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Sample records for mutation confers increased

  1. Cooperativity of Negative Autoregulation Confers Increased Mutational Robustness

    PubMed Central

    Marciano, David C.; Lua, Rhonald C.; Herman, Christophe; Lichtarge, Olivier

    2016-01-01

    Negative autoregulation is universally found across organisms. In the bacterium Escherichia coli, transcription factors often repress their own expression to form a negative feedback network motif that enables robustness to changes in biochemical parameters. Here we present a simple phenomenological model of a negative feedback transcription factor repressing both itself and another target gene. The strength of the negative feedback is characterized by three parameters: the cooperativity in self-repression, the maximal expression rate of the transcription factor, and the apparent dissociation constant of the transcription factor binding to its own promoter. Analysis of the model shows that the target gene levels are robust to mutations in the transcription factor, and that the robustness improves as the degree of cooperativity in self-repression increases. The prediction is tested in the LexA transcriptional network of E. coli by altering cooperativity in self-repression and promoter strength. Indeed, we find robustness is correlated with the former. Considering the proposed importance of gene regulation in speciation, parameters governing a transcription factor’s robustness to mutation may have significant influence on a cell or organism’s capacity to evolve. PMID:27391757

  2. Cooperativity of Negative Autoregulation Confers Increased Mutational Robustness

    NASA Astrophysics Data System (ADS)

    Marciano, David C.; Lua, Rhonald C.; Herman, Christophe; Lichtarge, Olivier

    2016-06-01

    Negative autoregulation is universally found across organisms. In the bacterium Escherichia coli, transcription factors often repress their own expression to form a negative feedback network motif that enables robustness to changes in biochemical parameters. Here we present a simple phenomenological model of a negative feedback transcription factor repressing both itself and another target gene. The strength of the negative feedback is characterized by three parameters: the cooperativity in self-repression, the maximal expression rate of the transcription factor, and the apparent dissociation constant of the transcription factor binding to its own promoter. Analysis of the model shows that the target gene levels are robust to mutations in the transcription factor, and that the robustness improves as the degree of cooperativity in self-repression increases. The prediction is tested in the LexA transcriptional network of E. coli by altering cooperativity in self-repression and promoter strength. Indeed, we find robustness is correlated with the former. Considering the proposed importance of gene regulation in speciation, parameters governing a transcription factor's robustness to mutation may have significant influence on a cell or organism's capacity to evolve.

  3. Mutations in the Drosophila Pushover Gene Confer Increased Neuronal Excitability and Spontaneous Synaptic Vesicle Fusion

    PubMed Central

    Richards, S.; Hillman, T.; Stern, M.

    1996-01-01

    We describe the identification of a gene called pushover (push), which affects both behavior and synaptic transmission at the neuromuscular junction. Adults carrying either of two mutations in push exhibit sluggishness, uncoordination, a defective escape response, and male sterility. Larvae defective in push exhibit increased release of transmitter at the neuromuscular junction. In particular, the frequency of spontaneous transmitter release and the amount of transmitter release evoked by nerve stimulation are each increased two- to threefold in push mutants at the lowest external [Ca(2+)] tested (0.15 mM). Furthermore, these mutants are more sensitive than wild type to application of the potassium channel-blocking drug quinidine: following qunidine application, push mutants, but not wild-type, display repetitive firing of the motor axon, leading to repetitive muscle postsynaptic potentials. The push gene thus might affect both neuronal excitability and the transmitter release process. Complementation tests and recombinational mapping suggest that the push mutations are allelic to a previously identified P-element-induced mutation, which also causes behavioral abnormalities and male sterility. PMID:8846899

  4. Registration of Durum Wheat Germplasm Lines with Combined Mutations in SBEIIa and SBEIIb Genes Conferring Increased Amylose and Resistant Starch.

    PubMed

    Hazard, Brittany; Zhang, Xiaoqin; Naemeh, Mahmoudreza; Dubcovsky, Jorge

    2014-08-25

    Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.], used in pasta, couscous, and flatbread production, is an important source of starch food products worldwide. The amylose portion of the starch forms resistant starch complexes that resist digestion and contribute to dietary fiber. Increasing the amount of amylose and resistant starch in wheat by mutating the STARCH BRANCHING ENZYME II (SBEII) genes has potential to provide human health benefits. Ethyl methane sulfonate mutations in the linked SBEIIa and SBEIIb paralogs were combined on chromosomes 2A (SBEIIa/b-A; Reg. No. GP-968, PI 670159), 2B (SBEIIa/b-B; Reg. No. GP-970, PI 670161), and on both chromosomes (SBEIIa/b-AB; Reg. No. GP-969, PI 670160) in the tetraploid wheat cultivar Kronos, a semidwarf durum wheat cultivar that has high yield potential and excellent pasta quality. These three double and quadruple SBEII-mutant lines were compared with a control sib line with no SBEII mutations in two field locations in California. The SBEIIa/b-AB line with four mutations showed dramatic increases in amylose (average 66%) and resistant starch (average 753%) relative to the control. However, the SBEIIa/b-AB line also showed an average 7% decrease in total starch and an 8% decrease in kernel weight. The release by the University of California-Davis of the durum wheat germplasm combining four SBEIIa and SBEIIb mutations will accelerate the deployment of these mutations in durum wheat breeding programs and the development of durum wheat varieties with increased resistant starch.

