nuclear Information System (INIS)

    Lapade, A.G.; Asencion, A.B.; Santos, I.S.; Grafia, A.O.; Veluz, AM.S.; Barrida, A.C.; Marbella, L.J.

    1996-01-01

    This paper summarizes the accomplishments, prospects and future plans of mutation breeding for crop improvement at the Philippine Nuclear Research Institute (PNRI). Mutation induction has become a proven way creating variation within a crop variety and inducing desired attributes that cannot be found in nature or have been lost during evolution. Several improved varieties with desirable traits were successfully developed through induced mutation breeding at our research institute. In rice, mutation breeding has resulted in the development of new varieties: (1) PARC 2, (2) Milagrosa mutant, (3) Bengawan mutant and (4) Azmil mutant. Mutation breeding in leguminous crops has led to the induction of an improved L 114 soybean mutant that is shorter that the original variety but yield about 40% more. Several PAEC mungbean varieties characterized with long pods that are non-shattering were also induced. In asexually propagated crops, an increase in yield and chlorophyll mutants were obtained in sweet potatos. Likewise, chlorophyll mutant which look-like 'ornamental bromeliads' and a mutant with reduced spines have been developed in pineapple Queen variety. At present, we have started a new project in mutation breeding in ornamentals. Tissue culture is being utilized in our mutation breeding program. In the near future, radiation induced mutagenesis coupled with in vitro culture techniques on protoplast culture and somatic hybridization will be integrated into our mutation breeding program to facilitate the production of new crop varieties. (author)

  19. A comparison of ARMS and mutation specific IHC for common activating EGFR mutations analysis in small biopsy and cytology specimens of advanced non small cell lung cancer

    OpenAIRE

    Wang, Xueqing; Wang, Guoqing; Hao, Yueyue; Xu, Yinhong; Zhang, Lihua

    2014-01-01

    We have compared mutation analysis by Amplification Refractory Mutation System (ARMS) and epidermal growth factor receptor (EGFR) mutant-specific antibodies for their ability to detect two common activating EGFR mutations in a cohort of 115 advanced non-small cell lung cancer (NSCLC), including cytology material, core biopsy, and bronchoscopic biopsies. Assessment of EGFR mutation status was performed by using antibodies and ARMS assay specific to the two major forms of mutant EGFR, exon 19 d...

  20. Germline activating TYK2 mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences.

    Science.gov (United States)

    Waanders, E; Scheijen, B; Jongmans, M C J; Venselaar, H; van Reijmersdal, S V; van Dijk, A H A; Pastorczak, A; Weren, R D A; van der Schoot, C E; van de Vorst, M; Sonneveld, E; Hoogerbrugge, N; van der Velden, V H J; Gruhn, B; Hoogerbrugge, P M; van Dongen, J J M; Geurts van Kessel, A; van Leeuwen, F N; Kuiper, R P

    2017-04-01

    The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

  1. Error-prone polymerase activity causes multinucleotide mutations in humans.

    Science.gov (United States)

    Harris, Kelley; Nielsen, Rasmus

    2014-09-01

    About 2% of human genetic polymorphisms have been hypothesized to arise via multinucleotide mutations (MNMs), complex events that generate SNPs at multiple sites in a single generation. MNMs have the potential to accelerate the pace at which single genes evolve and to confound studies of demography and selection that assume all SNPs arise independently. In this paper, we examine clustered mutations that are segregating in a set of 1092 human genomes, demonstrating that the signature of MNM becomes enriched as large numbers of individuals are sampled. We estimate the percentage of linked SNP pairs that were generated by simultaneous mutation as a function of the distance between affected sites and show that MNMs exhibit a high percentage of transversions relative to transitions, findings that are reproducible in data from multiple sequencing platforms and cannot be attributed to sequencing error. Among tandem mutations that occur simultaneously at adjacent sites, we find an especially skewed distribution of ancestral and derived alleles, with GC → AA, GA → TT, and their reverse complements making up 27% of the total. These mutations have been previously shown to dominate the spectrum of the error-prone polymerase Pol ζ, suggesting that low-fidelity DNA replication by Pol ζ is at least partly responsible for the MNMs that are segregating in the human population. We develop statistical estimates of MNM prevalence that can be used to correct phylogenetic and population genetic inferences for the presence of complex mutations. © 2014 Harris and Nielsen; Published by Cold Spring Harbor Laboratory Press.

  2. Gain-Of-Function Mutational Activation of Human TRNA Synthetase Procytokine

    Energy Technology Data Exchange (ETDEWEB)

    Yang, X.L.; Kapoor, M.; Otero, F.J.; Slike, B.M.; Tsuruta, H.; Frausto, R.; Bates, A.; Ewalt, K.L.; Cheresh, D.A.; Schimmel, P.; /Scripps Res. Inst. /SLAC, SSRL

    2009-04-30

    Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

  3. De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

    Science.gov (United States)

    Thai, Alesha; Chia, Puey L; Russell, Prudence A; Do, Hongdo; Dobrovic, Alex; Mitchell, Paul; John, Thomas

    2017-09-01

    In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features. © 2017 Royal Australasian College of Physicians.

  4. 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

  5. Effect of the G375C and G346E achondroplasia mutations on FGFR3 activation.

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    Lijuan He

    Full Text Available Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism.

  6. Effect of the G375C and G346E achondroplasia mutations on FGFR3 activation.

    Science.gov (United States)

    He, Lijuan; Serrano, Christopher; Niphadkar, Nitish; Shobnam, Nadia; Hristova, Kalina

    2012-01-01

    Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism.

  7. A secondary RET mutation in the activation loop conferring resistance to vandetanib.

    Science.gov (United States)

    Nakaoku, Takashi; Kohno, Takashi; Araki, Mitsugu; Niho, Seiji; Chauhan, Rakhee; Knowles, Phillip P; Tsuchihara, Katsuya; Matsumoto, Shingo; Shimada, Yoko; Mimaki, Sachiyo; Ishii, Genichiro; Ichikawa, Hitoshi; Nagatoishi, Satoru; Tsumoto, Kouhei; Okuno, Yasushi; Yoh, Kiyotaka; McDonald, Neil Q; Goto, Koichi

    2018-02-12

    Resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic lung adenocarcinoma harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in a serine-to-phenylalanine substitution at codon 904 in the activation loop of the RET kinase domain. The S904F mutation confers resistance to vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase. A reduced interaction with the drug is also observed in vitro for the S904F mutant by thermal shift assay. A crystal structure of the S904F mutant reveals a small hydrophobic core around F904 likely to enhance basal kinase activity by stabilizing an active conformer. Our findings indicate that missense mutations in the activation loop of the kinase domain are able to increase kinase activity and confer drug resistance through allosteric effects.

  8. Activities of different fluoroquinolones against Bacillus anthracis mutants selected in vitro and harboring topoisomerase mutations.

    Science.gov (United States)

    Grohs, Patrick; Podglajen, Isabelle; Gutmann, Laurent

    2004-08-01

    Three sets of mutants of Bacillus anthracis resistant to fluoroquinolones were selected on ciprofloxacin and moxifloxacin in a stepwise manner from a nalidixic acid-resistant but fluoroquinolone-susceptible plasmidless strain harboring a Ser85Leu GyrA mutation. A high level of resistance to fluoroquinolones could be obtained in four or five selection steps. In each case, ParC was the secondary target. However, in addition to the GyrA mutation, expression of high-level resistance required (i) in the first set of mutants, active drug efflux associated with a mutation in the QRDR of ParC; (ii) in the second set, two mutations in the QRDR of ParC associated with a mutation in GyrB; and (iii) in the third set, two QRDR mutations, one in ParC and one in GyrA. Interestingly, several selection steps occurred without obvious mutations in the QRDR of any topoisomerase, thereby implying the existence of other resistance mechanisms. Among the fluoroquinolones tested, garenoxacin showed the best activity.

  9. Use of human tissue to assess the oncogenic activity of melanoma-associated mutations.

    Science.gov (United States)

    Chudnovsky, Yakov; Adams, Amy E; Robbins, Paul B; Lin, Qun; Khavari, Paul A

    2005-07-01

    Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

  10. Impact of kinase activating and inactivating patient mutations on binary PKA interactions.

    Science.gov (United States)

    Röck, Ruth; Mayrhofer, Johanna E; Bachmann, Verena; Stefan, Eduard

    2015-01-01

    The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions.

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

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    Paolo Cossu-Rocca

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

  12. Controlling the activity of the Tec kinase Itk by mutation of the phenylalanine gatekeeper residue.

    Science.gov (United States)

    Joseph, Raji E; Andreotti, Amy H

    2011-01-18

    The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2-kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or by removal of the amino-terminal SH2-kinase linker region or by mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2-kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases.

  13. Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn's disease.

    Science.gov (United States)

    Mao, Liming; Kitani, Atsushi; Similuk, Morgan; Oler, Andrew J; Albenberg, Lindsey; Kelsen, Judith; Aktay, Atiye; Quezado, Martha; Yao, Michael; Montgomery-Recht, Kim; Fuss, Ivan J; Strober, Warren

    2018-02-06

    In these studies we evaluated the contribution of the NLRP3 inflammasome to Crohn's disease (CD) in a kindred containing individuals having a missense mutation in CARD8, a protein known to inhibit this inflammasome. Whole exome sequencing and PCR studies identified that the affected individuals had a V44I mutation in a single allele of the T60 isoform of CARD8. The serum levels of IL-1β in the affected individuals were increased compared with that in healthy controls and their peripheral monocytes produced increased amounts of IL-1β when stimulated by NLRP3 activators. Immunoblot studies probing the basis of these findings showed that mutated T60 CARD8 fails to down-regulate the NLRP3 inflammasome because it does not bind to NLRP3 and inhibit its oligomerization. In addition, these studies showed that mutated T60 CARD8 exerts a dominant negative effect by its capacity to bind to and form oligomers with unmutated T60 or T48 CARD8 that impede their binding to NLRP3. Finally, inflammasome activation studies revealed that intact but not mutated CARD8 prevents NLRP3 deubiquitination and serine dephosphorylation. CD due to a CARD8 mutation was not effectively treated by anti-TNF-α, but did respond to IL-1β inhibitors. Thus, patients with anti-TNF-α-resistant CD may respond to this treatment option.

  14. 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.

  15. Activating mutations of GNAS in canine cortisol-secreting adrenocortical tumors.

    Science.gov (United States)

    Kool, M M J; Galac, S; Spandauw, C G; Kooistra, H S; Mol, J A

    2013-01-01

    Cushing's syndrome or hypercortisolism is a common endocrinopathy in dogs. In approximately 15% of cases, the disorder is caused by adrenocorticotropin (ACTH)-independent hypersecretion of cortisol by an adrenocortical tumor (AT). Without other explanation, the cortisol hypersecretion has been referred to as autonomous. To investigate whether ACTH-independent hypersecretion of cortisol may be associated with aberrant activation of the melanocortin 2 receptor (MC2R)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway. All analyses were performed on 44 cortisol-secreting ATs (14 adenomas and 30 carcinomas) derived from dogs diagnosed with ACTH-independent hypercortisolism. Mutation analysis was performed of genes encoding the stimulatory G protein alpha subunit (GNAS), MC2R, and PKA regulatory subunit 1A (PRKAR1A) in all ATs. Approximately one-third of all ATs harbored an activating mutation of GNAS. Missense mutations, known to result in constitutive activation, were present in codon 201 in 11 ATs, in codon 203 (1 AT), and in codon 227 (3 ATs). No functional mutations were found in MC2R and PRKAR1A. Activation of cAMP signaling is a frequent event in canine cortisol-secreting ATs and may play a crucial role in both ACTH-independent cortisol production and tumor formation. To the best of our knowledge, this is the first report of potentially causative mutations in canine cortisol-secreting ATs. Copyright © 2013 by the American College of Veterinary Internal Medicine.

  16. Computational identification of harmful mutation regions to the activity of transposable elements.

    Science.gov (United States)

    Jin, Lingling; McQuillan, Ian; Li, Longhai

    2017-11-17

    Transposable elements (TEs) are interspersed DNA sequences that can move or copy to new positions within a genome. TEs are believed to promote speciation and their activities play a significant role in human disease. In the human genome, the 22 AluY and 6 AluS TE subfamilies have been the most recently active, and their transposition has been implicated in many inherited human diseases and in various forms of cancer. Therefore, understanding their transposition activity is very important and identifying the factors that affect their transpositional activity is of great interest. Recently, there has been some work done to quantify the activity levels of active Alu TEs based on variation in the sequence. Given this activity data, an analysis of TE activity based on the position of mutations is conducted. A method/simulation is created to computationally predict so-called harmful mutation regions in the consensus sequence of a TE; that is, mutations that occur in these regions decrease the transpositional activity dramatically. The methods are applied to the most active subfamily, AluY, to identify the harmful regions, and seven harmful regions are identified within the AluY consensus with q-values less than 0.05. A supplementary simulation also shows that the identified harmful regions covering the AluYa5 RNA functional regions are not occurring by chance. This method is then applied to two additional TE families: the Alu family and the L1 family, to computationally detect the harmful regions in these elements. We use a computational method to identify a set of harmful mutation regions. Mutations within the identified harmful regions decrease the transpositional activity of active elements. The correlation between the mutations within these regions and the transpositional activity of TEs are shown to be statistically significant. Verifications are presented using the activity of AluY elements and the secondary structure of the AluYa5 RNA, providing evidence that the

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket......'-deoxythymidine (AZT) compared with the natural substrate thymidine. The crystal structure of the T163S-mutated HuTK1 reveals a less ordered conformation of the ligand thymidine triphosphate compared with the wild-type structure but the cause of the changed specificity towards AZT is not obvious. Based on its...... highly increased AZT activity relative to thymidine activity this TK1 mutant could be suitable for suicide gene therapy....

  18. 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.

  19. Cantú Syndrome Resulting from Activating Mutation in the KCNJ8 Gene

    OpenAIRE

    Cooper, Paige E.; Reutter, Heiko; Woelfle, Joachim; Engels, Hartmut; Grange, Dorothy K.; van Haaften, Gijs; van Bon, Bregje W.; Hoischen, Alexander; Nichols, Colin G.

    2014-01-01

    ATP-sensitive potassium (KATP) channels, composed of inward-rectifying potassium channel subunits (Kir6.1 and Kir6.2, encoded by KCNJ8 and KCNJ11, respectively) and regulatory sulfonylurea receptor (SUR1 and SUR2, encoded by ABCC8 and ABCC9, respectively), couple metabolism to excitability in multiple tissues. Mutations in ABCC9 cause Cantú syndrome, a distinct multi-organ disease, potentially via enhanced KATP channel activity. We screened KCNJ8 in an ABCC9 mutation-negative patient who also...

  20. Mutational analysis of the active site of human insulin-regulated aminopeptidase

    DEFF Research Database (Denmark)

    Laustsen, P G; Vang, S; Kristensen, T

    2001-01-01

    -length transmembrane form of human IRAP was expressed in HEK293 cells and recombinant wild-type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Mutational analysis using single amino-acid substitutions in the GAMEN motif (G428A...... acids N-terminal to the Zn(2+)-coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importance of the G428AMEN and H464ELAH-(18X)-E487 motifs for the activity of IRAP, mutational analysis was carried out. cDNA encoding the full...

  1. Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

    Directory of Open Access Journals (Sweden)

    Korvala Johanna

    2012-04-01

    Full Text Available Abstract Background Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in the Low-density lipoprotein receptor-related protein 5 (LRP5 gene, and the role of LRP5 is further investigated here. Methods LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis (manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression fractures but who lacked the clinical features of osteogenesis imperfecta (OI or other known syndromes linked to low BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex ligation-dependent probe amplification (MLPA. In vitro studies were performed using luciferase assay and quantitative real-time polymerase chain reaction (qPCR to examine the effect of two novel and three previously identified mutations on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1 and 5-hydroxytryptamine (5-Htr1b. Results Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R were identified in two patients and their affected family members. In vitro analyses showed that one of these novel mutations together with two previously reported mutations (p.C913fs, p.R1036Q significantly reduced the activity of the canonical Wnt signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.L1149Q reduced expression of serotonin receptor 5-Htr1b (p Conclusions Our results provide additional information on the role of LRP5 mutations and their

  2. Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity.

    Science.gov (United States)

    Korvala, Johanna; Jüppner, Harald; Mäkitie, Outi; Sochett, Etienne; Schnabel, Dirk; Mora, Stefano; Bartels, Cynthia F; Warman, Matthew L; Deraska, Donald; Cole, William G; Hartikka, Heini; Ala-Kokko, Leena; Männikkö, Minna

    2012-04-10

    Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in the Low-density lipoprotein receptor-related protein 5 (LRP5) gene, and the role of LRP5 is further investigated here. LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis (manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression fractures) but who lacked the clinical features of osteogenesis imperfecta (OI) or other known syndromes linked to low BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex ligation-dependent probe amplification (MLPA). In vitro studies were performed using luciferase assay and quantitative real-time polymerase chain reaction (qPCR) to examine the effect of two novel and three previously identified mutations on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1) and 5-hydroxytryptamine (5-Htr1b). Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R) were identified in two patients and their affected family members. In vitro analyses showed that one of these novel mutations together with two previously reported mutations (p.C913fs, p.R1036Q) significantly reduced the activity of the canonical Wnt signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.L1149Q) reduced expression of serotonin receptor 5-Htr1b (p LRP5 mutations and their effects on the development of juvenile-onset primary osteoporosis, and hence the pathogenesis of the disorder. The

  3. Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

    Science.gov (United States)

    Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

    2003-02-01

    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

  4. Mild thyroid peroxidase deficiency caused by TPO mutations with residual activity: Correlation between clinical phenotypes and enzymatic activity.

    Science.gov (United States)

    Narumi, Satoshi; Fox, Larry A; Fukudome, Keisuke; Sakaguchi, Zenichi; Sugisawa, Chiho; Abe, Kiyomi; Kameyama, Kaori; Hasegawa, Tomonobu

    2017-11-29

    Thyroid peroxidase (TPO) deficiency, caused by biallelic TPO mutations, is a well-established genetic form of congenital hypothyroidism (CH). More than 100 patients have been published, and the patients have been diagnosed mostly in the frame of newborn screening (NBS) programs. Correlation between clinical phenotypes and TPO activity remains unclear. Here, we report clinical and molecular findings of two unrelated TPO mutation-carrying mildly hypothyroid patients. The two patients were born at term after an uneventful pregnancy and delivery, and were NBS negative. They sought medical attention due to goiter at age 8 years. Evaluation of the thyroid showed mild elevation of serum TSH levels, normal or slightly low serum T 4 levels, high serum T 3 to T 4 molar ratio, high serum thyroglobulin levels, and high thyroidal 123 I uptake. We performed next-generation sequencing-based genetic screening, and found that one patient was compound heterozygous for two novel TPO mutations (p.Asp224del; c.820-2A>G), and the other was homozygous for a previously known mutation (p.Trp527Cys). In vitro functional analyses using HEK293 cells showed that the two amino acid-altering mutations (p.Asp224del and p.Trp527Cys) caused partial loss of the enzymatic activity. In conclusion, we report that TPO mutations with residual activity are associated with mild TPO deficiency, which is clinically characterized by marked goiter, mild TSH elevation, high serum T 3 to T 4 molar ratio, and high serum thyroglobulin levels. Our findings illuminate the hitherto under-recognized correlation between clinical phenotypes and residual enzymatic activity among patients with TPO deficiency.

  5. Structural Mutations that Probe the Interactions between the Catalytic and Dianion Activation Sites at Triosephosphate Isomerase‡

    Science.gov (United States)

    Zhai, Xiang; Amyes, Tina L.; Wierenga, Rik K.; Loria, J. Patrick; Richard, John P.

    2013-01-01

    Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form D-glyceraldehyde 3-phosphate. The effects of two structural mutations at TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50-fold and 80-fold decreases, respectively, in (kcat/Km)E and (kcat/Km)E•HPi for deprotonation of GA catalyzed by free TIM and by the TIM•HPO32− complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy, or on the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG for 208-YGGS. The L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E•HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy, and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation, but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provide support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst. PMID:23909928

  6. The second activating glucokinase mutation (A456V)

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  7. 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.

  8. 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.

  9. 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.

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

    Science.gov (United States)

    Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B; Wu, Chia-Chin; Akdemir, Kadir C; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T; Welch, Heidi C E; Garraway, Levi A; Chin, Lynda

    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 (PREX2(E824)*) 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 p57(KIP2)). 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.

  11. 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.

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

    Science.gov (United States)

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

    2007-10-01

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

  13. 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.

  14. Distinct biological properties of two RET isoforms activated by MEN 2A and MEN 2B mutations.

    Science.gov (United States)

    Rossel, M; Pasini, A; Chappuis, S; Geneste, O; Fournier, L; Schuffenecker, I; Takahashi, M; van Grunsven, L A; Urdiales, J L; Rudkin, B B; Lenoir, G M; Billaud, M

    1997-01-23

    Germline mutations of the RET proto-oncogene, which codes for a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). MEN 2 mutations have been shown to result in RET oncogenic activation. The RET gene encodes several isoforms whose biological properties, when altered by MEN 2 mutations, have not been thoroughly addressed yet. In this study, we have introduced a MEN 2A mutation (Cys634-->Arg) and the unique MEN 2B mutation (Met918-->Thr) in two RET isoforms of 1114 and 1072 amino acids which differ in the carboxy-terminus part. Herein, we report that each RET isoform activated by MEN 2A or MEN 2B mutation was transforming in fibroblasts and induced neuronal differentiation of pheochromocytoma PC12 cells. However, among the different RET-MEN 2 mutants, the long RET isoform activated by the MEN 2B mutation stimulated the most prominent neurite outgrowth in PC12 cells, while the short RET isoform counterpart elicited a very weak differentiation effect in PC12 cells. We further demonstrate that the morphological changes of PC12 cells caused by constitutively activated RET oncoproteins involved the engagement of a Ras-dependent pathway. These findings provide evidence that the biological properties of RET-MEN 2 mutants depend on the interplay between the RET isoforms and the nature of the activating MEN 2 mutation.

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

    Science.gov (United States)

    Ledoux, Sarah; Guthrie, Christine

    2016-06-03

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity

    Science.gov (United States)

    Salvat, Regina S.; Parker, Andrew S.; Kirsch, Jack R.; Brooks, Seth A.

    2017-01-01

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide–MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening. PMID:28607051

  17. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

    Science.gov (United States)

    Salvat, Regina S; Verma, Deeptak; Parker, Andrew S; Kirsch, Jack R; Brooks, Seth A; Bailey-Kellogg, Chris; Griswold, Karl E

    2017-06-27

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10 9 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

  18. Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity.

    Science.gov (United States)

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

    2017-01-01

    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 intramelanosomal 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 showed very low protein expression and 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 inactivate tyrosinase and result in OCA1B albinism. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  19. PROGRANULIN MUTATIONS AFFECTS BRAIN OSCILLATORY ACTIVITY IN FRONTO-TEMPORAL DEMENTIA

    Directory of Open Access Journals (Sweden)

    Davide Vito Moretti

    2016-02-01

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

  20. 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

    Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperek......Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage...... 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...... a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X...

  1. Mutation of Chinese hamster cells by near-UV activation of promutagens

    International Nuclear Information System (INIS)

    Barnhart, B.J.; Cox, S.H.

    1980-01-01

    A tissue-culture assay for mutagenesis and cytotoxicity incorporating near ultraviolet (NUV) light activation of polyaromatic hydrocarbons (PAH) has been developed. Cultures of Chinese hamster cells (line CHO) growing in suspension culture were inoculated with benzo[a]pyrene (B[a]P), 7,12-dimethylbenzanthracene (DMBA) or shale-oil retort-water and exposed to light from a high-pressure mercury lamp fitted with a Corning NUV bandpass filter. This light source both permitted activation of PAH and the shale-oil water and precluded detectable damage to DNA. Neither the PAH nor the NUV alone had any effect on cell survival or mutation frequencies but the chemicals plus NUV were extremely effective in producing mutations to 6-thioguanine resistance (hgprt gene). (orig.)

  2. Use of Human Tissue to Assess the Oncogenic Activity of Melanoma-Associated Mutations

    OpenAIRE

    Chudnovsky, Yakov; Adams, Amy E.; Robbins, Paul B.; Lin, Qun; Khavari, Paul A.

    2005-01-01

    Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence1,2. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Ras and Raf induction can occur via active N-Ras and B-Raf mutants as well as by gene amplification3–5. Activation of PI3K pathway components occurs by PTEN loss and by AKT amplification6–8. Melanomas also commonly display impairment of p16INK4A-CDK4-Rb and ARF-HDM2-p53 tumor s...

  3. An activating mutation of interferon regulatory factor 4 (IRF4) in adult T cell leukemia.

    Science.gov (United States)

    Cherian, Mathew A; Olson, Sydney; Sundaramoorthi, Hemalatha; Cates, Kitra; Cheng, Xiaogang; Harding, John; Martens, Andrew; Challen, Grant A; Tyagi, Manoj; Ratner, Lee; Rauch, Daniel

    2018-03-14

    The human T cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, likely as a result of specific immuno-editing, Tax expression is downregulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL, and the K59R mutation is the most common single-nucleotide variation in IRF4 and is found exclusively in ATL. Here high throughput whole-exome sequencing revealed recurrent activating genetic alterations in the T cell receptor, CD28, and NF-kB pathways. Moreover, we found that IRF4, which is transcriptionally activated downstream of these pathways, is frequently mutated in ATL. IRF4 RNA, protein, and IRF4 transcriptional targets are uniformly elevated in HTLV transformed cells and ATL cell lines, and IRF4 was bound to genomic regulatory DNA of many of these transcriptional targets in HTLV-1 transformed cell lines. We further noted that the K59R IRF4 mutant is expressed at higher levels in the nucleus than is wild-type IRF4, and is transcriptionally more active. Expression of both wild-type and the K59R mutant of IRF4 from a constitutive promoter in retrovirally transduced murine bone marrow cells increased the abundance of T lymphocytes but not myeloid cells or B lymphocytes in mice. IRF4 may represent a therapeutic target in ATL since ATL cells select for a mutant of IRF4 with higher nuclear expression and transcriptional activity, and over-expression of IRF4 induces the expansion of T lymphocytes in vivo. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Enzyme-activity mutations detected in mice after paternal fractionated irradiation

    International Nuclear Information System (INIS)

    Charles, D.J.; Pretsch, W.

    1986-01-01

    (101/E1 X C3H/E1)F 1 -hybrid male mice were exposed in a 24-h fractionation interval to either 3.0 + 3.0-Gy or 5.1 + 5.1-Gy X-irradiation, and mated to untreated Test-stock females. The offspring were examined for mutations at 7 recessive specific loci and for activity alterations of erythrocyte enzymes controlled presumably by 12 loci. No enzyme-activity mutant was found in 3610 F 1 -offspring of the control group. In the experimental groups, no mutant was detected in 533 (3.0 + 3.0 Gy) and 173 (5.1 + 5.1 Gy) offspring from postspermatogonial germ cells treated. After treatment of spermatogonia, 1 mutant in 3388 F 1 -offspring of the 3.0 + 3.0-Gy group, and 5 mutants in 3187 F 1 offspring of the 5.1 + 5.1-Gy group were found. The mutants were all genetically confirmed. The frequency (expressed as mutants/locus/gamete) of enzyme-activity mutations is 2 (5.1 + 5.1-Gy group) to 10 (3.0 + 3.0-Gy group) times lower than the frequency of recessive specific-locus mutations. (Auth.)

  5. Mex3c mutation reduces adiposity partially through increasing physical activity.

    Science.gov (United States)

    Han, Changjie; Jiao, Yan; Zhao, Qingguo; Lu, Baisong

    2014-06-01

    MEX3C is an RNA-binding protein with unknown physiological function. We have recently reported that a Mex3c mutation in mice causes growth retardation and reduced adiposity, but how adiposity is reduced remains unclear. Herein, we show that homozygous Mex3c gene trap mice have increased physical activity. The Mex3c mutation consistently conferred full protection from diet-induced obesity, hyperglycemia, insulin resistance, hyperlipidemia, and hepatic steatosis. In ob/ob mice with leptin deficiency, the Mex3c mutation also increased physical activity and improved glucose and lipid profiles. Expressing cre in the neurons of Mex3c gene trap mice, an attempt to partially restoring neuronal Mex3c expression, significantly increased white adipose tissue deposition, but had no effects on body length. Our data suggest that one way in which Mex3c regulates adiposity is through controlling physical activity, and that neuronal Mex3c expression could play an important role in this process. © 2014 Society for Endocrinology.

  6. Dopamine Induces Oscillatory Activities in Human Midbrain Neurons with Parkin Mutations.

    Science.gov (United States)

    Zhong, Ping; Hu, Zhixing; Jiang, Houbo; Yan, Zhen; Feng, Jian

    2017-05-02

    Locomotor symptoms in Parkinson's disease (PD) are accompanied by widespread oscillatory neuronal activities in basal ganglia. Here, we show that activation of dopamine D1-class receptors elicits a large rhythmic bursting of spontaneous excitatory postsynaptic currents (sEPSCs) in midbrain neurons differentiated from induced pluripotent stem cells (iPSCs) of PD patients with parkin mutations, but not normal subjects. Overexpression of wild-type parkin, but not its PD-causing mutant, abolishes the oscillatory activities in patient neurons. Dopamine induces a delayed enhancement in the amplitude of spontaneous, but not miniature, EPSCs, thus increasing quantal content. The results suggest that presynaptic regulation of glutamatergic transmission by dopamine D1-class receptors is significantly potentiated by parkin mutations. The aberrant dopaminergic regulation of presynaptic glutamatergic transmission in patient-specific iPSC-derived midbrain neurons provides a mechanistic clue to PD pathophysiology, and it demonstrates the usefulness of this model system in understanding how mutations of parkin cause movement symptoms in Parkinson's disease. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations.

    Science.gov (United States)

    Osada, Takuya; Chen, Minyong; Yang, Xiao Yi; Spasojevic, Ivan; Vandeusen, Jeffrey B; Hsu, David; Clary, Bryan M; Clay, Timothy M; Chen, Wei; Morse, Michael A; Lyerly, H Kim

    2011-06-15

    Wnt/β-catenin pathway activation caused by adenomatous polyposis coli (APC) mutations occurs in approximately 80% of sporadic colorectal cancers (CRC). The antihelminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream β-catenin signaling. In this study, we determined whether niclosamide could inhibit the Wnt/β-catenin pathway in human CRCs and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/β-catenin pathway activation, downregulated Dvl2, decreased downstream β-catenin signaling, and exerted antiproliferative effects in human colon cancer cell lines and CRC cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-κB or mTOR did not exert similar antiproliferative effects in these CRC model systems. In mice implanted with human CRC xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity, and led to tumor control. Our findings support clinical explorations to reposition niclosamide for the treatment of CRC.

  8. Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

    Science.gov (United States)

    Zhuang, P L; Yu, L X; Tao, Y; Zhou, Y; Zhi, Q H; Lin, H C

    2016-04-11

    Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans. The point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. SrtA△N40(UA159) and the mutant enzymes, SrtA△N40(D56E) and SrtA△N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA△N40(D56E) and SrtA△N40(R157H) remained approximately equal to the affinity of SrtA△N40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the k cat and k cat /K m values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159). The findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.

  9. Novel Oncogenic Mutations of CBL in Human Acute Myeloid Leukemia That Activate Growth and Survival Pathways Depend on Increased Metabolism*

    Science.gov (United States)

    Fernandes, Margret S.; Reddy, Mamatha M.; Croteau, Nicole J.; Walz, Christoph; Weisbach, Henry; Podar, Klaus; Band, Hamid; Carroll, Martin; Reiter, Andreas; Larson, Richard A.; Salgia, Ravi; Griffin, James D.; Sattler, Martin

    2010-01-01

    Acute myeloid leukemia (AML) is characterized by multiple mutagenic events that affect proliferation, survival, as well as differentiation. Recently, gain-of-function mutations in the α helical structure within the linker sequence of the E3 ubiquitin ligase CBL have been associated with AML. We identified four novel CBL mutations, including a point mutation (Y371H) and a putative splice site mutation in AML specimens. Characterization of these two CBL mutants revealed that coexpression with the receptor tyrosine kinases FLT3 (Fms-like tyrosine kinase 3) or KIT-induced ligand independent growth or ligand hyperresponsiveness, respectively. Growth of cells expressing mutant CBL required expression and kinase activity of FLT3. In addition to the CBL-dependent phosphorylation of FLT3 and CBL itself, transformation was associated with activation of Akt and STAT5 and required functional expression of the small GTPases Rho, Rac, and Cdc42. Furthermore, the mutations led to constitutively elevated intracellular reactive oxygen species levels, which is commonly linked to increased glucose metabolism in cancer cells. Inhibition of hexokinase with 2-deoxyglucose blocked the transforming activity of CBL mutants and reduced activation of signaling mechanisms. Overall, our data demonstrate that mutations of CBL alter cellular biology at multiple levels and require not only the activation of receptor proximal signaling events but also an increase in cellular glucose metabolism. Pathways that are activated by CBL gain-of-function mutations can be efficiently targeted by small molecule drugs. PMID:20622007

  10. Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.

    Science.gov (United States)

    McDonald, Charles J; Acheff, Eric; Kennedy, Ryan; Taylor, Lynn; Curthoys, Norman P

    2015-09-01

    The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Mechanism for activation of mutated epidermal growth factor receptors in lung cancer.

    Science.gov (United States)

    Red Brewer, Monica; Yun, Cai-Hong; Lai, Darson; Lemmon, Mark A; Eck, Michael J; Pao, William

    2013-09-17

    The initiation of epidermal growth factor receptor (EGFR) kinase activity proceeds via an asymmetric dimerization mechanism in which a "donor" tyrosine kinase domain (TKD) contacts an "acceptor" TKD, leading to its activation. In the context of a ligand-induced dimer, identical wild-type EGFR TKDs are thought to assume the donor or acceptor roles in a random manner. Here, we present biochemical reconstitution data demonstrating that activated EGFR mutants found in lung cancer preferentially assume the acceptor role when coexpressed with WT EGFR. Mutated EGFRs show enhanced association with WT EGFR, leading to hyperphosphorylation of the WT counterpart. Mutated EGFRs also hyperphosphorylate the related erythroblastic leukemia viral oncogene (ErbB) family member, ErbB-2, in a similar manner. This directional "superacceptor activity" is particularly pronounced in the drug-resistant L834R/T766M mutant. A 4-Å crystal structure of this mutant in the active conformation reveals an asymmetric dimer interface that is essentially the same as that in WT EGFR. Asymmetric dimer formation induces an allosteric conformational change in the acceptor subunit. Thus, superacceptor activity likely arises simply from a lower energetic cost associated with this conformational change in the mutant EGFR compared with WT, rather than from any structural alteration that impairs the donor role of the mutant. Collectively, these findings define a previously unrecognized mode of mutant-specific intermolecular regulation for ErbB receptors, knowledge of which could potentially be exploited for therapeutic benefit.

  12. Deep intronic mutation and pseudo exon activation as a novel muscular hypertrophy modifier in cattle.

    Directory of Open Access Journals (Sweden)

    Claire Bouyer

    Full Text Available Myostatin is essential for proper regulation of myogenesis, and inactivation of Myostatin results in muscle hypertrophy. Here, we identified an unexpected mutation in the myostatin gene which is almost fixed in Blonde d'Aquitaine cattle. In skeletal muscle, the mutant allele was highly expressed leading to an abnormal transcript consisting of a 41-bp inclusion and premature termination codons and to residual levels of a correctly spliced transcript. This expression pattern, caused by a leaky intronic mutation with regard to spliceosome activity and its apparent stability with regard to surveillance mechanisms, could contribute to the moderate muscle hypertrophy in this cattle breed. This finding is of importance for genetic counseling for meat quantity and quality in livestock production and possibly to manipulate myostatin pre-mRNA in human muscle diseases.

  13. 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...

  14. Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway.

    Directory of Open Access Journals (Sweden)

    George Dialynas

    2015-05-01

    Full Text Available Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel

  15. Novel somatic mutations in large granular lymphocytic leukemia affecting the STAT-pathway and T-cell activation

    International Nuclear Information System (INIS)

    Andersson, E I; Rajala, H L M; Eldfors, S; Ellonen, P; Olson, T; Jerez, A; Clemente, M J; Kallioniemi, O; Porkka, K; Heckman, C; Loughran, T P Jr; Maciejewski, J P; Mustjoki, S

    2013-01-01

    T-cell large granular lymphocytic (T-LGL) leukemia is a clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene, whereas STAT5 mutations are present in 2% of patients. In order to identify putative disease-causing genetic alterations in the remaining T-LGL patients, we performed exome sequencing from three STAT mutation-negative patients and validated the findings in 113 large granular lymphocytic (LGL) leukemia patients. On average, 11 CD8+ LGL leukemia cell-specific high-confidence nonsynonymous somatic mutations were discovered in each patient. Interestingly, all patients had at least one mutation that affects either directly the STAT3-pathway (such as PTPRT) or T-cell activation (BCL11B, SLIT2 and NRP1). In all three patients, the STAT3 pathway was activated when studied by RNA expression or pSTAT3 analysis. Screening of the remaining 113 LGL leukemia patients did not reveal additional patients with same mutations. These novel mutations are potentially biologically relevant and represent rare genetic triggers for T-LGL leukemia, and are associated with similar disease phenotype as observed in patients with mutations in the STAT3 gene

  16. 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.

    2013-01-01

    Summary 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. PMID:24206670

  17. Effect of Vandetanib on Lung Tumorigenesis in Transgenic Mice Carrying an Activating Egfr Gene Mutation.

    Science.gov (United States)

    Osawa, Masahiro; Ohashi, Kadoaki; Kubo, Toshio; Ichihara, Eiki; Takata, Saburo; Takigawa, Nagio; Takata, Minoru; Tanimoto, Mitsune; Kiura, Katsuyuki

    2016-08-01

    Vandetanib (ZactimaTM) is a novel, orally available inhibitor of both vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinase. In the present study, a line of transgenic mice with a mouse Egfr gene mutation (delE748-A752) corresponding to a human EGFR mutation (delE746-A750) was established. The transgenic mice developed atypical adenomatous hyperplasia to adenocarcinoma of the lung at around 5 weeks of age and died of lung tumors at approximately 17 weeks of age. In the mice treated with vandetanib (6mg/kg/day), these lung tumors disappeared and the phosphorylations of EGFR and VEGFR-2 were reduced in lung tissues to levels comparable to those of non-transgenic control mice. The median overall survival time of the transgenic mice was 28 weeks in the vandetanib-treated group and 17 weeks in the vehicle-treated group. Vandetanib significantly prolonged the survival of the transgenic mice (log-rank test, p< 0.01); resistance to vandetanib occurred at 20 weeks of age and the animals died from their lung tumors at about 28 weeks of age. These data suggest that vandetanib could suppress the progression of tumors harboring an activating EGFR mutation.

  18. 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.

  19. Potential late-onset Alzheimer's disease-associated mutations in the ADAM10 gene attenuate {alpha}-secretase activity.

    Science.gov (United States)

    Kim, Minji; Suh, Jaehong; Romano, Donna; Truong, Mimy H; Mullin, Kristina; Hooli, Basavaraj; Norton, David; Tesco, Giuseppina; Elliott, Kathy; Wagner, Steven L; Moir, Robert D; Becker, K David; Tanzi, Rudolph E

    2009-10-15

    ADAM10, a member of a disintegrin and metalloprotease family, is an alpha-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. Here, we present evidence for genetic association of ADAM10 with Alzheimer's disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated alpha-secretase activity of ADAM10 (>70% decrease), and elevated Abeta levels (1.5-3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD.

  20. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-09-04

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

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ...... activation of the receptor kinase in skeletal muscle was reduced about 70%. Selection of only those receptors that bound to anti-phosphotyrosine antibody showed that these receptors had normal kinase activity and that the reduction in overall kinase activity was due to the inability of about 70......% 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms.

    Science.gov (United States)

    Mackenzie, Robertson; Kommoss, Stefan; Winterhoff, Boris J; Kipp, Benjamin R; Garcia, Joaquin J; Voss, Jesse; Halling, Kevin; Karnezis, Anthony; Senz, Janine; Yang, Winnie; Prigge, Elena-Sophie; Reuschenbach, Miriam; Doeberitz, Magnus Von Knebel; Gilks, Blake C; Huntsman, David G; Bakkum-Gamez, Jamie; McAlpine, Jessica N; Anglesio, Michael S

    2015-05-19

    Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell

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

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available 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.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.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.

  6. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    Science.gov (United States)

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    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. PMID:24392141

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

    Science.gov (United States)

    Dolinska, Monika B; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2014-01-01

    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. 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. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  9. Transcriptionally Silenced Transgenes in Maize Are Activated by Three Mutations Defective in Paramutation

    Science.gov (United States)

    McGinnis, Karen M.; Springer, Catherine; Lin, Yan; Carey, Charles C.; Chandler, Vicki

    2006-01-01

    Plants with mutations in one of three maize genes, mop1, rmr1, and rmr2, are defective in paramutation, an allele-specific interaction that leads to meiotically heritable chromatin changes. Experiments reported here demonstrate that these genes are required to maintain the transcriptional silencing of two different transgenes, suggesting that paramutation and transcriptional silencing of transgenes share mechanisms. We hypothesize that the transgenes are silenced through an RNA-directed chromatin mechanism, because mop1 encodes an RNA-dependent RNA polymerase. In all the mutants, DNA methylation was reduced in the active transgenes relative to the silent transgenes at all of the CNG sites monitored within the transgene promoter. However, asymmetrical methylation persisted at one site within the reactivated transgene in the rmr1-1 mutant. With that one mutant, rmr1-1, the transgene was efficiently resilenced upon outcrossing to reintroduce the wild-type protein. In contrast, with the mop1-1 and rmr2-1 mutants, the transgene remained active in a subset of progeny even after the wild-type proteins were reintroduced by outcrossing. Interestingly, this immunity to silencing increased as the generations progressed, consistent with a heritable chromatin state being formed at the transgene in plants carrying the mop1-1 and rmr2-1 mutations that becomes more resistant to silencing in subsequent generations. PMID:16702420

  10. 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.