  5. Registration of Durum Wheat Germplasm Lines with Combined Mutations in SBEIIa and SBEIIb Genes Conferring Increased Amylose and Resistant Starch

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Naemeh, Mahmoudreza; Dubcovsky, Jorge

    2016-01-01

    Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.], used in pasta, couscous, and flatbread production, is an important source of starch food products worldwide. The amylose portion of the starch forms resistant starch complexes that resist digestion and contribute to dietary fiber. Increasing the amount of amylose and resistant starch in wheat by mutating the STARCH BRANCHING ENZYME II (SBEII) genes has potential to provide human health benefits. Ethyl methane sulfonate mutations in the linked SBEIIa and SBEIIb paralogs were combined on chromosomes 2A (SBEIIa/b-A; Reg. No. GP-968, PI 670159), 2B (SBEIIa/b-B; Reg. No. GP-970, PI 670161), and on both chromosomes (SBEIIa/b-AB; Reg. No. GP-969, PI 670160) in the tetraploid wheat cultivar Kronos, a semidwarf durum wheat cultivar that has high yield potential and excellent pasta quality. These three double and quadruple SBEII-mutant lines were compared with a control sib line with no SBEII mutations in two field locations in California. The SBEIIa/b-AB line with four mutations showed dramatic increases in amylose (average 66%) and resistant starch (average 753%) relative to the control. However, the SBEIIa/b-AB line also showed an average 7% decrease in total starch and an 8% decrease in kernel weight. The release by the University of California–Davis of the durum wheat germplasm combining four SBEIIa and SBEIIb mutations will accelerate the deployment of these mutations in durum wheat breeding programs and the development of durum wheat varieties with increased resistant starch. PMID:27110322

  6. Registration of Common Wheat Germplasm with Mutations in SBEII Genes Conferring Increased Grain Amylose and Resistant Starch Content

    PubMed Central

    Schönhofen, André; Hazard, Brittany; Zhang, Xiaoqin; Dubcovsky, Jorge

    2016-01-01

    Starch present in the endosperm of common wheat (Triticum aestivum L.) grains is an important source of carbohydrates worldwide. Starches with a greater proportion of amylose have increased levels of resistant starch, a dietary fiber that can provide human health benefits. Induced mutations in STARCH BRANCHING ENZYME II (SBEII) genes in wheat are associated with increased amylose and resistant starch. Ethyl methane sulfonate mutations in SBEIIa and SBEIIb paralogs were combined in the hexaploid wheat cultivar Lassik. Four mutant combinations were generated: SBEIIa/b-AB (Reg. No. GP-997, PI 675644); SBEIIa/b-A, SBEIIa-D (Reg. No. GP-998, PI 675645); SBEIIa/b-B, SBEIIa-D (Reg. No. GP-999, PI 675646); and SBEIIa/b-AB, SBEIIa-D (Reg. No. GP-1000, PI 675647). The SBEII mutant lines were compared with a wild-type control in a greenhouse and field experiment. The quintuple mutant line (SBEIIa/b-AB, SBEIIa-D) presented significant increases in both amylose (51% greenhouse; 63% field) and resistant starch (947% greenhouse; 1057% field) relative to the control. A decrease in total starch content (7.8%) was observed in the field experiment. The quintuple mutant also differed in starch viscosity parameters. Registration of the hexaploid wheat SBEII-mutant lines by University of California, Davis can help expedite the development of common wheat cultivars with increased amylose and resistant starch content. PMID:27818720

  7. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    PubMed Central

    Alemán, Fernando; Caballero, Fernando; Ródenas, Reyes; Rivero, Rosa M.; Martínez, Vicente; Rubio, Francisco

    2014-01-01

    Potassium (K+) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter. PMID:25228905

  8. Heterozygosity increases microsatellite mutation rate

    PubMed Central

    Amos, William

    2016-01-01

    Whole genome sequencing of families of Arabidopsis has recently lent strong support to the heterozygote instability (HI) hypothesis that heterozygosity locally increases mutation rate. However, there is an important theoretical difference between the impact on base substitutions, where mutation rate increases in regions surrounding a heterozygous site, and the impact of HI on sequences such as microsatellites, where mutations are likely to occur at the heterozygous site itself. At microsatellite loci, HI should create a positive feedback loop, with heterozygosity and mutation rate mutually increasing each other. Direct support for HI acting on microsatellites is limited and contradictory. I therefore analysed AC microsatellites in 1163 genome sequences from the 1000 genomes project. I used the presence of rare alleles, which are likely to be very recent in origin, as a surrogate measure of mutation rate. I show that rare alleles are more likely to occur at locus-population combinations with higher heterozygosity even when all populations carry exactly the same number of alleles. PMID:26740567

  9. Increased pilus production conferred by a naturally occurring mutation alters host-pathogen interaction in favor of carriage in Streptococcus pyogenes.