  11. Germline activating MTOR mutation arising through gonadal mosaicism in two brothers with megalencephaly and neurodevelopmental abnormalities.

    Science.gov (United States)

    Mroske, Cameron; Rasmussen, Kristen; Shinde, Deepali N; Huether, Robert; Powis, Zoe; Lu, Hsiao-Mei; Baxter, Ruth M; McPherson, Elizabeth; Tang, Sha

    2015-11-05

    In humans, Mammalian Target of Rapamycin (MTOR) encodes a 300 kDa serine/ threonine protein kinase that is ubiquitously expressed, particularly at high levels in brain. MTOR functions as an integrator of multiple cellular processes, and in so doing either directly or indirectly regulates the phosphorylation of at least 800 proteins. While somatic MTOR mutations have been recognized in tumors for many years, and more recently in hemimegalencephaly, germline MTOR mutations have rarely been described. We report the successful application of family-trio Diagnostic Exome Sequencing (DES) to identify the underlying molecular etiology in two brothers with multiple neurological and developmental lesions, and for whom previous testing was non-diagnostic. The affected brothers, who were 6 and 23 years of age at the time of DES, presented symptoms including but not limited to mild Autism Spectrum Disorder (ASD), megalencephaly, gross motor skill delay, cryptorchidism and bilateral iris coloboma. Importantly, we determined that each affected brother harbored the MTOR missense alteration p.E1799K (c.5395G>A). This exact variant has been previously identified in multiple independent human somatic cancer samples and has been shown to result in increased MTOR activation. Further, recent independent reports describe two unrelated families in whom p.E1799K co-segregated with megalencephaly and intellectual disability (ID); in both cases, p.E1799K was shown to have originated due to germline mosaicism. In the case of the family reported herein, the absence of p.E1799K in genomic DNA extracted from the blood of either parent suggests that this alteration most likely arose due to gonadal mosaicism. Further, the p.E1799K variant exerts its effect by a gain-of-function (GOF), autosomal dominant mechanism. Herein, we describe the use of DES to uncover an activating MTOR missense alteration of gonadal mosaic origin that is likely to be the causative mutation in two brothers who present

  12. Physiochemical and Thermodynamic Characterization of Highly Active Mutated Aspergillus niger β-glucosidase for Lignocellulose Hydrolysis.

    Science.gov (United States)

    Javed, Muhammad Rizwan; Rashid, Muhammad Hamid; Riaz, Muhammad; Nadeem, Habibullah; Qasim, Muhammad; Ashiq, Nourin

    2018-01-30

    The extracellular β-glucosidases (BGL) from Aspergillus niger NIBGE (P) and its 2-Deoxy-D-glucose resistant mutant derivative NIBGE-06 (M-6), produced under submerged growth conditions were purified to homogeneity level by ammonium sulfate precipitation and FPLC system. The BGLs from both strains were dimeric in nature, with subunit and native molecular masses of 130 kDa and 252 kDa, respectively. The comparative analysis of nucleotides of bgl genes revealed 8 point mutations within the bgl gene of mutant strain as compared to parental strain. Out of these 8 mutations 2 were true substitutions, 2 were semi-conserved and 3 were conserved substitutions, while one was silent mutation. The enzyme belongs to family 3 of glycoside hydrolases with retaining catalytic mechanism. Significant improvement was observed in the kinetic properties of the mutant BGL relative to the wild type enzyme. Arrhenius plot for energy of activation (Ea) showed a biphasic trend and ES-complex formation required Ea of 50 and 42 kJ mol-1 by BGL from parent and mutant, respectively. The pKa1 and pKa2 of the active site residues were 3.4 & 5.5 and 3.2 & 5.6, respectively. The heat of ionization for the acidic limb (ΔHI-AL) and the basic limb (ΔHI-BL) of BGL from both strains were equal to 56 & 41 and 71 & 45 kJ mol-1, respectively. Kinetic constants of cellobiose hydrolysis for BGL from both strains were determined as follows: kcat = 2,589 and 4,135 s-1, Km = 0.24 and 0.26 mM cellobiose, kcat/Km = 10,872 and 15,712 s-1 mM-1 cellobiose, respectively. Thermodynamic parameters for cellobiose hydrolysis also suggested that mutant BGL is more efficient compared to the parent enzyme. Comparative analysis of Ea(d), ΔH* and ΔG* for irreversible thermostability indicated that the thermostabilization of mutant enzyme was due to higher functional energy (free energy), which enabled the enzyme to resist against unfolding of its transition state. Hence, directed mutation resulted in the engineering of

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

    Science.gov (United States)

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

    2016-01-01

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

  14. Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

    Science.gov (United States)

    Griffin, Carol E.; Hoke, Jonathan M.; Samarakoon, Upeka; Duan, Junhui; Mu, Jianbing; Ferdig, Michael T.; Warhurst, David C.

    2012-01-01

    The Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (−)-quinine and (+)-quinidine. The potency of (+)-isomers is greater than the (−)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC50 ratio of (−)/(+) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (−) and (+) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids. PMID:22869567

  15. High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation

    International Nuclear Information System (INIS)

    Yamakawa, Nobuhiro; Takahashi, Akihisa; Mori, Eiichiro; Imai, Yuichiro; Ohnishi, Ken; Kirita, Tadaaki; Ohnishi, Takeo; Furusawa, Yoshiya

    2008-01-01

    Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation-induced apoptosis. Human gingival cancer cells (Ca9-22 cells) containing a mutated p53 (mp53) gene were irradiated with X-rays, C-ion (13-100 KeV/μm), or Fe-ion beams (200 KeV/μm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase-3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET-dependent manner. Both RBE curves reached a peak at 100 KeV/μm, and then decreased at values over 100 KeV/μm. When cells were irradiated with high LET radiation, Caspase-3 was cleaved and activated, leading to poly (ADP-ribose) polymerase (PARP) cleavage. In addition, Caspase-9 inhibitor suppressed Caspase-3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase-8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase-3 through Caspase-9, even in the presence of mp53. (author)

  16. A strong loss-of-function mutation in RAN1 results in constitution activation of the ethylene response pathway as well as a rosette-lethal phenotype

    Science.gov (United States)

    Keith Woeste; Joseph J. Kieber

    2000-01-01

    A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resuited in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a...

  17. 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.

  18. Detection of an activating c-kit mutation by real-time PCR in patients with anaphylaxis.

    Science.gov (United States)

    Lawley, Wendy; Hird, Heather; Mallinder, Philip; McKenna, Sue; Hargadon, Beverley; Murray, Alistair; Bradding, Peter

    2005-05-02

    A well-characterised gain-of-function point mutation within exon 17 of the c-kit proto-oncogene known as Asp816Val is present in patients with mastocytosis. Activation of mast cells through this receptor primes them for IgE-dependent activation, and patients with mastocytosis are at increased risk of anaphylaxis. We hypothesised that the Asp816Val mutation is associated with a history of anaphylaxis in the general population. A mismatch amplification real-time PCR assay was developed and validated to test for the Asp816Val mutation. Subjects were recruited to four subject groups: normal non-atopics, atopics without anaphylaxis, food-induced anaphylactics and non-food anaphylactics. Blood samples collected from forty subjects were tested for the presence of Asp816Val. Thirteen subjects were found to carry the mutation; normals (2/9), atopics (2/10), food anaphylactics (5/11) and non-food anaphylactics (4/10). Statistical analysis of the data determined that there was no significant difference between the numbers of subjects found to carry the Asp816Val mutation in each of the groups although a trend towards an increased occurrence in anaphylactics was observed. In summary, the hypothesis that the presence of the Asp816Val mutation is linked to the occurrence of anaphylaxis was not supported, but interestingly, we have shown for the first time Asp816Val may occur more frequently than previously reported within the general population.

  19. 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.

  20. Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize

    Directory of Open Access Journals (Sweden)

    Susan Gabay-Laughnan

    2018-01-01

    Full Text Available Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds, Enhancer/Suppressor-mutator (En/Spm, and Mutator (Mu transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development.

  1. 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.

  2. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Association of mutator activity with UV sensitivity in an aphidicolin-resistant mutant of Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Liu, P.K.; Chang, C.; Trosko, J.E.

    1982-01-01

    The spontaneous mutation rates of an ultraviolet light (UV)-sensitive aphidicolin-resistant mutant (aphsup(r)-4-2) and its revertants have been determined by 2 techniques. By using the fluctuation analysis, the mutant and its thymidine (TdR)-prototrophic 'revertant' were found to exhibit elevated spontaneous mutation rates at the 6-thioguanine- and diphtheria-toxin-resistant loci. In contrast, the TdR-auxotrophic 'revertant' did not show this property. Similar results were obtained by the multiple replating technique. From these comparative studies and other previous characterizations, it appears that a single gene mutation is responsible for the following pleiotropic phenotype: slow growth, UV sensitivity, high UV-induced mutability, high frequency of site-specific bromodeoxyuridine (BrdU)-dependent chromosome breaks and enhanced spontaneous mutation rate. Recent studies indicate that the mutation may be on the gene for DNA polymerase α. The results further indicate that thymidine auxotrophy or imbalance in nucleotide pools is not necessarily associated with the mutator activity in mammalian cells. (orig.)

  4. MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia

    Science.gov (United States)

    Pikman, Yana; Lee, Benjamin H; Mercher, Thomas; McDowell, Elizabeth; Ebert, Benjamin L; Gozo, Maricel; Cuker, Adam; Wernig, Gerlinde; Moore, Sandra; Galinsky, Ilene; DeAngelo, Daniel J; Clark, Jennifer J; Lee, Stephanie J; Golub, Todd R; Wadleigh, Martha; Gilliland, D. Gary; Levine, Ross L

    2006-01-01

    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 × 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-mediated myeloproliferative

  5. 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 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.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.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-mediated myeloproliferative disorders (MPD exhibits certain features of

  6. Activity of the Escherichia coli mutT mutator allele in an anaerobic environment.

    OpenAIRE

    Fowler, R G; Erickson, J A; Isbell, R J

    1994-01-01

    Mutation frequencies for an Escherichia coli mutT strain were measured in both aerobic and anaerobic environments. When cells were grown in a rich medium (L broth), mutation frequencies were similar in both aerobic and anaerobic conditions. In contrast, when grown in a minimal medium, mutT anaerobic mutation frequencies were reduced dramatically compared with aerobic values, which were similar to L broth frequencies. L broth mutT cultures treated with a commercial enzyme complex that reduces ...

  7. Leukemia-associated activating mutation of Flt3 expands dendritic cells and alters T cell responses.

    Science.gov (United States)

    Lau, Colleen M; Nish, Simone A; Yogev, Nir; Waisman, Ari; Reiner, Steven L; Reizis, Boris

    2016-03-07

    A common genetic alteration in acute myeloid leukemia is the internal tandem duplication (ITD) in FLT3, the receptor for cytokine FLT3 ligand (FLT3L). Constitutively active FLT3-ITD promotes the expansion of transformed progenitors, but also has pleiotropic effects on hematopoiesis. We analyzed the effect of FLT3-ITD on dendritic cells (DCs), which express FLT3 and can be expanded by FLT3L administration. Pre-leukemic mice with the Flt3(ITD) knock-in allele manifested an expansion of classical DCs (cDCs) and plasmacytoid DCs. The expansion originated in DC progenitors, was cell intrinsic, and was further enhanced in Flt3(ITD/ITD) mice. The mutation caused the down-regulation of Flt3 on the surface of DCs and reduced their responsiveness to Flt3L. Both canonical Batf3-dependent CD8(+) cDCs and noncanonical CD8(+) cDCs were expanded and showed specific alterations in their expression profiles. Flt3(ITD) mice showed enhanced capacity to support T cell proliferation, including a cell-extrinsic expansion of regulatory T (T reg) cells. Accordingly, these mice restricted alloreactive T cell responses during graft-versus-host reaction, but failed to control autoimmunity without T reg cells. Thus, the FLT3-ITD mutation directly affects DC development, indirectly modulating T cell homeostasis and supporting T reg cell expansion. We hypothesize that this effect of FLT3-ITD might subvert immunosurveillance and promote leukemogenesis in a cell-extrinsic manner. © 2016 Lau et al.

  8. Influence of Drug Resistance Mutations on the Activity of HIV-1 Subtypes A and B Integrases: a Comparative Study.

    Science.gov (United States)

    Shadrina, O A; Zatsepin, T S; Agapkina, Yu Yu; Isaguliants, M G; Gottikh, M B

    2015-01-01

    Integration of human immunodeficiency virus (HIV-1) DNA into the genome of an infected cell is one of the key steps in the viral replication cycle. The viral enzyme integrase (IN), which catalyzes the integration, is an attractive target for the development of new antiviral drugs. However, the HIV-1 therapy often results in the IN gene mutations inducing viral resistance to integration inhibitors. To assess the impact of drug resistance mutations on the activity of IN of HIV-1 subtype A strain FSU-A, which is dominant in Russia, variants of the consensus IN of this subtype containing the primary resistance mutations G118R and Q148K and secondary compensatory substitutions E138K and G140S were prepared and characterized. Comparative study of these enzymes with the corresponding mutants of IN of HIV-1 subtype B strains HXB-2 was performed. The mutation Q148K almost equally reduced the activity of integrases of both subtypes. Its negative effect was partially compensated by the secondary mutations E138K and G140S. Primary substitution G118R had different influence on the activity of proteins of the subtypes A and B, and the compensatory effect of the secondary substitution E138K also depended on the viral subtype. Comparison of the mutants resistance to the known strand transfer inhibitors raltegravir and elvitegravir, and a new inhibitor XZ-259 (a dihydro-1H-isoindol derivative), showed that integrases of both subtypes with the Q148K mutation were insensitive to raltegravir and elvitegravir but were effectively inhibited by XZ-259. The substitution G118R slightly reduced the efficiency of IN inhibition by raltegravir and elvitegravir and caused no resistance to XZ_259.

  9. 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.

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

    DEFF Research Database (Denmark)

    Vissing, John; Duno, Morten; Schwartz, Marianne

    2009-01-01

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

  11. Potential late-onset Alzheimer's disease-associated mutations in the ADAM10 gene attenuate α-secretase activity

    Science.gov (United States)

    Kim, Minji; Suh, Jaehong; Romano, Donna; Truong, Mimy H.; Mullin, Kristina; Hooli, Basavaraj; Norton, David; Tesco, Giuseppina; Elliott, Kathy; Wagner, Steven L.; Moir, Robert D.; Becker, K. David; Tanzi, Rudolph E.

    2009-01-01

    ADAM10, a member of a disintegrin and metalloprotease family, is an α-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. Here, we present evidence for genetic association of ADAM10 with Alzheimer's disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated α-secretase activity of ADAM10 (>70% decrease), and elevated Aβ levels (1.5–3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD. PMID:19608551

  12. 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.

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

    Science.gov (United States)

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

    2015-10-01

    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 diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9' position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22' position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

    Energy Technology Data Exchange (ETDEWEB)

    Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

    2010-01-01

    Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

  15. A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst.

    Science.gov (United States)

    Koch, Aaron A; Hansen, Douglas A; Shende, Vikram V; Furan, Lawrence R; Houk, K N; Jiménez-Osés, Gonzalo; Sherman, David H

    2017-09-27

    Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper , we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TE S148C ) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TE S148C and TE WT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition-elimination (TE WT ) to a lower energy concerted acyl substitution (TE S148C ), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones.

  16. KRAS exon 2 mutations influence activity of regorafenib in an SW48-based disease model of colorectal cancer.

    Science.gov (United States)

    Camaj, Peter; Primo, Stefano; Wang, Yan; Heinemann, Volker; Zhao, Yue; Laubender, Ruediger Paul; Stintzing, Sebastian; Giessen-Jung, Clemens; Jung, Andreas; Gamba, Sebastian; Bruns, Christiane Josephine; Modest, Dominik Paul

    2015-01-01

    To investigate the impact of KRAS mutation variants on the activity of regorafenib in SW48 colorectal cancer cells. Activity of regorafenib was evaluated in isogenic SW48 KRAS wild-type (WT) and mutant cells. Subcutaneous xenografts (KRAS WT and G12C mutant variants) in NOD/SCID mice were analyzed to elucidate the effect of regorafenib treatment in vivo. Compared with KRAS WT cells, all mutant variants seemed associated with some degree of resistance to regorafenib-treatment in vitro. In vivo, activation of apoptosis (TUNEL) and reduction of proliferation (Ki67) after treatment with regorafenib were more pronounced in KRAS WT tumors as compared with G12C variants. In SW48 cells, exon 2 mutations of the KRAS gene may influence antitumor effects of regorafenib.

  17. 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.

  18. Mutation in cytochrome b gene of mitochondrial DNA in a family with fibromyalgia is associated with NLRP3-inflammasome activation.

    Science.gov (United States)

    Cordero, Mario D; Alcocer-Gómez, Elísabet; Marín-Aguilar, Fabiola; Rybkina, Tatyana; Cotán, David; Pérez-Pulido, Antonio; Alvarez-Suarez, José Miguel; Battino, Maurizio; Sánchez-Alcazar, José Antonio; Carrión, Angel M; Culic, Ognjen; Navarro-Pando, José M; Bullón, Pedro

    2016-02-01

    Fibromyalgia (FM) is a worldwide diffuse musculoskeletal chronic pain condition that affects up to 5% of the general population. Many symptoms associated with mitochondrial diseases are reported in patients with FM such as exercise intolerance, fatigue, myopathy and mitochondrial dysfunction. In this study, we report a mutation in cytochrome b gene of mitochondrial DNA (mtDNA) in a family with FM with inflammasome complex activation. mtDNA from blood cells of five patients with FM were sequenced. We clinically and genetically characterised a patient with FM and family with a new mutation in mtCYB. Mitochondrial mutation phenotypes were determined in skin fibroblasts and transmitochondrial cybrids. After mtDNA sequence in patients with FM, we found a mitochondrial homoplasmic mutation m.15804T>C in the mtCYB gene in a patient and family, which was maternally transmitted. Mutation was observed in several tissues and skin fibroblasts showed a very significant mitochondrial dysfunction and oxidative stress. Increased NLRP3-inflammasome complex activation was observed in blood cells from patient and family. We propose further studies on mtDNA sequence analysis in patients with FM with evidences for maternal inheritance. The presence of similar symptoms in mitochondrial myopathies could unmask mitochondrial diseases among patients with FM. On the other hand, the inflammasome complex activation by mitochondrial dysfunction could be implicated in the pathophysiology of mitochondrial diseases. 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/

  19. Investigating mutation-specific biological activities of small molecules using quantitative structure-activity relationship for epidermal growth factor receptor in cancer.

    Science.gov (United States)

    Anoosha, P; Sakthivel, R; Gromiha, M Michael

    2017-12-01

    Epidermal Growth Factor Receptor (EGFR) is a potential drug target in cancer therapy. Missense mutations play major roles in influencing the protein function, leading to abnormal cell proliferation and tumorigenesis. A number of EGFR inhibitor molecules targeting ATP binding domain were developed for the past two decades. Unfortunately, they become inactive due to resistance caused by new mutations in patients, and previous studies have also reported noticeable differences in inhibitor binding to distinct known driver mutants as well. Hence, there is a high demand for identification of EGFR mutation-specific inhibitors. In our present study, we derived a set of anti-cancer compounds with biological activities against eight typical EGFR known driver mutations and developed quantitative structure-activity relationship (QSAR) models for each separately. The compounds are grouped based on their functional scaffolds, which enhanced the correlation between compound features and respective biological activities. The models for different mutants performed well with a correlation coefficient, (r) in the range of 0.72-0.91 on jack-knife test. Further, we analyzed the selected features in different models and observed that hydrogen bond and aromaticity-related features play important roles in predicting the biological activity of a compound. This analysis is complimented with docking studies, which showed the binding patterns and interactions of ligands with EGFR mutants that could influence their activities. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Dan Lu

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

  2. Absence of activating mutations in ras and gsp oncogenes in a cohort of nine patients with sporadic pediatric thyroid tumors.

    Science.gov (United States)

    Pauws, E; Tummers, R F; Ris-Stalpers, C; de Vijlder, J J; Voûte, T

    2001-06-01

    Characterization of the genetic background of pediatric thyroid carcinomas could aid in distinguishing between differently staged tumors with respect to treatment and prognosis. Two known genetic factors associated with thyroid carcinoma, the proto-oncogenes gsp and ras were investigated. DNA was extracted from paraffin sections from both tumor and normal thyroid tissue of nine patients (ages 9-16 years). Of these patients, eight were diagnosed with papillary carcinoma and one with follicular adenoma. The coding exons of gsp and the three known ras genes (H, K, and N-ras) were screened for mutations using SSCP-analysis. There were no mutations present in the ras and gsp proto-oncogenes hot spots, however, LOH of H-ras (chromosome location 11p15.5) was found in tumor tissue from one patient and a homozygous mutation in exon 12 of gsp causing a Pro-->Ser conversion was present in the thyroid tumor tissue from another patient. Two silent polymorphisms were detected, H-ras exon1, 86T-->C and gsp exon 5, 81T-->C. Our results indicate that the ras/gsp mutations found are probably late events in the tumorigenesis representing general oncogenic stress. In conclusion, it seems that ras/gsp activation is not a factor in the mechanism causing sporadic thyroid carcinoma in children. Copyright 2001 Wiley-Liss, Inc.

  3. Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF).

    Science.gov (United States)

    Vancauwenberghe, Eric; Noyer, Lucile; Derouiche, Sandra; Lemonnier, Loïc; Gosset, Pierre; Sadofsky, Laura R; Mariot, Pascal; Warnier, Marine; Bokhobza, Alexandre; Slomianny, Christian; Mauroy, Brigitte; Bonnal, Jean-Louis; Dewailly, Etienne; Delcourt, Philippe; Allart, Laurent; Desruelles, Emilie; Prevarskaya, Natalia; Roudbaraki, Morad

    2017-08-01

    Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells, and cancer-associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis. © 2017 Wiley Periodicals, Inc.

  4. Tyrosine agonists reverse the molecular defects associated with dominant-negative mutations in human peroxisome proliferator-activated receptor gamma.

    Science.gov (United States)

    Agostini, Maura; Gurnell, Mark; Savage, David B; Wood, Emily M; Smith, Aaron G; Rajanayagam, Odelia; Garnes, Keith T; Levinson, Sidney H; Xu, H Eric; Schwabe, John W R; Willson, Timothy M; O'Rahilly, Stephen; Chatterjee, V Krishna

    2004-04-01

    Loss-of-function mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) are associated with a novel syndrome characterized by partial lipodystrophy and severe insulin resistance. Here we have further characterized the properties of natural dominant-negative PPARgamma mutants (P467L, V290M) and evaluated the efficacy of putative natural ligands and synthetic thiazolidinedione (TZD) or tyrosine-based (TA) receptor agonists in rescuing mutant receptor function. A range of natural ligands failed to activate the PPARgamma mutants and their transcriptional responses to TZDs (e.g. pioglitazone, rosiglitazone) were markedly attenuated, whereas TAs (e.g. farglitazar) corrected defects in ligand binding and coactivator recruitment by the PPARgamma mutants, restoring transcriptional function comparable with wild-type receptor. Transcriptional silencing via recruitment of corepressor contributes to dominant-negative inhibition of wild type by the P467L and V290M mutants and the introduction of an artificial mutation (L318A) disrupting corepressor interaction abrogated their dominant-negative activity. More complete ligand-dependent corepressor release and reversal of dominant-negative inhibition was achieved with TA than TZD agonists. Modeling suggests a structural basis for these observations: both mutations destabilize helix 12 to favor receptor-corepressor interaction; conversely, farglitazar makes more extensive contacts than rosiglitazone within the ligand-binding pocket, to stabilize helix 12, facilitating corepressor release and transcriptional activation. Farglitazar was a more potent inducer of PPARgamma target gene (aP2) expression in peripheral blood mononuclear cells with the P467L mutation. Having shown that rosiglitazone is of variable and limited efficacy in these subjects, we suggest that TAs may represent a more rational therapeutic approach.

  5. 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.

    long QT syndrome (LQTS) (D95V and D129G)2, on spontaneous Ca2+ release in HEK293 cells expressing the RyR2 channel. Furthermore, we studied the impact of these mutations on the interactions between CaM and a peptide corresponding to the RyR2 CaM binding domain (CaMBD) residue number 3581......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...... in the other two CaM genes (CALM2 and CALM3). All CaM mutations are associated with severe ventricular arrhythmias. CaM regulates several key proteins governing cardiac excitation-contraction coupling (ECC), including the cardiac ryanodine receptor (RyR2) Ca2+ release channel. RyR2 mutations also dominantly...

  6. Mutations that abrogate transactivational activity of the feline leukemia virus long terminal repeat do not affect virus replication

    International Nuclear Information System (INIS)

    Abujamra, Ana L.; Faller, Douglas V.; Ghosh, Sajal K.

    2003-01-01

    The U3 region of the LTR of oncogenic Moloney murine leukemia virus (Mo-MuLV) and feline leukemia viruses (FeLV) have been previously reported to activate expression of specific cellular genes in trans, such as MHC class I, collagenase IV, and MCP-1, in an integration-independent manner. It has been suggested that transactivation of these specific cellular genes by leukemia virus U3-LTR may contribute to the multistage process of leukemogenesis. The U3-LTR region, necessary for gene transactivational activity, also contains multiple transcription factor-binding sites that are essential for normal virus replication. To dissect the promoter activity and the gene transactivational activity of the U3-LTR, we conducted mutational analysis of the U3-LTR region of FeLV-A molecular clone 61E. We identified minimal nucleotide substitution mutants on the U3 LTR that did not disturb transcription factor-binding sites but abrogated its ability to transactivate the collagenase gene promoter. To determine if these mutations actually have altered any uncharacterized important transcription factor-binding site, we introduced these U3-LTR mutations into the full-length infectious molecular clone 61E. We demonstrate that the mutant virus was replication competent but could not transactivate cellular gene expression. These results thus suggest that the gene transactivational activity is a distinct property of the LTR and possibly not related to its promoter activity. The cellular gene transactivational activity-deficient mutant FeLV generated in this study may also serve as a valuable reagent for testing the biological significance of LTR-mediated cellular gene activation in the tumorigenesis caused by leukemia viruses

  7. A screen for mutations that prevent lethality caused by expression of activated sevenless and Ras1 in the Drosophila embryo.

    Science.gov (United States)

    Maixner, A; Hecker, T P; Phan, Q N; Wassarman, D A

    1998-01-01

    Ras1 plays a critical role in receptor tyrosine kinase (RTK) signal transduction pathways that function during Drosophila development. We demonstrate that mis-expression of constitutively active forms of Ras1 (Ras1V12) and the Sevenless (Sev) RTK (SevS11) during embryogenesis causes lethality due to inappropriate activation of RTK/Ras1 signaling pathways. Genetic and molecular data indicate that the rate of SevS11/sev-Ras1V12 lethality is sensitive to the expression level of both transgenes. To identify genes that encode components of RTK/Ras1 signaling pathways or modulators of RNA polymerase II transcription, we took advantage of the dose-sensitivity of the system and screened for second site mutations that would dominantly suppress the lethality. The collection of identified suppressors includes the PR55 subunit of Protein Phosphatase 2A indicating that downstream of Sev and Ras1 this subunit acts as a negative regulator of phosphatase activity. The isolation of mutations in the histone deacetylase RPD3 suggests that it functions as positive regulator of sev enhancer-driven transcription. Finally, the isolation of mutations in the Trithorax group gene devenir and the characterized allelism with the Breathless RTK encoding gene provides evidence for Ras1-mediated regulation of homeotic genes.

  8. BGB-283, a Novel RAF Kinase and EGFR Inhibitor, Displays Potent Antitumor Activity in BRAF-Mutated Colorectal Cancers.

    Science.gov (United States)

    Tang, Zhiyu; Yuan, Xi; Du, Rong; Cheung, Shing-Hu; Zhang, Guoliang; Wei, Jing; Zhao, Yuan; Feng, Yingcai; Peng, Hao; Zhang, Yi; Du, Yunguang; Hu, Xiaoxia; Gong, Wenfeng; Liu, Yong; Gao, Yajuan; Liu, Ye; Hao, Rui; Li, Shengjian; Wang, Shaohui; Ji, Jiafu; Zhang, Lianhai; Li, Shuangxi; Sutton, David; Wei, Min; Zhou, Changyou; Wang, Lai; Luo, Lusong

    2015-10-01

    Oncogenic BRAF, which drives cell transformation and proliferation, has been detected in approximately 50% of human malignant melanomas and 5% to 15% of colorectal cancers. Despite the remarkable clinical activities achieved by vemurafenib and dabrafenib in treating BRAF(V600E) metastatic melanoma, their clinical efficacy in BRAF(V600E) colorectal cancer is far less impressive. Prior studies suggested that feedback activation of EGFR and MAPK signaling upon BRAF inhibition might contribute to the relative unresponsiveness of colorectal cancer to the first-generation BRAF inhibitors. Here, we report characterization of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently under clinical investigation. In vitro, BGB-283 potently inhibits BRAF(V600E)-activated ERK phosphorylation and cell proliferation. It demonstrates selective cytotoxicity and preferentially inhibits proliferation of cancer cells harboring BRAF(V600E) and EGFR mutation/amplification. In BRAF(V600E) colorectal cancer cell lines, BGB-283 effectively inhibits the reactivation of EGFR and EGFR-mediated cell proliferation. In vivo, BGB-283 treatment leads to dose-dependent tumor growth inhibition accompanied by partial and complete tumor regressions in both cell line-derived and primary human colorectal tumor xenografts bearing BRAF(V600E) mutation. These findings support BGB-283 as a potent antitumor drug candidate with clinical potential for treating colorectal cancer harboring BRAF(V600E) mutation. ©2015 American Association for Cancer Research.

  9. Anti-helminth compound niclosamide downregulates Wnt Signaling and elicits antitumor responses in tumors with activating APC mutations

    Science.gov (United States)

    Osada, Takuya; Chen, Minyong; Yang, Xiao Yi; Spasojevic, Ivan; Vandeusen, Jeffrey B.; Hsu, David; Clary, Bryan M.; Clay, Timothy M.; Chen, Wei; Morse, Michael A.; Lyerly, H. Kim

    2011-01-01

    Wnt/β-catenin pathway activation caused by APC mutations occurs in approximately 80% of sporadic colorectal cancers. The anti-helminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream β-catenin signaling. In this study, we determined if niclosamide could inhibit the Wnt/ β-catenin pathway in human colorectal cancers and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/ β-catenin pathway activation, downregulated Dvl2, decreased downstream β-catenin signaling and exerted anti-proliferative effects in human colon cancer cell lines and colorectal cancer cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-κB or mTOR did not exert similar anti-proliferative effects in these colorectal cancer model systems. In mice implanted with human colorectal cancer xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity and led to tumor control. Our findings support clinical explorations to reposition niclosamide for treatment of colorectal cancer. PMID:21531761

  10. 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

  11. Cutaneous onchocerciasis in Dumbu, a pastoral area in the North ...

    African Journals Online (AJOL)

    ... bone in extra-skeletal sites leading to severe disability and eventually death. We present a sporadic case from Northern Tanzania with a minor unilateral hallux anomaly and the common ACVR1 c.617G>A mutation. Key words: Fibrodysplasia ossificans progressiva, heterotopic ossification, hallux valgus, recurrent ACVR1 ...

  12. 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

    2013-01-01

    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. PMID:23582645

  13. 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.

  14. Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and Patients HarboringMETExon 14 Mutations and Overcomes Mutation-mediated Resistance to Type I MET Inhibitors in Nonclinical Models.

    Science.gov (United States)

    Engstrom, Lars D; Aranda, Ruth; Lee, Matthew; Tovar, Elizabeth A; Essenburg, Curt J; Madaj, Zachary; Chiang, Harrah; Briere, David; Hallin, Jill; Lopez-Casas, Pedro P; Baños, Natalia; Menendez, Camino; Hidalgo, Manuel; Tassell, Vanessa; Chao, Richard; Chudova, Darya I; Lanman, Richard B; Olson, Peter; Bazhenova, Lyudmilla; Patel, Sandip Pravin; Graveel, Carrie; Nishino, Mizuki; Shapiro, Geoffrey I; Peled, Nir; Awad, Mark M; Jänne, Pasi A; Christensen, James G

    2017-11-01

    Purpose: MET exon 14 deletion ( MET ex14 del) mutations represent a novel class of non-small cell lung cancer (NSCLC) driver mutations. We evaluated glesatinib, a spectrum-selective MET inhibitor exhibiting a type II binding mode, in MET ex14 del-positive nonclinical models and NSCLC patients and assessed its ability to overcome resistance to type I MET inhibitors. Experimental Design: As most MET inhibitors in clinical development bind the active site with a type I binding mode, we investigated mechanisms of acquired resistance to each MET inhibitor class utilizing in vitro and in vivo models and in glesatinib clinical trials. Results: Glesatinib inhibited MET signaling, demonstrated marked regression of MET ex14 del-driven patient-derived xenografts, and demonstrated a durable RECIST partial response in a MET ex14 del mutation-positive patient enrolled on a glesatinib clinical trial. Prolonged treatment of nonclinical models with selected MET inhibitors resulted in differences in resistance kinetics and mutations within the MET activation loop (i.e., D1228N, Y1230C/H) that conferred resistance to type I MET inhibitors, but remained sensitive to glesatinib. In vivo models exhibiting MET ex14 del/A-loop double mutations and resistance to type I inhibitors exhibited a marked response to glesatinib. Finally, a MET ex14 del mutation-positive NSCLC patient who responded to crizotinib but later relapsed, demonstrated a mixed response to glesatinib including reduction in size of a MET Y1230H mutation-positive liver metastasis and concurrent loss of detection of this mutation in plasma DNA. Conclusions: Together, these data demonstrate that glesatinib exhibits a distinct mechanism of target inhibition and can overcome resistance to type I MET inhibitors. Clin Cancer Res; 23(21); 6661-72. ©2017 AACR . ©2017 American Association for Cancer Research.

  15. USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination

    Directory of Open Access Journals (Sweden)

    Laura Novellasdemunt

    2017-10-01

    Full Text Available The tumor suppressor gene adenomatous polyposis coli (APC is mutated in most colorectal cancers (CRCs, resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs.

  16. Insights into the activity change of spore photoproduct lyase induced by mutations at a peripheral glycine residue

    Science.gov (United States)

    Yang, Linlin; Li, Lei

    2017-03-01

    UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that was later found to be > 15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3 4 fold while the G168R mutant by 80 fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2-; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

  17. Distribution of Gs-alpha activating mutations in human thyroid tumors measured by subcloning.

    Science.gov (United States)

    Gorelov, V N; Gyenes, M; Neser, F; Röher, H D; Goretzki, P E

    1996-01-01

    In the search for the prevalence and distribution pattern of Gs-alpha gene mutations in differentiated thyroid tumors we examined 66 tumor tissue samples for the presence of mutations at "hot-spot" codons 201 and 227 using methods based on the polymerase chain reaction, subcloning and sequencing. The prevailing type of single-base substitution at codon 201 (71.4%) corresponded to the replacement of the wild-type sequence CGT (Arg) with TGT (Cys). The fragments of the Gs-alpha gene, including codon 201 or 227 from five follicular carcinomas and one follicular adenoma, were subcloned in Escherichia coli and it was found that the proportion of alleles with mutated codon 201 varied from 3.2% to 43%. Sequencing of the corresponding region has confirmed preliminary data indicating that the single-base changes CGT (Arg) to TGT (Cys) or CGT to CAT (His) occurred. There was only a weak correlation between the prevalence of cells bearing a mutation in the Gs-alpha gene and the level of Gs-alpha protein expression in the corresponding thyroid tumors.

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

    NARCIS (Netherlands)

    de Winter, J.M.; Buck, D.; Hidalgo, C.; Jasper, J.R.; Malik, F.I.; Clarke, N.F.; Stienen, G.J.M.; Lawlor, M.W.; Beggs, A.H.; Ottenheijm, C.A.C.; Granzier, H.

    2013-01-01

    Background: 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 nebuli which plays a crucial role in skeletal muscle

  19. Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping

    Czech Academy of Sciences Publication Activity Database

    Divina, Petr; Kvitkovicova, Andrea; Buratti, E.; Vorechovsky, I.

    2009-01-01

    Roč. 17, č. 6 (2009), s. 759-765 ISSN 1018-4813 Institutional research plan: CEZ:AV0Z50520514 Keywords : mutation * cryptic splice site * exon skipping Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.564, year: 2009

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

    African Journals Online (AJOL)

    Acute Myeloid Leukemia (AML)-M0 is a cancer of blood-forming cells in the bone marrow. KIT gene is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. Mutations of KIT receptor ...

  1. Two CGD Families with a Hypomorphic Mutation in the Activation Domain of p67(phox)

    NARCIS (Netherlands)

    Roos, Dirk; van Buul, Jaap D.; Tool, Anton Tj; Matute, Juan D.; Marchal, Christophe M.; Hayee, Bu'Hussain; Köker, M. Yavuz; de Boer, Martin; van Leeuwen, Karin; Segal, Anthony W.; Pick, Edgar; Dinauer, Mary C.

    2014-01-01

    Chronic granulomatous Disease (CGD) is a rare immunodeficiency caused by a defect in the leukocyte NADPH oxidase. This enzyme generates superoxide, which is needed for the killing of bacteria and fungi by phagocytic leukocytes. Most CGD patients have mutations in CYBB, the X-linked gene that encodes

  2. Alterations of the immunosuppressive IL4I1 enzyme activity induced by naturally occurring SNP/mutations.

    Science.gov (United States)

    Molinier-Frenkel, V; Mestivier, D; Castellano, F

    2016-03-01

    The immunosuppressive phenylalanine oxidase interleukin 4-induced gene 1 (IL4I1), primarily produced by antigen-presenting cells, inhibits T-cell proliferation and promotes the generation of Foxp3(+) regulatory T cells in vitro. Highly expressed by tumour-associated macrophages from human cancers, IL4I1 has a potential role in immune evasion from the anti-tumour immune response. We have reviewed single-nucleotide polymorphisms (SNPs) and mutations described for the exon 4 of the IL4I1 isoform 1, which is expressed in lymphoid tissue. Two of them were expressed in an exogenous system to analyse their effect on the enzymatic activity. The N92D SNP leads to a hyperactive enzyme, while the R102G mutation is hypomorphic. Moreover, we show that IL4I1 activity is not only directed against phenylalanine, as initially described, but also at a lower level against arginine. These data pave the way to more extensive analyses of the mutational state of IL4I1 in pathological conditions such as cancer, where its participation in immune system dysfunctions may have therapeutic implications.

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

    Directory of Open Access Journals (Sweden)

    Anice Moumen

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

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

    Science.gov (United States)

    Moumen, Anice; Virard, Isabelle; Raoul, Cédric

    2011-01-01

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

  5. Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

    Science.gov (United States)

    Bernasconi-Elias, P; Hu, T; Jenkins, D; Firestone, B; Gans, S; Kurth, E; Capodieci, P; Deplazes-Lauber, J; Petropoulos, K; Thiel, P; Ponsel, D; Hee Choi, S; LeMotte, P; London, A; Goetcshkes, M; Nolin, E; Jones, M D; Slocum, K; Kluk, M J; Weinstock, D M; Christodoulou, A; Weinberg, O; Jaehrling, J; Ettenberg, S A; Buckler, A; Blacklow, S C; Aster, J C; Fryer, C J

    2016-11-24

    Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

  6. Mutations in linker for activation of T cells (LAT) lead to a novel form of severe combined immunodeficiency.

    Science.gov (United States)

    Bacchelli, Chiara; Moretti, Federico A; Carmo, Marlene; Adams, Stuart; Stanescu, Horia C; Pearce, Kerra; Madkaikar, Manisha; Gilmour, Kimberly C; Nicholas, Adeline K; Woods, C Geoffrey; Kleta, Robert; Beales, Phil L; Qasim, Waseem; Gaspar, H Bobby

    2017-02-01

    Signaling through the T-cell receptor (TCR) is critical for T-cell development and function. Linker for activation of T cells (LAT) is a transmembrane adaptor signaling molecule that is part of the TCR complex and essential for T-cell development, as demonstrated by LAT-deficient mice, which show a complete lack of peripheral T cells. We describe a pedigree affected by a severe combined immunodeficiency phenotype with absent T cells and normal B-cell and natural killer cell numbers. A novel homozygous frameshift mutation in the gene encoding for LAT was identified in this kindred. Genetic, molecular, and functional analyses were used to identify and characterize the LAT defect. Clinical and immunologic analysis of patients was also performed and reported. Homozygosity mapping was used to identify potential defective genes. Sanger sequencing of the LAT gene showed a mutation that resulted in a premature stop codon and protein truncation leading to complete loss of function and loss of expression of LAT in the affected family members. We also demonstrate loss of LAT expression and lack of TCR signaling restoration in LAT-deficient cell lines reconstituted with a synthetic LAT gene bearing this severe combined immunodeficiency mutation. For the first time, the results of this study show that inherited LAT deficiency should be considered in patients with combined immunodeficiency with T-cell abnormalities. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  7. 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

    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...