    PubMed

    Flores, Anthony R; Olsen, Randall J; Cantu, Concepcion; Pallister, Kyler B; Guerra, Fermin E; Voyich, Jovanka M; Musser, James M

    2017-03-06

    Studies of the human pathogen group A Streptococcus (GAS) define the carrier phenotype as increased ability to adhere to and persist on epithelial surfaces and decreased ability to cause disease. We tested the hypothesis that a single amino acid change (Arg135Gly) in a highly conserved sensor kinase (LiaS) of a poorly defined GAS regulatory system contributes to a carrier phenotype through increased pilus production. When introduced into an emm serotype-matched invasive strain, the carrier allele (liaS(R135G)) recapitulated a carrier phenotype defined by increased ability to adhere to mucosal surfaces and decreased ability to cause disease. Gene transcript analyses revealed that the liaS mutation significantly altered transcription of the genes encoding pilus when in the presence of bacitracin. Elimination of pilus production in the isogenic carrier mutant decreased ability to colonize the mouse nasopharynx, adhere to and be internalized by cultured human epithelial cells, and restored a virulence phenotype in a mouse model of necrotizing fasciitis. We also observed significantly reduced survival of the isogenic carrier mutant compared to the parental invasive strain after exposure to human neutrophils. Elimination of pilus in the isogenic carrier mutant increased neutrophil survival to the parental invasive strain level. Together, our data demonstrate that the carrier mutation (liaS(R135G)) affects pilus expression. Our data suggest new mechanisms of pilus gene regulation in GAS and differs from the enhanced invasiveness associated with increased pilus production in other bacterial pathogens.

  10. A mutation in the uvi4 gene promotes progression of endo-reduplication and confers increased tolerance towards ultraviolet B light.

    PubMed

    Hase, Yoshihiro; Trung, Khuat Huu; Matsunaga, Tsukasa; Tanaka, Atsushi

    2006-04-01

    We have isolated and characterized a new ultraviolet B (UV-B)-resistant mutant, uvi4 (UV-B-insensitive 4), of Arabidopsis. The fresh weight (FW) of uvi4 plants grown under supplemental UV-B light was more than twice that of the wild-type. No significant difference was found in their ability to repair the UV-B-induced cyclobutane pyrimidine dimers, or in the amount of UV-B absorptive compounds, both of which are well-known factors that contribute to UV sensitivity. Positional cloning revealed that the UVI4 gene encodes a novel basic protein of unknown function. We found that the hypocotyl cells in uvi4 undergo one extra round of endo-reduplication. The uvi4 mutation also promoted the progression of endo-reduplication during leaf development. The UVI4 gene is expressed mainly in actively dividing cells. In the leaves of P(UVI4)::GUS plants, the GUS signal disappeared in basipetal fashion as the leaf developed. The total leaf blade area was not different between uvi4 and the wild-type through leaf development, while the average cell area in the adaxial epidermis was considerably larger in uvi4, suggesting that the uvi4 leaves have fewer but larger epidermal cells. These results suggest that UVI4 is necessary for the maintenance of the mitotic state, and the loss of UVI4 function stimulated endo-reduplication. Tetraploid Arabidopsis was hyper-resistant to UV-B compared to diploid Arabidopsis, suggesting that the enhanced polyploidization is responsible for the increased UV-B tolerance of the uvi4 mutant.

  11. Fitness cost of chromosomal drug resistance-conferring mutations.

    PubMed

    Sander, Peter; Springer, Burkhard; Prammananan, Therdsak; Sturmfels, Antje; Kappler, Martin; Pletschette, Michel; Böttger, Erik C

    2002-05-01

    To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with defined Mycobacterium smegmatis laboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.

  12. Multidrug resistance conferred by novel DNA polymerase mutations in human cytomegalovirus isolates.

    PubMed

    Scott, Gillian M; Weinberg, Adriana; Rawlinson, William D; Chou, Sunwen

    2007-01-01

    The emergence of antiviral-resistant cytomegalovirus (CMV) strains is a continuing clinical problem, with increased numbers of immunocompromised patients given longer-duration antiviral prophylaxis. Two previously unrecognized CMV DNA polymerase mutations (N408K and A834P) identified separately and together in at-risk lung and kidney transplant recipients and a third mutation (L737M) identified in a liver transplant recipient were characterized by marker transfer to antiviral-sensitive laboratory strains AD169 and Towne. Subsequent phenotypic analyses of recombinant strains demonstrated the ability of mutation N408K to confer ganciclovir (GCV) and cidofovir (CDV) resistance and of mutation A834P to confer GCV, foscarnet, and CDV resistance. Mutation L737M did not confer resistance to any of the antiviral agents tested. A recombinant strain containing both N408K and A834P demonstrated increased GCV and CDV resistance compared to the levels of resistance of the virus containing only the A834P mutation. The addition of mutation N408K in combination with A834P also partially reconstituted the replication impairment of recombinant virus containing only A834P. This suggests that perturbation of both DNA polymerization (A834P) and exonuclease (N408K) activities contributes to antiviral resistance and altered replication kinetics in these mutant strains. The identification of these multidrug-resistant CMV strains in at-risk seronegative recipients of organs from seropositive donors suggests that improved prophylactic and treatment strategies are required. The additive effect of multiple mutations on antiviral susceptibility suggests that increasing antiviral-resistant phenotypes can result from different virus-antiviral interactions.

  13. Ribosomal Mutations in Arcanobacterium pyogenes Confer a Unique Spectrum of Macrolide Resistance

    PubMed Central

    Jost, B. Helen; Trinh, Hien T.; Songer, J. Glenn; Billington, Stephen J.

    2004-01-01

    Four macrolide-resistant Arcanobacterium pyogenes isolates contained A2058T, A2058G, or C2611G (Escherichia coli numbering) mutations in their 23S rRNA genes. While these mutations conferred resistance to erythromycin, oleandomycin, and spiramycin, they did not confer resistance to tylosin. PMID:14982799

  14. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates

    PubMed Central

    Straimer, Judith; Gnädig, Nina F.; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D.; Urnov, Fyodor D.; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M.; Ménard, Didier; Fidock, David A.