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

    Directory of Open Access Journals (Sweden)

    Catherine E Smith

    2013-10-01

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

  9. Activating mutations in c-KIT and PDGFRalpha are exclusively found in gastrointestinal stromal tumors and not in other tumors overexpressing these imatinib mesylate target genes.

    Science.gov (United States)

    Burger, Herman; den Bakker, Michael A; Kros, Johan M; van Tol, Hans; de Bruin, Alex M; Oosterhuis, Wolter; van den Ingh, Harry F G M; van der Harst, Erwin; de Schipper, Hans P; Wiemer, Erik A C; Nooter, Kees

    2005-11-01

    Previous studies have shown that Imatinib mesylate (Gleevec), a selective tyrosine kinase inhibitor of c-KIT and platelet-derived growth factor receptors (PDGFR), is highly effective in c-KIT/CD117-positive gastrointestinal stromal tumors (GIST), especially in those having activating mutations in c-kit exon 11. In addition, gain-of-function mutations in the juxtamembrane domain (exon 12) and the kinase activation loop (exon 18) of PDGFRalpha were found in GISTs. Importantly, the presence and type of these mutually exclusive c-KIT or PDGFRalpha mutations were found to be associated with the response to imatinib. Here, we examined the prevalence of c-kit exon 11 and PDGFRalpha exons 12 and 18 mutations in other tumor types known to express these tyrosine kinase receptors in order to explore which other cancer types may potentially benefit from imatinib treatment. We determined the mutational status of these commonly mutated exons by direct sequencing in 11 different tumor types (in total: 215 unrelated cases), including GIST, chordoma, and various distinct tumors of lung, brain and its coverings, and skin cancer. Of the 579 exons examined (211 c-kit exon 11, 192 PDGFRalpha exon 12, 142 PDGFRalpha exon18, 17 PDGFRbeta exon 12 and 17 PDGFRbeta exon 18), only 12 (all GIST) harbored mutations (10 c-kit exon 11 and 2 PDGFRalpha exon18). From these data we conclude that activating c-KIT and PDGFR mutations are sporadic in human cancers known to overexpress these tyrosine kinase receptor genes and suggest that, except in GIST, this overexpression is not correlated with activating mutations. The latter may imply that these wild-type c-KIT and PDGFR tumor types will probably not benefit from imatinib treatment.

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

    Directory of Open Access Journals (Sweden)

    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

  11. Genetic heterogeneity of activating mutations of the luteinizing hormone receptor gene in familial male-limited precocious puberty

    Energy Technology Data Exchange (ETDEWEB)

    Laue, L.; Chan, W.Y.; Wu, S.M. [Georgetown Univ. Medical Center, Washington, DC (United States)] [and others

    1994-09-01

    Familial male-limited precocious puberty (FMPP) is an autosomal dominant disorder characterized by elevated serum levels of testosterone, low levels of gonadotropins, and Leydig cell hyperplasia. Recently, 3 mutations have been found in FMPP families which encode substitution of Gly for Asp 578, Ile for Met 571, and Ile for Thr 577 in transmembrane helix 6 (TM 6) of the luteinizing hormone receptor (LHR). We have studied 28 additional unrelated FMPP families. Genomic DNA was isolated from affected males and PCR was performed to amplify a fragment of the LHR gene encoding amino acid residues 441 to 594. MspI restriction enzyme digests were positive for the Asp 578 to Gly mutation in 22 families. Four new mutations were found in the remaining 6 families: an A to C transition encoding substitution of Leu for Ile 542 in transmembrane helix 5 (TM 5), an A to G transition encoding substitution of Gly for Asp 564 in the third cytoplasmic loop, a G to T transition encoding substitution of Try for Asp 578 in TM 6, and a T to C transition encoding substitution of Arg for Cys 581 in TM 6 of the LHR. 293 cells transfected with cDNAs for each of the 4 mutant LHRs, created by site-directed mutagenesis of the wild-type LHR cDNA, exhibited markedly increased levels of basal cAMP production in the absence of agonist, indicating constitutive activation of the mutant LHRs. We conclude that substitution of residues at multiple sites with TM 5, TM 6, and the intervening third cytoplasmic loop of the LHR cause constitutive receptor activation resulting in FMPP. These findings allow future diagnosis of affected patients and provide the basis to study the receptor domains involved in G-protein activation.

  12. Tumor-biopsy stratification based on mTOR-pathway activity and functional mutations in the upstream genes PIK3CA and PTEN.

    Science.gov (United States)

    Laes, Jean-François; Sauvage, Sebastien; Ghitti, Gregori

    2017-10-13

    The mechanistic target of the rapamycin (mTOR) pathway is frequently activated in human cancers. Our objective was to evaluate relationships between mTOR-pathway activity and functional mutations in the upstream genes PIK3CA and PTEN in solid-tumor biopsies from a broad selection of cancer types. Formalin-fixed paraffin-embedded (FFPE) tumor samples were analyzed by immunohistochemistry (IHC) and next-generation sequencing (NGS). TOR-pathway activation was identified by expression (by IHC) of the downstream effector p-4E-BP1. Activating PIK3CA mutations and null PTEN mutations were identified by NGS, and for PTEN , confirmed by IHC. Overall, mTOR-pathway activation was identified in 444/538 (83%) samples representing 40 different cancer types. Functional mutations in either or both PIK3CA and PTEN genes were identified in 173/538 (32%) samples. PIK3CA mutations were identified in 60/538 (11%) samples, PTEN mutations were identified in 155/538 (29%) samples and mutations in both PIK3CA and PTEN were identified in 18/538 (3%) samples. Overall, mTOR-pathway activation was not significantly associated with the PIK3CA and PTEN genotypes. However, all 18 samples with both PIK3CA and PTEN mutations also displayed mTOR-pathway activation (χ 2 p =0.0471). Also, out of a total of 95 breast cancer samples, there were 5 breast-cancer samples which did not have mTOR-pathway activation, and all 5 (100%) of these had PIK3CA and PTEN mutations compared to 51/90 (57%) in the breast-cancer samples with mTOR-pathway activation ( χ 2 p =0.0134). Finally, the percentages of PIK3CA mutations were higher in colorectal-cancer samples which had mTOR-pathway activation (9/27, 33%) than in colorectal-cancer samples without mTOR-pathway activation (6/44; 14%; χ 2 p =0.0484). Therefore, tumor-biopsy analyses based on combined mTOR-pathway biomarkers (and combined NGS and IHC assessments) could potentially provide treatment-informative stratification for particular cancer types.

  13. Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro.

    OpenAIRE

    Fredman, J N; Pettit, S C; Horwitz, M S; Engler, J A

    1991-01-01

    Eighteen linker insertion mutants with mutations in the adenovirus precursor to terminal protein (pTP), which were originally constructed and tested in virions by Freimuth and Ginsberg (Proc. Natl. Acad. Sci. USA 83:7816-7820, 1986), were transferred to expression plasmids for assay of the various functions of the isolated pTP. Function was measured by the ability of individual pTP mutant proteins to participate in the initiation of replication from an adenovirus DNA end, by their activity in...

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

    International Nuclear Information System (INIS)

    Bandyopadhyay, R.; Sengupta, A.; Das, J.

    1989-01-01

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

  15. Oncogenic Effects of High MAPK Activity in Colorectal Cancer Mark Progenitor Cells and Persist Irrespective of RAS Mutations.

    Science.gov (United States)

    Blaj, Cristina; Schmidt, Eva Marina; Lamprecht, Sebastian; Hermeking, Heiko; Jung, Andreas; Kirchner, Thomas; Horst, David

    2017-04-01

    About 40% of colorectal cancers have mutations in KRAS accompanied by downstream activation of MAPK signaling, which promotes tumor invasion and progression. Here, we report that MAPK signaling shows strong intratumoral heterogeneity and unexpectedly remains regulated in colorectal cancer irrespective of KRAS mutation status. Using primary colorectal cancer tissues, xenograft models, and MAPK reporter constructs, we showed that tumor cells with high MAPK activity resided specifically at the leading tumor edge, ceased to proliferate, underwent epithelial-mesenchymal transition (EMT), and expressed markers related to colon cancer stem cells. In KRAS-mutant colon cancer, regulation of MAPK signaling was preserved through remaining wild-type RAS isoforms. Moreover, using a lineage tracing strategy, we provide evidence that high MAPK activity marked a progenitor cell compartment of growth-fueling colon cancer cells in vivo Our results imply that differential MAPK signaling balances EMT, cancer stem cell potential, and tumor growth in colorectal cancer. Cancer Res; 77(7); 1763-74. ©2017 AACR . ©2017 American Association for Cancer Research.

  16. 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.

  17. 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.

  18. The Presenilin-1 ΔE9 Mutation Results in Reduced γ-Secretase Activity, but Not Total Loss of PS1 Function, in Isogenic Human Stem Cells

    Directory of Open Access Journals (Sweden)

    Grace Woodruff

    2013-11-01

    Full Text Available Presenilin 1 (PS1 is the catalytic core of γ-secretase, which cleaves type 1 transmembrane proteins, including the amyloid precursor protein (APP. PS1 also has γ-secretase-independent functions, and dominant PS1 missense mutations are the most common cause of familial Alzheimer’s disease (FAD. Whether PS1 FAD mutations are gain- or loss-of-function remains controversial, primarily because most studies have relied on overexpression in mouse and/or nonneuronal systems. We used isogenic euploid human induced pluripotent stem cell lines to generate and study an allelic series of PS1 mutations, including heterozygous null mutations and homozygous and heterozygous FAD PS1 mutations. Rigorous analysis of this allelic series in differentiated, purified neurons allowed us to resolve this controversy and to conclude that FAD PS1 mutations, expressed at normal levels in the appropriate cell type, impair γ-secretase activity but do not disrupt γ-secretase-independent functions of PS1. Thus, FAD PS1 mutations do not act as simple loss of PS1 function but instead dominantly gain an activity toxic to some, but not all, PS1 functions.

  19. Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor.

    Science.gov (United States)

    Haas, Ann-Karin; Kleinau, Gunnar; Hoyer, Inna; Neumann, Susanne; Furkert, Jens; Rutz, Claudia; Schülein, Ralf; Gershengorn, Marvin C; Krause, Gerd

    2011-01-01

    The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.

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

    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.

  1. Troponin C Mutations Partially Stabilize the Active State of Regulated Actin and Fully Stabilize the Active State When Paired with Δ14 TnT.

    Science.gov (United States)

    Baxley, Tamatha; Johnson, Dylan; Pinto, Jose R; Chalovich, Joseph M

    2017-06-13

    Striated muscle contraction is regulated by the actin-associated proteins tropomyosin and troponin. The extent of activation of myosin ATPase activity is lowest in the absence of both Ca 2+ and activating cross-bridges (i.e., S1-ADP or rigor S1). Binding of activating species of myosin to actin at a saturating Ca 2+ concentration stabilizes the most active state (M state) of the actin-tropomyosin-troponin complex (regulated actin). Ca 2+ binding alone produces partial stabilization of the active state. The extent of stabilization at a saturating Ca 2+ concentration depends on the isoform of the troponin subunits, the phosphorylation state of troponin, and, in the case of cardiac muscle, the presence of hypertrophic cardiomyopathy-producing mutants of troponin T and troponin I. Cardiac dysfunction is also associated with mutations of troponin C (TnC). Troponin C mutants A8V, C84Y, and D145E increase the Ca 2+ sensitivity of ATPase activity. We show that these mutants change the distribution of regulated actin states. The A8V and C84Y TnC mutants decreased the inactive B state distribution slightly at low Ca 2+ concentrations, but the D145E mutants had no effect on that state. All TnC mutants increased the level of the active M state compared to that of the wild type, at a saturating Ca 2+ concentration. Troponin complexes that contained two mutations that stabilize the active M state, A8V TnC and Δ14 TnT, appeared to be completely in the active state in the presence of only Ca 2+ . Because Ca 2+ gives full activation, in this situation, troponin must be capable of positioning tropomyosin in the active M state without the need for rigor myosin binding.

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

    Science.gov (United States)

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

    2016-06-01

    Chronic activation of AMP-activated protein kinase (AMPK) increases glycogen content in skeletal muscle. Previously, we demonstrated that a mutation in the ryanodine receptor (RyR1(R615C)) blunts AMPK phosphorylation in longissimus muscle of pigs with a gain of function mutation in the AMPKγ3 subunit (AMPKγ3(R200Q)); this may decrease the glycogen storage capacity of AMPKγ3(R200Q) + RyR1(R615C) muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPKγ3(R200Q) and AMPK activation contribute to glycogen storage and metabolism in muscle. We selected and bred pigs in order to generate offspring with naturally occurring AMPKγ3(R200Q), RyR1(R615C), and AMPKγ3(R200Q) + RyR1(R615C) mutations, and also retained wild-type littermates (control). We assessed glycogen content and parameters of glycogen metabolism in longissimus muscle. Regardless of RyR1(R615C), AMPKγ3(R200Q) increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6-phosphate (G6P) was decreased in AMPKγ3(R200Q) relative to all other genotypes, whereas both AMPKγ3(R200Q) and AMPKγ3(R200Q) + RyR1(R615C) muscle exhibited increased GS activity with G6P. Increased activity of GS with G6P was not associated with increased abundance of GS or hexokinase 2. However, AMPKγ3(R200Q) enhanced UDP-glucose pyrophosphorylase 2 (UGP2) expression approximately threefold. Although UGP2 is not generally considered a rate-limiting enzyme for glycogen synthesis, our model suggests that UGP2 plays an important role in increasing flux to glycogen synthase. Moreover, we have shown that the capacity for glycogen storage is more closely related to the AMPKγ3(R200Q) mutation than activity. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  3. 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)

    Dhar, Alok; Polev, Dmitrii E.; Masharsky, Alexey E.; Rogozin, Igor B.; Pavlov, Youri I.

    2015-01-01

    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. PMID:25941824

  4. CYP2U1 activity is altered by missense mutations in hereditary spastic paraplegia 56.

    Science.gov (United States)

    Durand, Christelle M; Dhers, Laura; Tesson, Christelle; Tessa, Alessandra; Fouillen, Laetitia; Jacqueré, Stéphanie; Raymond, Laure; Coupry, Isabelle; Benard, Giovanni; Darios, Frédéric; El-Hachimi, Khalid H; Astrea, Guja; Rivier, François; Banneau, Guillaume; Pujol, Claire; Lacombe, Didier; Durr, Alexandra; Babin, Patrick J; Santorelli, Filippo M; Pietrancosta, Nicolas; Boucher, Jean-Luc; Mansuy, Daniel; Stevanin, Giovanni; Goizet, Cyril

    2018-01-01

    Hereditary spastic paraplegia (HSP) is an inherited disorder of the central nervous system mainly characterized by gradual spasticity and weakness of the lower limbs. SPG56 is a rare autosomal recessive early onset complicated form of HSP caused by mutations in CYP2U1. The CYP2U1 enzyme was shown to catalyze the hydroxylation of arachidonic acid. Here, we report two further SPG56 families carrying three novel CYP2U1 missense variants and the development of an in vitro biochemical assay to determine the pathogenicity of missense variants of uncertain clinical significance. We compared spectroscopic, enzymatic, and structural (from a 3D model) characteristics of the over expressed wild-type or mutated CYP2U1 in HEK293T cells. Our findings demonstrated that most of the tested missense variants in CYP2U1 were functionally inactive because of a loss of proper heme binding or destabilization of the protein structure. We also showed that functional data do not necessarily correlate with in silico predictions of variants pathogenicity, using different bioinformatic phenotype prediction tools. Our results therefore highlight the importance to use biological tools, such as the enzymatic test set up in this study, to evaluate the effects of newly identified variants in clinical settings. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2016-08-04

    Cardiospondylocarpofacial (CSCF) syndrome is characterized by growth retardation, dysmorphic facial features, brachydactyly with carpal-tarsal fusion and extensive posterior cervical vertebral synostosis, cardiac septal defects with valve dysplasia, and deafness with inner ear malformations. Whole-exome sequencing identified heterozygous MAP3K7 mutations in six distinct CSCF-affected individuals from four families and ranging in age from 5 to 37 years. MAP3K7 encodes transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), which is involved in the mitogen-activated protein kinase (MAPK)-p38 signaling pathway. MAPK-p38 signaling was markedly altered when expression of non-canonical TGF-β-driven target genes was impaired. These findings support the loss of transcriptional control of the TGF-β-MAPK-p38 pathway in fibroblasts obtained from affected individuals. Surprisingly, although TAK1 is located at the crossroad of inflammation, immunity, and cancer, this study reports MAP3K7 mutations in a developmental disorder affecting mainly cartilage, bone, and heart. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  6. Mutations in the PA Protein of Avian H5N1 Influenza Viruses Affect Polymerase Activity and Mouse Virulence.

    Science.gov (United States)

    Zhong, Gongxun; Le, Mai Quynh; Lopes, Tiago J S; Halfmann, Peter; Hatta, Masato; Fan, Shufang; Neumann, Gabriele; Kawaoka, Yoshihiro

    2018-02-15

    To study the influenza virus determinants of pathogenicity, we characterized two highly pathogenic avian H5N1 influenza viruses isolated in Vietnam in 2012 (A/duck/Vietnam/QT1480/2012 [QT1480]) and 2013 (A/duck/Vietnam/QT1728/2013 [QT1728]) and found that the activity of their polymerase complexes differed significantly, even though both viruses were highly pathogenic in mice. Further studies revealed that the PA-S343A/E347D (PA with the S-to-A change at position 343 and the E-to-D change at position 347) mutations reduced viral polymerase activity and mouse virulence when tested in the genetic background of QT1728 virus. In contrast, the PA-343S/347E mutations increased the polymerase activity of QT1480 and the virulence of a low-pathogenic H5N1 influenza virus. The PA-343S residue (which alone increased viral polymerase activity and mouse virulence significantly relative to viral replication complexes encoding PA-343A) is frequently found in H5N1 influenza viruses of several subclades; infection with a virus possessing this amino acid may pose an increased risk to humans. IMPORTANCE H5N1 influenza viruses cause severe infections in humans with a case fatality rate that exceeds 50%. The factors that determine the high virulence of these viruses in humans are not fully understood. Here, we identified two amino acid changes in the viral polymerase PA protein that affect the activity of the viral polymerase complex and virulence in mice. Infection with viruses possessing these amino acid changes may pose an increased risk to humans. Copyright © 2018 American Society for Microbiology.

  7. Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity.

    Directory of Open Access Journals (Sweden)

    Yang Shu

    Full Text Available Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2 gene with Parkinson's disease (PD. Among the mutations, LRRK2 c.4883G>C (R1628P variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn't alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5. Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+ phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

  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. Bisphosphonates enhance EGFR-TKIs efficacy in advanced NSCLC patients with EGFR activating mutation: A retrospective study.

    Science.gov (United States)

    Huang, Chu-Ying; Wang, Li; Feng, Cheng-Jun; Yu, Ping; Cai, Xiao-Hong; Yao, Wen-Xiu; Xu, Yong; Liu, Xiao-Ke; Zhu, Wen-Jiang; Wang, Yan; Zhou, Jin; Lu, You; Wang, Yong-Sheng

    2016-10-11

    Bisphosphonates have exhibited anti-tumor activity in non-small cell lung cancer (NSCLC). We aimed to evaluate whether the combination of bisphosphonates with tyrosine kinase inhibitors of EGFR (EGFR-TKIs) could obtain a synergistic effect on advanced NSCLC patients with EGFR mutations. Between January 2008 and October 2013, 114 advanced EGFR mutations NSCLC patients who received EGFR-TKIs as first-line therapy were recruited from two cancer centers. Patients were separated into EGFR-TKIs alone or EGFR-TKIs plus bisphosphonates (combination) group. Median progression free survival (mPFS), median overall survival (mOS) distributions and survival curves were analyzed. Among the 114 patients, 62 had bone metastases (19 patients treated with EGFR-TKIs, 43 patients treated with EGFR-TKIs + bisphosphonates). Median PFS and OS were significantly improved in combination group compared with EGFR-TKIs group (mPFS: 15.0 vs 7.3 months, P = 0.0017; mOS: 25.2 vs 10.4 months, P = 0.0015) in patients with bone metastases. Among the 71 patients (19 patients with bone metastases) treated with EGFR-TKIs alone, patients with bone metastases had poor survival prognosis (mPFS:7.3 vs 12.1 months, P = 0.0434; mOS:10.4 vs 22.0 months, P = 0.0036). The survival of patients with bone metastases who received EGFR-TKIs plus bisphosphonates therapy was non-inferior to patients without bone metastases treated with EGFR-TKIs alone (mPFS: 15.0 vs 12.1 months, p = 0.1871; mOS: 25.2 vs 22.0 months, p = 0.9798). Concomitant use of bisphosphonates and EGFR-TKIs improves therapeutic efficacy and brings survival benefits to NSCLC patients with EGFR mutation and bone metastases.

  11. Mutation of katG in a clinical isolate of Mycobacterium tuberculosis: effects on catalase-peroxidase for isoniazid activation.

    Science.gov (United States)

    Purkan; Ihsanawati; Natalia, D; Syah, Y M; Retnoningrum, D S; Kusuma, H S

    2016-01-01

    Mutations in katG gene are often associated with isoniazid (INH) resistance in Mycobacterium tuberculosis strain. This research was perfomed to identify the katG mutation in clinical isolate (L8) that is resistant to INH at 1 μg/ml. In addition to characterize the catalase-peroxidase of KatG L8 and perform the ab initio structural study of the protein to get a more complete understanding in drug activation and the resistan­ce mechanism. The katG gene was cloned and expressed in Escherichia coli, then followed by characterization of catalase-peroxidase of KatG. The structure modelling was performed to know a basis of alterations in enzyme activity. A substitution of A713G that correspond to Asn238Ser replacement was found in the L8 katG. The Asn238Ser modification leads to a decline in the activity of catalase-peroxidase and INH oxidation of the L8 KatG protein. The catalytic efficiency (Kcat/KM) of mutant KatGAsn238Ser respectively decreases to 41 and 52% for catalase and peroxidase. The mutant KatGAsn238Ser also shows a decrease of 62% in INH oxidation if compared to a wild type KatG (KatGwt). The mutant Asn238Ser might cause instability in the substrate binding­ site of KatG, because of removal of a salt bridge connecting the amine group of Asn238 to the carbo­xyl group of Glu233, which presents in KatGwt. The lost of the salt bridge in the substrate binding site in mutant KatGAsn238Ser created changes unfavorable for enzyme activities, which in turn emerge as INH resistan­ce in the L8 isolate of M. tuberculosis.

  12. Mutagenic and antimutagenic activities of Artemisia absinthium volatile oil by the bacterial reverse mutation assay in Salmonella typhimurium strains TA98 and TA100

    Directory of Open Access Journals (Sweden)

    Mahboubeh Taherkhani

    2014-09-01

    Full Text Available Objective: To investigate the mutagenic and antimutagenic activities of Artemisia absinthium L. (A. absinthium essential oil by the bacterial reverse mutation assay in Salmonella typhimurium (S. typhimurium strains. Methods: Water-distilled essential oil of A. absinthium collected from Ardabil, NorthWestern Iran, was investigated for mutagenic and antimutagenic activities. In present study, the mutagenic and antimutagenic activities of A. absinthium oil were investigated by the bacterial revere mutation assay in S. typhimurium TA98 and TA100 strains with and without S9 (microsomal mutagenesis assay. Results: The comparative mutagenicity effect was seen in 1.5 mg/plate by the bacterial reverse mutation assay in S. typhimurium TA98 strains, without S9 and the excellent antimutagenicity effect was seen in 1.5 mg/plate against S. typhimurium TA100, without S9. Conclusions: The mutagenicity and antimutagenicity effects of the volatile oil of A. absinthium were seen without the presence of metabolic activation.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  14. Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.

    Science.gov (United States)

    Yang, W; Punyadarsaniya, D; Lambertz, R L O; Lee, D C C; Liang, C H; Höper, D; Leist, S R; Hernández-Cáceres, A; Stech, J; Beer, M; Wu, C Y; Wong, C H; Schughart, K; Meng, F; Herrler, G

    2017-04-15

    The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs. IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by

  15. A complicated pregnancy in a patient with lipodystrophic diabetes attributable to a peroxisome proliferator-activated receptor gamma (PPARG) mutation.

    Science.gov (United States)

    Madhra, M; Noh, R M; Zammitt, N N; Patrick, A W; Love, C D B

    2012-10-01

    We describe an unplanned pregnancy in a 19-year-old with lipodystrophic diabetes caused by a mutation in the peroxisome proliferator-activated receptor gamma (PPARG) gene. The pregnancy was complicated by poor compliance with treatment, severe hypertriglyceridaemia and pancreatitis. The patient presented at 6 weeks' gestation with an HbA(1c) of 140 mmol/mol (15%), cholesterol 8.1 mmol/l and triglycerides 20.1 mmol/l. She wished to continue the pregnancy so lipid-lowering therapy was discontinued. She was severely insulin resistant and poorly compliant with diet and medication. A continuous subcutaneous insulin infusion was required for efficient delivery of large doses of basal insulin, alongside injected mealtime boluses, (up to 300 units insulin per day). At 17 weeks' gestation she developed acute pancreatitis secondary to hypertriglyceridaemia (triglycerides > 100 mmol/l) and required plasmapheresis. Lipid-lowering therapy was reinstated in the third trimester and plasmapheresis was required repeatedly to maintain triglycerides pancreatitis in pregnancy reviewed. There are few documented cases of pregnancy in women with PPARG mutations. The notable features of this case include the consequences of non-concordance with treatment, the use of continuous subcutaneous insulin infusion to treat insulin-resistant diabetes and the need for repeated plasmapheresis during pregnancy to avert pancreatitis. © 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.

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

    Directory of Open Access Journals (Sweden)

    Ludovica Ciuffreda

    2009-08-01

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

  17. 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

  18. Activating mutations in β-catenin in colon cancer cells alter their interaction with macrophages; the role of snail.

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    Pawan Kaler

    Full Text Available Tumor cells become addicted to both activated oncogenes and to proliferative and pro-survival signals provided by the abnormal tumor microenvironment. Although numerous soluble factors have been identified that shape the crosstalk between tumor cells and stroma, it has not been established how oncogenic mutations in the tumor cells alter their interaction with normal cells in the tumor microenvironment.We showed that the isogenic HCT116 and Hke-3 cells, which differ only by the presence of the mutant kRas allele, both stimulate macrophages to produce IL1β. In turn, macrophages enhanced Wnt signaling, proliferation and survival in both HCT116 and Hke-3 cells, demonstrating that signaling by oncogenic kRas in tumor cells does not impact their interaction with macrophages. HCT116 cells are heterozygous for β-catenin (HCT116(WT/MT, harboring one wild type (WT and one mutant (MT allele, but isogenic lines that carry only the WT (HCT116(WT or MT β-catenin allele (HCT116(MT have been generated. We showed that macrophages promoted Wnt signaling in cells that carry the MT β-catenin allele, but not in HCT116(WT cells. Consistent with this observation, macrophages and IL1β failed to stabilize Snail in HCT116(WT cells, and to protect these cells from TRAIL-induced apoptosis. Finally, we demonstrated that HCT116 cells expressing dominant negative TCF4 (dnTCF4 or HCT116 cells with silenced Snail failed to stimulate IL1β production in macrophages, demonstrating that tumor cells activate macrophages via a Wnt-dependent factor.Our data demonstrate that oncogenic β-catenin mutations in tumor cells, and subsequent activation of Wnt signaling, not only trigger cell-intrinsic alterations, but also have a significant impact on the crosstalk of tumor cells with the tumor associated macrophages.

  19. 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.

  20. Comparative proteomic analysis to dissect differences in signal transduction in activating TSH receptor mutations in the thyroid.

    Science.gov (United States)

    Krause, Kerstin; Boisnard, Alexandra; Ihling, Christian; Ludgate, Marian; Eszlinger, Markus; Krohn, Knut; Sinz, Andrea; Fuhrer, Dagmar

    2012-02-01

    In the thyroid, cAMP controls both thyroid growth and function. Gain-of-function mutations in the thyroid-stimulating hormone receptor (TSHR) lead to constitutive cAMP formation and are a major cause of autonomous thyroid adenomas. The impact of activating TSHR mutations on the signal transduction network of the thyrocyte is not fully understood. To gain more insights into constitutive TSHR signaling, rat thyrocytes (FRTL-5 cells) with stable expression of three activating TSHR mutants (mutTSHR: A623I, L629F and Del613-621), which differ in their functional characteristics in vitro, were analyzed by a quantitative proteomic approach and compared to the wild-type TSHR (WT-TSHR). This study revealed (1) differences in the expression of Rab proteins suggesting an increased TSHR internalization in mutTSHR but not in the WT-TSHR; (2) differential stimulation of PI3K/Akt signaling in mutTSHR vs. WT-TSHR cells, (3) activation of Epac, impairing short-time Akt phosphorylation in both, mutTSHR and WT-TSHR cells. Based on the analysis of global changes in protein expression patterns, our findings underline the complexity of gain-of-function TSHR signaling in thyrocytes, which extends beyond pure cAMP and/or IP formation. Moreover, evidence for augmented endocytosis in the mutTSHR, adds to a new concept of TSHR signaling in thyroid autonomy. Further studies are required to clarify whether the observed differences in Rab, PI3K and Epac signaling may contribute to differences in the phenotypic presentation, i.e. stimulation of function and growth of thyroid autonomy in vivo. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. A Point Mutation in the RNA-Binding Domain of Human Parainfluenza Virus Type 2 Nucleoprotein Elicits Abnormally Enhanced Polymerase Activity.

    Science.gov (United States)

    Matsumoto, Yusuke; Ohta, Keisuke; Kolakofsky, Daniel; Nishio, Machiko

    2017-05-01

    The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as the template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA were identified and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (which contacts a base rather than the RNA backbone) to various amino acids resulted in an over 30-fold increase of polymerase activity as evidenced by a minireplicon assay, even though the RNA-binding affinity was unaltered. Using various modified minireplicons, we found that the enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA. IMPORTANCE We examined the importance of amino acids in the putative RNA-binding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed upon substitution mutation of the NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes that were partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for the infectious life cycle. This report highlights the potential of the polymerase complex with point mutations in NP and helps our detailed understanding of the molecular basis of gene expression. Copyright © 2017 American Society for Microbiology.

  2. Contribution of novel ATGL missense mutations to the clinical phenotype of NLSD-M: a strikingly low amount of lipase activity may preserve cardiac function.

    Science.gov (United States)

    Tavian, Daniela; Missaglia, Sara; Redaelli, Chiara; Pennisi, Elena M; Invernici, Gloria; Wessalowski, Ruediger; Maiwald, Robert; Arca, Marcello; Coleman, Rosalind A

    2012-12-15

    The lack of adipose triglyceride lipase (ATGL), a patatin-like phospholipase domain-containing enzyme that hydrolyzes fatty acids from triacylglycerol (TAG) stored in multiple tissues, causes the autosomal recessive disorder neutral lipid storage disease with myopathy (NLSD-M). In two families of Lebanese and Italian origin presenting with NLSD-M, we identified two new missense mutations in highly conserved regions of ATGL (p.Arg221Pro and p.Asn172Lys) and a novel nonsense mutation (p.Trp8X). The Lebanese patients harbor homozygous p.Arg221Pro, whereas the Italian patients are heterozygotes for p.Asn172Lys and the p.Trp8X mutation. The p.Trp8X mutation results in a complete absence of ATGL protein, while the p.Arg221Pro and p.Asn172Lys mutations result in proteins with minimal lipolytic activity. Although these mutations did not affect putative catalytic residues or the lipid droplet (LD)-binding domain of ATGL, cytosolic LDs accumulated in cultured skin fibroblasts from the patients. The missense mutations might destabilize a random coil (p.Asn172Lys) or a helix (p.Arg221Pro) structure within or proximal to the patatin domain of the lipase, thereby interfering with the enzyme activity, while leaving intact the residues required to localize the protein to LDs. Overexpressing wild-type ATGL in one patient's fibroblasts corrected the metabolic defect and effectively reduced the number and area of cellular LDs. Despite the poor lipase activity in vitro, the Lebanese siblings have a mild myopathy and not clinically evident myocardial dysfunction. The patients of Italian origin show a late-onset and slowly progressive skeletal myopathy. These findings suggest that a small amount of correctly localized lipase activity preserves cardiac function in NLSD-M.

  3. 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.

  4. 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.

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

    International Nuclear Information System (INIS)

    Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V.

    2010-01-01

    Research highlights: → Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). → Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. → We are reporting that mutations in POR may reduce CYP3A4 activity. → POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. → 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.

  6. A T9G mutation in the prototype TATA-box TCACTATATATAG determines nucleosome formation and synergy with upstream activator sequences in plant promoters.

    Science.gov (United States)

    Ranjan, Amol; Ansari, Suraiya A; Srivastava, Rakesh; Mantri, Shrikant; Asif, Mehar H; Sawant, Samir V; Tuli, Rakesh

    2009-12-01

    We had earlier reported that mutations to G and C at the seventh and eighth positions in the prototype TATA-box TCACTATATATAG inhibited light-dependent activation of transcription from the promoter. In this study, we characterized mutations at the ninth position of the prototype TATA-box. Substitution of T at the ninth position with G or C enhanced transcription from the promoter in transgenic tobacco (Nicotiana tabacum) plants. The effect of T9G/C mutations was not light dependent, although the 9G/C TATA-box showed synergy with the light-responsive element (lre). However, the 9G/C mutants in the presence of lre failed to respond to phytochromes, sugar, and calcium signaling, in contrast to the prototype TATA-box with lre. The 9G/C mutation shifted the point of initiation of transcription, and transcription activation was dependent upon the type of activating element present upstream. The synergy in activation was noticed with lre and legumin activators but not with rbcS, Pcec, and PR-1a activators. The 9G mutation resulted in a micrococcal nuclease-sensitive region over the TATA-box, suggesting a nucleosome-free region, in contrast to the prototype promoter, which had a distinct nucleosome on the TATA-box. Thus, the transcriptional augmentation with mutation at the ninth position might be because of the loss of a repressive nucleosomal structure on the TATA-box. In agreement with our findings, the promoters containing TATAGATA as identified by genome-wide analysis of Arabidopsis (Arabidopsis thaliana) are not tightly repressed.

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

    Science.gov (United States)

    Hubler, Tina; Adams, Patti; Scammell, Jonathan

    2015-01-01

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

  8. 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.

  9. A novel de novo mutation of SCN8A (Nav1.6) with enhanced channel activation in a child with epileptic encephalopathy.

    Science.gov (United States)

    Estacion, Mark; O'Brien, Janelle E; Conravey, Allison; Hammer, Michael F; Waxman, Stephen G; Dib-Hajj, Sulayman D; Meisler, Miriam H

    2014-09-01

    Rare de novo mutations of sodium channels are thought to be an important cause of sporadic epilepsy. The well established role of de novo mutations of sodium channel SCN1A in Dravet Syndrome supports this view, but the etiology of many cases of epileptic encephalopathy remains unknown. We sought to identify the genetic cause in a patient with early onset epileptic encephalopathy by whole exome sequencing of genomic DNA. The heterozygous mutation c. 2003C>T in SCN8A, the gene encoding sodium channel Nav1.6, was detected in the patient but was not present in either parent. The resulting missense substitution, p.Thr767Ile, alters an evolutionarily conserved residue in the first transmembrane segment of channel domain II. The electrophysiological effects of this mutation were assessed in neuronal cells transfected with mutant or wildtype cDNA. The mutation causes enhanced channel activation, with a 10mV depolarizing shift in voltage dependence of activation as well as increased ramp current. In addition, pyramidal hippocampal neurons expressing the mutant channel exhibit increased spontaneous firing with PDS-like complexes as well as increased frequency of evoked action potentials. The identification of this new gain-of-function mutation of Nav1.6 supports the inclusion of SCN8A as a causative gene in infantile epilepsy, demonstrates a novel mechanism for hyperactivity of Nav1.6, and further expands the role of de novo mutations in severe epilepsy. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  10. The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in Escherichia coli.

    Science.gov (United States)

    Hosaka, T; Tamehiro, N; Chumpolkulwong, N; Hori-Takemoto, C; Shirouzu, M; Yokoyama, S; Ochi, K

    2004-04-01

    Resistance to streptomycin in bacterial cells often results from a mutation in the rpsL gene that encodes the ribosomal protein S12. We found that a particular rpsL mutation (K87E), newly identified in Escherichia coli, causes aberrant protein synthesis activity late in the growth phase. While protein synthesis decreased with age in cells in the wild-type strain, it was sustained at a high level in the mutant, as determined using living cells. This was confirmed using an in vitro protein synthesis system with poly(U) and natural mRNAs (GFP mRNA and CAT mRNA). Other classical rpsL mutations (K42N and K42T) tested did not show such an effect, indicating that this novel characteristic is typical of ribosomes bearing the K87E mutant form of S12, although the K87E mutation conferred the streptomycin resistance and error-restrictive phenotypes also seen with the K42N and K42T mutations. The K87E (but not K42N or K42T) mutant ribosomes exhibited increased stability of the 70S complex in the presence of low concentrations of magnesium. We propose that the aberrant activation of protein synthesis at the late growth phase is caused by the increased stability of the ribosome.

  11. Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity

    Science.gov (United States)

    Roberts, Gareth A; Chen, Kai; Bower, Edward K M; Madrzak, Julia; Woods, Arcadia; Barker, Amy M; Cooper, Laurie P; White, John H; Blakely, Garry W; Manfield, Iain; Dryden, David T F

    2013-01-01

    ArdA antirestriction proteins are encoded by genes present in many conjugative plasmids and transposons within bacterial genomes. Antirestriction is the ability to prevent cleavage of foreign incoming DNA by restriction-modification (RM) systems. Antimodification, the ability to inhibit modification by the RM system, can also be observed with some antirestriction proteins. As these mobile genetic elements can transfer antibiotic resistance genes, the ArdA proteins assist their spread. The consequence of antirestriction is therefore the enhanced dissemination of mobile genetic elements. ArdA proteins cause antirestriction by mimicking the DNA structure bound by Type I RM enzymes. The crystal structure of ArdA showed it to be a dimeric protein with a highly elongated curved cylindrical shape [McMahon SA et al. (2009) Nucleic Acids Res37, 4887–4897]. Each monomer has three domains covered with negatively charged side chains and a very small interface with the other monomer. We investigated the role of the domain forming the dimer interface for ArdA activity via site-directed mutagenesis. The antirestriction activity of ArdA was maintained when up to seven mutations per monomer were made or the interface was disrupted such that the protein could only exist as a monomer. The antimodification activity of ArdA was lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests, first, that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme, and second, that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction. Structured digital abstract ArdA and ArdA bind by molecular sieving (1, 2) ArdA and ArdA bind by cosedimentation in solution (1, 2) PMID:23910724

  12. Mutations in FLS2 Ser-938 dissect signaling activation in FLS2-mediated Arabidopsis immunity.

    Directory of Open Access Journals (Sweden)

    Yangrong Cao

    Full Text Available Flagellin-sensing 2 (FLS2 is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2(S938A, while the acidic phosphomimic mutants FLS2(S938D and FLS2(S938E conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2(S938A, demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2(S938A and FLS2(S938D mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2(S938A or FLS2(D997A (a kinase catalytic site mutant, but was normally induced in FLS2(S938D plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.

  13. 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.

  14. A novel mutation in the lysyl hydroxylase 1 gene causes decreased lysyl hydroxylase activity in an ehlers-danlos VIA patient

    NARCIS (Netherlands)

    Walker, L.C.; Overstreet, M.A.; Siddiqui, A.; Paepe, A. de; Ceylaner, G.; Malfait, F.; Symoens, S.; Atsawasuwan, P.; Yamauchi, M.; Ceylaner, S.; Bank, R.A.; Yeowell, H.N.

    2005-01-01

    The clinical diagnosis of a patient with the phenotype of Ehlers-Danlos syndrome type VI was confirmed biochemically by the severely diminished level of lysyl hydroxylase (LH) activity in the patient's skin fibroblasts. A novel homozygous mutation, a single base change of T1360 → G in exon 13 of the

  15. Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas.

    Science.gov (United States)

    Bal, Elodie; Park, Hyun-Sook; Belaid-Choucair, Zakia; Kayserili, Hülya; Naville, Magali; Madrange, Marine; Chiticariu, Elena; Hadj-Rabia, Smail; Cagnard, Nicolas; Kuonen, Francois; Bachmann, Daniel; Huber, Marcel; Le Gall, Cindy; Côté, Francine; Hanein, Sylvain; Rosti, Rasim Özgür; Aslanger, Ayca Dilruba; Waisfisz, Quinten; Bodemer, Christine; Hermine, Olivier; Morice-Picard, Fanny; Labeille, Bruno; Caux, Frédéric; Mazereeuw-Hautier, Juliette; Philip, Nicole; Levy, Nicolas; Taieb, Alain; Avril, Marie-Françoise; Headon, Denis J; Gyapay, Gabor; Magnaldo, Thierry; Fraitag, Sylvie; Crollius, Hugues Roest; Vabres, Pierre; Hohl, Daniel; Munnich, Arnold; Smahi, Asma

    2017-10-01

    Basal cell carcinoma (BCC), the most common human cancer, results from aberrant activation of the Hedgehog signaling pathway. Although most cases of BCC are sporadic, some forms are inherited, such as Bazex-Dupré-Christol syndrome (BDCS)-a cancer-prone genodermatosis with an X-linked, dominant inheritance pattern. We have identified mutations in the ACTRT1 gene, which encodes actin-related protein T1 (ARP-T1), in two of the six families with BDCS that were examined in this study. High-throughput sequencing in the four remaining families identified germline mutations in noncoding sequences surrounding ACTRT1. These mutations were located in transcribed sequences encoding enhancer RNAs (eRNAs) and were shown to impair enhancer activity and ACTRT1 expression. ARP-T1 was found to directly bind to the GLI1 promoter, thus inhibiting GLI1 expression, and loss of ARP-T1 led to activation of the Hedgehog pathway in individuals with BDCS. Moreover, exogenous expression of ACTRT1 reduced the in vitro and in vivo proliferation rates of cell lines with aberrant activation of the Hedgehog signaling pathway. In summary, our study identifies a disease mechanism in BCC involving mutations in regulatory noncoding elements and uncovers the tumor-suppressor properties of ACTRT1.