    2015-01-01

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

  15. Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila.

    PubMed

    Descours, Ghislaine; Ginevra, Christophe; Jacotin, Nathalie; Forey, Françoise; Chastang, Joëlle; Kay, Elisabeth; Etienne, Jerome; Lina, Gérard; Doublet, Patricia; Jarraud, Sophie

    2017-03-01

    Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

  16. Mutations in the Pneumocystis jirovecii DHPS gene confer cross-resistance to sulfa drugs.

    PubMed

    Iliades, Peter; Meshnick, Steven R; Macreadie, Ian G

    2005-02-01

    Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP) and results in a high degree of mortality in immunocompromised individuals. The drug of choice for PCP is typically sulfamethoxazole (SMX) or dapsone in conjunction with trimethoprim. Drug treatment failure and sulfa drug resistance have been implicated epidemiologically with point mutations in dihydropteroate synthase (DHPS) of P. jirovecii. P. jirovecii cannot be cultured in vitro; however, heterologous complementation of the P. jirovecii trifunctional folic acid synthesis (PjFAS) genes with an E. coli DHPS-disrupted strain was recently achieved. This enabled the evaluation of SMX resistance conferred by DHPS mutations. In this study, we sought to determine whether DHPS mutations conferred sulfa drug cross-resistance to 15 commonly available sulfa drugs. It was established that the presence of amino acid substitutions (T(517)A or P(519)S) in the DHPS domain of PjFAS led to cross-resistance against most sulfa drugs evaluated. The presence of both mutations led to increased sulfa drug resistance, suggesting cooperativity and the incremental evolution of sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine [SCP] and sulfamethoxypyridazine [SMP]) that had a higher inhibitory potential than SMX were identified. In addition, SCP, SMP, and sulfadiazine (SDZ) were found to be capable of inhibiting the clinically observed drug-resistant mutants. We propose that SCP, SMP, and SDZ should be considered for clinical evaluation against PCP or for future development of novel sulfa drug compounds.

  17. Molecular survey of turfgrass species for mutations conferring resistance to ACCase inhibiting herbicides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The control of grassy weeds in turfgrass is often problematic due to lack of herbicide selectivity. Seven different naturally occurring mutation sites have been reported to confer resistance to Acetyl coenzyme A carboxylase inhibiting herbicides. One or more of these mutation sites may hold potentia...

  18. Increased transversions in a novel mutator colon cancer cell line.

    PubMed

    Eshleman, J R; Donover, P S; Littman, S J; Swinler, S E; Li, G M; Lutterbaugh, J D; Willson, J K; Modrich, P; Sedwick, W D; Markowitz, S D; Veigl, M L

    1998-03-05

    We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.

  19. Bactobolin Resistance Is Conferred by Mutations in the L2 Ribosomal Protein

    PubMed Central

    Chandler, Josephine R.; Truong, Thao T.; Silva, Patricia M.; Seyedsayamdost, Mohammad R.; Carr, Gavin; Radey, Matthew; Jacobs, Michael A.; Sims, Elizabeth H.; Clardy, Jon; Greenberg, E. Peter

    2012-01-01

    ABSTRACT Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). PMID:23249812

  20. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation.

    PubMed

    Prudhomme, M; Méjean, V; Martin, B; Claverys, J P

    1991-11-01

    DNA repair systems able to correct base pair mismatches within newly replicated DNA or within heteroduplex molecules produced during recombination are widespread among living organisms. Evidence that such generalized mismatch repair systems evolved from a common ancestor is particularly strong for two of them, the Hex system of the gram-positive Streptococcus pneumoniae and the Mut system of the gram-negative Escherichia coli and Salmonella typhimurium. The homology existing between HexA and MutS and between HexB and MutL prompted us to investigate the effect of expressing hex genes in E. coli. Complementation of mutS or mutL mutations, which confer a mutator phenotype, was assayed by introducing on a multicopy plasmid the hexA and hexB genes, under the control of an inducible promoter, either individually or together in E. coli strains. No decrease in mutation rate was conferred by either hexA or hexB gene expression. However, a negative complementation effect was observed in wild-type E. coli cells: expression of hexA resulted in a typical Mut- mutator phenotype. hexB gene expression did not increase the mutation rate either individually or in conjunction with hexA. Since expression of hexA did not affect the mutation rate in mutS mutant cells and the hexA-induced mutator effect was recA independent, it is concluded that this effect results from inhibition of the Mut system. We suggest that HexA, like its homolog MutS, binds to mismatches resulting from replication errors, but in doing so it protects them from repair by the Mut system. In agreement with this hypothesis, an increase in mutS gene copy number abolished the hexA-induced mutator phenotype. HexA protein could prevent repair either by being unable to interact with Mut proteins or by producing nonfunctional repair complexes.

  1. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations.