  16. 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

    +). 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......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...

  17. BRAF mutations and phosphorylation status of mitogen-activated protein kinases in the development of flat and depressed-type colorectal neoplasias

    Science.gov (United States)

    Konishi, K; Takimoto, M; Kaneko, K; Makino, R; Hirayama, Y; Nozawa, H; Kurahashi, T; Kumekawa, Y; Yamamoto, T; Ito, H; Yoshikawa, N; Kusano, M; Nakayama, K; Rembacken, B J; Ota, H; Imawari, M

    2006-01-01

    Although some molecular differences between flat-depressed neoplasias (FDNs) and protruding neoplasias (PNs) have been reported, it is uncertain if the BRAF mutations or the status of phosphorylated mitogen-activated protein kinase (p-MAPK) are different between theses two groups. We evaluated the incidence of BRAF and KRAS mutations, high-frequency microsatellite instability (MSI-H), and the immunohistochemical status of p-MAPK in the nonserrated neoplasias (46 FDNs and 57 PNs). BRAF mutations were detected in four FDNs (9%) and none of PNs (P=0.0369 by Fisher's exact test). KRAS mutations were observed in none of FDNs and in 14 PNs (25%; P=0.0002 by Fisher's exact test). MSI-H was detected in seven out of 44 FDNs (16%) and in one out of 52 of PNs (2%) (P=0.022 by Fisher's exact test). Type B and C immunostaining for p-MAPK was observed in 34 out of 46 FDNs (72%), compared with 24 out of 55 PNs (44%; P=0.0022 by χ2 test). There was no significant difference in the type B and C immunostaining of p-MAPK between FDNs with and without BRAF mutations. BRAF and KRAS mutations are mutually exclusive in the morphological characteristics of colorectal nonserrated neoplasia. Abnormal accumulation of p-MAPK protein is more likely to be implicated in the tumorigenesis of FDNs than of PNs. However, this abnormality in FDNs might occur via the genetic alteration other than BRAF or KRAS mutation. PMID:16404419

  18. 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.

  19. A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase.

    Science.gov (United States)

    Zhou, Zheng; Li, Min; Xu, Jian-He; Zhang, Zhi-Jun

    2018-03-02

    Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g prot -1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ 0 ) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ 1 ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    NARCIS (Netherlands)

    Bleeker, Fonnet E.; Atai, Nadia A.; Lamba, Simona; Jonker, Ard; Rijkeboer, Denise; Bosch, Klazien S.; Tigchelaar, Wikky; Troost, Dirk; Vandertop, W. Peter; Bardelli, Alberto; van Noorden, Cornelis 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,

  1. Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome

    DEFF Research Database (Denmark)

    Thorsen, Kasper; Dam, Vibeke S.; Kjaer-Sorensen, Kasper

    2017-01-01

    unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses...

  2. 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....

  3. 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. Copyright © 2014 Elsevier GmbH. All rights reserved.

  4. Identification of a novel mutation (Ala66Thr) of SRY gene causes XY pure gonadal dysgenesis by affecting DNA binding activity and nuclear import.

    Science.gov (United States)

    Wang, Xiang; Xue, Mei; Zhao, Minggang; He, Fang; Li, Cui; Li, Xu

    2018-04-20

    Sex-determining region of the Y chromosome (SRY) gene plays a crucial role in male sexual differentiation and development. Several mutations in the SRY gene have been reported in the high mobility group (HMG) box domain and can cause gonadal dysgenesis symptoms. In this study, we report that a novel missense mutation in the SRY gene, a G to A transition within the HMG box, causes the Ala66Thr amino acid substitution in a female patient presenting 46,XY karyotype with pure gonadal dysgenesis. The G to A base transition was not found in the SRY sequence after the screening of 100 normal males. Furthermore, Ala66Thr mutation drastically reduced the binding capacity of SRY to DNA sequences, whereas wild-type SRY protein showed the normal binding capacity to DNA sequences in vitro. We also found that the mutant SRY protein was partly localized in cytoplasm, whereas wild-type SRY protein was strictly localized in cell nucleus. In addition, we analyzed the three-dimensional structure of SRY protein by homology modeling methods. In conclusion, we identified a novel SRY mutation in a 46,XY female patient with pure gonadal dysgenesis, demonstrating the importance of the Ala66Thr mutation in DNA binding activity and nuclear transport. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. alpha-Adducin mutations increase Na/K pump activity in renal cells by affecting constitutive endocytosis: implications for tubular Na reabsorption.

    Science.gov (United States)

    Torielli, Lucia; Tivodar, Simona; Montella, Rosa Chiara; Iacone, Roberto; Padoani, Gloria; Tarsini, Paolo; Russo, Ornella; Sarnataro, Daniela; Strazzullo, Pasquale; Ferrari, Patrizia; Bianchi, Giuseppe; Zurzolo, Chiara

    2008-08-01

    Genetic variation in alpha-adducin cytoskeletal protein is implicated in the polymerization and bundling of actin and alteration of the Na/K pump, resulting in abnormal renal sodium transport and hypertension in Milan hypertensive rats and humans. To investigate the molecular involvement of alpha-adducin in controlling Na/K pump activity, wild-type or mutated rat and human alpha-adducin forms were, respectively, transfected into several renal cell lines. Through multiple experimental approaches (microscopy, enzymatic assays, coimmunoprecipitation), we showed that rat and human mutated forms increased Na/K pump activity and the number of pump units; moreover, both variants coimmunoprecipitate with Na/K pump. The increased Na/K pump activity was not due to changes in its basolateral localization, but to an alteration of Na/K pump residential time on the plasma membrane. Indeed, both rat and human mutated variants reduced constitutive Na/K pump endocytosis and similarly affected transferrin receptor trafficking and fluid-phase endocytosis. In fact, alpha-adducin was detected in clathrin-coated vesicles and coimmunoprecipitated with clathrin. These results indicate that adducin, besides its modulatory effects on actin cytoskeleton dynamics, might play a direct role in clathrin-dependent endocytosis. The constitutive reduction of the Na/K pump endocytic rate induced by mutated adducin variants may be relevant in Na-dependent hypertension.

  6. Human Connexin43E42K mutation from a sudden infant death victim leads to impaired ventricular activation and neonatal death in mice.

    Science.gov (United States)

    Lübkemeier, Indra; Bosen, Felicitas; Kim, Jung-Sun; Sasse, Philipp; Malan, Daniela; Fleischmann, Bernd K; Willecke, Klaus

    2015-02-01

    Sudden infant death syndrome (SIDS) describes the sudden, unexplained death of a baby during its first year of age and is the third leading cause of infant mortality. It is assumed that ≤20% of all SIDS cases are because of cardiac arrhythmias resulting from mutations in ion channel proteins. Besides ion channels also cardiac gap junction channels are important for proper conduction of cardiac electric activation. In the mammalian heart Connexin43 (Cx43) is the major gap junction protein expressed in ventricular cardiomyocytes. Recently, a novel Connexin43 loss-of-function mutation (Cx43E42K) was identified in a 2-month-old SIDS victim. We have generated Cx43E42K-expressing mice as a model for SIDS. Heterozygous cardiac-restricted Cx43E42K-mutated mice die neonatally without major cardiac morphological defects. Electrocardiographic recordings of embryonic Cx43+/E42K mice reveal severely disturbed ventricular activation, whereas immunohistochemical analyses show normal localization and expression patterns of gap junctional Connexin43 protein in the Cx43E42K-mutated newborn mouse heart. Because we did not find heterogeneous gap junction loss in Cx43E42K mouse hearts, we conclude that the Cx43E42K gap junction channel creates an arrhythmogenic substrate leading to lethal ventricular arrhythmias. The strong cardiac phenotype of Cx43E42K expressing mice supports the association between the human Cx43E42K mutation and SIDS and indicates that Connexin43 mutations should be considered in future studies when SIDS cases are to be molecularly explained. © 2014 American Heart Association, Inc.

  7. 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

  8. The Presence of Telomere Fusion in Sporadic Colon Cancer Independently of Disease Stage, TP53/KRAS Mutation Status, Mean Telomere Length, and Telomerase Activity

    Directory of Open Access Journals (Sweden)

    Hiromi Tanaka

    2014-10-01

    Full Text Available Defects in telomere maintenance can result in telomere fusions that likely play a causative role in carcinogenesis by promoting genomic instability. However, this proposition remains to be fully understood in human colon carcinogenesis. In the present study, the temporal sequence of telomere dysfunction dynamics was delineated by analyzing telomere fusion, telomere length, telomerase activity, hotspot mutations in KRAS or BRAF, and TP53 of tissue samples obtained from 18 colon cancer patients. Our results revealed that both the deficiency of p53 and the shortening of mean telomere length were not necessary for producing telomere fusions in colon tissue. In five cases, telomere fusion was observed even in tissue adjacent to cancerous lesions, suggesting that genomic instability is initiated in pathologically non-cancerous lesions. The extent of mean telomere attrition increased with lymph node invasiveness of tumors, implying that mean telomere shortening correlates with colon cancer progression. Telomerase activity was relatively higher in most cancer tissues containing mutation(s in KRAS or BRAF and/or TP53 compared to those without these hotspot mutations, suggesting that telomerase could become fully active at the late stage of colon cancer development. Interestingly, the majority of telomere fusion junctions in colon cancer appeared to be a chromatid-type containing chromosome 7q or 12q. In sum, this meticulous correlative study not only highlights the concept that telomere fusion is present in the early stages of cancer regardless of TP53/KRAS mutation status, mean telomere length, and telomerase activity, but also provides additional insights targeting key telomere fusion junctions which may have significant implications for colon cancer diagnoses.

  9. Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases.

    Science.gov (United States)

    Farhat, M; Poissonnier, A; Hamze, A; Ouk-Martin, C; Brion, J-D; Alami, M; Feuillard, J; Jayat-Vignoles, C

    2014-05-01

    Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated.

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

    International Nuclear Information System (INIS)

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

    2003-01-01

    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→T:A transversion and G:C→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→T:A transversion and G:C→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

  11. 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

  12. Identification of a PTEN mutation with reduced protein stability, phosphatase activity, and nuclear localization in Hong Kong patients with autistic features, neurodevelopmental delays, and macrocephaly.

    Science.gov (United States)

    Wong, Chi Wai; Or, Penelope Mei Yu; Wang, Yubing; Li, Lisha; Li, Jing; Yan, Mingfei; Cao, Ye; Luk, Ho Ming; Tong, Tony Ming For; Leslie, Nick R; Lo, Ivan Fai-Man; Choy, Kwong Wai; Chan, Andrew Man Lok

    2018-04-02

    PTEN is a tumor suppressor gene inactivated in over 30% of human cancers. It encodes a lipid phosphatase that serves as a gatekeeper of the phosphoinositide 3-kinase signaling pathway. Germline mutation frequently occurs in this gene in patients diagnosed with PTEN Hamartoma Tumor Syndrome (PHTS). PHTS individuals are characterized by macrocephaly, benign growth of multiple tissues and increased tumor risk. In addition, autistic phenotypes are found in 10-20% of individuals carrying the germline PTEN mutation with macrocephaly. In this report, 13 suspected PHTS patients were screened for mutation in the PTEN gene. A missense variant (c. 302T > C) substituting the isoleucine at codon 101 to a threonine, a single nucleotide insertion (c. 327-328insC) causing a frame shift mutation and termination at codon 109, and a nonsense variant (c. 1003C > T) truncated the protein at codon 335 were identified. The I101T mutation significantly reduced PTEN protein expression levels by 2.5- to 4.0-fold. Mechanistically, I101T reduced the protein half-life of PTEN possibly due to enhanced polyubiquitination at Lysine 13. However, the I101T mutant retained almost 30% of the lipid phosphatase activity of the wild-type protein. Finally, the I101T mutant has reduced phosphorylation at a PTEN auto-dephosphorylation site at Threonine 366 and a lowered ratio of nuclear to cytosolic protein level. These partial losses of multiple PTEN biochemical functions may contribute to the tissue overgrowth and autistic features of this PHTS patient. Autism Res 2018. © 2018 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc. The genetics of autism spectrum disorders is highly complex with individual risk influenced by both genetic and environmental factors. Mutation in the human PTEN gene confers a high risk of developing autistic behavior. This report revealed that PTEN mutations occurred in 23% of a selected group of Hong Kong

  13. 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

    Colorectal cancers (CRC) often show activating mutations of the KRAS or BRAF genes, which stimulate the extracellular signal-regulated kinase (ERK) pathway, thus increasing cell proliferation and inhibiting apoptosis. However, immunohistochemical results on ERK activation in such tumors differ gr...

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

    Science.gov (United States)

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

    2012-12-01

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

  15. Human xanthine oxidase changes its substrate specificity to aldehyde oxidase type upon mutation of amino acid residues in the active site: roles of active site residues in binding and activation of purine substrate.

    Science.gov (United States)

    Yamaguchi, Yuichiro; Matsumura, Tomohiro; Ichida, Kimiyoshi; Okamoto, Ken; Nishino, Takeshi

    2007-04-01

    Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion of the substrate preference to AO type. Human XOR and its Glu803-to-valine (E803V) and Arg881-to-methionine (R881M) mutants were expressed in an Escherichia coli system. The E803V mutation almost completely abrogated the activity towards hypoxanthine as a substrate, but very weak activity towards xanthine remained. On the other hand, the R881M mutant lacked activity towards xanthine, but retained slight activity towards hypoxanthine. Both mutants, however, exhibited significant aldehyde oxidase activity. The crystal structure of E803V mutant of human XOR was determined at 2.6 A resolution. The overall molybdopterin domain structure of this mutant closely resembles that of bovine milk XOR; amino acid residues in the active centre pocket are situated at very similar positions and in similar orientations, except that Glu803 was replaced by valine, indicating that the decrease in activity towards purine substrate is not due to large conformational change in the mutant enzyme. Unlike wild-type XOR, the mutants were not subject to time-dependent inhibition by allopurinol.

  16. 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.

  17. Mutational analysis of metacaspase CaMca1 and decapping activator Edc3 in the pathogenicity of Candida albicans.

    Science.gov (United States)

    Jeong, Jeong-Hoon; Lee, Seok-Eui; Kim, Jinmi

    2016-12-01

    Candida albicans, an opportunistic fungal pathogen, displays apoptotic cell death in response to various stresses and a wide range of antifungal treatments. CaMca1, which is the only metacaspase in C. albicans, has been described as a key player in apoptotic cell death. Edc3 is an mRNA decapping activator and a scaffold protein of processing bodies. Edc3 was previously shown to regulate CaMCA1 expression and oxidative stress-induced apoptosis. In this study, we analyzed the contribution of the catalytic residues of the CaMca1 to the oxidative stress-induced apoptosis and pathogenicity of C. albicans. The CaMCA1 C292A mutation decreased caspase activity to a level similar to that observed in the Camca1/Camca1 deletion strain and over-expression of CaMCA1 C292A failed to suppress the oxidative-stress phenotypes of the edc3/edc3 mutant strain. The edc3/edc3, Camca1/Camca1, and CaMCA1 C292A mutant strains were not virulent in a murine candidiasis model. Filamentation defects were observed in the Camca1/Camca1 mutant cells, whereas this defect was only partial in CaMCA1 C292A mutant cells. These results suggest that CaMca1 and Edc3 play essential roles in the oxidative stress-induced apoptosis and virulence of C. albicans, and also support the notion that Edc3 is a key regulator of CaMca1 expression. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Influence of the RDL A301S mutation in the brown planthopper Nilaparvata lugens on the activity of phenylpyrazole insecticides.

    Science.gov (United States)

    Garrood, William T; Zimmer, Christoph T; Gutbrod, Oliver; Lüke, Bettina; Williamson, Martin S; Bass, Chris; Nauen, Ralf; Emyr Davies, T G

    2017-10-01

    We discovered the A301S mutation in the RDL GABA-gated chloride channel of fiprole resistant rice brown planthopper, Nilaparvata lugens populations by DNA sequencing and SNP calling via RNASeq. Ethiprole selection of two field N. lugens populations resulted in strong resistance to both ethiprole and fipronil and resulted in fixation of the A301S mutation, as well as the emergence of another mutation, Q359E in one of the selected strains. To analyse the roles of these mutations in resistance to phenylpyrazoles, three Rdl constructs: wild type, A301S and A301S+Q359E were expressed in Xenopus laevis oocytes and assessed for their sensitivity to ethiprole and fipronil using two-electrode voltage-clamp electrophysiology. Neither of the mutant Rdl subtypes significantly reduced the antagonistic action of fipronil, however there was a significant reduction in response to ethiprole in the two mutated subtypes compared with the wild type. Bioassays with a Drosophila melanogaster strain carrying the A301S mutation showed strong resistance to ethiprole but not fipronil compared to a strain without this mutation, thus further supporting a causal role for the A301S mutation in resistance to ethiprole. Homology modelling of the N. lugens RDL channel did not suggest implications of Q359E for fiprole binding in contrast to A301S located in transmembrane domain M2 forming the channel pore. Synergist bioassays provided no evidence of a role for cytochrome P450s in N. lugens resistance to fipronil and the molecular basis of resistance to this compound remains unknown. In summary this study provides strong evidence that target-site resistance underlies widespread ethiprole resistance in N. lugens populations. Copyright © 2017 Rothamsted Research Ltd. Published by Elsevier Inc. All rights reserved.

  19. Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome

    DEFF Research Database (Denmark)

    Thorsen, Kasper; Dam, Vibeke S.; Kjaer-Sorensen, Kasper

    2017-01-01

    Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain ... unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses...

  20. H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production ofEscherichia coliStx2 Phage Lysogen.

    Science.gov (United States)

    Fu, Qiang; Li, Shiyu; Wang, Zhaofei; Shan, Wenya; Ma, Jingjiao; Cheng, Yuqiang; Wang, Hengan; Yan, Yaxian; Sun, Jianhe

    2017-01-01

    Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli ( E. coli ) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δ hns mutant (MG1655Δ hns ) of the E. coli K-12 strain MG1655 was obtained. The Δ hns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δ hns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.

  1. WNT Inhibitory Activity of Malus Pumila miller cv Annurca and Malus domestica cv Limoncella Apple Extracts on Human Colon-Rectal Cells Carrying Familial Adenomatous Polyposis Mutations

    Directory of Open Access Journals (Sweden)

    Gennaro Riccio

    2017-11-01

    Full Text Available Inhibitors of the Wingless-related Integration site (WNT/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP. This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples, Malus pumila Miller cv. ‘Annurca’ and Malus domestica cv ‘Limoncella’; (ii identify the mechanisms underpinning their activities and; (iii evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract.

  2. WNT Inhibitory Activity of Malus Pumila miller cv Annurca and Malus domestica cv Limoncella Apple Extracts on Human Colon-Rectal Cells Carrying Familial Adenomatous Polyposis Mutations.

    Science.gov (United States)

    Riccio, Gennaro; Maisto, Maria; Bottone, Sara; Badolati, Nadia; Rossi, Giovanni Battista; Tenore, Gian Carlo; Stornaiuolo, Mariano; Novellino, Ettore

    2017-11-18

    Inhibitors of the Wingless-related Integration site (WNT)/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP). This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i) test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples, Malus pumila Miller cv. 'Annurca' and Malus domestica cv 'Limoncella'; (ii) identify the mechanisms underpinning their activities and; (iii) evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract.

  3. Erlotinib and bevacizumab in patients with advanced non-small-cell lung cancer and activating EGFR mutations (BELIEF): an international, multicentre, single-arm, phase 2 trial.

    Science.gov (United States)

    Rosell, Rafael; Dafni, Urania; Felip, Enriqueta; Curioni-Fontecedro, Alessandra; Gautschi, Oliver; Peters, Solange; Massutí, Bartomeu; Palmero, Ramon; Aix, Santiago Ponce; Carcereny, Enric; Früh, Martin; Pless, Miklos; Popat, Sanjay; Kotsakis, Athanasios; Cuffe, Sinead; Bidoli, Paolo; Favaretto, Adolfo; Froesch, Patrizia; Reguart, Noemí; Puente, Javier; Coate, Linda; Barlesi, Fabrice; Rauch, Daniel; Thomas, Michael; Camps, Carlos; Gómez-Codina, Jose; Majem, Margarita; Porta, Rut; Shah, Riyaz; Hanrahan, Emer; Kammler, Roswitha; Ruepp, Barbara; Rabaglio, Manuela; Kassapian, Marie; Karachaliou, Niki; Tam, Rachel; Shames, David S; Molina-Vila, Miguel A; Stahel, Rolf A

    2017-05-01

    The tyrosine kinase inhibitor erlotinib improves the outcomes of patients with advanced non-small-cell lung carcinoma (NSCLC) harbouring epidermal growth factor receptor (EGFR) mutations. The coexistence of the T790M resistance mutation with another EGFR mutation in treatment-naive patients has been associated with a shorter progression-free survival to EGFR inhibition than in the absence of the T790M mutation. To test this hypothesis clinically, we developed a proof-of-concept study, in which patients with EGFR-mutant NSCLC were treated with the combination of erlotinib and bevacizumab, stratified by the presence of the pretreatment T790M mutation. BELIEF was an international, multicentre, single-arm, phase 2 trial done at 29 centres in eight European countries. Eligible patients were aged 18 years or older and had treatment-naive, pathologically confirmed stage IIIB or stage IV lung adenocarcinoma with a confirmed, activating EGFR mutation (exon 19 deletion or L858R mutation). Patients received oral erlotinib 150 mg per day and intravenous bevacizumab 15 mg/kg every 21 days and were tested centrally for the pretreatment T790M resistance mutation with a peptide nucleic acid probe-based real-time PCR. The primary endpoint was progression-free survival. The primary efficacy analysis was done in the intention-to-treat population and was stratified into two parallel substudies according to the centrally confirmed pretreatment T790M mutation status of enrolled patients (T790M positive or negative). The safety analysis was done in all patients that have received at least one dose of trial treatment. This trial was registered with ClinicalTrials.gov, number NCT01562028. Between June 11, 2012, and Oct 28, 2014, 109 patients were enrolled and included in the efficacy analysis. 37 patients were T790M mutation positive and 72 negative. The overall median progression-free survival was 13·2 months (95% CI 10·3-15·5), with a 12 month progression-free survival of 55% (95% CI

  4. Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers.

    Science.gov (United States)

    Tesauro, Cinzia; Graziani, Grazia; Arnò, Barbara; Zuccaro, Laura; Muzi, Alessia; D'Annessa, Ilda; Santori, Elettra; Tentori, Lucio; Leonetti, Carlo; Fiorani, Paola; Desideri, Alessandro

    2014-09-17

    DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects. Using site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures. Mutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity. It can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.

  5. 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.

  6. [Frequency of Helicobacter pylori nitroreductase RdxA mutations for metronidazole activation in a population in the Cauca Department, Colombia].

    Science.gov (United States)

    Acosta, Claudia Patricia; Quiroga, Andrés Javier; Sierra, Carlos H; Trespalacios, Alba Alicia

    2017-06-01

    Resistance to metronidazole is a key factor associated with Helicobacter pylori treatment failure. Even though resistance is mostly associated with RdxA nitroreductase mutations, studies of this H. pylori protein in Popayán (Colombia) are still incipient. To evaluate the frequency of mutations in the RdxA nitroreductase in a population of patients with H. pylori-positive gastrointestinal disease. We amplified the DNA of 170 gastric biopsies by PCR to detect mutations in the RdxA nitroreductase. An analysis of DNA sequences translated into amino acid sequences was done and then compared to the reference strain 26695. The frequency of RdxA nitroreductase mutations in this study population was 78%. Its most frequent distribution was found in positions D59N (153 samples), R131K (101 samples), R90K (97 samples), A118T (42 samples), I160F (32 samples) and H97T (26 samples), and meaningful stop codons Q50*, D59*; E75*, C159* and I160* in five, one, three, ten and six samples, respectively. The most common virulence genotype was vacAs1/m1 cagA negative (48.6 %). The high frequency of RdxA nitroreductase mutations in H. pylori isolates in Popayán (Colombia) indicates that empirical therapy with metronidazole may not be a valid option for the eradication of H. pylori in patients of the studied population.

  7. 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.

  8. Mother-to-son transmission of a luteinizing hormone receptor activating mutation in a prepubertal child with testotoxicosis.

    Science.gov (United States)

    Eunice, Marumudi; Philibert, Pascal; Kulshreshtha, Bindu; Audran, Françoise; Paris, Françoise; Sultan, Charles; Ammini, Ariachery C

    2009-03-01

    To identify the LHR gene mutation in a prepubertal child with testotoxicosis. Standard RIA procedure was used for estimating LH, FSH and testosterone levels. Molecular analysis was done by standard PCR using different sets of primers and reaction conditions specific for the LHR gene. Direct sequencing was done using the ABI Prism Dye terminator sequencing kit and the ABI 310 sequencing apparatus. We found a heterozygous mutation of the LHR gene in exon 11 of the second transmembrane region, Met-->Thr at the 398 position (M398T). The same mutation was also found in the proband's mother. To our knowledge, this is the first molecular characterization of maternally inherited testotoxicosis in a 5 1/2-year-old boy from the Indian subcontinent.

  9. 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.

  10. [From gene to disease; achondroplasia and other skeletal dysplasias due to an activating mutation in the fibroblast growth factor

    NARCIS (Netherlands)

    Ravenswaaij-Arts, C.M.A. van; Losekoot, M.

    2001-01-01

    Achondroplasia, the most common and best known skeletal dysplasia, is inherited in an autosomal dominant fashion. Like a number of other skeletal dysplasias, among which hypochondroplasia and thanatophoric dysplasia, achondroplasia is caused by mutations in the fibroblast growth factor receptor 3

  11. A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin-biased signaling

    DEFF Research Database (Denmark)

    Gorvin, Caroline M.; Babinsky, Valerie N.; Malinauskas, Tomas

    2018-01-01

    SR mutation selectively enhanced β-arrestin signaling by disrupting a salt bridge formed between Arg680 and Glu767, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through β-arrestin and the importance of the Arg680-Glu767...... salt bridge in mediating signaling bias....

  12. Development of drug resistance mutations in patients on highly active antiretroviral therapy: does competitive advantage drive evolution.

    Science.gov (United States)

    Kolber, Michael A

    2007-01-01

    Most physicians that treat individuals with HIV-1 disease are able to successfully suppress viral replication with the pharmacologic armamentarium available today. For the majority of patients this results in immune reconstitution and improved quality of life. However, a large fraction of these patients have transient elevations in their viral burden and even persistence of low-level viremia. In fact, many individuals whose viral load is suppressed to < 50 c/ml have evidence of low-level viral replication. The impact of low-level viremia and persistent viral replication is an area of significant study and interest owing to the potential for the development of drug resistance mutations. Here the fundamental question is whether and perhaps what factors provide a venue for the development of resistant virus. The concern is clearly the eventual progression of disease with the exhaustion of treatment options. The purpose of this review is to evaluate the current literature regarding the effect of low-level viremia on the development of drug resistance mutations. Herein, we discuss the impact of different levels of viral suppression on the development of mutations. In addition, we look at the role that resistance and fitness play in determining the survival of a breakthrough mutation within the background of drug.

  13. 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.

  14. Disseminated Tuberculosis and Chronic Mucocutaneous Candidiasis in a Patient with a Gain-of-Function Mutation in Signal Transduction and Activator of Transcription 1

    Science.gov (United States)

    Pedraza-Sánchez, Sigifredo; Lezana-Fernández, Jose Luis; Gonzalez, Yolanda; Martínez-Robles, Luis; Ventura-Ayala, María Laura; Sadowinski-Pine, Stanislaw; Nava-Frías, Margarita; Moreno-Espinosa, Sarbelio; Casanova, Jean-Laurent; Puel, Anne; Boisson-Dupuis, Stephanie; Torres, Martha

    2017-01-01

    In humans, recessive loss-of-function mutations in STAT1 are associated with mycobacterial and viral infections, whereas gain-of-function (GOF) mutations in STAT1 are associated with a type of primary immunodeficiency related mainly, but not exclusively, to chronic mucocutaneous candidiasis (CMC). We studied and established a molecular diagnosis in a pediatric patient with mycobacterial infections, associated with CMC. The patient, daughter of a non-consanguineous mestizo Mexican family, had axillary adenitis secondary to BCG vaccination and was cured with resection of the abscess at 1-year old. At the age of 4 years, she had a supraclavicular abscess with acid-fast-staining bacilli identified in the soft tissue and bone, with clinical signs of disseminated infection and a positive Gene-X-pert test, which responded to anti-mycobacterial drugs. Laboratory tests of the IL-12/interferon gamma (IFN-γ) circuit showed a higher production of IL-12p70 in the whole blood from the patient compared to healthy controls, when stimulated with BCG and BCG + IFN-γ. The whole blood of the patient produced 35% less IFN-γ compared to controls assessed by ELISA and flow cytometry, but IL-17 producing T cells from patient were almost absent in PBMC stimulated with PMA plus ionomycin. Signal transduction and activator of transcription 1 (STAT1) was hyperphosphorylated at tyrosine 701 in response to IFN-γ and -α, as demonstrated by flow cytometry and Western blotting in fresh blood mononuclear cells and in Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs); phosphorylation of STAT1 in EBV-LCLs from the patient was resistant to inhibition by staurosporine but sensitive to ruxolitinib, a Jak phosphorylation inhibitor. Genomic DNA sequencing showed a de novo mutation in STAT1 in cells from the patient, absent in her parents and brother; a known T385M missense mutation in the DNA-binding domain of the transcription factor was identified, and it is a GOF mutation. Therefore

  15. Disseminated Tuberculosis and Chronic Mucocutaneous Candidiasis in a Patient with a Gain-of-Function Mutation in Signal Transduction and Activator of Transcription 1

    Directory of Open Access Journals (Sweden)

    Sigifredo Pedraza-Sánchez

    2017-12-01

    Full Text Available In humans, recessive loss-of-function mutations in STAT1 are associated with mycobacterial and viral infections, whereas gain-of-function (GOF mutations in STAT1 are associated with a type of primary immunodeficiency related mainly, but not exclusively, to chronic mucocutaneous candidiasis (CMC. We studied and established a molecular diagnosis in a pediatric patient with mycobacterial infections, associated with CMC. The patient, daughter of a non-consanguineous mestizo Mexican family, had axillary adenitis secondary to BCG vaccination and was cured with resection of the abscess at 1-year old. At the age of 4 years, she had a supraclavicular abscess with acid-fast-staining bacilli identified in the soft tissue and bone, with clinical signs of disseminated infection and a positive Gene-X-pert test, which responded to anti-mycobacterial drugs. Laboratory tests of the IL-12/interferon gamma (IFN-γ circuit showed a higher production of IL-12p70 in the whole blood from the patient compared to healthy controls, when stimulated with BCG and BCG + IFN-γ. The whole blood of the patient produced 35% less IFN-γ compared to controls assessed by ELISA and flow cytometry, but IL-17 producing T cells from patient were almost absent in PBMC stimulated with PMA plus ionomycin. Signal transduction and activator of transcription 1 (STAT1 was hyperphosphorylated at tyrosine 701 in response to IFN-γ and -α, as demonstrated by flow cytometry and Western blotting in fresh blood mononuclear cells and in Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs; phosphorylation of STAT1 in EBV-LCLs from the patient was resistant to inhibition by staurosporine but sensitive to ruxolitinib, a Jak phosphorylation inhibitor. Genomic DNA sequencing showed a de novo mutation in STAT1 in cells from the patient, absent in her parents and brother; a known T385M missense mutation in the DNA-binding domain of the transcription factor was identified, and it is a GOF

  16. 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.

  17. A De Novo Mutation in the Sodium-Activated Potassium Channel KCNT2 Alters Ion Selectivity and Causes Epileptic Encephalopathy

    Directory of Open Access Journals (Sweden)

    Sushmitha Gururaj

    2017-10-01

    Full Text Available Early infantile epileptic encephalopathies (EOEE are a debilitating spectrum of disorders associated with cognitive impairments. We present a clinical report of a KCNT2 mutation in an EOEE patient. The de novo heterozygous variant Phe240Leu SLICK was identified by exome sequencing and confirmed by Sanger sequencing. Phe240Leu rSlick and hSLICK channels were electrophysiologically, heterologously characterized to reveal three significant alterations to channel function. First, [Cl−]i sensitivity was reversed in Phe240Leu channels. Second, predominantly K+-selective WT channels were made to favor Na+ over K+ by Phe240Leu. Third, and consequent to altered ion selectivity, Phe240Leu channels had larger inward conductance. Further, rSlick channels induced membrane hyperexcitability when expressed in primary neurons, resembling the cellular seizure phenotype. Taken together, our results confirm that Phe240Leu is a “change-of-function” KCNT2 mutation, demonstrating unusual altered selectivity in KNa channels. These findings establish pathogenicity of the Phe240Leu KCNT2 mutation in the reported EOEE patient.

  18. Cryptic splice activation but not exon skipping is observed in minigene assays of dystrophin c.9361+1G>A mutation identified by NGS.

    Science.gov (United States)

    Niba, Emma Tabe Eko; Nishida, Atsushi; Tran, Van Khanh; Vu, Dung Chi; Matsumoto, Masaaki; Awano, Hiroyuki; Lee, Tomoko; Takeshima, Yasuhiro; Nishio, Hisahide; Matsuo, Masafumi

    2017-04-01

    Next-generation sequencing (NGS) discloses nucleotide changes in the genome. Mutations at splicing regulatory elements are expected to cause splicing errors, such as exon skipping, cryptic splice site activation, partial exon loss or intron retention. In dystrophinopathy patients, prediction of splicing outcomes is essential to determine the phenotype: either severe Duchenne or mild Becker muscular dystrophy, based on the reading frame rule. In a Vietnamese patient, NGS identified a c.9361+1G>A mutation in the dystrophin gene and an additional DNA variation of A>G at +117 bases in intron 64. To ascertain the consequences of these DNA changes on dystrophin splicing, minigene constructs were prepared inserting dystrophin exon 64 plus various lengths of intron 64. Exon 64 skipping was observed in the minigene construct with 160 nucleotide (nt) of intron 64 sequence with both c.9361+1A and +117G. In contrast, minigene constructs with larger flanking intronic domains resulted in cryptic splice site activation rather than exon skipping. Meanwhile, the cryptic splice site activation was induced even in +117G when intron 64 was elongated to 272 nt and longer. It was expected that cryptic splice site activation is an in vivo splicing outcome.

  19. 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.

  20. Common Co-activation of AXL and CDCP1 in EGFR-mutation-positive Non-smallcell Lung Cancer Associated With Poor Prognosis

    Directory of Open Access Journals (Sweden)

    Niki Karachaliou

    2018-03-01

    Full Text Available Epidermal growth factor receptor (EGFR-mutation-positive non-smallcell lung cancer (NSCLC is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs. We investigated receptor tyrosine kinases (RTKs, Src family kinases and focal adhesion kinase (FAK as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2 of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2 inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1 was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p = 0.0407 and overall survival (hazard ratio of 2.23, p = 0.0192. A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing. Keywords: Lung cancer, EGFR, Resistance, AXL, CDCP1, Combination therapies

  1. Common Co-activation of AXL and CDCP1 in EGFR-mutation-positive Non-smallcell Lung Cancer Associated With Poor Prognosis.

    Science.gov (United States)

    Karachaliou, Niki; Chaib, Imane; Cardona, Andres Felipe; Berenguer, Jordi; Bracht, Jillian Wilhelmina Paulina; Yang, Jie; Cai, Xueting; Wang, Zhigang; Hu, Chunping; Drozdowskyj, Ana; Servat, Carles Codony; Servat, Jordi Codony; Ito, Masaoki; Attili, Ilaria; Aldeguer, Erika; Capitan, Ana Gimenez; Rodriguez, July; Rojas, Leonardo; Viteri, Santiago; Molina-Vila, Miguel Angel; Ou, Sai-Hong Ignatius; Okada, Morihito; Mok, Tony S; Bivona, Trever G; Ono, Mayumi; Cui, Jean; Cajal, Santiago Ramón Y; Cao, Peng; Rosell, Rafael

    2018-03-01

    Epidermal growth factor receptor (EGFR)-mutation-positive non-smallcell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p=0.0407) and overall survival (hazard ratio of 2.23, p=0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Characterization of vanadate-dependent NADH oxidation activity and isolation of yeast DNA which complements a class 1 vanadate resistance mutation

    International Nuclear Information System (INIS)

    Minasi, L.E.

    1989-01-01

    A vanadate-dependent NADH oxidation activity has been characterized in plasma membranes from the yeast S cerevisiae. NADH oxidation activity was maximally stimulated at pH 5.0 in phosphate buffer. NADH oxidation was not dependent on the concentration of plasma membranes. The vanadate-dependent NADH oxidation activity was abolished under anaerobic conditions and the concomitant uptake of oxygen occurred during NADH oxidation. The activity was inhibited by superoxide dismutase and stimulated by the presence of paraquat. These results indicate that the vanadate stimulation of NADH oxidation in yeast plasma membranes occurs as a result of the vanadate-dependent oxidation of NADH by superoxide, generated by a plasma membrane NADH oxidase. 51 V-NMR results indicated that a phosphate-vanadate anhydride was the stimulatory species in pH 5.0 and pH 7.0 phosphate buffer. Yeast DNA has been isolated which complements a class 1 vanadate resistance mutation

  3. Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes

    OpenAIRE

    Tiffen, Jessamy C.; Gunatilake, Dilini; Gallagher, Stuart J.; Gowrishankar, Kavitha; Heinemann, Anja; Cullinane, Carleen; Dutton-Regester, Ken; Pupo, Gulietta M.; Strbenac, Dario; Yang, Jean Y.; Madore, Jason; Mann, Graham J.; Hayward, Nicholas K.; McArthur, Grant A.; Filipp, Fabian V.

    2015-01-01

    The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant hum...

  4. 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.

    Science.gov (United States)

    Arimoto-Kobayashi, Sakae; Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko

    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 phiX174 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

  5. 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

  6. Biochanin A partially restores the activity of ofloxacin and ciprofloxacin against topoisomerase IV mutation-associated fluoroquinolone-resistant Ureaplasma species.

    Science.gov (United States)

    Jin, Hong; Qi, Chao; Zou, Yanping; Kong, Yingying; Ruan, Zhi; Ding, Honghui; Xie, Xinyou; Zhang, Jun

    2017-11-01

    This study aims to investigate the synergistic antimicrobial activity of four phytoalexins in combination with fluoroquinolones against Ureaplasma spp., a genus of cell wall-free bacteria that are intrinsically resistant to many available antibiotics, making treatment inherently difficult. A total of 22 958 urogenital tract specimens were assessed for Ureaplasma spp. identification and antimicrobial susceptibility. From these, 31 epidemiologically unrelated strains were randomly selected for antimicrobial susceptibility testing to determine the minimum inhibitory concentration (MIC) of four fluoroquinolones and the corresponding quinolone resistance-determining regions (QRDRs). Synergistic effects between fluoroquinolones and four phytoalexins (reserpine, piperine, carvacrol and biochanin A) were evaluated by fractional inhibitory concentration indices (FICIs). Analysis of the QRDRs suggested a vital role for the mutation of Ser-83→Leu in ParC in fluoroquinolone-resistant strains, and the occurrence of mutations in QRDRs showed significant associations with the breakpoint of levofloxacin. Moreover, diverse synergistic effects of the four phytoalexins with ofloxacin or ciprofloxacin were observed and biochanin A was able to enhance the antimicrobial activity of fluoroquinolones significantly. This is the first report of the antimicrobial activity of biochanin A in combination with fluoroquinolones against a pathogenic mycoplasma, and opens up the possibility of using components of biochanin A as a promising therapeutic option for treating antibiotic-resistant Ureaplasma spp. infections.

  7. 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.

  8. CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas.

    Science.gov (United States)

    Oberst, Michael D; Fuhrmann, Stacy; Mulgrew, Kathy; Amann, Maria; Cheng, Lily; Lutterbuese, Petra; Richman, Laura; Coats, Steve; Baeuerle, Patrick A; Hammond, Scott A

    2014-01-01

    Individual or combinations of somatic mutations found in genes from colorectal cancers can redirect the effects of chemotherapy and targeted agents on cancer cell survival and, consequently, on clinical outcome. Novel therapeutics with mechanisms of action that are independent of mutational status would therefore fulfill a current unmet clinical need. Here the CEA and CD3 bispecific single-chain antibody MEDI-565 (also known as MT111 and AMG 211) was evaluated for its ability to activate T cells both in vitro and in vivo and to kill human tumor cell lines harboring various somatic mutations commonly found in colorectal cancers. MEDI-565 specifically bound to normal and malignant tissues in a CEA-specific manner, and only killed CEA positive cells. The BiTE® antibody construct mediated T cell-directed killing of CEA positive tumor cells within 6 hours, at low effector-to-target ratios which were independent of high concentrations of soluble CEA. The potency of in vitro lysis was dependent on CEA antigen density but independent of the mutational status in cancer cell lines. Importantly, individual or combinations of mutated KRAS and BRAF oncogenes, activating PI3KCA mutations, loss of PTEN expression, and loss-of-function mutations in TP53 did not reduce the activity in vitro. MEDI-565 also prevented growth of human xenograft tumors which harbored various mutations. These findings suggest that MEDI-565 represents a potential treatment option for patients with CEA positive tumors of diverse origin, including those with individual or combinations of somatic mutations that may be less responsive to chemotherapy and other targeted agents.