  2. Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer

    PubMed Central

    Tavtigian, Sean V.; Oefner, Peter J.; Babikyan, Davit; Hartmann, Anne; Healey, Sue; Le Calvez-Kelm, Florence; Lesueur, Fabienne; Byrnes, Graham B.; Chuang, Shu-Chun; Forey, Nathalie; Feuchtinger, Corinna; Gioia, Lydie; Hall, Janet; Hashibe, Mia; Herte, Barbara; McKay-Chopin, Sandrine; Thomas, Alun; Vallée, Maxime P.; Voegele, Catherine; Webb, Penelope M.; Whiteman, David C.; Sangrajrang, Suleeporn; Hopper, John L.; Southey, Melissa C.; Andrulis, Irene L.; John, Esther M.; Chenevix-Trench, Georgia

    2009-01-01

    The susceptibility gene for ataxia telangiectasia, ATM, is also an intermediate-risk breast-cancer-susceptibility gene. However, the spectrum and frequency distribution of ATM mutations that confer increased risk of breast cancer have been controversial. To assess the contribution of rare variants in this gene to risk of breast cancer, we pooled data from seven published ATM case-control mutation-screening studies, including a total of 1544 breast cancer cases and 1224 controls, with data from our own mutation screening of an additional 987 breast cancer cases and 1021 controls. Using an in silico missense-substitution analysis that provides a ranking of missense substitutions from evolutionarily most likely to least likely, we carried out analyses of protein-truncating variants, splice-junction variants, and rare missense variants. We found marginal evidence that the combination of ATM protein-truncating and splice-junction variants contribute to breast cancer risk. There was stronger evidence that a subset of rare, evolutionarily unlikely missense substitutions confer increased risk. On the basis of subset analyses, we hypothesize that rare missense substitutions falling in and around the FAT, kinase, and FATC domains of the protein may be disproportionately responsible for that risk and that a subset of these may confer higher risk than do protein-truncating variants. We conclude that a comparison between the graded distributions of missense substitutions in cases versus controls can complement analyses of truncating variants and help identify susceptibility genes and that this approach will aid interpretation of the data emerging from new sequencing technologies. PMID:19781682

  3. A new point mutation in the iron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum.

    PubMed

    Wang, Yong; Duan, Yabing; Wang, Jianxin; Zhou, Mingguo

    2015-09-01

    Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid-resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid-sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild-type strains, BR and GSM (SdhB gene in the wild-type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron-sulfur subunit of SDH. These results suggest that resistance based on non-conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs.

  4. Increased human AP endonuclease 1 level confers protection against the paternal age effect in mice

    PubMed Central

    Sanchez, Jamila R.; Reddick, Traci L.; Perez, Marissa; Centonze, Victoria E.; Mitra, Sankar; Izumi, Tadahide; McMahan, C. Alex; Walter, Christi A.

    2015-01-01

    Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells. PMID:26201249

  5. Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    PubMed Central

    Utt, Age; Das, Pratyush Kumar; Varjak, Margus; Lulla, Valeria; Lulla, Aleksei

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCE CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the

  6. Misfolding Ectodomain Mutations of the Lutropin Receptor Increase Efficacy of Hormone Stimulation

    PubMed Central

    Charmandari, E.; Guan, R.; Zhang, M.; Silveira, L. G.; Fan, Q. R.; Chrousos, G. P.; Sertedaki, A. C.; Latronico, A. C.

    2016-01-01

    We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated. PMID:26554443

  7. Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

    PubMed

    Mito, Takayuki; Kikkawa, Yoshiaki; Shimizu, Akinori; Hashizume, Osamu; Katada, Shun; Imanishi, Hirotake; Ota, Azusa; Kato, Yukina; Nakada, Kazuto; Hayashi, Jun-Ichi

    2013-01-01

    Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0)) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

  8. Mitochondrial DNA Mutations in Mutator Mice Confer Respiration Defects and B-Cell Lymphoma Development

    PubMed Central

    Mito, Takayuki; Kikkawa, Yoshiaki; Shimizu, Akinori; Hashizume, Osamu; Katada, Shun; Imanishi, Hirotake; Ota, Azusa; Kato, Yukina; Nakada, Kazuto; Hayashi, Jun-Ichi

    2013-01-01

    Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ0) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan. PMID:23418460

  9. Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

    PubMed Central

    Almeida, Deepak; Ioerger, Thomas; Tyagi, Sandeep; Li, Si-Yang; Mdluli, Khisimuzi; Andries, Koen; Grosset, Jacques; Sacchettini, Jim

    2016-01-01

    The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene. PMID:27185800

  10. Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis.

    PubMed

    Almeida, Deepak; Ioerger, Thomas; Tyagi, Sandeep; Li, Si-Yang; Mdluli, Khisimuzi; Andries, Koen; Grosset, Jacques; Sacchettini, Jim; Nuermberger, Eric

    2016-08-01

    The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.

  11. Mutations in the aph(2")-Ic Gene Are Responsible for Increased Levels of Aminoglycoside Resistance

    PubMed Central

    Lee, Hae Kyung; Vakulenko, Sergei B.; Clewell, Don B.; Lerner, Stephen A.; Chow, Joseph W.