  9. 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

    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...... 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...

  10. Escape Mutations in NS4B Render Dengue Virus Insensitive to the Antiviral Activity of the Paracetamol Metabolite AM404.

    Science.gov (United States)

    van Cleef, Koen W R; Overheul, Gijs J; Thomassen, Michael C; Marjakangas, Jenni M; van Rij, Ronald P

    2016-04-01

    Despite the enormous disease burden associated with dengue virus infections, a licensed antiviral drug is lacking. Here, we show that the paracetamol (acetaminophen) metabolite AM404 inhibits dengue virus replication. Moreover, we find that mutations in NS4B that were previously found to confer resistance to the antiviral compounds NITD-618 and SDM25N also render dengue virus insensitive to AM404. Our work provides further support for NS4B as a direct or indirect target for antiviral drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Linker-scanning mutational analysis of the transcriptional activity of the human immunodeficiency virus type 1 long terminal repeat.

    OpenAIRE

    Zeichner, S L; Kim, J Y; Alwine, J C

    1991-01-01

    We have compared the relative importance of transcription regulatory regions in the U3 and R regions of the human immunodeficiency virus type 1 long terminal repeat (LTR) by using linker-scanning mutational analysis. Twenty-six mutant LTR-chloramphenicol acetyltransferase (CAT) transient expression plasmids were prepared in which consecutive 18-bp regions of wild-type LTR were replaced with an NdeI-XhoI-SalI (NXS) polylinker. The mutant LTR-CAT plasmids were transfected into unstimulated Jurk...

  12. Hemophilia B with mutations at glycine-48 of factor IX exhibited delayed activation by the factor VIIa-tissue factor complex.

    Science.gov (United States)

    Wu, P C; Hamaguchi, N; Yu, Y S; Shen, M C; Lin, S W

    2000-10-01

    Gly-48 is in the conserved DGDQC sequence (residues 47-51 of human factor IX) of the first EGF (EGF-1)-like domain of factor IX. The importance of the Gly-48 is manifested by two hemophilia B patients; factor IXTainan and factor IXMalmo27, with Gly-48 replaced by arginine (designated IXG48R) and valine (IXG48V), respectively. Both patients were CRM+ exhibiting mild hemophilic episodes with 25% (former) and 19% (latter) normal clotting activities. We characterize both factor IX variants to show the roles of Gly-48 and the conservation of the DGDQC sequence in factor IX. Purified plasma and recombinant factor IX variants exhibited approximately 26%-27% normal factor IX's clotting activities with G48R or G48V mutation. Both variants depicted normal quenching of the intrinsic fluorescence by increasing concentrations of calcium ions and Tb3+, indicating that arginine and valine substitution for Gly-48 did not perturb the calcium site in the EGF-1 domain. Activation of both mutants by factor XIa appeared normal. The reduced clotting activity of factors IXG48R and IXG48V was attributed to the failure of both mutants to cleavage factor X: in the presence of only phospholipids and calcium ions, both mutants showed a 4 to approximately 7-fold elevation in Km, and by adding factor VIIIa to the system, although factor VIIIa potentiated the activation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2 to approximately 3-fold decrease in the catalytic function was observed with the mutant factor IXa's, despite that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced affinity when compared to wild-type factor IXa. The apparent Kd for factor VIIIa binding was 0.83 nM for normal factor IXa, 1.74 nM for IXaG48R and 1.4 nM for IXaG48V. Strikingly, when interaction with the factor VIIa-TF complex was examined, both mutations were barely activated by the VIIa-TF complex and they also showed abnormal interaction with VIIa-TF in bovine

  13. 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....

  14. New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Dutreix, M.; Moreau, P.L.; Bailone, A.; Galibert, F.; Battista, J.R.; Walker, G.C.; Devoret, R.

    1989-05-01

    To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+.

  15. MPL mutations in myeloproliferative disorders

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  16. The ability of AIF-1 to activate human vascular smooth muscle cells is lost by mutations in the EF-hand calcium-binding region

    International Nuclear Information System (INIS)

    Autieri, Michael V.; Chen Xing

    2005-01-01

    Allograft Inflammatory Factor-1 (AIF-1) is a cytoplasmic calcium-binding protein expressed in vascular smooth muscle cells (VSMC) in response to injury or cytokine stimulation. AIF-1 contains a partially conserved EF-hand calcium-binding domain, and participates in VSMC activation by activation of Rac1 and induction of Granulocyte-Colony Stimulating Factor (G-CSF) expression; however, the mechanism whereby AIF-1 mediates these effects is presently uncharacterized. To determine if calcium binding plays a functional role in AIF-1 activity, a single site-specific mutation was made in the EF-hand calcium-binding domain to abrogate binding of calcium (AIF-1ΔA), which was confirmed by calcium overlay. Functionally, similar to wild-type AIF-1, AIF-1ΔA was able to polymerize F-actin in vitro. However, in contrast to wild-type AIF-1, over-expression of AIF-1ΔA was unable to increase migration or proliferation of primary human VSMC. Further, it was unable to activate Rac1, or induce G-CSF expression to the degree as wild-type AIF-1. Taken together, modification of the wild-type EF-hand domain and native calcium-binding activity results in a loss of AIF-1 function. We conclude that appropriate calcium-binding potential is critical in AIF-1-mediated effects on VSMC pathophysiology, and that AIF-1 activity is mediated by Rac1 activation and G-CSF expression

  17. 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

    2016-03-03

    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. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  18. 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

  19. A Homozygous [Cys25]PTH(1-84) Mutation That Impairs PTH/PTHrP Receptor Activation Defines a Novel Form of Hypoparathyroidism.

    Science.gov (United States)

    Lee, Sihoon; Mannstadt, Michael; Guo, Jun; Kim, Seul Min; Yi, Hyon-Seung; Khatri, Ashok; Dean, Thomas; Okazaki, Makoto; Gardella, Thomas J; Jüppner, Harald

    2015-10-01

    Hypocalcemia and hyperphosphatemia are encountered in idiopathic hypoparathyroidism (IHP) and pseudohypoparathyroidism type Ib (PHP1B). In contrast to PHP1B, which is caused by resistance toward parathyroid hormone (PTH), the genetic defects leading to IHP impair production of this important regulator of mineral ion homeostasis. So far, only five PTH mutations were shown to cause IHP, each of which is located in the hormone's pre-pro leader segment and thus impair hormone secretion. In three siblings affected by IHP, we now identified a homozygous arginine-to-cysteine mutation at position 25 (R25C) of the mature PTH(1-84) polypeptide; heterozygous family members are healthy. Depending on the assay used for evaluating these patients, plasma PTH levels were either low or profoundly elevated, thus leading to ambiguities regarding the underlying diagnosis, namely IHP or PHP1B. Consistent with increased PTH levels, recombinant [Cys25]PTH(1-84) and wild-type PTH(1-84) were secreted equally well by transfected COS-7 cells. However, synthetic [Cys25]PTH(1-34) was found to have a lower binding affinity for the PTH receptor type-1 (PTH1R) than PTH(1-34) and consequently a lower efficiency for stimulating cAMP formation in cells expressing this receptor. Consistent with these in vitro findings, long-term infusion of [Cys25]PTH(1-34) resulted only in minimal calcemic and phosphaturic responses, despite readily detectable levels of [Cys25]PTH(1-34) in plasma. The mineral ion abnormalities observed in the three IHP patients are thus most likely caused by the inherited homozygous missense PTH mutation, which reduces bioactivity of the secreted hormone. Based on these findings, screening for PTH(1-84) mutations should be considered when clinical and laboratory findings are consistent with PHP1B, but GNAS methylation changes have been excluded. Differentiating between IHP and PHP1B has considerable implications for genetic counseling, therapy, and long-term outcome because

  20. Interaction of reverse transcriptase (RT) mutations conferring resistance to lamivudine and etravirine: effects on fitness and RT activity of human immunodeficiency virus type 1.

    Science.gov (United States)

    Hu, Zixin; Kuritzkes, Daniel R

    2011-11-01

    Resistance to the nonnucleoside reverse transcriptase inhibitors etravirine and rilpivirine (RPV) is conferred by the E138K mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Clinical trials of RPV administered with lamivudine or emtricitabine showed the emergence of E138K together with M184I, which confers lamivudine and emtricitabine resistance in most patients with virologic failure. To understand why M184I was favored over M184V, we determined the drug susceptibility, infectivity, relative fitness, and reverse transcriptase activity of HIV-1 carrying E138K/M184I or E138K/M184V mutations. Whereas the replication capacity (RC) of the single mutants was reduced compared to that of the wild type (WT), the RC of the two double mutants was comparable to that of the WT in the absence of drug. The RC of the E138K/M184I mutant in the presence of etravirine was significantly greater than that of the E138K and E138K/M184V mutants; the RC of the double mutants was greater than that of the M184I or M184V mutant. Fitness profiles and growth competition experiments showed that the E138K/M184I mutant had a significant replicative advantage over the E138K/M184V mutant in the presence of etravirine and lamivudine. The virion-associated RT activity of the E138K, M184I, or M184V virus was significantly reduced compared to that of the WT, whereas the RT activity of the E138K/M184I virus was significantly greater than that of the WT or E138K/M184V virus. These results suggest that the E138K and M184I/V mutations are mutually compensatory and may explain the frequent occurrence of E138K/M184I after the virologic failure of rilpivirine-, lamivudine-, and emtricitabine-containing regimens.

  1. Interaction of Reverse Transcriptase (RT) Mutations Conferring Resistance to Lamivudine and Etravirine: Effects on Fitness and RT Activity of Human Immunodeficiency Virus Type 1 ▿

    Science.gov (United States)

    Hu, Zixin; Kuritzkes, Daniel R.

    2011-01-01

    Resistance to the nonnucleoside reverse transcriptase inhibitors etravirine and rilpivirine (RPV) is conferred by the E138K mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Clinical trials of RPV administered with lamivudine or emtricitabine showed the emergence of E138K together with M184I, which confers lamivudine and emtricitabine resistance in most patients with virologic failure. To understand why M184I was favored over M184V, we determined the drug susceptibility, infectivity, relative fitness, and reverse transcriptase activity of HIV-1 carrying E138K/M184I or E138K/M184V mutations. Whereas the replication capacity (RC) of the single mutants was reduced compared to that of the wild type (WT), the RC of the two double mutants was comparable to that of the WT in the absence of drug. The RC of the E138K/M184I mutant in the presence of etravirine was significantly greater than that of the E138K and E138K/M184V mutants; the RC of the double mutants was greater than that of the M184I or M184V mutant. Fitness profiles and growth competition experiments showed that the E138K/M184I mutant had a significant replicative advantage over the E138K/M184V mutant in the presence of etravirine and lamivudine. The virion-associated RT activity of the E138K, M184I, or M184V virus was significantly reduced compared to that of the WT, whereas the RT activity of the E138K/M184I virus was significantly greater than that of the WT or E138K/M184V virus. These results suggest that the E138K and M184I/V mutations are mutually compensatory and may explain the frequent occurrence of E138K/M184I after the virologic failure of rilpivirine-, lamivudine-, and emtricitabine-containing regimens. PMID:21849432

  2. 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

    -dead, had marginal ATPase activity and may be structurally compromised, while the other two showed greatly reduced helicase and ATPase activities. The remaining biochemical activities and ability to recruit to damage sites were not significantly impaired for any of the mutants. Our findings demonstrate...

  3. Mutational destabilization of the critical interface water cluster in Scapharca dimeric hemoglobin: structural basis for altered allosteric activity.

    Science.gov (United States)

    Pardanani, A; Gambacurta, A; Ascoli, F; Royer, W E

    1998-12-04

    A cluster of interface ordered water molecules has been proposed to act as a key mediator of intersubunit communication in the homodimeric hemoglobin of Scapharca inaequivalvis. Mutations of Thr72 to Val and Ile, which lack the hydroxyl group to hydrogen bond the deoxy interface water molecules, result in sharply altered functional properties. We have determined the high resolution (1.6-1. 8 A) crystal structures of these two mutants in both the deoxygenated and CO-liganded states. These structures show minimal protein structural changes relative to the same native derivatives, despite greater than 40-fold increases in oxygen affinity. In the deoxy state of both mutants two water molecules at the periphery of the water cluster are lost, and the remaining cluster water molecules are destabilized. The CO-liganded structures show key differences between the two mutants including a more optimal interface packing involving Ile72 that acts to stabilize its high affinity (R) state. This additional stabilization allows rationalization of its lowered cooperativity within the context of a two-state model. These studies support a key role of ordered water in cooperative functioning and illustrate how subtle structural alterations can result in significantly altered functional properties in an allosteric molecule. Copyright 1998 Academic Press

  4. 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

    . 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...... factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1...

  5. The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction-dependent manner.

    Directory of Open Access Journals (Sweden)

    Aniko Keller-Pinter

    Full Text Available The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs. Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu mutant of syndecan-4 (SDC4. SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1 to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1-RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways.

  6. The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction–dependent manner

    Science.gov (United States)

    Keller-Pinter, Aniko; Ughy, Bettina; Domoki, Monika; Pettko-Szandtner, Aladar; Letoha, Tamas; Tovari, Jozsef; Timar, Jozsef

    2017-01-01

    The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1–RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways. PMID:29121646

  7. The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction-dependent manner.

    Science.gov (United States)

    Keller-Pinter, Aniko; Ughy, Bettina; Domoki, Monika; Pettko-Szandtner, Aladar; Letoha, Tamas; Tovari, Jozsef; Timar, Jozsef; Szilak, Laszlo

    2017-01-01

    The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1-RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways.

  8. The effect of aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations on the expression and activity of vasopressin V2 receptor gene.

    Science.gov (United States)

    Najafzadeh, Hossein; Safaeian, Leila; Mirmohammad Sadeghi, Hamid; Rabbani, Mohammad; Jafarian, Abbas

    2010-01-01

    Vasopressin type 2 receptor (V2R) plays an important role in the water reabsorption in the kidney collecting ducts. V2R is a G protein coupled receptor (GPCR) and the triplet of amino acids aspartate-arginine-histidine (DRH) in this receptor might significantly influence its activity similar to other GPCR. However, the role of this motif has not been fully confirmed. Therefore, the present study attempted to shed some more light on the role of DRH motif in G protein coupling and V2R function with the use of site-directed mutagenesis. Nested PCR using specific primers was used to produce DNA fragments containing aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations with replacements of the arginine to lysine and histidine to tyrosine, respectively. After digestion, these inserts were ligated into the pcDNA3 vector and transformation into E. coli HB101 was performed using heat shock method. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using diethylaminoethyl-dextran method, the adenylyl cyclase activity assay was performed for functional study. The cell surface expression was analyzed by indirect ELISA method. The functional assay indicated that none of these mutations significantly altered cAMP production and cell surface expression of V2R in these cells. Since some substitutions in arginine residue have shown to lead to the inactive V2 receptor, further studies are required to define the role of this residue more precisely. However, it seems that the role of the histidine residue is not critical in the V2 receptor function.

  9. 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.

  10. An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State.

    Science.gov (United States)

    Davlieva, Milya; Tovar-Yanez, Angel; DeBruler, Kimberly; Leonard, Paul G; Zianni, Michael R; Arias, Cesar A; Shamoo, Yousif

    2016-11-06

    The cyclic antimicrobial lipopeptide daptomycin (DAP) triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence-specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, non-susceptibility to DAP by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaR W73C ). We have determined the structure of the activated E. faecium LiaR protein at 3.2Å resolution and, in combination with solution studies, show that the activation of LiaR induces the formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaR W73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to DAP by the upregulation of the LiaFSR regulon. A comparison of the E. faecalis LiaR, E. faecium LiaR, and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with DAP resistance suggests that physicochemical properties such as oligomerization state and DNA specificity, although tuned to the biology of each organism, share some features that could be targeted for new antimicrobials. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. 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...

  12. Analysis of active site residues of botulinum neurotoxin E by mutational, functional, and structural studies: Glu335Gln is an apoenzyme.

    Science.gov (United States)

    Agarwal, Rakhi; Binz, Thomas; Swaminathan, Subramanyam

    2005-06-14

    Clostridial neurotoxins comprising the seven serotypes of botulinum neurotoxins and tetanus neurotoxin are the most potent toxins known to humans. Their potency coupled with their specificity and selectivity underscores the importance in understanding their mechanism of action in order to develop a strategy for designing counter measures against them. To develop an effective vaccine against the toxin, it is imperative to achieve an inactive form of the protein which preserves the overall conformation and immunogenicity. Inactive mutants can be achieved either by targeting active site residues or by modifying the surface charges farther away from the active site. The latter affects the long-range forces such as electrostatic potentials in a subtle way without disturbing the structural integrity of the toxin causing some drastic changes in the activity/environment. Here we report structural and biochemical analysis on several mutations on Clostridium botulinum neurotoxin type E light chain with at least two producing dramatic effects: Glu335Gln causes the toxin to transform into a persistent apoenzyme devoid of zinc, and Tyr350Ala has no hydrolytic activity. The structural analysis of several mutants has led to a better understanding of the catalytic mechanism of this family of proteins. The residues forming the S1' subsite have been identified by comparing this structure with a thermolysin-inhibitor complex structure.

  13. 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. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Point mutation in activated c-Ha-ras gene of a chemically induced transplantable human pancreas carcinoma

    International Nuclear Information System (INIS)

    Maheshwari, K.K.; Parsa, I.

    1986-01-01

    The authors have reported a model of human pancreas carcinogenesis where repeated treatment with MNU of explants results in the development of transplantable carcinoma. This report compares the endonuclease digests of DNAs from normal human pancreas (HP) and MNU-induced transplantable tumor (HP-T1) analyzed with 32 P-labelled Ha-ras probe prepared from clone BS-9. The hybridization patterns of BamHI, BglII, EcoRI and HindIII digests of HP were significantly different from those of HP-T1. In EcoRI digests a 3.0 kb fragments of HP-T1 DNA hybridized with Ha-ras probe instead of a 4.3 kb fragments seen in HP DNA. The pattern for HindIII digests was similar to those of EcoRI. The BgIII digests of HP DNA revealed two hybridizing fragments of 8.0 and 4.3 kb whereas those of HP-T1 DNA fragments measured 8.5 and 4.0 kb. BamHI treated HP DNA showed only hybridizing fragments of 6.6 kb while the HP-T1 DNA showed to hybridizing fragments of 6.8 and 7.2 kb. The digested DNAs by HhaI, HinfI, KpnI, pstI, PvuII, SaII, SstI, TaqI and XbaI showed similar hybridization profiles. The point mutation in c-Ha-ras was examined in the HpaII and MspI double digests of both DNAs by 0.6 Kb SmaI fragments of pEJ. The hybridized fragments measured 412 and 355 bp in DNA digests from tumor and normal pancreas respectively

  15. Differential Effects of HRAS Mutation on LTP-Like Activity Induced by Different Protocols of Repetitive Transcranial Magnetic Stimulation.

    Science.gov (United States)

    Dileone, Michele; Ranieri, Federico; Florio, Lucia; Capone, Fioravante; Musumeci, Gabriella; Leoni, Chiara; Mordillo-Mateos, Laura; Tartaglia, Marco; Zampino, Giuseppe; Di Lazzaro, Vincenzo

    2016-01-01

    Costello syndrome (CS) is a rare congenital disorder due to a G12S amino acid substitution in HRAS protoncogene. Previous studies have shown that Paired Associative Stimulation (PAS), a repetitive brain stimulation protocol inducing motor cortex plasticity by coupling peripheral nerve stimulation with brain stimulation, leads to an extremely pronounced motor cortex excitability increase in CS patients. Intermittent Theta Burst Stimulation (iTBS) represents a protocol able to induce motor cortex plasticity by trains of stimuli at 50 Hz. In healthy subjects PAS and iTBS produce similar after-effects in motor cortex excitability. Experimental models showed that HRAS-dependent signalling pathways differently affect LTP induced by different patterns of repetitive synaptic stimulation. We aimed to compare iTBS-induced after-effects on motor cortex excitability with those produced by PAS in CS patients and to observe whether HRAS mutation differentially affects two different forms of neuromodulation protocols. We evaluated in vivo after-effects induced by PAS and iTBS applied over the right motor cortex in 4 CS patients and in 21 healthy age-matched controls. Our findings confirmed HRAS-dependent extremely pronounced PAS-induced after-effects and showed for the first time that iTBS induces no change in MEP amplitude in CS patients whereas both protocols lead to an increase of about 50% in controls. CS patients are characterized by an impairment of iTBS-related LTP-like phenomena besides enhanced PAS-induced after-effects, suggesting that HRAS-dependent signalling pathways have a differential influence on PAS- and iTBS-induced plasticity in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. In vivo mutational analysis of YtvA from Bacillus subtilis: Mechanism of light activation of the general stress response

    NARCIS (Netherlands)

    Avila-Pérez, M.; Vreede, J.; Tang, Y.; Bende, O.; Losi, A.; Gärtner, W.; Hellingwerf, K.

    2009-01-01

    The general stress response of Bacillus subtilis can be activated by stimuli such as the addition of salt or ethanol and with blue light. In the latter response, YtvA activates sigma(B) through a cascade of Rsb proteins, organized in stressosomes. YtvA is composed of an N-terminal LOV (light,

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

    DEFF Research Database (Denmark)

    Pedersen, Margit; Hammer, Karin

    2007-01-01

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

  18. Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities

    Directory of Open Access Journals (Sweden)

    Daisuke Matsui

    2014-01-01

    Full Text Available In this study, it was shown for the first time that l-amino acid oxidase of Pseudomonas sp. AIU813, renamed as l-amino acid oxidase/monooxygenase (l-AAO/MOG, exhibits l-lysine 2-monooxygenase as well as oxidase activity. l-Lysine oxidase activity of l-AAO/MOG was increased in a p-chloromercuribenzoate (p-CMB concentration-dependent manner to a final level that was five fold higher than that of the non-treated enzyme. In order to explain the effects of modification by the sulfhydryl reagent, saturation mutagenesis studies were carried out on five cysteine residues, and we succeeded in identifying l-AAO/MOG C254I mutant enzyme, which showed five-times higher specific activity of oxidase activity than that of wild type. The monooxygenase activity shown by the C254I variant was decreased significantly. Moreover, we also determined a high-resolution three-dimensional structure of l-AAO/MOG to provide a structural basis for its biochemical characteristics. The key residue for the activity conversion of l-AAO/MOG, Cys-254, is located near the aromatic cage (Trp-418, Phe-473, and Trp-516. Although the location of Cys-254 indicates that it is not directly involved in the substrate binding, the chemical modification by p-CMB or C254I mutation would have a significant impact on the substrate binding via the side chain of Trp-516. It is suggested that a slight difference of the binding position of a substrate can dictate the activity of this type of enzyme as oxidase or monooxygenase.

  19. A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

    Science.gov (United States)

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-02-20

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. 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.

  1. 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

    2015-10-23

    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.

  2. 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...... (EMS) on a transgenic line that harbours the activation-tagged LEP gene and exhibits the leafy petiole phenotype. A collection of revertant for leafy petiole (rlp lines was isolated that form petiolated rosette leaves in the presence of the activated LEP gene, and could be classified into three groups...

  3. 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.

  4. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    Directory of Open Access Journals (Sweden)

    Takuji Oyama

    Full Text Available CpMan5B is a glycoside hydrolase (GH family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196 in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  5. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    Science.gov (United States)

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  6. A single amino acid mutation affects elicitor and expansins-like activities of cerato-platanin, a non-catalytic fungal protein.

    Directory of Open Access Journals (Sweden)

    Simone Luti

    Full Text Available Cerato-platanin (CP is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response. Indeed, CP is an elicitor of primary defences acting as a Pathogen-Associated Molecular Patterns (PAMP. So far, structure-function relationship study has been mainly performed on the bacterial BsEXLX1 expansin, probably due to difficulties in expressing plant expansins in heterologous systems. Here, we report a subcloning and purification method of CP in the engineered E. coli SHuffle cells, which proved to be suitable to obtain the properly folded and biologically active protein. The method also enabled the production of the mutant D77A, rationally designed to be inactive. The wild-type and the mutated CP were characterized for cellulose weakening activity and for PAMP activity (i.e. induction of Reactive Oxygen Species synthesis and phytoalexins production. Our analysis reveals that the carboxyl group of D77 is crucial for expansin-like and PAMP activities, thus permitting to establish a correlation between the ability to weaken cellulose and the capacity to induce defence responses in plants. Our results enable the structural and functional characterization of a mono-domain eukaryotic expansin and identify the essential role of a specific aspartic residue in cellulose weakening.

  7. The ducky2J mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression

    Science.gov (United States)

    Donato, Roberta; Page, Karen M.; Koch, Dietlind; Nieto-Rostro, Manuela; Foucault, Isabelle; Davies, Anthony; Wilkinson, Tonia; Rees, Michele; Edwards, Frances A.; Dolphin, Annette C.

    2006-01-01

    The mouse mutant ducky and its allele ducky2J represent a model for absence epilepsy characterized by spike-wave seizures, and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the α2δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2du2J mutation results in a two base-pair deletion in the coding region and a complete loss of α2δ-2 protein. Here we show that du2J/du2J mice have a 30% reduction in somatic calcium current, and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C du2J/du2J PCs show no spontaneous intrinsic activity. Du2J/du2J mice also have alterations in the cerebellar expression of several genes related to PC function. At P21 there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du2J/+ mice have a marked reduction in α2δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However, du2J/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α2δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α2δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma. PMID:17135419

  8. The ducky(2J) mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression.

    Science.gov (United States)

    Donato, Roberta; Page, Karen M; Koch, Dietlind; Nieto-Rostro, Manuela; Foucault, Isabelle; Davies, Anthony; Wilkinson, Tonia; Rees, Michele; Edwards, Frances A; Dolphin, Annette C

    2006-11-29

    The mouse mutant ducky and its allele ducky(2J) represent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the alpha2delta-2 calcium channel subunit. Of relevance to the ataxic phenotype, alpha2delta-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2(du2J) mutation results in a 2 bp deletion in the coding region and a complete loss of alpha2delta-2 protein. Here we show that du(2J)/du(2J) mice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22 degrees C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34 degrees C, du(2J)/du(2J) PCs show no spontaneous intrinsic activity. Du(2J)/du(2J) mice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du(2J)/+ mice have a marked reduction in alpha2delta-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tyrosine hydroxylase gene expression. However, du(2J)/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in alpha2delta-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of alpha2delta-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma.

  9. 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.

  10. Human beta-galactosidase gene mutations in morquio B disease.

    OpenAIRE

    Oshima, A; Yoshida, K; Shimmoto, M; Fukuhara, Y; Sakuraba, H; Suzuki, Y

    1991-01-01

    Three different beta-galactosidase gene mutations--a 273Trp----Leu (mutation F) in both families, 482Arg----His (mutation G) in one family, and 509Trp----Cys (mutation H) in the other family--were identified in three patients with Morquio B disease who were from two unrelated families. Restriction-site analysis using StuI, Nsp(7524)I or RsaI confirmed these mutations. In human fibroblasts, mutation F expressed as much as 8% of the normal allele's enzyme activity, but the other mutations expre...

  11. The involvement of ataxia-telangiectasia mutated protein activation in nucleotide excision repair-facilitated cell survival with cisplatin treatment.

    Science.gov (United States)

    Colton, Stephanie L; Xu, Xiaoxin S; Wang, Y Alan; Wang, Gan

    2006-09-15

    DNA damage can lead to either DNA repair with cell survival or to apoptotic cell death. Although the biochemical processes underlying DNA repair and apoptosis have been extensively studied, the mechanisms by which cells determine whether the damage will be repaired or the apoptotic pathway will be activated is largely unknown. We have studied the role of nucleotide excision repair (NER) in cisplatin DNA damage-induced apoptotic cell death using both normal human fibroblasts and NER-defective xeroderma pigmentosum (XP) XPA and XPG cells. The caspase-3 activation experiment demonstrated a greatly increased casapse-3 activation in the NER-defective cells following cisplatin treatment. The flow cytometry experiment revealed an altered cell cycle arrest pattern of the NER-defective cells following cisplatin treatment. The results obtained from the Western blot experiment showed that NER defects resulted in enhanced CHK1 phosphorylation and p21 induction after cisplatin treatment. The cisplatin treatment-induced ATM phosphorylation, however, was attenuated in NER-defective cells. The results obtained from our immunoprecipitation experiment further demonstrated that the ATM protein interacted with the TFIIH basal transcription factor and the XPG protein of the NER pathway. It also showed that a functional XPC protein was required for the association of the ATM protein to genomic DNA. These results suggest that the NER process may prevent the cisplatin treatment-induced apoptosis by activating the ATM protein, and that the presence of the XPC protein is essential for recruiting the ATM protein to the DNA template.

  12. 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

  13. 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.

  14. 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.

  15. Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis.

    Science.gov (United States)

    Zhang, Nan; Schäfer, Jorrit; Sharma, Amit; Rayner, Lucy; Zhang, Xiaodong; Tuma, Roman; Stockley, Peter; Buck, Martin

    2015-11-06

    In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ(70)-dependent and the contrasting σ(54)-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ(54)-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ(70)-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ(54)-regulated promoters. Strikingly, removal of the σ(54) Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Effect of active-site mutation at Asn67 on the proton transfer mechanism of human carbonic anhydrase II.

    Science.gov (United States)

    Maupin, C Mark; Zheng, Jiayin; Tu, Chingkuang; McKenna, Robert; Silverman, David N; Voth, Gregory A

    2009-08-25

    The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonic anhydrase II (HCA II) has been examined by kinetic, X-ray, and simulation approaches. The X-ray crystallography studies, which were previously reported, and molecular dynamics (MD) simulations indicate that the proton shuttling residue, His64, predominantly resides in the outward orientation with a significant disruption of the ordered water in the active site for the dehydration pathway. While disorder is seen in the active-site water, water cluster analysis indicates that the N67L mutant may form water clusters similar to those seen in the wild-type (WT). For the hydration pathway of the enzyme, the active site water cluster analysis reveals an inability of the N67L mutant to stabilize water clusters when His64 is in the inward orientation, thereby favoring PT when His64 is in the outward orientation. The preference of the N67L mutant to carry out the PT when His64 is in the outward orientation for both the hydration and dehydration pathway is reasoned to be the main cause of the observed reduction in the overall rate. To probe the mechanism of PT, solvent H/D kinetic isotope effects (KIEs) were experimentally studied with catalysis measured by the exchange of (18)O between CO(2) and water. The values obtained from the KIEs were determined as a function of the deuterium content of solvent, using the proton inventory method. No differences were detected in the overarching mechanism of PT between WT and N67L HCA II, despite changes in the active-site water structure and/or the orientation of His64.

  17. PTEN mutation cooperates with EGFR activation in human glioblastoma cells to increase VEGF mRNA levels by transactivating an element in the proximal promoter

    International Nuclear Information System (INIS)

    Maity, A.; Pore, N.; Haas-Kogan, D.A.; O'Rourke, D.M.

    2001-01-01

    Purpose: Glioblastomas often express high levels of vascular endothelial growth factor (VEGF), even under normoxic conditions. Genetic alterations that commonly occur in these tumors, including PTEN mutations and epidermal growth factor receptor (EGFR) overexpression, may contribute to this increased VEGF expression. Our previous work showed that, compared to parental U87MG human glioblastoma cells, VEGF mRNA levels are decreased in U87/T691, a derivative line in which EGFR signaling is inhibited by introduction of a truncated p185Neu protein (Cancer Research 60: 5879-5886, 2000). In that study, we also showed that the effect of EGFR activation on VEGF was mediated at the level of transcription via a PI3K dependent pathway. The purpose of the current study was to assess the role of PTEN, a negative regulator of PI3K, and its interaction with EGFR activation in regulating VEGF expression. Materials and Methods: Protein lysates were obtained from U87MG and U87/T691 cells for use in Western blotting for phosphorylated Akt. U87MG and U87/T691 cells were infected with adenovirus expressing either wildtype PTEN or phosphatase dead PTEN, then RNA was harvested for Northern blotting for VEGF. Nuclear extracts were also obtained from these cells for use in gel shift assays. Luciferase assays were performed with lysates of U87MG cells transfected with luciferase reporter constructs containing fragments of the VEGF promoter. Results: The level of phosphorylated Akt, a marker for PI3K activation, was lower in U87/T691 cells compared to U87MG cells, an expected result because of inhibition of the EGFR in the former. Treatment of U87/T691 cells with the PI3K inhibitor LY294002 further decreased the level of phosphorylated Akt and VEGF mRNA. Therefore, in spite of EGFR inhibition, U87/T691 cells contain residual PI3K activity that regulates VEGF expression. To determine whether the PTEN status of these cells influences VEGF mRNA levels, wildtype PTEN was introduced into U87MG and

  18. Functional dissection of a trigger enzyme: mutations of the bacillus subtilis glutamate dehydrogenase RocG that affect differentially its catalytic activity and regulatory properties.

    Science.gov (United States)

    Gunka, Katrin; Newman, Joseph A; Commichau, Fabian M; Herzberg, Christina; Rodrigues, Cecilia; Hewitt, Lorraine; Lewis, Richard J; Stülke, Jörg

    2010-07-23

    Any signal transduction requires communication between a sensory component and an effector. Some enzymes engage in signal perception and transduction, as well as in catalysis, and these proteins are known as "trigger" enzymes. In this report, we detail the trigger properties of RocG, the glutamate dehydrogenase of Bacillus subtilis. RocG not only deaminates the key metabolite glutamate to form alpha-ketoglutarate but also interacts directly with GltC, a LysR-type transcription factor that regulates glutamate biosynthesis from alpha-ketoglutarate, thus linking the two metabolic pathways. We have isolated mutants of RocG that separate the two functions. Several mutations resulted in permanent inactivation of GltC as long as a source of glutamate was present. These RocG proteins have lost their ability to catabolize glutamate due to a strongly reduced affinity for glutamate. The second class of mutants is exemplified by the replacement of aspartate residue 122 by asparagine. This mutant protein has retained enzymatic activity but has lost the ability to control the activity of GltC. Crystal structures of glutamate dehydrogenases that permit a molecular explanation of the properties of the various mutants are presented. Specifically, we may propose that D122N replacement affects the surface of RocG. Our data provide evidence for a correlation between the enzymatic activity of RocG and its ability to inactivate GltC, and thus give insights into the mechanism that couples the enzymatic activity of a trigger enzyme to its regulatory function. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  19. Two Gαi1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity

    Science.gov (United States)

    ZIELINSKI, THOMAS; KIMPLE, ADAM J.; HUTSELL, STEPHANIE Q.; KOEFF, MARK D.; SIDEROVSKI, DAVID P.; LOWERY, ROBERT G.

    2009-01-01

    RGS proteins are critical modulators of G protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via “GTPase-accelerating protein” (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow GDP release from Gα and lack of solution-phase assays for detecting free GDP in the presence of excess GTP. To overcome these hurdles, we developed a Gαi1 mutant with increased GDP dissociation and decreased GTP hydrolysis, enabling detection of GAP activity using steady-state GTP hydrolysis. Gαi1(R178M/A326S) GTPase activity was stimulated 6~12 fold by RGS proteins known to act on Gαi subunits, and not affected by those unable to act on Gαi, demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. Gαi1(R178M/A326S) interacted with RGS proteins with expected binding specificity and affinities. To enable non-radioactive, homogenous detection of RGS protein effects on Gαi1(R178M/A326S), we developed a Transcreener® fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gαi1(R178M/A326S) with a homogenous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling. PMID:19820068

  20. The King's Return: The Mutation In Classical Division Of Powers By Judicialization Of Social Relations And Judicial Activism

    Directory of Open Access Journals (Sweden)

    Kelly de Souza Barbosa

    2017-02-01

    Full Text Available Separation of powers is one of strongest aspects of contemporary constitutionalism, mostly to rationalize the exercise of state power. In Brazil, the 1988 Constitution provides as entrenchment clause to tripartition of powers. However, there is a change in paradigms, especially at the level of constitutional jurisdiction, through the phenomena of judicialization and judicial activism because the intervention of the Judiciary in the primary functions of other powers. Using deductive and descriptive method, bibliographical and documentary research, we tried to point out the harms that the invasive action of the Judiciary causes to the functional balance between the powers and democracy.

  1. PF-114, a potent and selective inhibitor of native and mutated BCR/ABL is active against Philadelphia chromosome-positive (Ph+) leukemias harboring the T315I mutation.

    Science.gov (United States)

    Mian, A A; Rafiei, A; Haberbosch, I; Zeifman, A; Titov, I; Stroylov, V; Metodieva, A; Stroganov, O; Novikov, F; Brill, B; Chilov, G; Hoelzer, D; Ottmann, O G; Ruthardt, M

    2015-05-01

    Targeting BCR/ABL with tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Resistance attributable to either kinase mutations in BCR/ABL or nonmutational mechanisms remains the major clinical challenge. With the exception of ponatinib, all approved TKIs are unable to inhibit the 'gatekeeper' mutation T315I. However, a broad spectrum of kinase inhibition increases the off-target effects of TKIs and may be responsible for cardiovascular issues of ponatinib. Thus, there is a need for more selective options for the treatment of resistant Ph+ leukemias. PF-114 is a novel TKI developed with the specifications of (i) targeting T315I and other resistance mutations in BCR/ABL; (ii) achieving a high selectivity to improve safety; and (iii) overcoming nonmutational resistance in Ph+ leukemias. PF-114 inhibited BCR/ABL and clinically important mutants including T315I at nanomolar concentrations. It suppressed primary Ph+ acute lymphatic leukemia-derived long-term cultures that either displayed nonmutational resistance or harbor the T315I. In BCR/ABL- or BCR/ABL-T315I-driven murine leukemia as well as in xenograft models of primary Ph+ leukemia harboring the T315I, PF-114 significantly prolonged survival to a similar extent as ponatinib. Our work supports clinical evaluation of PF-114 for the treatment of resistant Ph+ leukemia.

  2. ampR Gene Mutations That Greatly Increase Class C β-Lactamase Activity in Enterobacter cloacae

    OpenAIRE

    Kuga, Akio; Okamoto, Ryoichi; Inoue, Matsuhisa

    2000-01-01

    The ampC and ampR genes of Enterobacter cloacae GN7471 were cloned into pMW218 to yield pKU403. Four mutant plasmids derived from pKU403 (pKU404, pKU405, pKU406, and pKU407) were isolated in an AmpD mutant of Escherichia coli ML4953 by selection with ceftazidime or aztreonam. The β-lactamase activities expressed by pKU404, pKU405, pKU406, and pKU407 were about 450, 75, 160, and 160 times higher, respectively, than that expressed by the original plasmid, pKU403. These mutant plasmids all carri...

  3. 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 ...

  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. Osteoprotegerin (OPG), The Endogenous Inhibitor of Receptor Activator of NF-κB Ligand (RANKL), is Dysregulated in BRCA Mutation Carriers

    Science.gov (United States)

    Widschwendter, Martin; Burnell, Matthew; Fraser, Lindsay; Rosenthal, Adam N.; Philpott, Sue; Reisel, Daniel; Dubeau, Louis; Cline, Mark; Pan, Yang; Yi, Ping-Cheng; Gareth Evans, D.; Jacobs, Ian J.; Menon, Usha; Wood, Charles E.; Dougall, William C.

    2015-01-01

    Breast cancer development in BRCA1/2 mutation carriers is a net consequence of cell-autonomous and cell nonautonomous factors which may serve as excellent targets for cancer prevention. In light of our previous data we sought to investigate the consequences of the BRCA-mutation carrier state on RANKL/osteoprotegerin (OPG) signalling. We analysed serum levels of RANKL, OPG, RANKL/OPG complex, oestradiol (E2), and progesterone (P) during menstrual cycle progression in 391 BRCA1/2-mutation carriers and 782 noncarriers. These studies were complemented by analyses of RANKL and OPG in the serum and mammary tissues of female cynomolgus macaques (n = 88) and serum RANKL and OPG in postmenopausal women (n = 150). BRCA-mutation carriers had lower mean values of free serum OPG in particular in BRCA1-mutation carriers (p = 0.018) compared with controls. Among BRCA1/2 mutation carriers, lower OPG levels were associated with germline mutation locations known to confer an increased breast cancer risk (p = 0.003). P is associated with low OPG levels in serum and tissue, particularly in BRCA-mutation carriers (rho = − 0.216; p = 0.002). Serum OPG levels were inversely correlated (rho = − 0.545, p < 0.001) with mammary epithelial proliferation measured by Ki67 expression and increased (p = 0.01) in postmenopause. The P–RANKL/OPG system is dysregulated in BRCA-mutation carriers. These and previously published data provide a strong rationale for further investigation of antiprogestogens or an anti-RANKL antibody such as denosumab for breast cancer prevention. PMID:26629528

  6. Mutating RBF can enhance its pro-apoptotic activity and uncovers a new role in tissue homeostasis.

    Directory of Open Access Journals (Sweden)

    Cécile Milet

    Full Text Available The tumor suppressor retinoblastoma protein (pRb is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD is found in the Drosophila homologue of pRb(RBF at a position similar to the site generating the p76Rb form in mammals. Thus, we generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBFD253A, conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. We show that this overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo.

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

    Science.gov (United States)

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. 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.

  9. 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

    2010-06-01

    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.

  10. The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage

    International Nuclear Information System (INIS)

    Yoon, Jung-Hoon; Qiu Junzhuan; Cai Sheng; Chen Yuan; Cheetham, Michael E.; Shen Binghui; Pfeifer, Gerd P.

    2006-01-01

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease

  11. Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis

    Science.gov (United States)

    Verhoeven, Maarten D.; Lee, Misun; Kamoen, Lycka; van den Broek, Marcel; Janssen, Dick B.; Daran, Jean-Marc G.; van Maris, Antonius J. A.; Pronk, Jack T.

    2017-01-01

    Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d-xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic growth on d-xylose. However, anaerobic growth required a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1, encoding a Golgi Ca2+/Mn2+ ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d-xylose. In pmr1 strains, intracellular Mn2+ concentrations were much higher than in the parental strain. XI activity assays in cell extracts and reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn2+ relative to other divalent metal ions. This study indicates engineering of metal homeostasis as a relevant approach for optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologous XIs as an underexplored aspect of engineering xylose metabolism in yeast. PMID:28401919

  12. 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

  13. Osteoprotegerin (OPG, The Endogenous Inhibitor of Receptor Activator of NF-κB Ligand (RANKL, is Dysregulated in BRCA Mutation Carriers

    Directory of Open Access Journals (Sweden)

    Martin Widschwendter

    2015-10-01

    The P–RANKL/OPG system is dysregulated in BRCA-mutation carriers. These and previously published data provide a strong rationale for further investigation of antiprogestogens or an anti-RANKL antibody such as denosumab for breast cancer prevention.

  14. 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.

  15. 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.

  16. Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities.

    Science.gov (United States)

    Uson, Maria Loressa; Carl, Ayala; Goldgur, Yehuda; Shuman, Stewart

    2018-04-09

    Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5' exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA's activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface β-loop that coordinates a fourth manganese ion (M4).

  17. Mapping Mutations on Phylogenies

    DEFF Research Database (Denmark)

    Nielsen, Rasmus

    2005-01-01

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

  18. 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.

  19. 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.

  20. Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline.

    Science.gov (United States)

    Giannoulatou, Eleni; McVean, Gilean; Taylor, Indira B; McGowan, Simon J; Maher, Geoffrey J; Iqbal, Zamin; Pfeifer, Susanne P; Turner, Isaac; Burkitt Wright, Emma M M; Shorto, Jennifer; Itani, Aysha; Turner, Karen; Gregory, Lorna; Buck, David; Rajpert-De Meyts, Ewa; Looijenga, Leendert H J; Kerr, Bronwyn; Wilkie, Andrew O M; Goriely, Anne

    2013-12-10

    The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline.

  1. Activity of dasatinib, a dual SRC/ABL kinase inhibitor, and IPI-504, a heat shock protein 90 inhibitor, against gastrointestinal stromal tumor-associated PDGFRAD842V mutation.

    Science.gov (United States)

    Dewaele, Barbara; Wasag, Bartosz; Cools, Jan; Sciot, Raf; Prenen, Hans; Vandenberghe, Peter; Wozniak, Agnieszka; Schöffski, Patrick; Marynen, Peter; Debiec-Rychter, Maria

    2008-09-15

    Activating mutations in platelet-derived growth factor receptor-alpha (PDGFRA) have been reported in approximately 5% to 10% of patients with gastrointestinal stromal tumors (GIST). Imatinib efficiently inhibits the juxtamembrane PDGFRA mutations, whereas many tyrosine kinase domain activation loop PDGFRA mutations confer primary resistance to imatinib. In this study, we compared the efficacy of second-line tyrosine kinase inhibitors such as dasatinib, sorafenib, and nilotinib against two GIST-related PDGFRA mutants, PDGFRA(D842V) and PDGFRA(DeltaDIM842-844). In addition, we sought to investigate the inhibitory effect of the heat shock protein 90 inhibitor, IPI-504, on these mutants. Primary imatinib-resistant tumor cells and cell lines expressing imatinib-resistant PDGFRA(D842V) or imatinib-sensitive PDGFRA(DeltaDIM842-844) mutants were treated with different concentrations of dasatinib, sorafenib, nilotinib, and IPI-504. The effect of treatment on proliferation, survival, and signaling was determined. All inhibitors tested exhibited a high efficacy toward the PDGFRA(DeltaDIM842-844) mutant. In contrast, ex vivo and in vitro assays revealed that only dasatinib potently inhibited the PDGFRA(D842V) isoform with an IC(50) value of 62 nmol/L. Sorafenib and nilotinib were significantly less efficacious against this mutation, inhibiting the PDGFRA kinase activity at >1,000 and >5,000 nmol/L, and suppressing the proliferation of the cells expressing the PDGFRA(D842V) mutant with an IC(50) value of 239 and 1,310 nmol/L, respectively. IPI-504 treatment potently inhibited PDGFRA kinase activity by inducing the degradation of PDGFRA(D842V) and PDGFRA(DeltaDIM842-844) at 256 and 182 nmol/L, respectively. Treatment with dasatinib or the heat shock protein 90 inhibitor IPI-504 may provide a therapeutic alternative for GIST patients whose tumors carry the imatinib-resistant PDGFRA(D842V) mutant isoform.

  2. Topoisomerase mutations that are associated with high-level resistance to earlier fluoroquinolones in Staphylococcus aureus have less effect on the antibacterial activity of besifloxacin.

    Science.gov (United States)

    Sanfilippo, Christine M; Hesje, Christine K; Haas, Wolfgang; Morris, Timothy W

    2011-01-01

    The impact of mutations in DNA gyrase and topoisomerase IV on minimum inhibitory concentrations (MICs) was investigated to better understand why besifloxacin has a higher potency against Staphylococcus aureus when compared to other fluoroquinolones, which was especially pronounced against ciprofloxacin-resistant isolates. MICs were determined for 52 clinical isolates against besifloxacin, moxifloxacin, gatifloxacin, ciprofloxacin, and levofloxacin. The genes encoding GyrA, GyrB, ParC, and ParE were sequenced and the potential impact of mutations assessed in light of recent structural data. For all fluoroquinolones tested, the MICs increased with the number of mutations in the quinolone resistance-determining regions. However, this increase was the smallest for besifloxacin and the largest for ciprofloxacin and levofloxacin. In addition to the commonly observed mutations in ParC and GyrA, more unusual mutations in ParE, such as Asp-432→His or Pro-585→Ser, were also detected. Compared to earlier fluoroquinolones, the higher potency of besifloxacin suggests that the drug's unique combination of a 7-azepinyl ring and an 8-chloro-substituent results in unique interactions with DNA gyrase and topoisomerase IV. Copyright © 2011 S. Karger AG, Basel.

  3. Human beta-galactosidase gene mutations in morquio B disease.

    Science.gov (United States)

    Oshima, A; Yoshida, K; Shimmoto, M; Fukuhara, Y; Sakuraba, H; Suzuki, Y

    1991-11-01

    Three different beta-galactosidase gene mutations--a 273Trp----Leu (mutation F) in both families, 482Arg----His (mutation G) in one family, and 509Trp----Cys (mutation H) in the other family--were identified in three patients with Morquio B disease who were from two unrelated families. Restriction-site analysis using StuI, Nsp(7524)I or RsaI confirmed these mutations. In human fibroblasts, mutation F expressed as much as 8% of the normal allele's enzyme activity, but the other mutations expressed no detectable enzyme activity. We conclude that the unique clinical manifestations are specifically associated with mutation F, a common two-base substitution, in this disease.

  4. Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a.

    Science.gov (United States)

    Hearn, Elizabeth M; Gray, Murray R; Foght, Julia M

    2006-01-01

    The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.

  5. UV Signature Mutations

    Science.gov (United States)

    2014-01-01

    Sequencing complete tumor genomes and exomes has sparked the cancer field's interest in mutation signatures for identifying the tumor's carcinogen. This review and meta-analysis discusses signatures and their proper use. We first distinguish between a mutagen's canonical mutations – deviations from a random distribution of base changes to create a pattern typical of that mutagen – and the subset of signature mutations, which are unique to that mutagen and permit inference backward from mutations to mutagen. To verify UV signature mutations, we assembled literature datasets on cells exposed to UVC, UVB, UVA, or solar simulator light (SSL) and tested canonical UV mutation features as criteria for clustering datasets. A confirmed UV signature was: ≥60% of mutations are C→T at a dipyrimidine site, with ≥5% CC→TT. Other canonical features such as a bias for mutations on the non-transcribed strand or at the 3' pyrimidine had limited application. The most robust classifier combined these features with criteria for the rarity of non-UV canonical mutations. In addition, several signatures proposed for specific UV wavelengths were limited to specific genes or species; non-signature mutations induced by UV may cause melanoma BRAF mutations; and the mutagen for sunlight-related skin neoplasms may vary between continents. PMID:25354245

  6. 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.

  7. 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

    . 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...

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    BACKGROUND: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to i...

  9. A Crohn's disease-associated NOD2 mutation suppresses transcription of human IL10 by inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1.

    NARCIS (Netherlands)

    Noguchi, E.; Homma, Y.; Kang, X.; Netea, M.G.; Ma, X.

    2009-01-01

    A common mutation in the gene encoding the cytoplasmic sensor Nod2, involving a frameshift insertion at nucleotide 3020 (3020insC), is strongly associated with Crohn's disease. How 3020insC contributes to this disease is a controversial issue. Clinical studies have identified defective production of

  10. Mutations affecting gyrase in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-11-01

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

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

    International Nuclear Information System (INIS)

    Hirano, Ken-ichi; Tanaka, Tatsuya; Ikeda, Yoshihiko; Yamaguchi, Satoshi; Zaima, Nobuhiro; Kobayashi, Kazuhiro; Suzuki, Akira; Sakata, Yasuhiko

    2014-01-01

    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

  12. 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

  13. Isocitrate dehydrogenase mutations in gliomas

    Science.gov (United States)

    Waitkus, Matthew S.; Diplas, Bill H.; Yan, Hai

    2016-01-01

    Over the last decade, extraordinary progress has been made in elucidating the underlying genetic causes of gliomas. In 2008, our understanding of glioma genetics was revolutionized when mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were identified in the vast majority of progressive gliomas and secondary glioblastomas (GBMs). IDH enzymes normally catalyze the decarboxylation of isocitrate to generate α-ketoglutarate (αKG), but recurrent mutations at Arg132 of IDH1 and Arg172 of IDH2 confer a neomorphic enzyme activity that catalyzes reduction of αKG into the putative oncometabolite D-2-hydroxyglutate (D2HG). D2HG inhibits αKG-dependent dioxygenases and is thought to create a cellular state permissive to malignant transformation by altering cellular epigenetics and blocking normal differentiation processes. Herein, we discuss the relevant literature on mechanistic studies of IDH1/2 mutations in gliomas, and we review the potential impact of IDH1/2 mutations on molecular classification and glioma therapy. PMID:26188014

  14. Better plants through mutations

    International Nuclear Information System (INIS)

    1988-01-01

    This is a public relations film describing problems associated with the genetic improvement of crop plants through induced mutations. Mutations are the ultimate source of genetic variation in plants. Mutation induction is now established as a practical tool in plant breeding. The Joint FAO/IAEA Division and the IAEA's laboratory at Seibersdorf have supported research and practical implementation of mutation breeding of both seed propagated and vegetatively propagated plants. Plant biotechnology based on in vitro culture and recombinant DNA technology will make a further significant contribution to plant breeding

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    BACKGROUND: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study...... was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only...... a nonspecific but also a specific pharmacogenetic risk factor for anesthesia. METHODS: The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp...

  16. Late onset of neutral lipid storage disease due to novel PNPLA2 mutations causing total loss of lipase activity in a patient with myopathy and slight cardiac involvement.

    Science.gov (United States)

    Missaglia, Sara; Maggi, Lorenzo; Mora, Marina; Gibertini, Sara; Blasevich, Flavia; Agostoni, Piergiuseppe; Moro, Laura; Cassandrini, Denise; Santorelli, Filippo Maria; Gerevini, Simonetta; Tavian, Daniela

    2017-05-01

    Neutral lipid storage disease with myopathy (NLSDM) presents with skeletal muscle myopathy and severe dilated cardiomyopathy in nearly 40% of cases. NLSDM is caused by mutations in the PNPLA2 gene, which encodes the adipose triglyceride lipase (ATGL). Here we report clinical and genetic findings of a patient carrying two novel PNPLA2 mutations (c.696+4A>G and c.553_565delGTCCCCCTTCTCG). She presented at age 39 with right upper limb abduction weakness slowly progressing over the years with asymmetric involvement of proximal upper and lower limb muscles. Cardiological evaluation through ECG and heart echo scan was normal until the age 53, when mild left ventricular diastolic dysfunction was detected. Molecular analysis revealed that only one type of PNPLA2 transcript, with exon 5 skipping, was expressed in patient cells. Such aberrant mRNA causes the production of a shorter ATGL protein, lacking part of the catalytic domain. This is an intriguing case, displaying severe PNPLA2 mutations with clinical presentation characterized by slight cardiac impairment and full expression of severe asymmetric myopathy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Mutations in ARSB in MPS VI patients in India

    Directory of Open Access Journals (Sweden)

    Juby Mathew

    2015-09-01

    Full Text Available Mucopolysaccharidosis VI (MPS VI is an autosomal recessive inborn error of metabolism caused by mutations in the arylsulfatase B gene (ARSB and consequent deficient activity of ARSB, a lysosomal enzyme. We present here the results of a study undertaken to identify the mutations in ARSB in MPS VI patients in India. Around 160 ARSB mutations, of which just 4 are from India, have been reported in the literature. Our study covered nine MPS VI patients from eight families. Both familial mutations were found in seven families, and only one mutation was found in one family. Seven mutations were found — four novel (p.G38_G40del3, p.C91R, p.L98R and p.R315P, two previously reported from India (p.D53N and p.W450C, and one reported from outside India (p.R160Q. One mutation, p.W450C, was present in two families, and the other six mutations were present in one family each. Analysis of the molecular structure of the enzyme revealed that most of these mutations either cause loss of an active site residue or destabilize the structure of the enzyme. The only previous study on mutations in ARSB in Indian MPS VI patients, by Kantaputra et al. 2014 [1], reported four novel mutations of which two (p.D53N and p.W450C were found in our study as well. Till date, nine mutations have been reported from India, through our study and the Kantaputra study. Eight out of these nine mutations have been found only in India. This suggests that the population studied by us might have its own typical set of mutations, with other populations equally likely to have their own set of mutations.

  18. Mutational robustness of gene regulatory networks.

    Directory of Open Access Journals (Sweden)

    Aalt D J van Dijk

    Full Text Available Mutational robustness of gene regulatory networks refers to their ability to generate constant biological output upon mutations that change network structure. Such networks contain regulatory interactions (transcription factor-target gene interactions but often also protein-protein interactions between transcription factors. Using computational modeling, we study factors that influence robustness and we infer several network properties governing it. These include the type of mutation, i.e. whether a regulatory interaction or a protein-protein interaction is mutated, and in the case of mutation of a regulatory interaction, the sign of the interaction (activating vs. repressive. In addition, we analyze the effect of combinations of mutations and we compare networks containing monomeric with those containing dimeric transcription factors. Our results are consistent with available data on biological networks, for example based on evolutionary conservation of network features. As a novel and remarkable property, we predict that networks are more robust against mutations in monomer than in dimer transcription factors, a prediction for which analysis of conservation of DNA binding residues in monomeric vs. dimeric transcription factors provides indirect evidence.

  19. Hepatosplenomegalic lipidosis: what unless Gaucher? Adult cholesteryl ester storage disease (CESD) with anemia, mesenteric lipodystrophy, increased plasma chitotriosidase activity and a homozygous lysosomal acid lipase -1 exon 8 splice junction mutation.

    Science.gov (United States)

    vom Dahl, S; Harzer, K; Rolfs, A; Albrecht, B; Niederau, C; Vogt, C; van Weely, S; Aerts, J; Müller, G; Häussinger, D

    1999-10-01

    A 36-year-old woman was admitted for hepatosplenomegaly and anemia. Bone marrow cytology showed "sea-blue histiocytes", vacuolated macrophages and plasma cells. As primary liver disease, malignancy or hematologic disorders were excluded, and plasma chitotriosidase activity was increased 27-fold over control, the presence of a lysosomal storage disease was suspected. Biochemical analysis of skin fibroblasts revealed normal glucocerebrosidase and sphingomyelinase activity, but lipid analysis showed a more than 15-fold accumulation of cholesterol esters within the cells. The activity of lysosomal acid lipase (LAL) in fibroblast homogenates was decreased to 12% of control subjects. Mutational analysis of the patient's blood showed the homozygous G-->A mutation at position -1 of the exon 8 splice donor site (E8SJM-allele) known for adult cholesteryl ester storage disease (CESD); the polymorphic background was that of the complex haplotype -6Thr, 2Gly, 894 G-->A. Based on clinical, laboratory, cytological and and biochemical findings, CESD can clearly be separated from other more frequent inherited lysosomal storage diseases, e.g. atypical forms of Gaucher disease.

  20. RET haplotype, not linked to the C620R activating mutation, associated with Hirschsprung disease in a novel MEN2 family

    Directory of Open Access Journals (Sweden)

    Elisangela P. S. Quedas

    2012-01-01

    Full Text Available Hirschsprung disease is a congenital form of aganglionic megacolon that results from cristopathy. Hirschsprung disease usually occurs as a sporadic disease, although it may be associated with several inherited conditions, such as multiple endocrine neoplasia type 2. The rearranged during transfection (RET proto-oncogene is the major susceptibility gene for Hirschsprung disease, and germline mutations in RET have been reported in up to 50% of the inherited forms of Hirschsprung disease and in 15-20% of sporadic cases of Hirschsprung disease. The prevalence of Hirschsprung disease in multiple endocrine neoplasia type 2 cases was recently determined to be 7.5% and the cooccurrence of Hirschsprung disease and multiple endocrine neoplasia type 2 has been reported in at least 22 families so far. It was initially thought that Hirschsprung disease could be due to disturbances in apoptosis or due to a tendency of the mutated RET receptor to be retained in the Golgi apparatus. Presently, there is strong evidence favoring the hypothesis that specific inactivating haplotypes play a key role in the fetal development of congenital megacolon/Hirschsprung disease. In the present study, we report the genetic findings in a novel family with multiple endocrine neoplasia type 2: a specific RET haplotype was documented in patients with Hirschsprung disease associated with medullary thyroid carcinoma, but it was absent in patients with only medullary thyroid carcinoma. Despite the limited number of cases, the present data favor the hypothesis that specific haplotypes not linked to RET germline mutations are the genetic causes of Hirschsprung disease.

  1. Splice, insertion-deletion and nonsense mutations that perturb the phenylalanine hydroxylase transcript cause phenylketonuria in India.

    Science.gov (United States)

    Bashyam, Murali D; Chaudhary, Ajay K; Kiran, Manjari; Nagarajaram, Hampapathalu A; Devi, Radha Rama; Ranganath, Prajnya; Dalal, Ashwin; Bashyam, Leena; Gupta, Neerja; Kabra, Madhulika; Muranjan, Mamta; Puri, Ratna D; Verma, Ishwar C; Nampoothiri, Sheela; Kadandale, Jayarama S

    2014-03-01

    Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutational inactivation of the phenylalanine hydroxylase (PAH) gene. Missense mutations are the most common PAH mutation type detected in PKU patients worldwide. We performed PAH mutation analysis in 27 suspected Indian PKU families (including 7 from our previous study) followed by structure and function analysis of specific missense and splice/insertion-deletion/nonsense mutations, respectively. Of the 27 families, disease-causing mutations were detected in 25. A total of 20 different mutations were identified of which 7 "unique" mutations accounted for 13 of 25 mutation positive families. The unique mutations detected exclusively in Indian PKU patients included three recurrent mutations detected in three families each. The 20 mutations included only 5 missense mutations in addition to 5 splice, 4 each nonsense and insertion-deletion mutations, a silent variant in coding region and a 3'UTR mutation. One deletion and two nonsense mutations were characterized to confirm significant reduction in mutant transcript levels possibly through activation of nonsense mediated decay. All missense mutations affected conserved amino acid residues and sequence and structure analysis suggested significant perturbations in the enzyme activity of respective mutant proteins. This is probably the first report of identification of a significantly low proportion of missense PAH mutations from PKU families and together with the presence of a high proportion of splice, insertion-deletion, and nonsense mutations, points to a unique PAH mutation profile in Indian PKU patients. © 2013 Wiley Periodicals, Inc.

  2. Novel missense MTTP gene mutations causing abetalipoproteinemia.

    Science.gov (United States)

    Miller, Sharon A; Burnett, John R; Leonis, Mike A; McKnight, C James; van Bockxmeer, Frank M; Hooper, Amanda J

    2014-10-01

    The microsomal triglyceride transfer protein (MTTP) plays a critical role in the formation of hepatic very low density lipoprotein. Abetalipoproteinemia (ABL) is a rare, naturally occurring extreme form of MTTP inhibition, which is characterized by the virtual absence of apolipoprotein (apo) B-containing lipoproteins in blood. The goal of this study was to examine the effect that four novel MTTP missense mutations had on protein interactions, expression and lipid-transfer activity, and to determine which mutations were responsible for the ABL phenotype observed in two patients. In two patients with ABL, we identified in MTTP a novel frameshift mutation (K35Ffs*37), and four novel missense mutations, namely, G264R, Y528H, R540C, and N649S. When transiently expressed in COS-7 cells, all missense MTTP mutations interacted with apoB17, apoB48, and protein disulfide isomerase. Mutations Y528H and R540C, however, displayed negligible levels of MTTP activity and N649S displayed a partial reduction relative to the wild-type MTTP. In contrast, G264R retained full lipid-transfer activity. These studies indicate that missense mutations Y528H, R540C, and N649S appear to cause ABL by reducing MTTP activity rather than by reducing binding of MTTP with protein disulfide isomerase or apoB. The region of MTTP containing amino acids 528 and 540 constitutes a critical domain for its lipid-transfer activity. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Molecular evaluation of a novel missense mutation & an insertional truncating mutation in SUMF1 gene

    Directory of Open Access Journals (Sweden)

    Udhaya H Kotecha

    2014-01-01

    Full Text Available Background & objectives: Multiple suphphatase deficiency (MSD is an autosomal recessive disorder affecting the post translational activation of all enzymes of the sulphatase family. To date, approximately 30 different mutations have been identified in the causative gene, sulfatase modifying factor 1 (SUMF1. We describe here the mutation analysis of a case of MSD. Methods: The proband was a four year old boy with developmental delay followed by neuroregression. He had coarse facies, appendicular hypertonia, truncal ataxia and ichthyosis limited to both lower limbs. Radiographs showed dysostosis multiplex. Clinical suspicion of MSD was confirmed by enzyme analysis of four enzymes of the sulphatase group. Results: The patient was compound heterozygote for a c.451A>G (p.K151E substitution in exon 3 and a single base insertion mutation (c.690_691 InsT in exon 5 in the SUMF1 gene. The bioinformatic analysis of the missense mutation revealed no apparent effect on the overall structure. However, the mutated 151-amino acid residue was found to be adjacent to the substrate binding and the active site residues, thereby affecting the substrate binding and/or catalytic activity, resulting in almost complete loss of enzyme function. Conclusions: The two mutations identified in the present case were novel. This is perhaps the first report of an insertion mutation in SUMF1 causing premature truncation of the protein.

  4. High rates of human immunodeficiency virus type 1 mutational profiles by single-genome amplification after 48-hour propagation in peripheral blood mononuclear cells at different levels of cell activation.

    Science.gov (United States)

    Russo, Cristiano Teodoro; Alkmim, Wagner; Munerato, Patricia; Zukurov, Jean; Maricato, Juliana T; Sucupira, M Cecília; Diaz, Ricardo S; Janini, Luiz Mário

    2014-01-01

    Human immunodeficiency virus type 1 (HIV-1) genetic diversity is one of the most important features of HIV-1 infections and the result of error accumulation during reverse transcription and of high viral turnover. HIV-1 reverse transcription is influenced by factors such as the level of nucleotides and/or the cellular activation state. HIV-1 diversity was investigated after 48 h of viral propagation in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors in three different cell culture conditions: (1) resting PBMCs, (2) simultaneous infection and PBMC activation, and (3) PBMC activation 72 h before infection. Cellular DNA was extracted and proviruses of each culture condition were amplified. Single-genome PCR clones were obtained and the protease and reverse transcriptase of the pol gene were sequenced. An elevated number of nucleotide substitutions in all three culture conditions were observed. In condition 1, the mutational rate observed ranged from 1.0 × 10(-3) to 2.1 × 10(-2), the genetic diversity was 0.6%, and hypermutation was observed in 7.1% of sequenced clones. In condition 2, the mutational rate ranged from 1.0 × 10(-3) to 1.0 × 10(-2), the genetic diversity was 0.8%, and hypermutation affected 6.7% of clones. In condition 3, the mutational rate ranged from 2.8 × 10(-3) to 1.1 × 10(-2), the genetic diversity was 1%, and 5.9% of clones were hypermutated. Substitutions occurred more frequently in some specific nucleotide stretches, and a common pattern for substitutions in all the different conditions was identified. There was a significant accumulation of mutations during the initial periods of in vitro HIV-1 propagation irrespective of culture conditions. The rapid accumulation of virus diversity might represent a viral strategy when colonizing new hosts. Complementary studies are necessary to allow for a better understanding of the initial periods of infection, which represent a crucial event related to disease progression.

  5. Antitumor activity and safety of the PARP inhibitor rucaparib in patients with high-grade ovarian carcinoma and a germline or somatic BRCA1 or BRCA2 mutation: Integrated analysis of data from Study 10 and ARIEL2.

    Science.gov (United States)

    Oza, Amit M; Tinker, Anna V; Oaknin, Ana; Shapira-Frommer, Ronnie; McNeish, Iain A; Swisher, Elizabeth M; Ray-Coquard, Isabelle; Bell-McGuinn, Katherine; Coleman, Robert L; O'Malley, David M; Leary, Alexandra; Chen, Lee-May; Provencher, Diane; Ma, Ling; Brenton, James D; Konecny, Gottfried E; Castro, Cesar M; Giordano, Heidi; Maloney, Lara; Goble, Sandra; Lin, Kevin K; Sun, James; Raponi, Mitch; Rolfe, Lindsey; Kristeleit, Rebecca S

    2017-11-01

    An integrated analysis was undertaken to characterize the antitumor activity and safety profile of the oral poly(ADP-ribose) polymerase inhibitor rucaparib in patients with relapsed high-grade ovarian carcinoma (HGOC). Eligible patients from Study 10 (NCT01482715) and ARIEL2 (NCT01891344) who received a starting dose of oral rucaparib 600mg twice daily (BID) with or without food were included in these analyses. The integrated efficacy population included patients with HGOC and a deleterious germline or somatic BRCA1 or BRCA2 (BRCA1/2) mutation who received at least two prior chemotherapies and were sensitive, resistant, or refractory to platinum-based chemotherapy. The primary endpoint was investigator-assessed confirmed objective response rate (ORR). Secondary endpoints included duration of response (DOR) and progression-free survival (PFS). The integrated safety population included patients with HGOC who received at least one dose of rucaparib 600mg BID, irrespective of BRCA1/2 mutation status and prior treatments. In the efficacy population (n=106), ORR was 53.8% (95% confidence interval [CI], 43.8-63.5); 8.5% and 45.3% of patients achieved complete and partial responses, respectively. Median DOR was 9.2months (95% CI, 6.6-11.6). In the safety population (n=377), the most frequent treatment-emergent adverse events (AEs) were nausea, asthenia/fatigue, vomiting, and anemia/hemoglobin decreased. The most common grade ≥3 treatment-emergent AE was anemia/hemoglobin decreased. Treatment-emergent AEs led to treatment interruption, dose reduction, and treatment discontinuation in 58.6%, 45.9%, and 9.8% of patients, respectively. No treatment-related deaths occurred. Rucaparib has antitumor activity in advanced BRCA1/2-mutated HGOC and a manageable safety profile. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. IDH1 and IDH2 Mutations in Gliomas

    Science.gov (United States)

    Cohen, Adam; Holmen, Sheri; Colman, Howard

    2014-01-01

    Mutations in isocitrate dehydrogenase (IDH) 1 and 2, originally discovered in 2009, occur in the vast majority of low grade gliomas and secondary high grade gliomas. These mutations, which occur early in gliomagenesis, change the function of the enzymes, causing them to produce 2-hydroxyglutarate, a possible oncometabolite, and to not produce NADPH. IDH mutations are oncogenic, although whether the mechanism is through alterations in hydroxylases, redox potential, cellular metabolism, or gene expression is not clear. The mutations also drive increased methylation in gliomas. Gliomas with mutated IDH1 and IDH2 have improved prognosis compared to gliomas with wild-type IDH. Mutated IDH can now be detected by immunohistochemistry and magnetic resonance spectroscopy. No drugs currently target mutated IDH, although this remains an area of active research. PMID:23532369

  7. [Study of gene mutation in 62 hemophilia A children].

    Science.gov (United States)

    Hu, Q; Liu, A G; Zhang, L Q; Zhang, A; Wang, Y Q; Wang, S M; Lu, Y J; Wang, X

    2017-11-02

    Objective: To analyze the mutation type of FⅧ gene in children with hemophilia A and to explore the relationship among hemophilia gene mutation spectrum, gene mutation and clinical phenotype. Method: Sixty-two children with hemophilia A from Department of Pediatric Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology between January 2015 and March 2017 were enrolled. All patients were male, aged from 4 months to 7 years and F Ⅷ activity ranged 0.2%-11.0%. Fifty cases had severe, 10 cases had moderate and 2 cases had mild hemophilia A. DNA was isolated from peripheral blood in hemophilia A children and the target gene fragment was amplified by PCR, in combination with the second generation sequencing, 22 and 1 introns were detected. Negative cases were detected by the second generation sequencing and results were compared with those of the international FⅧ gene mutation database. Result: There were 20 cases (32%) of intron 22 inversion, 2 cases (3%) of intron 1 inversion, 18 cases (29%) of missense mutation, 5 cases (8%) of nonsense mutation, 7 cases (11%) of deletion mutation, 1 case(2%)of splice site mutation, 2 cases (3%) of large fragment deletion and 1 case of insertion mutation (2%). No mutation was detected in 2 cases (3%), and 4 cases (7%) failed to amplify. The correlation between phenotype and genotype showed that the most common gene mutation in severe hemophilia A was intron 22 inversion (20 cases), accounting for 40% of severe patients, followed by 11 cases of missense mutation (22%). The most common mutation in moderate hemophilia A was missense mutation (6 cases), accounting for 60% of moderate patients. Conclusion: The most frequent mutation type in hemophilia A was intron 22 inversion, followed by missense mutation, again for missing mutation. The relationship between phenotype and genotype: the most frequent gene mutation in severe hemophilia A is intron 22 inversion, followed by missense

  8. Induced mutation of soy by ionization mutation

    Energy Technology Data Exchange (ETDEWEB)

    Li, C.L.; Hsu, H.L.

    1975-09-01

    This article presents the results of experiments dealing with how 14 different doses of three types of ionization irradiation-roentgen rays, /sup 60/Co gamma rays, and thermal neutrons affect mutation of 14 types of soy beans and their hybrids. It was learned that with an increased dose the coefficient of seed germination decreases, the cotyledon becomes increasingly thicker, shoots develop more and more slowly, various deformities arise in the stalk, and fertility decreases. As far as M/sub 2/ mutation is concerned, a great variety has been discovered with regard to the height of the stem, the leaf formation, the color of the bloom, the color of the edge, the characteristics of the pod, the size of the seed and the color of the cicatrix. At the same time some specific characteristics having an important economic significance are being revealed, as for example, dwarf stems, the ability to withstand lodging, great pod density, increased inflorescence and short sprouts.

  9. Mutations in GABRB3

    DEFF Research Database (Denmark)

    Møller, Rikke S; Wuttke, Thomas V; Helbig, Ingo

    2017-01-01

    OBJECTIVE: To examine the role of mutations in GABRB3 encoding the β3 subunit of the GABAA receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes. METHODS: We performed massive parallel sequencing...... of GABRB3 in 416 patients with a range of epileptic encephalopathies and childhood-onset epilepsies and recruited additional patients with epilepsy with GABRB3 mutations from other research and diagnostic programs. RESULTS: We identified 22 patients with heterozygous mutations in GABRB3, including 3...... probands from multiplex families. The phenotypic spectrum of the mutation carriers ranged from simple febrile seizures, genetic epilepsies with febrile seizures plus, and epilepsy with myoclonic-atonic seizures to West syndrome and other types of severe, early-onset epileptic encephalopathies...

  10. Mutation breeding in peas

    International Nuclear Information System (INIS)

    Jaranowski, J.; Micke, A.

    1985-01-01

    The pea as an ancient crop plant still today has wide uses and is an import source of food protein. It is also an important object for genetic studies and as such has been widely used in mutation induction experiments. However, in comparison with cereals this ancient crop plant (like several other grain legumes) has gained relatively little from advances in breeding. The review focuses on the prospects of genetic improvement of pea by induced mutations, discusses principles and gives methodological information. (author)

  11. Actionable mutations in canine hemangiosarcoma.

    Directory of Open Access Journals (Sweden)

    Guannan Wang

    Full Text Available Angiosarcomas (AS are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA, that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma.Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3 demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage.Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

  12. Actionable mutations in canine hemangiosarcoma.

    Science.gov (United States)

    Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A; Wojcik, John; Durham, Amy C; Mason, Nicola J; Roth, David B

    2017-01-01

    Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

  13. South African Medical Journal - Vol 102, No 7 (2012)

    African Journals Online (AJOL)

    Confirmation of the recurrent ACVR1 617G>A mutation in South Africans with fibrodysplasia ossificans progressiva · EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. Collet Dandara, Chris Scott, Mike Urban, Karen Fieggen, Regan Arendse, Peter Beighton ...

  14. Identification of new bacteria harboring qnrS and aac(6')-Ib/cr and mutations possibly involved in fluoroquinolone resistance in raw sewage and activated sludge samples from a full-scale WWTP.

    Science.gov (United States)

    Paiva, Magna C; Reis, Mariana P; Costa, Patrícia S; Dias, Marcela F; Bleicher, Lucas; Scholte, Larissa L S; Nardi, Regina M D; Nascimento, Andréa M A

    2017-03-01

    Wastewater treatment plants (WWTPs) harbor bacteria and antimicrobial resistance genes, favoring gene exchange events and resistance dissemination. Here, a culture-based and metagenomic survey of qnrA, qnrB, qnrS, and aac(6')-Ib genes from raw sewage (RS) and activated sludge (AS) of a full-scale municipal WWTP was performed. A total of 96 bacterial isolates were recovered from nalidixic acid-enrichment cultures. Bacteria harboring the aac(6')-Ib gene predominated in RS, whereas qnrS-positive isolates were specific to AS. Novel qnrS- and aac(6')-Ib-cr positive species were identified: Morganella morganii, Providencia rettgeri, and Pseudomonas guangdongensis (qnrS), and Alcaligenes faecalis and P. rettgeri (aac(6')-Ib-cr). Analysis of qnrS and aac(6')-Ib sequences from isolates and clone libraries suggested that the diversity of qnrS is wider than that of aac(6')-Ib. A large number of amino acid mutations were observed in the QnrS and AAC(6')-Ib proteins at previously undetected positions, whose structural implications are not clear. An accumulation of mutations at the C72, Q73, L74, A75 and M76 positions of QnrS, and D181 of AAC(6')-Ib might be important for resistance. These findings add significant information on bacteria harboring qnrS and aac(6')-Ib genes, and the presence of novel mutations that may eventually emerge in clinical isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. An Activity-Staining Method on Filtration Paper Enables High-Throughput Screening of Temperature-Sensitive and Inactive Mutations of Rice α-Amylase for Improvement of Rice Grain Quality.

    Science.gov (United States)

    Yamakawa, Hiromoto; Hirai-Kimura, Rieko; Nakata, Yuriko; Nakata, Masaru; Kuroda, Masaharu; Yamaguchi, Takeshi

    2017-04-01

    α-Amylase is a starch-hydrolyzing enzyme (EC 3.2.1.1) indispensable for germination of cereal seeds, but it is also expressed during the ripening stage. Previous studies demonstrated that the enzyme is activated in developing rice seeds under extremely hot weather and triggers a loss of grain quality by hindering the accumulation of storage starch in the endosperm. Since inactive or, preferably, heat-labile α-amylases are preferable for breeding premium rice, we developed a method for rapid screening of inactive and temperature-sensitive mutants of the enzyme by combining the random mutagenesis by error-prone PCR and an on-filter activity test of the recombinant enzyme expressed by Escherichia coli. This technique was applied to a major α-amylase in the developing seed, Amy3D, and the activity of the isolated mutant enzymes was verified with both the bacteria-expressed recombinant proteins and the extract from the endosperm overexpressing each of them. Then, we identified several substitutions leading to loss of the activity of amino acid residues (Leu28, Asp112, Cys149, Trp201, Asp204, Gly295, Leu300 and Cys342), as well as a variety of heat-sensitive substitutions of Asp83, Asp187 and Glu252. Furthermore, variations of the heat-labile enzymes were created by combining these heat-sensitive mutations. The effects of the respective mutations and their relationship to the structure of the enzyme molecule are discussed. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Heterogeneous ethnic distribution of the factor v leiden mutation

    OpenAIRE

    Franco, Rendrik F.; Elion, Jacques; Santos, Sidney E.B.; Araújo, Amélia G.; Tavella, Marli H.; Zago, Marco A.

    1999-01-01

    Inherited resistance to activated protein C caused by the factor V Leiden (FVL) mutation is the most common genetic cause of venous thrombosis yet described, being found in 20-60% of patients with venous thrombophilia. A relationship between the FVL mutation and an increased predisposition to arterial thrombosis in young women was recently reported. We assessed the prevalence of the FVL mutation in 440 individuals (880 chromosomes) belonging to four different ethnic groups: Caucasians, Africa...

  17. Carcinogenic oestrogens induce respiration deficiency mutation in yeast

    OpenAIRE

    Stopper, Helga; Metzler, M.

    2012-01-01

    In addition to hormonal activity, genetic darnage has been proposed as an important factor in oestrogen-mediated carcinogenesis. However, as short-term tests for oestrogens usually fail to show DNA mutations, lesions other than dassie nuclear DNA mutation have to be considered. Oestrogeninduced mitochondrial darnage was studied in the yeast Saccharomyces cerevisiae. Stilbene-type, but not steroidal, oestrogens were found to induce respiration-dcficient petite mutation. The effect was inversel...

  18. PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers.

    Directory of Open Access Journals (Sweden)

    Filip Janku

    Full Text Available Oncogenic mutations of PIK3CA, RAS (KRAS, NRAS, and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.Tumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS, and BRAF mutations using polymerase chain reaction-based DNA sequencing.PIK3CA mutations were found in 54 (11% of 504 patients tested; KRAS in 69 (19% of 367; NRAS in 19 (8% of 225; and BRAF in 31 (9% of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%, uterine (7/28, 25%, breast (6/29, 21%, and colorectal cancers (18/105, 17%; KRAS in pancreatic (5/9, 56%, colorectal (49/97, 51%, and uterine cancers (3/20, 15%; NRAS in melanoma (12/40, 30%, and uterine cancer (2/11, 18%; BRAF in melanoma (23/52, 44%, and colorectal cancer (5/88, 6%. Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wtPIK3CA (p = 0.001. In total, RAS (KRAS, NRAS or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001. PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001 and in 20% of patients with RAS (KRAS, NRAS or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS or wtBRAF (p = 0.001.PIK3CA, RAS (KRAS, NRAS, and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS and BRAF mutations.

  19. Statistical method on nonrandom clustering with application to somatic mutations in cancer

    Directory of Open Access Journals (Sweden)

    Rejto Paul A

    2010-01-01

    Full Text Available Abstract Background Human cancer is caused by the accumulation of tumor-specific mutations in oncogenes and tumor suppressors that confer a selective growth advantage to cells. As a consequence of genomic instability and high levels of proliferation, many passenger mutations that do not contribute to the cancer phenotype arise alongside mutations that drive oncogenesis. While several approaches have been developed to separate driver mutations from passengers, few approaches can specifically identify activating driver mutations in oncogenes, which are more amenable for pharmacological intervention. Results We propose a new statistical method for detecting activating mutations in cancer by identifying nonrandom clusters of amino acid mutations in protein sequences. A probability model is derived using order statistics assuming that the location of amino acid mutations on a protein follows a uniform distribution. Our statistical measure is the differences between pair-wise order statistics, which is equivalent to the size of an amino acid mutation cluster, and the probabilities are derived from exact and approximate distributions of the statistical measure. Using data in the Catalog of Somatic Mutations in Cancer (COSMIC database, we have demonstrated that our method detects well-known clusters of activating mutations in KRAS, BRAF, PI3K, and β-catenin. The method can also identify new cancer targets as well as gain-of-function mutations in tumor suppressors. Conclusions Our proposed method is useful to discover activating driver mutations in cancer by identifying nonrandom clusters of somatic amino acid mutations in protein sequences.