    2002-01-01

    Random PCR mutagenesis of the enterococcal aph(2")-Ic gene followed by selection for mutant enzymes that confer enhanced levels of aminoglycoside resistance resulted in mutants of APH(2")-Ic with His-258-Leu and Phe-108-Leu substitutions, all of which conferred rises in the MICs of several aminoglycosides. The mutated residues are located outside conserved regions of aminoglycoside phosphotransferases. PMID:12234853

  12. Germline mutation in the RAD51B gene confers predisposition to breast cancer

    PubMed Central

    2013-01-01

    Background Most currently known breast cancer predisposition genes play a role in DNA repair by homologous recombination. Recent studies conducted on RAD51 paralogs, involved in the same DNA repair pathway, have identified rare germline mutations conferring breast and/or ovarian cancer predisposition in the RAD51C, RAD51D and XRCC2 genes. The present study analysed the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) to estimate their contribution to breast and ovarian cancer predisposition. Methods The study was conducted on 142 unrelated patients with breast and/or ovarian cancer either with early onset or with a breast/ovarian cancer family history. Patients were referred to a French family cancer clinic and had been previously tested negative for a BRCA1/2 mutation. Coding sequences of the five genes were analysed by EMMA (Enhanced Mismatch Mutation Analysis). Detected variants were characterized by Sanger sequencing analysis. Results Three splicing mutations and two likely deleterious missense variants were identified: RAD51B c.452 + 3A > G, RAD51C c.706-2A > G, RAD51C c.1026 + 5_1026 + 7del, RAD51B c.475C > T/p.Arg159Cys and XRCC3 c.448C > T/p.Arg150Cys. No RAD51D and XRCC2 gene mutations were detected. These mutations and variants were detected in families with both breast and ovarian cancers, except for the RAD51B c.475C > T/p.Arg159Cys variant that occurred in a family with 3 breast cancer cases. Conclusions This study identified the first RAD51B mutation in a breast and ovarian cancer family and is the first report of XRCC3 mutation analysis in breast and ovarian cancer. It confirms that RAD51 paralog mutations confer breast and ovarian cancer predisposition and are rare events. In view of the low frequency of RAD51 paralog mutations, international collaboration of family cancer clinics will be required to more accurately estimate their penetrance and establish clinical guidelines in carrier individuals. PMID

  13. Inhibition of FAAH confers increased stem cell migration via PPARα.

    PubMed

    Wollank, Yvonne; Ramer, Robert; Ivanov, Igor; Salamon, Achim; Peters, Kirsten; Hinz, Burkhard

    2015-10-01

    Regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (MSCs). The present study focused on inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (N-oleoylethanolamine, N-palmitoylethanolamine). Boyden chamber assays, the FAAH inhibitors, URB597 and arachidonoyl serotonin (AA-5HT), were found to increase the migration of human adipose-derived MSCs. LC-MS analyses revealed increased levels of all four aforementioned FAAH substrates in MSCs incubated with either FAAH inhibitor. Following addition to MSCs, all FAAH substrates mimicked the promigratory action of FAAH inhibitors. Promigratory effects of FAAH inhibitors and substrates were causally linked to activation of p42/44 MAPKs, as well as to cytosol-to-nucleus translocation of the transcription factor, PPARα. Whereas PPARα activation by FAAH inhibitors and substrates became reversed upon inhibition of p42/44 MAPK activation, a blockade of PPARα left p42/44 MAPK phosphorylation unaltered. Collectively, these data demonstrate FAAH inhibitors and substrates to cause p42/44 MAPK phosphorylation, which subsequently activates PPARα to confer increased migration of MSCs. This novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by FAAH inhibitors.

  14. Inhibition of FAAH confers increased stem cell migration via PPARα

    PubMed Central

    Wollank, Yvonne; Ramer, Robert; Ivanov, Igor; Salamon, Achim; Peters, Kirsten; Hinz, Burkhard

    2015-01-01

    Regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (MSCs). The present study focused on inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (N-oleoylethanolamine, N-palmitoylethanolamine). Boyden chamber assays, the FAAH inhibitors, URB597 and arachidonoyl serotonin (AA-5HT), were found to increase the migration of human adipose-derived MSCs. LC-MS analyses revealed increased levels of all four aforementioned FAAH substrates in MSCs incubated with either FAAH inhibitor. Following addition to MSCs, all FAAH substrates mimicked the promigratory action of FAAH inhibitors. Promigratory effects of FAAH inhibitors and substrates were causally linked to activation of p42/44 MAPKs, as well as to cytosol-to-nucleus translocation of the transcription factor, PPARα. Whereas PPARα activation by FAAH inhibitors and substrates became reversed upon inhibition of p42/44 MAPK activation, a blockade of PPARα left p42/44 MAPK phosphorylation unaltered. Collectively, these data demonstrate FAAH inhibitors and substrates to cause p42/44 MAPK phosphorylation, which subsequently activates PPARα to confer increased migration of MSCs. This novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by FAAH inhibitors. PMID:26263913

  15. Novel K540N mutation in Plasmodium falciparum dihydropteroate synthetase confers a lower level of sulfa drug resistance than does a K540E mutation.

    PubMed

    Lumb, Vanshika; Sharma, Yagya D

    2011-05-01

    Sulfadoxine (SDX) and sulfamethoxazole (SMX) each inhibit the Plasmodium falciparum dihydropteroate synthetase (PfDHPS), and certain point mutations in this enzyme yield the drug-resistant parasite. Using a simple Escherichia coli model system, we describe here the effect of the recently reported novel K540N mutation in PfDHPS on the level of SDX/SMX resistance. The survival rate of the transformed E. coli (DHPS-deficient strain) under different SDX or SMX concentrations revealed that the K540N mutation confers a lower level of drug resistance than its contemporary K540E mutation. Further, SMX was more effective than SDX in the E. coli system.