  20. Mutational spectrum drives the rise of mutator bacteria.

    Science.gov (United States)

    Couce, Alejandro; Guelfo, Javier R; Blázquez, Jesús

    2013-01-01

    Understanding how mutator strains emerge in bacterial populations is relevant both to evolutionary theory and to reduce the threat they pose in clinical settings. The rise of mutator alleles is understood as a result of their hitchhiking with linked beneficial mutations, although the factors that govern this process remain unclear. A prominent but underappreciated fact is that each mutator allele increases only a specific spectrum of mutational changes. This spectrum has been speculated to alter the distribution of fitness effects of beneficial mutations, potentially affecting hitchhiking. To study this possibility, we analyzed the fitness distribution of beneficial mutations generated from different mutator and wild-type Escherichia coli strains. Using antibiotic resistance as a model system, we show that mutational spectra can alter these distributions substantially, ultimately determining the competitive ability of each strain across environments. Computer simulation showed that the effect of mutational spectrum on hitchhiking dynamics follows a non-linear function, implying that even slight spectrum-dependent fitness differences are sufficient to alter mutator success frequency by several orders of magnitude. These results indicate an unanticipated central role for the mutational spectrum in the evolution of bacterial mutation rates. At a practical level, this study indicates that knowledge of the molecular details of resistance determinants is crucial for minimizing mutator evolution during antibiotic therapy.

  1. Mutational spectrum drives the rise of mutator bacteria.

    Directory of Open Access Journals (Sweden)

    Alejandro Couce

    Full Text Available Understanding how mutator strains emerge in bacterial populations is relevant both to evolutionary theory and to reduce the threat they pose in clinical settings. The rise of mutator alleles is understood as a result of their hitchhiking with linked beneficial mutations, although the factors that govern this process remain unclear. A prominent but underappreciated fact is that each mutator allele increases only a specific spectrum of mutational changes. This spectrum has been speculated to alter the distribution of fitness effects of beneficial mutations, potentially affecting hitchhiking. To study this possibility, we analyzed the fitness distribution of beneficial mutations generated from different mutator and wild-type Escherichia coli strains. Using antibiotic resistance as a model system, we show that mutational spectra can alter these distributions substantially, ultimately determining the competitive ability of each strain across environments. Computer simulation showed that the effect of mutational spectrum on hitchhiking dynamics follows a non-linear function, implying that even slight spectrum-dependent fitness differences are sufficient to alter mutator success frequency by several orders of magnitude. These results indicate an unanticipated central role for the mutational spectrum in the evolution of bacterial mutation rates. At a practical level, this study indicates that knowledge of the molecular details of resistance determinants is crucial for minimizing mutator evolution during antibiotic therapy.

  2. Detecting clusters of mutations.

    Directory of Open Access Journals (Sweden)

    Tong Zhou

    Full Text Available Positive selection for protein function can lead to multiple mutations within a small stretch of DNA, i.e., to a cluster of mutations. Recently, Wagner proposed a method to detect such mutation clusters. His method, however, did not take into account that residues with high solvent accessibility are inherently more variable than residues with low solvent accessibility. Here, we propose a new algorithm to detect clustered evolution. Our algorithm controls for different substitution probabilities at buried and exposed sites in the tertiary protein structure, and uses random permutations to calculate accurate P values for inferred clusters. We apply the algorithm to genomes of bacteria, fly, and mammals, and find several clusters of mutations in functionally important regions of proteins. Surprisingly, clustered evolution is a relatively rare phenomenon. Only between 2% and 10% of the genes we analyze contain a statistically significant mutation cluster. We also find that not controlling for solvent accessibility leads to an excess of clusters in terminal and solvent-exposed regions of proteins. Our algorithm provides a novel method to identify functionally relevant divergence between groups of species. Moreover, it could also be useful to detect artifacts in automatically assembled genomes.

  3. Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.

    Science.gov (United States)

    Ukai, Akiko; Ishimaru, Konomi; Ouchida, Rika; Mori, Hiromi; Kano, Chie; Moritan, Toshiyuki; Wang, Ji-Yang

    2008-12-01

    Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase eta (POLH). We found that fibroblasts and GC B cells expressed similar levels of the mismatch repair components, but nonetheless the fibroblasts failed to generate a significant proportion of A:T mutations in a GFP reporter gene even after POLH overexpression. To investigate whether the ability to generate A:T mutations is dependent on the cellular environment (i.e., GC B cell or fibroblast) or the target gene (i.e., Ig or GFP), we developed a mutation detection system in a human GC-like cell line. We introduced a GFP gene with a premature stop codon into Ramos cells and compared the activation-induced cytidine deaminase-induced mutations in the endogenous V(H) and the transgenic GFP genes. Remarkably, a high proportion of A:T mutations was induced in both genes. Ectopic expression of POLH did not further increase the proportion of A:T mutations but diminished the strand bias of these mutations that is normally observed in V(H) genes. Intriguingly, the total mutation frequency in the GFP gene was consistently one-fifth of that in the V(H) gene. These results demonstrate that the ability to generate A:T mutations is dependent on the GC B cell environment but independent of the mutation frequency and target gene location.

  4. Cre recombinase activity is inhibited in vivo but not ex vivo by a mutation in the asymmetric spacer region of the distal loxP site.

    Science.gov (United States)

    Arguello, Tania; Moraes, Carlos T

    2015-11-01

    The cre/loxP recombination system is a valuable tool used to generate tissue specific genomic rearrangements in mouse models. The deletion of a region of interest flanked by two loxP sites is accomplished by the recombinase (cre) enzyme, which binds to the inverted repeat segments of two loxP sites and recognition of a conserved TA sequence in the asymmetric central spacer region "ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT. In vivo, we found that a single T to C mutation at position 4 of the central spacer region in the distal (3') loxP site, completely inhibited the recombination reaction in two conditional mouse models. These mice were generated using a mitochondrial methionyl-tRNA formyltransferase (Mtfmt) gene targeted construct and cre transgene under the control of tissue-specific promoters: calcium/calmodulin-dependent kinase II alpha (Camk2a-cre) and myosin light polypeptide 1 (Myl1-cre). Surprisingly, transient transfection of a plasmid expressing cre in dermal fibroblasts derived from the same mutant floxed Mtfmt((loxP/loxP)) mice line, successfully deleted the region of interest. This study demonstrates the sequence specificity required in vivo, the possibility of bypassing this limitation by expressing high levels of cre recombinase ex vivo and raises concerns related to the quality control of large scale production of gene targeted constructs and mice. genesis 53:695-700, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  5. Are There Mutator Polymerases?

    Directory of Open Access Journals (Sweden)

    Miguel Garcia-Diaz

    2003-01-01

    Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

  6. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder. The ...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  7. Mutations in Exons 9 and 13 of KIT Gene Are Rare Events in Gastrointestinal Stromal Tumors

    Science.gov (United States)

    Lasota, Jerzy; Wozniak, Agnieszka; Sarlomo-Rikala, Maarit; Rys, Janusz; Kordek, Radzislaw; Nassar, Aziza; Sobin, Leslie H.; Miettinen, Markku

    2000-01-01

    Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, typically express the KIT protein. Activating mutations in the juxtamembrane domain (exon 11) of the c-kit gene have been shown in a subset of GISTs. These mutations lead into ligand-independent activation of the tyrosine kinase of c-kit, and have a transforming effect in vitro. Several groups have studied the clinical implication of the c-kit mutation status of exon 11 in GISTs and a possible relationship between c-kit mutations and malignant behavior has been established. Recently, a 1530ins6 mutation in exon 9 and missense mutations, 1945A>G in exon 13 of the c-kit gene were reported. The frequency and clinical importance of these findings are unknown. In this study we evaluated 200 GISTs for the presence of mutations in exons 9 and 13 of c-kit. Six cases revealed 1530ins6 mutation in exon 9 and two cases 1945A>G mutation in exon 13. All tumors with mutations in exon 9 and 13 lacked mutations in exon 11 of c-kit. None of the analyzed tumors had more than one type of c-kit mutation. All but one of the eight tumors with mutations in exon 9 or 13 of the c-kit gene were histologically and clinically malignant. All four of six cases with exon 9 mutation of which location of primary tumor was known, were small intestinal, suggesting that this type of mutation could preferentially occur in small intestinal tumors. Exon 9 and 13 mutations seem to be rare, and they cover only a small portion (8%) of the balance of GISTs that do not have mutations in exon 11 of c-kit. This finding indicates that other genetic alterations may activate c-kit in GISTs, or that KIT is not activated by mutations in all cases. PMID:11021812

  8. High frequency of additional gene mutations in acute myeloid leukemia with MLL partial tandem duplication: DNMT3A mutation is associated with poor prognosis.

    Science.gov (United States)

    Kao, Hsiao-Wen; Liang, D Cherng; Kuo, Ming-Chung; Wu, Jin-Hou; Dunn, Po; Wang, Po-Nan; Lin, Tung-Liang; Shih, Yu-Shu; Liang, Sung-Tzu; Lin, Tung-Huei; Lai, Chen-Yu; Lin, Chun-Hui; Shih, Lee-Yung

    2015-10-20

    The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene (MLL-PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL-PTD AML to determine the mutation frequency and clinical correlations. MLL-PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3-ITD (44.9%) and FLT3-TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL-PTD AML patients had at least one additional gene mutation. Of 55 MLL-PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL-PTD AML.

  9. A common Greenlandic Inuit BRCA1 RING domain founder mutation

    DEFF Research Database (Denmark)

    Hansen, T.v.O.; Ejlertsen, B.; Albrechtsen, Anders

    2009-01-01

    Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We examined 32 breast and/or ovarian cancer patients from Greenland for mutations in BRCA1 and BRCA2. Whereas no mutations were identified in 19 families, 13 families exhibited a BRCA1...... exon 3 nucleotide 234 T > G mutation, which has not previously been reported in the breast cancer information core (BIC) database. The mutation changes a conserved cysteine 39 to a glycine in the Zn(2+) site II of the RING domain, which is essential for BRCA1 ubiquitin ligase activity. Eight...... of the families had members with ovarian cancer, suggesting that the RING domain may be an ovarian cancer hotspot. By SNP array analysis, we find that all 13 families share a 4.5 Mb genomic fragment containing the BRCA1 gene, showing that the mutation originates from a founder. Finally, analysis of 1152 Inuit...

  10. POLE mutations in families predisposed to cutaneous melanoma

    DEFF Research Database (Denmark)

    Aoude, Lauren G; Heitzer, Ellen; Johansson, Peter

    2015-01-01

    Germline mutations in the exonuclease domain of POLE have been shown to predispose to colorectal cancers and adenomas. POLE is an enzyme involved in DNA repair and chromosomal DNA replication. In order to assess whether such mutations might also predispose to cutaneous melanoma, we interrogated...... whole-genome and exome data from probands of 34 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, BAP1, TERT, POT1, ACD and TERF2IP. We found a novel germline mutation, POLE p.(Trp347Cys), in a 7-case cutaneous melanoma family....... Functional assays in S. pombe showed that this mutation led to an increased DNA mutation rate comparable to that seen with a Pol ε mutant with no exonuclease activity. We then performed targeted sequencing of POLE in 1243 cutaneous melanoma cases and found that a further ten probands had novel or rare...

  11. Mutations du gene de la filamine et syndromes malformatifs | Koffi ...

    African Journals Online (AJOL)

    Filamin is a cytoskeletal protein that occurs in the control of cytoskeleton structure and activity, the modulation of cell shape and migration as well as in the maintaining of cell shape. Mutations in the genes FLNA and FLNB provoke diverse malformative diseases in human. Mutations in the gene FLNA cause four X-Linked ...

  12. Analysis of TSC1 mutation spectrum in mucosal melanoma.

    Science.gov (United States)

    Ma, Meng; Dai, Jie; Xu, Tianxiao; Yu, Sifan; Yu, Huan; Tang, Huan; Yan, Junya; Wu, Xiaowen; Yu, Jiayi; Chi, Zhihong; Si, Lu; Cui, Chuanliang; Sheng, Xinan; Kong, Yan; Guo, Jun

    2018-02-01

    Mucosal melanoma is a relatively rare subtype of melanoma for which no clearly established therapeutic strategy exists. The genes of the mTOR signalling pathway have drawn great attention as key targets for cancer treatment, including melanoma. In this study, we aimed to investigate the mutation status of the upstream mTOR regulator TSC1 and evaluated its correlation with the clinicopathological features of mucosal melanoma. We collected 91 mucosal melanoma samples for detecting TSC1 mutations. All the coding exons of TSC1 were amplified by PCR and subjected to Sanger sequencing. Expression level of TSC1 encoding protein (hamartin) was detected by immunohistochemistry. The activation of mTOR pathway was determined by evaluating the phosphorylation status of S6RP and 4E-BP1. The overall mutation frequency of TSC1 was found to be 17.6% (16/91 patients). TSC1 mutations were more inclined to occur in advanced mucosal melanoma (stages III and IV). In the 16 patients with TSC1 mutations, 14 different mutations were detected, affecting 11 different exons. TSC1 mutations were correlated with upregulation of S6RP phosphorylation but were unrelated to 4E-BP1 phosphorylation or hamartin expression. Mucosal melanoma patients with TSC1 mutations had a worse outcome than patients without TSC1 mutations (24.0 versus 34.0 months, P = 0.007). Our findings suggest that TSC1 mutations are frequent in mucosal melanoma. TSC1 mutations can activate the mTOR pathway through phospho-S6RP and might be a poor prognostic predictor of mucosal melanoma. Our data implicate the potential significance of TSC1 mutations for effective and specific drug therapy for mucosal melanoma.

  13. Mutation, somatic mutation and diseases of man

    International Nuclear Information System (INIS)

    Burnet, F.M.

    1976-01-01

    The relevance of the intrinsic mutagenesis for the evolution process, genetic diseases and the process of aging is exemplified. The fundamental reaction is the function of the DNA and the DNA-enzymes like the DNA-polymerases in replication, repair, and transcription. These defects are responsible for the mutation frequency and the genetic drift in the evolution process. They cause genetic diseases like Xeroderma pigmentosum which is described here in detail. The accumulation of structural and functional mistakes leads to diseases of old age, for example to autoimmune diseases and immune suppression. There is a proportionality between the duration of life and the frequency of mistakes in the enzymatic repair system. No possibility of prophylaxis or therapy is seen. Methods for prognosis could be developed. (AJ) [de

  14. Germline KRAS mutations cause Noonan syndrome.

    NARCIS (Netherlands)

    Schubbert, S.; Zenker, M.; Rowe, S.L.; Boll, S.; Klein, C.; Bollag, G.; Burgt, I. van der; Musante, L.; Kalscheuer, V.M.M.; Wehner, L.E.; Nguyen, H.; West, B.; Zhang, K.Y.; Sistermans, E.A.; Rauch, A.; Niemeyer, C.M.; Shannon, K.; Kratz, C.P.

    2006-01-01

    Noonan syndrome (MIM 163950) is characterized by short stature, facial dysmorphism and cardiac defects. Heterozygous mutations in PTPN11, which encodes SHP-2, cause approximately 50% of cases of Noonan syndrome. The SHP-2 phosphatase relays signals from activated receptor complexes to downstream

  15. FLT3 mutation incidence and timing of origin in a population case series of pediatric leukemia

    Directory of Open Access Journals (Sweden)

    Chang Jeffrey

    2010-09-01

    Full Text Available Abstract Background Mutations in FLT3 result in activated tyrosine kinase activity, cell growth stimulation, and a poor prognosis among various subtypes of leukemia. The causes and timing of the mutations are not currently known. We evaluated the prevalence and timing of origin of FLT3 mutations in a population series of childhood leukemia patients from Northern California. Methods We screened and sequenced FLT3 mutations (point mutations and internal tandem duplications, ITDs among 517 childhood leukemia patients, and assessed whether these mutations occurred before or after birth using sensitive "backtracking" methods. Results We determined a mutation prevalence of 9 of 73 acute myeloid leukemias (AMLs, 12% and 9 of 441 acute lymphocytic leukemias (ALLs, 2%. Among AMLs, FLT3 mutations were more common in older patients, and among ALLs, FLT3 mutations were more common in patients with high hyperdiploidy (3.7% than those without this cytogenetic feature (1.4%. Five FLT3 ITDs, one deletion mutation, and 3 point mutations were assessed for their presence in neonatal Guthrie spots using sensitive real-time PCR techniques, and no patients were found to harbor FLT3 mutations at birth. Conclusions FLT3 mutations were not common in our population-based patient series in California, and patients who harbor FLT3 mutations most likely acquire them after they are born.

  16. Mutation breeding newsletter. No. 33

    International Nuclear Information System (INIS)

    1989-01-01

    This issue of the newsletter reports a number of research news and research abstracts on application of radiation induced mutation techniques to increase mutagenesis and mutation frequency in plant breeding projects

  17. Mutation breeding newsletter. No. 45

    International Nuclear Information System (INIS)

    2001-07-01

    This issue of the Mutation Breeding newsletter contains 39 articles dealing with radiation induced mutations and chemical mutagenesis techniques in plant breeding programs with the aims of improving crop productivity and disease resistance as well as exploring genetic variabilities

  18. PI3 kinase mutations and mutational load as poor prognostic markers in diffuse glioma patients.

    Science.gov (United States)

    Draaisma, Kaspar; Wijnenga, Maarten M J; Weenink, Bas; Gao, Ya; Smid, Marcel; Robe, P; van den Bent, Martin J; French, Pim J

    2015-12-23

    III or IV, even within defined molecular subtypes (e.g. ATRX mutated diffuse gliomas). We have identified PI3K mutations as novel prognostic markers in gliomas. We also demonstrate that the mutational load is associated with tumor grade. The increase in mutational load may partially explain the increased aggressiveness of higher grade diffuse gliomas when a subset of the affected genes actively contributes to gliomagenesis and/or progression.

  19. BRAF mutation in hairy cell leukemia

    Directory of Open Access Journals (Sweden)

    Ahmad Ahmadzadeh

    2014-09-01

    Full Text Available BRAF is a serine/threonine kinase with a regulatory role in the mitogen-activated protein kinase (MAPK signaling pathway. A mutation in the RAF gene, especially in BRAF protein, leads to an increased stimulation of this cascade, causing uncontrolled cell division and development of malignancy. Several mutations have been observed in the gene coding for this protein in a variety of human malignancies, including hairy cell leukemia (HCL. BRAF V600E is the most common mutation reported in exon15 of BRAF, which is observed in almost all cases of classic HCL, but it is negative in other B-cell malignancies, including the HCL variant. Therefore it can be used as a marker to differentiate between these B-cell disorders. We also discuss the interaction between miRNAs and signaling pathways, including MAPK, in HCL. When this mutation is present, the use of BRAF protein inhibitors may represent an effective treatment. In this review we have evaluated the role of the mutation of the BRAF gene in the pathogenesis and progression of HCL.

  20. Mutation analysis in 54 propionic acidemia patients.

    Science.gov (United States)

    Kraus, J P; Spector, E; Venezia, S; Estes, P; Chiang, P W; Creadon-Swindell, G; Müllerleile, S; de Silva, L; Barth, M; Walter, M; Walter, K; Meissner, T; Lindner, M; Ensenauer, R; Santer, R; Bodamer, O A; Baumgartner, M R; Brunner-Krainz, M; Karall, D; Haase, C; Knerr, I; Marquardt, T; Hennermann, J B; Steinfeld, R; Beblo, S; Koch, H G; Konstantopoulou, V; Scholl-Bürgi, S; van Teeffelen-Heithoff, A; Suormala, T; Ugarte, M; Sperl, W; Superti-Furga, A; Schwab, K O; Grünert, S C; Sass, J O

    2012-01-01

    Deficiency of propionyl CoA carboxylase (PCC), a dodecamer of alpha and beta subunits, causes inherited propionic acidemia. We have studied, at the molecular level, PCC in 54 patients from 48 families comprised of 96 independent alleles. These patients of various ethnic backgrounds came from research centers and hospitals in Germany, Austria and Switzerland. The thorough clinical characterization of these patients was described in the accompanying paper (Grünert et al. 2012). In all 54 patients, many of whom originated from consanguineous families, the entire PCCB gene was examined by genomic DNA sequencing and in 39 individuals the PCCA gene was also studied. In three patients we found mutations in both PCC genes. In addition, in many patients RT-PCR analysis of lymphoblast RNA, lymphoblast enzyme assays, and expression of new mutations in E.coli were carried out. Eight new and eight previously detected mutations were identified in the PCCA gene while 15 new and 13 previously detected mutations were found in the PCCB gene. One missense mutation, p.V288I in the PCCB gene, when expressed in E.coli, yielded 134% of control activity and was consequently classified as a polymorphism in the coding region. Numerous new intronic polymorphisms in both PCC genes were identified. This study adds a considerable amount of new molecular data to the studies of this disease.

  1. A rifampicin-resistant (rpoB) mutation in Pseudomonas protegens Pf-5 strain leads to improved antifungal activity and elevated production of secondary metabolites.

    Science.gov (United States)

    Xie, Yali; Liu, Zhengqiang; Zhang, Guoyong; Mo, Xiangtao; Ding, Xuezhi; Xia, Liqiu; Hu, Shengbiao

    2016-10-01

    Ribosome engineering has proven to be a practical method for increasing antibiotic production, and is extensively applied to strain improvement in antibiotic production and activation of silent genes in several prokaryotes. In this study, ribosome engineering was used to improve production of bioactive secondary metabolites produced by Pseudomonas protegens Pf-5. Rifampicin-resistant mutants that bear the H531N in the β-subunit of RNA polymerase showed improved antifungal activity and morphological changes. The production of several secondary metabolites in R55 mutant was significantly improved using high-performance liquid chromatography (HPLC) analysis. Two antibiotics with antifungal activity, 2, 4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt), which may contribute to the improved antifungal activity of the R55 mutant, were identified by mass spectrometer (MS) analysis. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

    International Nuclear Information System (INIS)

    Kato, T.; Nakano, E.

    1981-01-01

    The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr + or uvr - strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His + , ColE and Spc, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N'-nitro-N-nitrosoguanidine-induced His + mutations. In contrast, only 1 out of 13 trp - frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC + -dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp + mutations in most of the strains regardless of the umu + or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC + -dependent pathway of error-prone repair. (orig.)

  3. Frequent DPH3 promoter mutations in skin cancers.

    Science.gov (United States)

    Denisova, Evgeniya; Heidenreich, Barbara; Nagore, Eduardo; Rachakonda, P Sivaramakrishna; Hosen, Ismail; Akrap, Ivana; Traves, Víctor; García-Casado, Zaida; López-Guerrero, José Antonio; Requena, Celia; Sanmartin, Onofre; Serra-Guillén, Carlos; Llombart, Beatriz; Guillén, Carlos; Ferrando, Jose; Gimeno, Enrique; Nordheim, Alfred; Hemminki, Kari; Kumar, Rajiv

    2015-11-03

    Recent reports suggested frequent occurrence of cancer associated somatic mutations within regulatory elements of the genome. Based on initial exome sequencing of 21 melanomas, we report frequent somatic mutations in skin cancers in a bidirectional promoter of diphthamide biosynthesis 3 (DPH3) and oxidoreductase NAD-binding domain containing 1 (OXNAD1) genes. The UV-signature mutations occurred at sites adjacent and within a binding motif for E-twenty six/ternary complex factors (Ets/TCF), at -8 and -9 bp from DPH3 transcription start site. Follow up screening of 586 different skin lesions showed that the DPH3 promoter mutations were present in melanocytic nevi (2/114; 2%), melanoma (30/304; 10%), basal cell carcinoma of skin (BCC; 57/137; 42%) and squamous cell carcinoma of skin (SCC; 12/31; 39%). Reporter assays carried out in one melanoma cell line for DPH3 and OXNAD1 orientations showed statistically significant increased promoter activity due to -8/-9CC > TT tandem mutations; although, no effect of the mutations on DPH3 and OXNAD1 transcription in tumors was observed. The results from this study show occurrence of frequent somatic non-coding mutations adjacent to a pre-existing binding site for Ets transcription factors within the directional promoter of DPH3 and OXNAD1 genes in three major skin cancers. The detected mutations displayed typical UV signature; however, the functionality of the mutations remains to be determined.

  4. Phase 2 Study of Erlotinib in Combination With Linsitinib (OSI-906) or Placebo in Chemotherapy-Naive Patients With Non-Small-Cell Lung Cancer and Activating Epidermal Growth Factor Receptor Mutations.

    Science.gov (United States)

    Leighl, Natasha B; Rizvi, Naiyer A; de Lima, Lopes Gilberto; Arpornwirat, Wichit; Rudin, Charles M; Chiappori, Alberto A; Ahn, Myung-Ju; Chow, Laura Q M; Bazhenova, Lyudmila; Dechaphunkul, Arunee; Sunpaweravong, Patrapim; Eaton, Keith; Chen, Jihong; Medley, Sonja; Poondru, Srinivasu; Singh, Margaret; Steinberg, Joyce; Juergens, Rosalyn A; Gadgeel, Shirish M

    2017-01-01

    First-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor treatment of advanced non-small-cell lung cancer with EGFR-activating mutations improves outcomes compared with chemotherapy, but resistance develops in most patients. Compensatory signaling through type 1 insulin-like growth factor 1 receptor (IGF-1R) may contribute to resistance; dual blockade of IGF-1R and EGFR may improve outcomes. We performed a randomized, double-blind, placebo-controlled phase II study of linsitinib, a dual IGF-1R and insulin receptor tyrosine kinase inhibitor, plus erlotinib versus placebo plus erlotinib in chemotherapy-naive patients with EGFR-mutation positive, advanced non-small-cell lung cancer. Patients received linsitinib 150 mg twice daily or placebo plus erlotinib 150 mg once daily on continuous 21-day cycles. The primary end point was progression-free survival. After randomization of 88 patients (44 each arm), the trial was unblinded early owing to inferiority in the linsitinib arm. The median progression-free survival for the linsitinib versus the placebo group was 8.4 months versus 12.4 months (hazard ratio, 1.37; P = .29). Overall response rate (47.7% vs. 75.0%; P = .02) and disease control rate (77.3% vs. 95.5%; P = .03) were also inferior. Whereas most adverse events were ≤ grade 2, linsitinib plus erlotinib was associated with increased adverse events that led to decreased erlotinib exposure (median days, 228 vs. 305). No drug-drug interaction was suggested by pharmacokinetic and pharmacodynamic results. Adding linsitinib to erlotinib resulted in inferior outcomes compared with erlotinib alone. Further understanding of the signaling pathways and a biomarker that can predict efficacy is needed prior to further clinical development of IGF-1R inhibitors in lung cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Mutation breeding in pepper

    International Nuclear Information System (INIS)

    Daskalov, S.

    1986-01-01

    Pepper (Capsicum sp.) is an important vegetable and spice crop widely grown in tropical as well as in temperate regions. Until recently the improvement programmes were based mainly on using natural sources of germ plasma, crossbreeding and exploiting the heterosis of F 1 hybrids. However, interest in using induced mutations is growing. A great number of agronomically useful mutants as well as mutants valuable for genetic, cytological and physiological studies have been induced and described. In this review information is presented about suitable mutagen treatment procedures with radiation as well as chemicals, M 1 effects, handling the treated material in M 1 , M 2 and subsequent generations, and mutant screening procedures. This is supplemented by a description of reported useful mutants and released cultivars. Finally, general advice is given on when and how to incorporate mutation induction in Capsicum improvement programmes. (author)

  6. Mutations causative of familial hypercholesterolaemia

    DEFF Research Database (Denmark)

    Benn, Marianne; Watts, Gerald F; Tybjærg-Hansen, Anne

    2016-01-01

    causing mutations in 98 098 participants from the general population, the Copenhagen General Population Study. METHODS AND RESULTS: We genotyped for LDLR[W23X;W66G;W556S] and APOB[R3500Q] accounting for 38.7% of pathogenic FH mutations in Copenhagen. Clinical FH assessment excluded mutation information......-cholesterol concentration to discriminate between mutation carriers and non-carriers was 4.4 mmol/L. CONCLUSION: Familial hypercholesterolaemia-causing mutations are estimated to occur in 1:217 in the general population and are best identified by a definite or probable phenotypic diagnosis of FH based on the DLCN criteria...

  7. Association of H-ras mutations with adenocarcinomas of the parotid gland

    NARCIS (Netherlands)

    van Halteren, H. K.; Top, B.; Mooi, W. J.; Balm, A. J.; Rodenhuis, S.

    1994-01-01

    We have examined 17 adenocarcinomas and 2 mixed tumors of the salivary glands for mutational activation of the oncogenes H-ras, K-ras and N-ras. The presence of mutations was determined by in vitro amplification of gene fragments spanning codons 12, 13 and 61 and the use of mutation-specific

  8. Mutational analysis of the binding affinity and transport activity for N-acetylglucosamine of the novel ABC transporter Ngc in the chitin-degrader Streptomyces olivaceoviridis.

    Science.gov (United States)

    Saito, A; Schrempf, H

    2004-06-01

    The highly differentiated bacterium Streptomyces olivaceoviridis efficiently hydrolyses chitin, a highly abundant natural polysaccharide, to low molecular weight products including N-acetylglucosamine (NAG) and N,N' -diacetylchitobiose (chitobiose). NAG is taken up by a PTS (phosphoenolpyruvate-dependent phosphotransferase system) which includes the PtsC2 protein, and via the ABC (ATP-binding cassette) transporter Ngc, which itself includes the substrate-binding protein NgcE. This is at present the only ABC transporter which is known to mediate specific uptake of NAG (K(m) 0.48 microM, V(max) 1.3 nmol/min/mg dry weight) and is competitively inhibited by chitobiose (K(i) 0.68 microM). The latter finding suggests that the Ngc system transports both NAG and chitobiose efficiently. To identify amino acid residues required for the function of NgcE, either the wild-type or one of several mutant forms of the ngcE gene was introduced into the strain S. olivaceoviridis DeltaNgcE/DeltaPtsC1/DeltaPtsC2, which lacks both functional transport systems for NAG, and chromosomal recombinants were selected. Based on the in vivo transport parameters of the recombinants, and the in vitro binding characteristics of the corresponding purified proteins, the following conclusions can be drawn. (1) Replacement of the C-terminally located residue Y396 by A (Y396A) has little effect on ligand-binding or transport parameters. The W395A mutation also induced little change in the substrate affinity in vitro, but it led in vivo to a marked increase (11 fold) in K(m), and enhanced V(max) (by 1.5 fold). (2) The amino acids Y201 and W280 both contribute (51% and 38%) to the ligand-binding capacity of NgcE. They are both very important for the in vivo function of the complete transport apparatus; strains expressing either Y201A or W280A show drastically (100 or 150 times) enhanced K(m) values. (3) The concomitant presence of either Y200 and W280 or Y201 and W280 is essential for the function of Ngc

  9. Mutation selection of strawberries

    International Nuclear Information System (INIS)

    Repka, F.; Tsaganova, I.

    1986-01-01

    A brief account is given of the preliminary results of selection work carried out with the aim of deriving a variety of strawberry suitable for mechanized picking. Mutation selection based on irradiation by gamma rays, fast neutrons and a laser beam has been used. The irradiation was performed on strawberry seedlings grown under field conditions and on in vitro cultures at different stages of development. The studies are continuing. (author)

  10. A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes

    Science.gov (United States)

    The active site motif of proteins belonging to ‘Histidine Acid Phosphatase’ (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs has revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger phyA phytase. However,...

  11. Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation.

    Science.gov (United States)

    Lochhead, Pamela A; Clark, Jonathan; Wang, Lan-Zhen; Gilmour, Lesley; Squires, Matthew; Gilley, Rebecca; Foxton, Caroline; Newell, David R; Wedge, Stephen R; Cook, Simon J

    2016-01-01

    ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS(G12C/G13D) or BRAF(V600E). Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.

  12. Kinetics and thermodynamics of the native and mutated extracellular endo-glucanases from Cellulomonas biazotea.

    Science.gov (United States)

    Rajoka, M I; Ashraf, Yasmin; Rashid, Hamid; Khalid, A M

    2003-12-01

    The mutation had dramatic effect on the kinetic and thermodynamic parameters inferring thermostability of endo-glucanase from Cellulomonas biazotea mutant 51 SM(r). The denaturation activation energies of native and mutated enzymes were 73.3 and 68.8 kJ/mol respectively. They showed compensation effect at 55 degrees C. Both enthalpy and entropy values of irreversible thermal inactivation for mutated enzyme were decreased suggesting that the mutation partly stabilized the enzyme.

  13. Characterization of a novel KCNQ1 mutation for type 1 long QT syndrome and assessment of the therapeutic potential of a novel IKs activator using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Ma, Dongrui; Wei, Heming; Lu, Jun; Huang, Dou; Liu, Zhenfeng; Loh, Li Jun; Islam, Omedul; Liew, Reginald; Shim, Winston; Cook, Stuart A

    2015-03-19

    Type 1 long QT syndrome (LQT1) is a common type of cardiac channelopathy associated with loss-of-function mutations of KCNQ1. Currently there is a lack of drugs that target the defected slowly activating delayed rectifier potassium channel (IKs). With LQT1 patient-specific human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs), we tested the effects of a selective IKs activator ML277 on reversing the disease phenotypes. A LQT1 family with a novel heterozygous exon 7 deletion in the KCNQ1 gene was identified. Dermal fibroblasts from the proband and her healthy father were reprogrammed to hiPSCs and subsequently differentiated into hiPSC-CMs. Compared with the control, LQT1 patient hiPSC-CMs showed reduced levels of wild type KCNQ1 mRNA accompanied by multiple exon skipping mRNAs and a ~50% reduction of the full length Kv7.1 protein. Patient hiPSC-CMs showed reduced IKs current (tail current density at 30 mV: 0.33±0.02 vs. 0.92±0.21, PKCNQ1, we generated hiPSC-CMs that faithfully recapitulated the LQT1 phenotypes that are likely associated with haploinsufficiency and trafficking defect of KCNQ1/Kv7.1. The small molecule ML277 restored IKs function in hiPSC-CMs and could have therapeutic value for LQT1 patients.

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

    OpenAIRE

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

    2016-01-01

    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...

  15. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

    Directory of Open Access Journals (Sweden)

    Kayla A Boortz

    Full Text Available Elevated fasting blood glucose (FBG has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln, rs149663725 (Gly114Arg and rs2232326 (Ser324Pro SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu, rs138726309 (His177Tyr, rs2232323 (Tyr207Ser rs374055555 (Arg293Trp, rs2232326 (Ser324Pro, rs137857125 (Pro313Leu and rs2232327 (Pro340Leu SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

  16. Reducing the Levels of Akt Activation by PDK1 Knock-in Mutation Protects Neuronal Cultures against Synthetic Amyloid-Beta Peptides

    Directory of Open Access Journals (Sweden)

    Shaobin Yang

    2018-01-01

    Full Text Available The Akt kinase has been widely assumed for years as a key downstream effector of the PI3K signaling pathway in promoting neuronal survival. This notion was however challenged by the finding that neuronal survival responses were still preserved in mice with reduced Akt activity. Moreover, here we show that the Akt signaling is elevated in the aged brain of two different mice models of Alzheimer Disease. We manipulate the rate of Akt stimulation by employing knock-in mice expressing a mutant form of PDK1 (phosphoinositide-dependent protein kinase 1 with reduced, but not abolished, ability to activate Akt. We found increased membrane localization and activity of the TACE/ADAM17 α-secretase in the brain of the PDK1 mutant mice with concomitant TNFR1 processing, which provided neurons with resistance against TNFα-induced neurotoxicity. Opposite to the Alzheimer Disease transgenic mice, the PDK1 knock-in mice exhibited an age-dependent attenuation of the unfolding protein response, which protected the mutant neurons against endoplasmic reticulum stressors. Moreover, these two mechanisms cooperatively provide the mutant neurons with resistance against amyloid-beta oligomers, and might singularly also contribute to protect these mice against amyloid-beta pathology.

  17. Breast cancer-specific mutations in CK1ε inhibit Wnt/β-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration

    NARCIS (Netherlands)

    Foldynová-Trantírková, S.; Sekyrová, P.; Tmejová, K.; Brumovská, E.; Bernatík, O.; Blankenfeldt, W.; Krejčí, P.; Kozubík, A.; Doležal, T.; Trantirek, L.|info:eu-repo/dai/nl/326057072; Bryja, V.

    2010-01-01

    Introduction Breast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1ε). Because CK1ε is a crucial regulator of the Wnt signaling cascades, we determined how these CK1ε mutations interfere with the Wnt

  18. Mutations in phoB, the positive gene activator of the pho regulon in Escherichia coli, affect the carbohydrate phenotype on MacConkey indicator plates.

    Science.gov (United States)

    Hartmann, A; Boos, W

    1993-05-01

    Mutants defective in phoB, the positive gene activator of the Escherichia coli pho regulon, exhibit aberrant behaviour on MacConkey indicator plates. They appear pale in the presence of a fermentable carbon source such as trehalose, maltose or glucose. The addition of at least 5 mM phosphate corrects this defect. Colonies of phoB+ strains turn red on MacConkey indicator plates and derepress the pho regulon when the cells are able to ferment the carbon source. In contrast, the inability to ferment the carbon source maintains the pho regulon in the repressed state.

  19. 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

    by suppressing gut-serotonin synthesis. This function of Lrp5 occurring in the gut is independent of β-catenin activation in osteoblasts. However, it is unknown whether Lrp5 can act directly in osteoblast to influence other functions that require β-catenin signaling, particularly, the deregulation...... of β-catenin in osteoblasts (Ctnnb1(CAosb)), accumulation of early myeloid progenitors, a characteristic of AML, myeloid-blasts in blood, and segmented neutrophils or dysplastic megakaryocytes in the bone marrow, are not observed in Lrp5(A214V) mice. Likewise, peripheral blood count analysis in HBM...

  20. Carcinogenic oestrogens induce respiration deficiency mutation in yeast.

    Science.gov (United States)

    Stopper, H; Metzler, M

    1991-01-01

    In addition to hormonal activity, genetic damage has been proposed as an important factor in oestrogen-mediated carcinogenesis. However, as short-term tests for oestrogens usually fail to show DNA mutations, lesions other than classic nuclear DNA mutation have to be considered. Oestrogen-induced mitochondrial damage was studied in the yeast Saccharomyces cerevisiae. Stilbene-type, but not steroidal, oestrogens were found to induce respiration-deficient petite mutation. The effect was inversely correlated with cytotoxicity and required aromatic hydroxyl groups at the stilbene molecule. It only occurred under growth conditions and apparently was not due to the ATPase inhibitory qualities of stilbene oestrogens. Other studies have shown that petite mutation clones, which can be induced by a variety of substances, contain altered mitochondrial DNA. The mechanism of petite mutation induction might be important in tumorigenesis by also acting on nuclear DNA or facilitating carcinogenesis by disturbance of mitochondrial function.

  1. Mutations targeting the coagulation pathway are enriched in brain metastases.

    Science.gov (United States)

    Richichi, Cristina; Fornasari, Lorenzo; Melloni, Giorgio E M; Brescia, Paola; Patanè, Monica; Del Bene, Massimiliano; Mustafa, Dana A M; Kros, Johan M; Pollo, Bianca; Pruneri, Giancarlo; Sciandivasci, Angela; Munzone, Elisabetta; DiMeco, Francesco; Pelicci, Pier Giuseppe; Riva, Laura; Pelicci, Giuliana

    2017-07-26

    Brain metastases (BMs) are the most common malignancy of the central nervous system. Recently it has been demonstrated that plasminogen activator inhibitor serpins promote brain metastatic colonization, suggesting that mutations in serpins or other members of the coagulation cascade can provide critical advantages during BM formation. We performed whole-exome sequencing on matched samples of breast cancer and BMs and found mutations in the coagulation pathway genes in 5 out of 10 BM samples. We then investigated the mutational status of 33 genes belonging to the coagulation cascade in a panel of 29 BMs and we identified 56 Single Nucleotide Variants (SNVs). The frequency of gene mutations of the pathway was significantly higher in BMs than in primary tumours, and SERPINI1 was the most frequently mutated gene in BMs. These findings provide direction in the development of new strategies for the treatment of BMs.

  2. A mutation in the COX5 gene of the yeast Scheffersomyces stipitis alters utilization of amino acids as carbon source, ethanol formation and activity of cyanide insensitive respiration.

    Science.gov (United States)

    Freese, Stefan; Passoth, Volkmar; Klinner, Ulrich

    2011-04-01

    Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis, in contrast to Saccharomyces cerevisiae, where two homologous genes are present. Mutant cells had lost the ability to grow with the amino acids glutamate, proline or aspartate and other non-fermentable carbon sources, such as acetic acid and ethanol, as sole carbon sources. Biomass formation of the mutant cells in medium containing glucose or xylose as carbon source was lower compared with the wild-type cells. However, yields and specific ethanol formation of the mutant were much higher, especially under conditions of higher aeration. The mutant cells lacked both cytochrome c oxidase activity and cyanide-sensitive respiration, whereas ADH and PDC activities were distinctly enhanced. SHAM-sensitive respiration was obviously essential for the fermentative metabolism, because SHAM completely abolished growth of the mutant cells with both glucose or xylose as carbon source. Copyright © 2011 John Wiley & Sons, Ltd.

  3. Mutation breeding in chickpea

    International Nuclear Information System (INIS)

    2009-01-01

    Chickpea is an important food legume in Turkey. Turkey is one of the most important gene centers in the world for legumes. The most widely known characteristic of chickpea is that it is an important vegetable protein source used in human and animal nutrition. However, the dry grains of chickpea, has 2-3 times more protein than our traditional food of wheat. In addition, cheakpea is also energy source because of its high carbohydrate content. It is very rich in some vitamin and mineral basis. In the plant breeding, mutation induction has become an effective way of supplementing existing germplasm and improving cultivars. Many successful examples of mutation induction have proved that mutation breeding is an effective and important approach to food legume improvement. The induced mutation technique in chickpea has proved successful and good results have been attained. Realizing the potential of induced mutations, a mutation breeding programme was initiated at the Nuclear Agriculture Section of the Saraykoey Nuclear Research and Training Center in 1994. The purpose of the study was to obtain high yielding chickpea mutants with large seeds, good cooking quality and high protein content. Beside this some characters such as higher adaptation ability, tolerant to cold and drought, increased machinery harvest type, higher yield, resistant to diseases especially to antracnose and pest were investigated too. Parents varieties were ILC-482, AK-7114 and AKCIN-91 (9 % seed moisture content and germination percentage 98 %) in these experiments. The irradiation doses were 0 (control), 50, 100, 150, 200, 250, 300, 350, 400, 500 ve 600 Gy for greenhouse experiments and 0 (control), 50, 100, 150, 200, 250, 300, 350 ve 400 Gy for field experiments, respectively. One thousand seeds for per treatment were sown in the field for the M 1 . At maturity, 3500 single plants were harvested and 20 seeds were taken from each M 1 plant and planted in the following season. During plant growth

  4. Amplification-refractory mutation system (ARMS) analysis of point mutations.

    Science.gov (United States)

    Little, S

    2001-05-01

    The amplification-refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base changes or small deletions. ARMS is based on the use of sequence-specific PCR primers that allow amplification of test DNA only when the target allele is contained within the sample. Following an ARMS reaction the presence or absence of a PCR product is diagnostic for the presence or absence of the target allele. The protocols detailed here outline methods that can be used to analyze human genomic DNA for one or more mutations. The Basic Protocol describes the development and application of an ARMS test for a single mutation; the Alternate Protocol extends this to multiplex ARMS for the analysis of two or more mutations. The Support Protocol describes a rapid DNA extraction method from blood or mouthwash samples that yields DNA compatible with the type of tests described. The amplification-refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change The amplification-refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change.

  5. Induced mutations in citrus

    International Nuclear Information System (INIS)

    Spiegel-Roy, P.; Vardi, Aliza

    1990-01-01

    Full text: Parthenocarpic tendency is an important prerequisite for successful induction of seedlessness in breeding and especially in mutation breeding. A gene for asynapsis and accompanying seedless fruit has been found by us in inbred progeny of cv. 'Wilking'. Using budwood irradiation by gamma rays, seedless mutants of 'Eureka' and 'Villafranca' lemon (original clone of the latter has 25 seeds) and 'Minneola' tangelo have been obtained. Ovule sterility of the three mutants is nearly complete, with some pollen fertility still remaining. A semi-compact mutant of Shamouti orange has been obtained by irradiation. A programme for inducing seedlessness in easy peeling citrus varieties and selections has been initiated. (author)

  6. The impact of rare EGFR mutations on the treatment response of patients with non-small cell lung cancer.

    Science.gov (United States)

    Karachaliou, Niki; Molina-Vila, Miguel Angel; Rosell, Rafael

    2015-06-01

    Mutational activation of the epidermal growth factor receptor (EGFR) gene is implicated in lung cancer; clinical and cancer genome sequencing studies have identified hundreds of mutations in the protein kinase domain. EGFR mutation testing usually focuses on common mutations like the exon 19 deletion and exon 21 point mutation (L858R). However, molecular screening methods have started to extend beyond identification of classic EGFR mutations to prevent exclusion of patients with rare or complex mutations who may benefit from anti-EGFR therapy. Rare EGFR-mutated non-small-cell lung cancers are heterogeneous: exon 20 insertions lack sensitivity to tyrosine kinase inhibitors while exon 18 or complex mutations are more sensitive and require individual assessment. Until testing for uncommon EGFR mutations evolves and studies with large number of patients are performed, knowledge of this field will remain limited.

  7. Mutational analysis of the extracellular disulphide bridges of the atypical chemokine receptor ACKR3/CXCR7 uncovers multiple binding and activation modes for its chemokine and endogenous non-chemokine agonists.

    Science.gov (United States)

    Szpakowska, Martyna; Meyrath, Max; Reynders, Nathan; Counson, Manuel; Hanson, Julien; Steyaert, Jan; Chevigné, Andy

    2018-03-09

    The atypical chemokine receptor ACKR3/CXCR7 plays crucial roles in numerous physiological processes but also in viral infection and cancer. ACKR3 shows strong propensity for activation and, unlike classical chemokine receptors, can respond to chemokines from both the CXC and CC families as well as to the endogenous peptides BAM22 and adrenomedullin. Moreover, despite belonging to the G protein coupled receptor family, its function appears to be mainly dependent on β-arrestin. ACKR3 has also been shown to continuously cycle between the plasma membrane and the endosomal compartments, suggesting a possible role as a scavenging receptor. So far, the molecular basis accounting for these atypical binding and signalling properties remains elusive. Noteworthy, ACKR3 extracellular domains bear three disulphide bridges. Two of them lie on top of the two main binding subpockets and are conserved among chemokine receptors, and one, specific to ACKR3, forms an intra-N terminus four-residue-loop of so far unknown function. Here, by mutational and functional studies, we examined the impact of the different disulphide bridges for ACKR3 folding, ligand binding and activation. We showed that, in contrast to most classical chemokine receptors, none of the extracellular disulphide bridges was essential for ACKR3 function. However, the disruption of the unique ACKR3 N-terminal loop drastically reduced the binding of CC chemokines whereas it only had a mild impact on CXC chemokine binding. Mutagenesis also uncovered that chemokine and endogenous non-chemokine ligands interact and activate ACKR3 according to distinct binding modes characterized by different transmembrane domain subpocket occupancy and N-terminal loop contribution, with BAM22 mimicking the binding mode of CC chemokine N terminus. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Mutation Breeding Newsletter. No. 39

    International Nuclear Information System (INIS)

    1992-01-01

    This newsletter contains brief articles on the use of radiation to induce mutations in plants; radiation-induced mutants in Chrysanthemum; disrupting the association between oil and protein content in soybean seeds; mutation studies on bougainvillea; a new pepper cultivar; and the use of mutation induction to improve the quality of yam beans. A short review of the seminar on the use of mutation and related biotechnology for crop improvement in the Middle East and Mediterranean regions, and a description of a Co-ordinated Research Programme on the application of DNA-based marker mutations for the improvement of cereals and other sexually reproduced crop species are also included. Two tables are given: these are based on the ''FAO/IAEA Mutant Varieties Database'' and show the number of mutated varieties and the number of officially released mutant varieties in particular crops/species. Refs and tabs

  9. In silico mutation analysis of non-structural protein-5 (NS5) dengue virus

    Science.gov (United States)

    Puspitasari, R. D.; Tambunan, U. S. F.

    2017-04-01

    Dengue fever is a world disease. It is endemic in more than 100 countries. Information about the effect of mutations in the virus is important in drug design and development. In this research, we studied the effect of mutation on NS5 dengue virus. NS5 is the large protein containing 67% amino acid similarity in DENV 1-4 and has multifunctional enzymatic activities. Dengue virus is an RNA virus that has very high mutation frequency with an average of 100 times higher than DNA mutations, and the accumulation of mutations will be possible to generate the new serotype. In this study, we report that mutation occurs in NS5 of DENV serotype 3, glutamine mutates into methionine at position 10 and threonine mutates into isoleucine at position 55. These residues are part of the domain named S-Adenosyl-L-Methionine-Dependent Methyltransferase (IPR029063).

  10. Evaluation of the antibiotic activity and genetic mutation of microorganisms in the effluent treated with the electron-beam from waste-water treatment plant

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dong Hun; Nam, Ji Hyun; Shin, Ji Hye; Yun, Seo Yeon; Cho, Young Cheol; Oh, Kyoung hee [Chungbuk National University, Cheongju (Korea, Republic of)

    2011-04-15

    In this study, the residual concentrations and activities of antibiotics after UV or gamma-ray treatments were estimated, and the effect of irradiation of UV, gamma-ray, or electron beam was estimated on the survivability and less mutagenic effect on bacteria. The changes of bacterial communities and radiation resistant population in the effluent treated with UV and electron-beam were analyzed. The gamma-ray irradiation was more effective than UV in degradation of antibiotics. The extent of mutagenicity of electron-beam irradiation was less than those of UV or gamma-ray irradiations. The application of election-beam to the wastewater treatment system showed the high efficiency of destroying and removal effects on bacterial cells. The selective increase in population of radiation resistant bacteria was not observed. These results indicate that the application of ionizing radiation to the processes of wastewater treatment system will be suitable than UV irradiation because of its degradability of variable antibiotics, high removal rate of harmful bacteria, less mutagenicity of bacteria, and low selective effect on radiation resistant bacteria

  11. Evaluation of the antibiotic activity and genetic mutation of microorganisms in the effluent treated with the electron-beam from waste-water treatment plant

    International Nuclear Information System (INIS)

    Lee, Dong Hun; Nam, Ji Hyun; Shin, Ji Hye; Yun, Seo Yeon; Cho, Young Cheol; Oh, Kyoung hee

    2011-04-01

    In this study, the residual concentrations and activities of antibiotics after UV or gamma-ray treatments were estimated, and the effect of irradiation of UV, gamma-ray, or electron beam was estimated on the survivability and less mutagenic effect on bacteria. The changes of bacterial communities and radiation resistant population in the effluent treated with UV and electron-beam were analyzed. The gamma-ray irradiation was more effective than UV in degradation of antibiotics. The extent of mutagenicity of electron-beam irradiation was less than those of UV or gamma-ray irradiations. The application of election-beam to the wastewater treatment system showed the high efficiency of destroying and removal effects on bacterial cells. The selective increase in population of radiation resistant bacteria was not observed. These results indicate that the application of ionizing radiation to the processes of wastewater treatment system will be suitable than UV irradiation because of its degradability of variable antibiotics, high removal rate of harmful bacteria, less mutagenicity of bacteria, and low selective effect on radiation resistant bacteria

  12. Mutation breeding in mangosteen

    International Nuclear Information System (INIS)

    Mohd Khalid Mohd Zain

    2002-01-01

    Mangosteen the queen of the tropical fruits is apomitic and only a cultivar is reported and it reproduces asexually. Conventional breeding is not possible and the other methods to create variabilities are through genetic engineering and mutation breeding. The former technique is still in the infantry stage in mangosteen research while the latter has been an established tool in breeding to improve cultivars. In this mutation breeding seeds of mangosteen were irradiated using gamma rays and the LD 50 for mangosteen was determined and noted to be very low at 10 Gy. After sowing in the seedbed, the seedlings were transplanted in polybags and observed in the nursery bed for about one year before planted in the field under old oil palm trees in Station MARDI, Kluang. After evaluation and screening, about 120 mutant mangosteen plants were selected and planted in Kluang. The plants were observed and some growth data taken. There were some mutant plants that have good growth vigour and more vigorous that the control plants. The trial are now in the fourth year and the plants are still in the juvenile stage. (Author)

  13. Mutation breeding in chickpea

    International Nuclear Information System (INIS)

    Sagel, Z.; Tutluer, M. I.; Peskircioglu, H.; Kantoglu, Y.; Kunter, B.

    2009-01-01

    Chickpea is an important food legume in Turkey. Turkey is one of the most important gene centers in the world for legumes. Realizing the potential of induced mutations, a mutation breeding programme was initiated at the Nuclear Agriculture Section of the Saraykoy Nuclear Research and Training Center in 1994. The purpose of the study was to obtain high yielding chickpea mutants with large seeds, good cooking quality and high protein content. Beside this some characters such as higher adaptation ability, tolerant to cold and drought, increased machinery harvest type, higher yield, resistant to diseases especially to antracnose and pest were investigated too. Parent varieties were ILC-482, AK-7114 and AKCIN-91 had been used in these experiments. The irradiation doses were 0 (control), 50, 100, 150, 200, 250, 300, 350 and 400 Gy for field experiments, respectively. As a result of these experiments, two promising mutant lines were chosen and given to the Seed Registration and Certification Center for official registration These two promising mutants were tested at five different locations of Turkey, in 2004 and 2005 years. After 2 years of registration experiments one of outstanding mutants was officially released as mutant chickpea variety under the name TAEK-SAGEL, in 2006. Some basic characteristics of this mutant are; earliness (95-100 day), high yield capacity (180-220 kg/da), high seed protein (22-25 %), first pot height (20-25 cm), 100 seeds weight (42-48 g), cooking time (35-40 min) and resistance to Ascochyta blight.

  14. Mutation breeding newsletter. No. 43

    International Nuclear Information System (INIS)

    1997-10-01

    This issue of the Newsletter includes articles dealing with radiation induced mutation based plant breeding research findings aimed at improving productivity, disease resistance and tolerance of stress conditions

  15. Neighborhood properties are important determinants of temperature sensitive mutations.

    Directory of Open Access Journals (Sweden)

    Svetlana Lockwood

    Full Text Available Temperature-sensitive (TS mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method-logistic regression-to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations.

  16. Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.

    Science.gov (United States)

    Meier, Susan; Tavraz, Neslihan N; Dürr, Katharina L; Friedrich, Thomas

    2010-02-01

    The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the

  17. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  18. Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

    Science.gov (United States)

    Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

    2010-10-01

    Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

  19. Mutational properties of amino acid residues: implications for evolvability of phosphorylatable residues

    DEFF Research Database (Denmark)

    Creixell, Pau; Schoof, Erwin M.; Tan, Chris Soon Heng

    2012-01-01

    in terms of their mutational activity. Moreover, we highlight the importance of the genetic code and physico-chemical properties of the amino acid residues as likely causes of these inequalities and uncover serine as a mutational hot spot. Finally, we explore the consequences that these different......; it is typically assumed that all amino acid residues are equally likely to mutate or to result from a mutation. Here, by reconstructing ancestral sequences and computing mutational probabilities for all the amino acid residues, we refute this assumption and show extensive inequalities between different residues...... mutational properties have on phosphorylation site evolution, showing that a higher degree of evolvability exists for phosphorylated threonine and, to a lesser extent, serine in comparison with tyrosine residues. As exemplified by the suppression of serine's mutational activity in phosphorylation sites, our...

  20. Recurrent LDL-receptor mutation causes familial ...

    African Journals Online (AJOL)

    1995-05-05

    May 5, 1995 ... mutation detection. Haplotype analysis with polymorphisms on both sides of the FH2 mutation indicated that the identical LDLR gene mutations found in two different South ... amplification refractory mutation system (ARMS)" and single- .... point mutations that cause familial defective apolipoprotein. 8-100 ...

  1. Studies on mutation techniques in rice breeding

    International Nuclear Information System (INIS)

    Wang Cailian; Chen Qiufang; Jin Wei

    2001-01-01

    Synthetical techniques for improving rice mutation breeding efficiency were studied. The techniques consist of corresponding relationship between radiosensitivity and mutation frequency, choosing appropriate materials, combination of physical and chemical mutagens, mutagenic effects of the new mutagenic agents as proton, ions, synchronous irradiation and space mutation. These techniques and methods for inducing mutations are very valuable to increase inducing mutation efficiency and breeding level

  2. TP53 and Beta-catenin mutations in liver tumours

    Directory of Open Access Journals (Sweden)

    Pierre Hainaut

    2007-02-01

    Full Text Available

    HBV and HCV play key roles in the etiopathogenesis of Hepatocellular carcinoma (HCC . Studies mostly based on cases from Western countries suggest distinct genetic pathways of carcinogenesis involving either TP53 or CTTNB1 mutations. Inappropriate reactivation of Wnt pathway due to mutations in CTNNB1 (Beta-Catenin gene itself is also frequently reported. Mutant Beta-catenin escapes to ubiquitination and down regulation by GSK3-B, it accumulates and trans-activates variety of oncogenes involved in neoplasmic transformation mimicking Wnt pathway activation. Taking into consideration viral infection, chromosome instability and TP53 /Beta-catenin alterations, Laurent-Puig et al. described two distinct HCC profiles in a serie of 137 HCC cases , the first one associates HBV infection with frequent chromosomal alteration and distributes with TP53 mutations, the second would be observed in HBV negative large sized tumors and distributes with Beta-catenin mutations. We have investigated the status of HBV and HCV infections and of genetic alterations in TP53 and CTTNB1 in 26 patients with HCC from Thailand. In tumours, HBV DNA was found in 19 cases (73% and HCV RNA in 4 cases (15.4% cases, 3 of whom were co-infected. Among the 19 HBV positive cases, sequencing of S gene showed genotype C in 82% and genotype B in 18%. Furthermore, 5/19 cases were negative for HBsAg and were categorized as occult HBV infections. TP53 mutations were detected in 9 cases (34,6% including 7 mutations at codon 249 (AGG to AGT, arginine to serine, considered as ";fingerprint"; of mutagenesis by aflatoxin metabolites. All cases with 249ser mutation had overt HBV infection.

    CTNNB1 mutations were found in 6/26 cases (23%, 4 of whom also had TP53 mutation. There was no significant association between CTTNB1

  3. Mutation breeding in soybean

    International Nuclear Information System (INIS)

    Baradjanegara, A.A.

    1983-01-01

    In Indonesia, soybean is one of the important crop after rice. It is generally cultivated in the lowlands and rarely in the highlands. Seeds of soybean variety ORBA were treated with various doses of fast neutrons, gamma rays, EMS and NaN 3 with the aims of studying the mutagen effects in M-1 and M-2 generations and also to select mutants adapted to highland conditions. D-50 doses for gamma rays, fast neutrons and EMS were around 23 krad, 2,300 rad, 0.3%, respectively. Much higher chlorophyll mutation frequency was observed in EMS treatment of 0.3%. Seven mutants were shorter and four early mutants matured from 4 to 20 days earlier than the control plants. Two early mutants were quite adaptable in both the low and highlands and produced better yields than the parental material. (author)

  4. Studies of human mutation rates, December 1, 1985--November 30, 1986

    International Nuclear Information System (INIS)

    Neel, J.V.

    1985-01-01

    This program seeks to quantify native human mutation rates and to determine how man's activities may affect these rates. The program is divided into six tasks, i.e. The American Indian mutation rate, monitoring populations for frequency of mutation by electrophoresis of blood proteins, application of molecular biological approaches to the detection and study of mutational events in human populations, development of two-dimensional electrophoresis for identification of mutant proteins, co-operative program with the Radiation Effects Research Foundation in Hiroshima and Nagasaki, Japan, and statistical problems associated with the estimation of mutation rates. Progress of each of the above tasks is related in detail. (DT)

  5. Mutations induced by X-radiation in the yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Loprieno, N.; Barale, R.; Baroncelli, S.; Cammellini, A.; Melani, M.; Nieri, R.; Nozzolini, M.; Rossi, A.M.; Pisa Univ.

    1975-01-01

    Experiments on strains of yeast with different genetic backgrounds were done to evaluate the kinetics of inactivation and mutation induction by X-radiation. A system of forward mutation induction in five loci was used and a specific mutation rate was evaluated for the wild type. From a comparison of observations with wild type and radiation-sensitive strains, it may be assumed that in this yeast, mutations are mainly the result of a repair-active process. The range of genotypic and phenotypic influence upon the specific locus mutation rate was evaluated with appropriate biological material and experiments

  6. Single gene deletions of mrpA to mrpG and mrpE point mutations affect activity of the Mrp Na+/H+ antiporter of alkaliphilic Bacillus and formation of hetero-oligomeric Mrp complexes.

    Science.gov (United States)

    Morino, Masato; Natsui, Shinsuke; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

    2008-06-01

    Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.

  7. Mutation breeding in ornamental plants

    International Nuclear Information System (INIS)

    Datta, S.K.

    1990-01-01

    Full text: Mutation induction produced a large number of new promising varieties in ornamental species. 37 new mutants of Chrysanthemum and 14 of rose have been developed by mutations and released for commercialisation. The mutations in flower colour/shape were detected as chimeras in M 1 V 1 , M 1 V 2 , M 1 V 3 generations. The mutation frequency varied with the cultivar and exposure to gamma rays. Comparative analysis of original cultivars and their respective induced mutants on cytomorphological, anatomical and biochemical characters are being carried out for better understanding of the mechanism involved in the origin and evolution of somatic flower colour/shape mutations. Cytological analysis with reference to chromosomal aberrations, chromosome number, ICV, INV and DNA content gave no differences between the original and mutant cultivars. Analysis of florets/petal pigments by TLC and spectrophotometric methods indicated both qualitative and quantitative changes. (author)

  8. Kinetic studies of the native and mutated intracellular beta-glucosidases from Cellulomonas biazotea.

    Science.gov (United States)

    Rajoka, M I; Durrani, I S; Khalid, A M

    2005-04-01

    The mutation, conferring streptomycin and deoxyglucose resistance on cells, had profound effect on the kinetic and thermodynamic parameters inferring thermostabilization of beta-glucosidase from mutant 51 SM(r) of Cellulomonas biazotea. Free energy of activation for substrate binding, enthalpy and entropy of activation for irreversible denaturation of mutant-derived enzyme were decreased compared with enzyme from wild organism suggesting that the mutation partly stabilized the enzyme and that mutation made it more reactive.

  9. Evolutionary Accessibility of Mutational Pathways

    Science.gov (United States)

    Franke, Jasper; Klözer, Alexander; de Visser, J. Arjan G. M.; Krug, Joachim

    2011-01-01

    Functional effects of different mutations are known to combine to the total effect in highly nontrivial ways. For the trait under evolutionary selection (‘fitness’), measured values over all possible combinations of a set of mutations yield a fitness landscape that determines which mutational states can be reached from a given initial genotype. Understanding the accessibility properties of fitness landscapes is conceptually important in answering questions about the predictability and repeatability of evolutionary adaptation. Here we theoretically investigate accessibility of the globally optimal state on a wide variety of model landscapes, including landscapes with tunable ruggedness as well as neutral ‘holey’ landscapes. We define a mutational pathway to be accessible if it contains the minimal number of mutations required to reach the target genotype, and if fitness increases in each mutational step. Under this definition accessibility is high, in the sense that at least one accessible pathway exists with a substantial probability that approaches unity as the dimensionality of the fitness landscape (set by the number of mutational loci) becomes large. At the same time the number of alternative accessible pathways grows without bounds. We test the model predictions against an empirical 8-locus fitness landscape obtained for the filamentous fungus Aspergillus niger. By analyzing subgraphs of the full landscape containing different subsets of mutations, we are able to probe the mutational distance scale in the empirical data. The predicted effect of high accessibility is supported by the empirical data and is very robust, which we argue reflects the generic topology of sequence spaces. Together with the restrictive assumptions that lie in our definition of accessibility, this implies that the globally optimal configuration should be accessible to genome wide evolution, but the repeatability of evolutionary trajectories is limited owing to the presence of a

  10. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma.

    Science.gov (United States)

    Krauthammer, Michael; Kong, Yong; Ha, Byung Hak; Evans, Perry; Bacchiocchi, Antonella; McCusker, James P; Cheng, Elaine; Davis, Matthew J; Goh, Gerald; Choi, Murim; Ariyan, Stephan; Narayan, Deepak; Dutton-Regester, Ken; Capatana, Ana; Holman, Edna C; Bosenberg, Marcus; Sznol, Mario; Kluger, Harriet M; Brash, Douglas E; Stern, David F; Materin, Miguel A; Lo, Roger S; Mane, Shrikant; Ma, Shuangge; Kidd, Kenneth K; Hayward, Nicholas K; Lifton, Richard P; Schlessinger, Joseph; Boggon, Titus J; Halaban, Ruth

    2012-09-01

    We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1(P29S)) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1(P29S) showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

  11. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Krauthammer, Michael; Kong, Yong; Ha, Byung Hak; Evans, Perry; Bacchiocchi, Antonella; McCusker, James P.; Cheng, Elaine; Davis, Matthew J.; Goh, Gerald; Choi, Murim; Ariyan, Stephan; Narayan, Deepak; Dutton-Regester, Ken; Capatana, Ana; Holman, Edna C.; Bosenberg, Marcus; Sznol, Mario; Kluger, Harriet M.; Brash, Douglas E.; Stern, David F.; Materin, Miguel A.; Lo, Roger S.; Mane, Shrikant; Ma, Shuangge; Kidd, Kenneth K.; Hayward, Nicholas K.; Lifton, Richard P.; Schlessinger, Joseph; Boggon, Titus J.; Halaban, Ruth (Yale-MED); (UCLA); (Queens)

    2012-10-11

    We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1{sup P29S}) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1{sup P29S} showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

  12. Monoallelic mutation analysis (MAMA) for identifying germline mutations.

    Science.gov (United States)

    Papadopoulos, N; Leach, F S; Kinzler, K W; Vogelstein, B

    1995-09-01

    Dissection of germline mutations in a sensitive and specific manner presents a continuing challenge. In dominantly inherited diseases, mutations occur in only one allele and are often masked by the normal allele. Here we report the development of a sensitive and specific diagnostic strategy based on somatic cell hybridization termed MAMA (monoallelic mutation analysis). We have demonstrated the utility of this strategy in two different hereditary colorectal cancer syndromes, one caused by a defective tumour suppressor gene on chromosome 5 (familial adenomatous polyposis, FAP) and the other caused by a defective mismatch repair gene on chromosome 2 (hereditary non-polyposis colorectal cancer, HNPCC).

  13. Prevalence of EGFR Mutations in Lung Cancer in Uruguayan Population

    Directory of Open Access Journals (Sweden)

    Nora Berois

    2017-01-01

    Full Text Available Background. Incorporation of molecular analysis of the epidermal growth factor receptor (EGFR gene into routine clinical practice represents a milestone for personalized therapy of the non-small-cell lung cancer (NSCLC. However, the genetic testing of EGFR mutations has not yet become a routine clinical practice in developing countries. In view of different prevalence of such mutations among different ethnicities and geographic regions, as well as the limited existing data from Latin America, our aim was to study the frequency of major types of activating mutations of the EGFR gene in NSCLC patients from Uruguay. Methods. We examined EGFR mutations in exons 18 through 21 in 289 NSCLC Uruguayan patients by PCR-direct sequencing. Results. EGFR mutations were detected in 53 of the 289 (18.3% patients, more frequently in women (23.4% than in men (14.5%. The distribution by exon was similar to that generally reported in the literature. Conclusions. This first epidemiological study of EGFR mutations in Uruguay reveals a wide spectrum of mutations and an overall prevalence of 18.3%. The background ethnic structure of the Uruguayan population could play an important role in explaining our findings.

  14. Recurrent KIF2A mutations are responsible for classic lissencephaly.

    Science.gov (United States)

    Cavallin, Mara; Bijlsma, Emilia K; El Morjani, Adrienne; Moutton, Sébastien; Peeters, Els A J; Maillard, Camille; Pedespan, Jean Michel; Guerrot, Anne-Marie; Drouin-Garaud, Valérie; Coubes, Christine; Genevieve, David; Bole-Feysot, Christine; Fourrage, Cecile; Steffann, Julie; Bahi-Buisson, Nadia

    2017-04-01

    Kinesins play a critical role in the organization and dynamics of the microtubule cytoskeleton, making them central players in neuronal proliferation, neuronal migration, and postmigrational development. Recently, KIF2A mutations were identified in cortical malformation syndromes associated with microcephaly. Here, we detected two de novo p.Ser317Asn and p.His321Pro mutations in KIF2A in two patients with lissencephaly and microcephaly. In parallel, we re-evaluated the two previously reported cases showing de novo mutations of the same residues. The identification of mutations only in the residues Ser317 and His321 suggests these are hotspots for de novo mutations. Both mutations lead to a classic form of lissencephaly, with a posterior to anterior gradient, almost indistinguishable from LIS1-related lissencephaly. However, three fourths of patients also showed variable congenital and postnatal microcephaly, up to -5 SD. Located in the motor domain of the KIF2A protein, the Ser317 and His321 alterations are expected to disrupt binding or hydrolysis of ATP and consequently the MT depolymerizing activity. This report also establishes that KIF2A mutations represent significant causes of classic lissencephaly with microcephaly.

  15. Mutagenicity of cyclopenta-fused polynuclear aromatic hydrocarbons and a non-polar fraction from a fuel combustion sample in a Salmonella forward mutation assay without exogenous metabolic activation.

    Science.gov (United States)

    Busby, W F; Smith, H; Plummer, E F; Lafleur, A L; Mulder, P P; Boere, B B; Cornelisse, J; Lugtenburg, J

    1997-07-14

    A series of cyclopenta-fused polynuclear aromatic hydrocarbons (PAH) were tested for mutagenicity in a bacterial forward mutation assay based on resistance to 8-azaguanine (8-AG) in Salmonella typhimurium TM677 in the absence of Aroclor-induced rat liver postmitochondrial supernatant (PMS). All of the aceanthrylenes tested were mutagenic in the absence of PMS, whereas none of the acephenanthrylenes were active. The following mutagenic potency series expressed as the minimum detectable mutagen concentration (MDMC) in nmol/ml was obtained: aceanthrylene (AA) (5.5); cyclopent[h,i]aceanthrylene (CPAA)(18.2); 6-methylaceanthrylene (6-MeAA)(112); 1,2,6,7-tetrahydrocyclopent[h,i]aceanthrylene (THCPAA) (166); 1,2-dihydroaceanthrylene (DHAA) (298). Saturation of the cyclopenta rings or methylation at the 6-position of AA reduced, but did not eliminate, mutagenicity measured in the absence of PMS. AA was unusual because it was approximately 4-fold more mutagenic in the absence of PMS than in its presence. The other aceanthrylenes tested were 1.3-10.7 times more mutagenic in the presence of PMS than in its absence to give an MDMC potency series of: CPAA (3.8); 6-MeAA (10.5); AA (19.9); THCPAA (52.9); DHAA (229). Approximately 20% of the PMS-independent mutagenicity in a combustion sample from ethylene burned under fuel-rich conditions was found in a fraction containing only non-polar, 4-7 ring PAHs, widely attributed to be mutagenic only in the presence of PMS. None of this mutagenicity could be attributed to aceanthrylenes, thus other non-polar PAHs appear to possess significant PMS-independent mutagenicity as well.

  16. SETBP1 mutations drive leukemic transformation in ASXL1-mutated MDS.

    Science.gov (United States)

    Inoue, D; Kitaura, J; Matsui, H; Hou, H-A; Chou, W-C; Nagamachi, A; Kawabata, K C; Togami, K; Nagase, R; Horikawa, S; Saika, M; Micol, J-B; Hayashi, Y; Harada, Y; Harada, H; Inaba, T; Tien, H-F; Abdel-Wahab, O; Kitamura, T

    2015-04-01

    Mutations in ASXL1 are frequent in patients with myelodysplastic syndrome (MDS) and are associated with adverse survival, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) is not fully understood. Recently, it has been found that deletion of Asxl1 or expression of C-terminal-truncating ASXL1-MTs inhibit myeloid differentiation and induce MDS-like disease in mice. Here, we find that SET-binding protein 1 (SETBP1) mutations (SETBP1-MT) are enriched among ASXL1-mutated MDS patients and associated with increased incidence of leukemic transformation, as well as shorter survival, suggesting that SETBP1-MT play a critical role in leukemic transformation of MDS. We identify that SETBP1-MT inhibit ubiquitination and subsequent degradation of SETBP1, resulting in increased expression. Expression of SETBP1-MT, in turn, inhibited protein phosphatase 2A activity, leading to Akt activation and enhanced expression of posterior Hoxa genes in ASXL1-mutant cells. Biologically, SETBP1-MT augmented ASXL1-MT-induced differentiation block, inhibited apoptosis and enhanced myeloid colony output. SETBP1-MT collaborated with ASXL1-MT in inducing acute myeloid leukemia in vivo. The combination of ASXL1-MT and SETBP1-MT activated a stem cell signature and repressed the tumor growth factor-β signaling pathway, in contrast to the ASXL1-MT-induced MDS model. These data reveal that SETBP1-MT are critical drivers of ASXL1-mutated MDS and identify several deregulated pathways as potential therapeutic targets in high-risk MDS.

  17. Mutations in PIK3CA are infrequent in neuroblastoma

    International Nuclear Information System (INIS)

    Dam, Vincent; Morgan, Brian T; Mazanek, Pavel; Hogarty, Michael D

    2006-01-01

    Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain 'hot spots' where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. These data suggest that activating

  18. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  19. HNPCC: Six new pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Epplen Joerg T

    2004-06-01

    Full Text Available Abstract Background Hereditary non-polyposis colorectal cancer (HNPCC is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR. HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6. Methods Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out. Results We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6 resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702], three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298] and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]. All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H. Conclusions HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.

  20. Radiation mutation breeding

    Energy Technology Data Exchange (ETDEWEB)

    Song, Hi Sup; Kim, Jae Sung; Kim, Jin Kyu; Shin, In Chul; Lim, Young Taek

    1998-04-01

    In order to develop an advanced technical knowledge for the selection of better mutants, some of the crops were irradiated and the mutation rate, the survival rate and the method for selction of a mutant were studied. Furthermore, this study aimed to obtain basic data applicable to the development of genetic resources by evaluation and analysis the specific character for selection of the superior mutant and its plant breeding. 1. selection of the mutant with a superior resistance against environment in the principal crops 1) New varieties of mutant rices such as Wonpyeongbyeo, Wongwangbyeo, Winmibyeo, and heogseon chalbeyeo (sticky forma) were registered in the national variety list and made an application to crop variety protection right. They are under review now. 2) We also keep on studying on the number of a grain of 8 lines of excellent mutant rice for the purpose of improvement of breeding . 3) We selected 3 lines which have a resistance to pod and stem blight in large soybean, 31 lines with small grain size and higher yield, 112 lines of soybean of cooking, 7 lines of low lipoxygenase content, and 12 lines with decreased phytic acid content by 20 % compared to the previous level. 2. Selection of advanced Mugunwha (Rose of Sharon) mutant 1) Bagseul, a new variety of mutant, was developed and 30 plantlets of it are being proliferated. 2) Fifty-three lines of a mutant having a various morphologies were selected.

  1. Induced mutations in castor

    International Nuclear Information System (INIS)

    Ganesan, K.; Javad Hussain, H.S.; Vindhiyavarman, P.

    2001-01-01

    Castor (Ricinus communis L.) is an important oilseed crop in India. To create variability mutations were induced in two cultivars 'TMV5' (maturing in 130-140 days) and 'CO1' (perennial type). Gamma rays and diethyl sulphate and ethidium bromide were used for seed treatment. Ten doses, from 100 to 1000 Gy were employed. For chemical mutagenesis five concentrations of mutagenes from 10 to 50 mM were tried. No economic mutants could be isolated after treatment with the chemical mutagens. The following economic mutants were identified in the dose 300 Gy of gamma rays. Annual types from perennial CO 1 castor CO 1 is a perennial variety (8-10 years) with bold seeds (100 seed weight 90 g) and high oil content (57%). Twenty-one lines were isolated with annual types (160-180 days) with high yield potential as well as bold seeds and high oil content. These mutants, identified in M 3 generation were bred true in subsequent generations up to M 8 generation. Critical evaluation of the mutants in yield evaluation trials is in progress

  2. Mutational dichotomy in desmoplastic malignant melanoma corroborated by multigene panel analysis.

    Science.gov (United States)

    Jahn, Stephan W; Kashofer, Karl; Halbwedl, Iris; Winter, Gerlinde; El-Shabrawi-Caelen, Laila; Mentzel, Thomas; Hoefler, Gerald; Liegl-Atzwanger, Bernadette

    2015-07-01

    Desmoplastic malignant melanoma is a distinct melanoma entity histologically subtyped into mixed and pure forms due to significantly reduced lymph node metastases in the pure form. Recent reports investigating common actionable driver mutations have demonstrated a lack of BRAF, NRAS, and KIT mutation in pure desmoplastic melanoma. In search for alternative driver mutations next generation amplicon sequencing for hotspot mutations in 50 genes cardinal to tumorigenesis was performed and in addition the RET G691S polymorphism was investigated. Data from 21 desmoplastic melanomas (12 pure and 9 mixed) were retrieved. Pure desmoplastic melanomas were either devoid of mutations (50%) or displayed mutations in tumor suppressor genes (TP53, CDKN2A, and SMAD4) singularly or in combination with the exception of a PIK3CA double-mutation lacking established biological relevance. Mixed desmoplastic melanomas on the contrary were frequently mutated (89%), and 67% exhibited activating mutations similar to common-type cutaneous malignant melanomas (BRAF, NRAS, FGFR2, and ERBB2). Separate analysis of morphologically heterogeneous tumor areas in four mixed desmoplastic malignant melanomas displayed no difference in mutation status and RET G691 status. GNAQ and GNA11, two oncogenes in BRAF and NRAS wild-type uveal melanomas, were not mutated in our cohort. The RET G691S polymorphism was found in 25% of pure and 38% of mixed desmoplastic melanomas. Apart from RET G691S our findings demonstrate absence of activating driver mutations in pure desmoplastic melanoma beyond previously investigated oncogenes (BRAF, NRAS, and KIT). The findings underline the therapeutic dichotomy of mixed versus pure desmoplastic melanoma with regard to activating mutations primarily of the mitogen-activated protein kinase pathway.

  3. Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency

    DEFF Research Database (Denmark)

    Berryer, Martin H; Hamdan, Fadi F; Klitten, Laura L

    2013-01-01

    De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID...

  4. Variable clinical expressivity of STAT3 mutation in hyperimmunoglobulin E syndrome: genetic and clinical studies of six patients

    NARCIS (Netherlands)

    Wolach, Ofir; Kuijpers, Taco; Ben-Ari, Josef; Gavrieli, Ronit; Feinstein-Goren, Neta; Alders, Marielle; Garty, Ben Zion; Wolach, Baruch

    2014-01-01

    Autosomal dominant Hyper IgE syndrome (AD-HIES) is a rare and complex primary immunodeficiency that affects multiple systems. Mutations in signal transducer and activator of transcription 3 (STAT3) gene cause AD-HIES. These mutations have a dominant-negative effect and the presence of such mutations

  5. 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

    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...... oligonucleotide (SSO) mediated blocking of the pseudoexon 3'ss and 5'ss effectively restores normal GLA splicing. This indicates that SSO based splicing correction may be a therapeutic alternative in the treatment of Fabry disease....

  6. Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase δ mutation in human cells.

    Science.gov (United States)

    Mertz, T M; Baranovskiy, A G; Wang, J; Tahirov, T H; Shcherbakova, P V

    2017-08-01

    Mutations in the POLD1 and POLE genes encoding DNA polymerases δ (Polδ) and ɛ (Polɛ) cause hereditary colorectal cancer (CRC) and have been found in many sporadic colorectal and endometrial tumors. Much attention has been focused on POLE exonuclease domain mutations, which occur frequently in hypermutated DNA mismatch repair (MMR)-proficient tumors and appear to be responsible for the bulk of genomic instability in these tumors. In contrast, somatic POLD1 mutations are seen less frequently and typically occur in MMR-deficient tumors. Their functional significance is often unclear. Here we demonstrate that expression of the cancer-associated POLD1-R689W allele is strongly mutagenic in human cells. The mutation rate increased synergistically when the POLD1-R689W expression was combined with a MMR defect, indicating that the mutator effect of POLD1-R689W results from a high rate of replication errors. Purified human Polδ-R689W has normal exonuclease activity, but the nucleotide selectivity of the enzyme is severely impaired, providing a mechanistic explanation for the increased mutation rate in the POLD1-R689W-expressing cells. The vast majority of mutations induced by the POLD1-R689W are GC→︀TA transversions and GC→︀AT transitions, with transversions showing a strong strand bias and a remarkable preference for polypurine/polypyrimidine sequences. The mutational specificity of the Polδ variant matches that of the hypermutated CRC cell line, HCT15, in which this variant was first identified. The results provide compelling evidence for the pathogenic role of the POLD1-R689W mutation in the development of the human tumor and emphasize the need to experimentally determine the significance of Polδ variants present in sporadic tumors.

  7. Mutation breeding newsletter. No. 44

    International Nuclear Information System (INIS)

    1999-04-01

    This issue of the Newsletter presents research reports on the role of radiation induced mutation and chemical mutagens in improving productivity, disease resistance; cold and salinity tolerance of various crops and ornamental plants

  8. Mutation breeding newsletter. No. 18

    International Nuclear Information System (INIS)

    1981-07-01

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  9. Mutation breeding newsletter. No. 6

    International Nuclear Information System (INIS)

    1975-08-01

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  10. Mutation breeding newsletter. No. 2

    International Nuclear Information System (INIS)

    1973-02-01

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  11. Mutation breeding newsletter. No. 5

    International Nuclear Information System (INIS)

    1975-02-01

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  12. Mutation breeding newsletter. No. 32

    International Nuclear Information System (INIS)

    1988-07-01

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  13. Mutation breeding newsletter. No. 9

    International Nuclear Information System (INIS)

    1977-01-01

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  14. Mutation breeding newsletter. No. 8

    International Nuclear Information System (INIS)

    1976-09-01

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  15. Mutation breeding newsletter. No. 10

    International Nuclear Information System (INIS)

    1977-07-01

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  16. Mutation breeding newsletter. No. 7

    International Nuclear Information System (INIS)

    1976-01-01

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  17. Mutation breeding newsletter. No. 36

    International Nuclear Information System (INIS)

    1990-07-01

    This issue of the Newsletter presents abstracts and short communications of research results on radiation and chemical induced mutation breeding projects. Positive traits such as disease resistance and increased productivity are highlighted

  18. Mutation breeding newsletter. No. 16

    International Nuclear Information System (INIS)

    1980-07-01

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  19. Mutation Breeding Newsletter. No. 37

    International Nuclear Information System (INIS)

    1991-01-01

    This newsletter contains a brief account of FAO/IAEA meetings held in 1990 on plant breeding involving the use of induced mutations. It also features a list of commercially available plant cultivars produced by such techniques. Refs and tabs

  20. Mutation breeding newsletter. No. 3

    International Nuclear Information System (INIS)

    1974-01-01

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

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