  16. Is Increased Low-dose somatic Radiosensitivity Associated with Increased Transgenerational Germline Mutation

    SciTech Connect

    Brenner, David J.

    2008-10-02

    Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm+/–) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm+/– males did not significantly differ from that in wild-type BALB/c mice. Acute gamma-ray exposure did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm+/– and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analyzed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.

  17. Increasing Role of Titin Mutations in Neuromuscular Disorders

    PubMed Central

    Savarese, Marco; Sarparanta, Jaakko; Vihola, Anna; Udd, Bjarne; Hackman, Peter

    2016-01-01

    The TTN gene with 363 coding exons encodes titin, a giant muscle protein spanning from the Z-disk to the M-band within the sarcomere. Mutations in the TTN gene have been associated with different genetic disorders, including hypertrophic and dilated cardiomyopathy and several skeletal muscle diseases. Before the introduction of next generation sequencing (NGS) methods, the molecular analysis of TTN has been laborious, expensive and not widely used, resulting in a limited number of mutations identified. Recent studies however, based on the use of NGS strategies, give evidence of an increasing number of rare and unique TTN variants. The interpretation of these rare variants of uncertain significance (VOUS) represents a challenge for clinicians and researchers. The main aim of this review is to describe the wide spectrum of muscle diseases caused by TTN mutations so far determined, summarizing the molecular findings as well as the clinical data, and to highlight the importance of joint efforts to respond to the challenges arising from the use of NGS. An international collaboration through a clinical and research consortium and the development of a single accessible database listing variants in the TTN gene, identified by high throughput approaches, may be the key to a better assessment of titinopathies and to systematic genotype– phenotype correlation studies. PMID:27854229

  18. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP

    SciTech Connect

    Yun, C.H.; Mengwasser, K.E.; Toms, A.V.; Woo, M.S.; Greulich, H.; Wong, K.K.; Meyerson, M.; Eck, M.J.

    2008-07-15

    Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the 'gatekeeper' residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a 'generic' resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.

  19. FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

    PubMed Central

    Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.

    2014-01-01

    Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus. PMID:25403676

  20. Mutation in the Bimd Gene of Aspergillus Nidulans Confers a Conditional Mitotic Block and Sensitivity to DNA Damaging Agents

    PubMed Central

    Denison, S. H.; Kafer, E.; May, G. S.

    1993-01-01

    Mutation in the bimD gene of Aspergillus nidulans results in a mitotic block in anaphase characterized by a defective mitosis. Mutation in bimD also confers, at temperatures permissive for the mitotic arrest phenotype, an increased sensitivity to DNA damaging agents, including methyl methanesulfonate and ultraviolet light. In order to better understand the relationship between DNA damage and mitotic progression, we cloned the bimD gene from Aspergillus. A cosmid containing the bimD gene was identified among pools of cosmids by cotransformation with the nutritional selective pyrG gene of a strain carrying the recessive, temperature-sensitive lethal bimD6 mutation. The bimD gene encodes a predicted polypeptide of 166,000 daltons in mass and contains amino acid sequence motifs similar to those found in some DNA-binding transcription factors. These sequences include a basic domain followed by a leucine zipper, which together are called a bZIP motif, and a carboxyl-terminal domain enriched in acidic amino acids. Overexpression of the wild-type bimD protein resulted in an arrest of the nuclear division cycle that was reversible and determined to be in either the G(1) or S phase of the cell cycle. Our data suggest that bimD may play an essential regulatory role relating to DNA metabolism which is required for a successful mitosis. PMID:8375649

  1. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    PubMed Central

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam

    2016-01-01

    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  2. A single gene mutation that increases maize seed weight

    SciTech Connect

    Giroux, M.J.; Shaw, J.; Hannah, L.C. |

    1996-06-11

    The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3 feet to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts. 20 refs., 5 figs., 5 tabs.

  3. ON pathway mutations increase susceptibility to form-deprivation myopia

    PubMed Central

    Chakraborty, Ranjay; Park, Han na; Hanif, Adam M.; Sidhu, Curran; Iuvone, P. Michael; Pardue, Machelle T.

    2015-01-01

    The ON pathway mutation in nob mice is associated with altered refractive development, and an increased susceptibility to form-deprivation (FD) myopia. In this study, we used mGluR6−/− mice, another ON pathway mutant, to determine whether the nob phenotype was due to the Nyx mutation or abnormal ON pathway transmission. Refractive development under a normal visual environment for mGluR6−/− and age-matched wild-type (WT) mice was measured every 2 weeks from 4 to 16 weeks of age. The response to monocular FD from 4 weeks of age was measured weekly in a separate cohort of mice. Refraction and ocular biometry were obtained using a photorefractor and optical coherence tomography. Retinas were harvested at 16 weeks, and analyzed for dopamine (DA) and DOPAC using high-performance liquid chromatography. Under normal conditions, mGluR6−/− mice were significantly more myopic than their WT controls (refraction at 12 weeks; WT: 9.40 ± 0.16 D, mGluR6−/−: 6.91 ± 0.38 D). Similar to nob mice, two weeks of FD resulted in a significant myopic shift of −5.57 ± 0.72 D in mGluR6−/− mice compared to −1.66 ± 0.19 D in WT animals. No significant axial length changes were observed with either normal or FD visual conditions. At 16 weeks, mGluR6−/− retinas showed significantly lower DOPAC levels (111.2 ± 33.0 pg/mg) compared to their WT counterparts (197.5 ± 11.2 pg/mg). Retinal DA levels were similar between the different genotypes. Our results indicate that reduced retinal DA metabolism/turnover may be associated with increased susceptibility to myopia in mice with ON pathway defect mutations. PMID:26072023

  4. RET mutation and increased angiogenesis in medullary thyroid carcinomas.

    PubMed

    Verrienti, Antonella; Tallini, Giovanni; Colato, Chiara; Boichard, Amélie; Checquolo, Saula; Pecce, Valeria; Sponziello, Marialuisa; Rosignolo, Francesca; de Biase, Dario; Rhoden, Kerry; Casadei, Gian Piero; Russo, Diego; Visani, Michela; Acquaviva, Giorgia; Ferdeghini, Marco; Filetti, Sebastiano; Durante, Cosimo

    2016-08-01

    Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs. RET mutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somatic RET mutations (RETmut group) and 34 with wild-type RET (RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared with RETwt tumors, RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels in RETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis in RETmut MTCs may be more intense and complete than that found in RETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density, RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors.

  5. A germ-line-selective advantage rather than an increased mutation rate can explain some unexpectedly common human disease mutations.

    PubMed

    Choi, Soo-Kyung; Yoon, Song-Ro; Calabrese, Peter; Arnheim, Norman

    2008-07-22

    Two nucleotide substitutions in the human FGFR2 gene (C755G or C758G) are responsible for virtually all sporadic cases of Apert syndrome. This condition is 100-1,000 times more common than genomic mutation frequency data predict. Here, we report on the C758G de novo Apert syndrome mutation. Using data on older donors, we show that spontaneous mutations are not uniformly distributed throughout normal testes. Instead, we find foci where C758G mutation frequencies are 3-4 orders of magnitude greater than the remaining tissue. We conclude this nucleotide site is not a mutation hot spot even after accounting for possible Luria-Delbruck "mutation jackpots." An alternative explanation for such foci involving positive selection acting on adult self-renewing Ap spermatogonia experiencing the rare mutation could not be rejected. Further, the two youngest individuals studied (19 and 23 years old) had lower mutation frequencies and smaller foci at both mutation sites compared with the older individuals. This implies that the mutation frequency of foci increases as adults age, and thus selection could explain the paternal age effect for Apert syndrome and other genetic conditions. Our results, now including the analysis of two mutations in the same set of testes, suggest that positive selection can increase the relative frequency of premeiotic germ cells carrying such mutations, although individuals who inherit them have reduced fitness. In addition, we compared the anatomical distribution of C758G mutation foci with both new and old data on the C755G mutation in the same testis and found their positions were not correlated with one another.

  6. Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila melanogaster populations

    PubMed Central

    Menozzi, Philippe; Shi, Ming An; Lougarre, Andrée; Tang, Zhen Hua; Fournier, Didier

    2004-01-01

    Background Organophosphate and carbamate insecticides irreversibly inhibit acetylcholinesterase causing death of insects. Resistance-modified acetylcholinesterases(AChEs) have been described in many insect species and sequencing of their genes allowed several point mutations to be described. However, their relative frequency and their cartography had not yet been addressed. Results To analyze the most frequent mutations providing insecticide resistance in Drosophila melanogaster acetylcholinesterase, the Ace gene was cloned and sequenced in several strains harvested from different parts of the world. Sequence comparison revealed four widespread mutations, I161V, G265A, F330Y and G368A. We confirm here that mutations are found either isolated or in combination in the same protein and we show that most natural populations are heterogeneous, composed of a mixture of different alleles. In vitro expression of mutated proteins showed that combining mutations in the same protein has two consequences: it increases resistance level and provides a wide spectrum of resistance. Conclusion The presence of several alleles in natural populations, offering various resistance to carbamate and organophosphate compounds will complicate the establishment of resistance management programs. PMID:15018651

  7. The L1014F point mutation in the house fly Vssc1 sodium channel confers knockdown resistance to pyrethroids.

    PubMed

    Smith, T J; Lee, S H; Ingles, P J; Knipple, D C; Soderlund, D M

    1997-10-01

    Voltage-sensitive sodium channels encoded by a full-length cDNA corresponding to the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes either alone or in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1 cRNA alone produced very small (50-150 nA) sodium currents, whereas the combination of Vssc1 and tipE cRNAs produced robust (0.5-3 microA), rapidly inactivating sodium currents. The pyrethroid insecticide cismethrin prolonged the sodium current carried by Vssc1/tipE sodium channels during a depolarizing pulse and induced a tail current after repolarization. The Vssc1 cDNA was specifically mutated to substitute phenylalanine for leucine at position 1014 of the inferred amino acid sequence (L1014F), a polymorphism shown previously to be associated with the kdr (knockdown resistance) trait of the house fly. The L1014F substitution reduced the sensitivity of expressed house fly sodium channels to cismethrin at least 10-fold and increased the rate of decay of pyrethroid-induced sodium tail currents. These results demonstrate that the resistance-associated L1014F mutation confers a reduction in the sensitivity of house fly sodium channels to pyrethroids that is sufficient to account for the kdr resistance trait.

  8. Mutations in Dalpha1 or Dbeta2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster.

    PubMed

    Perry, Trent; Heckel, David G; McKenzie, John A; Batterham, Philip

    2008-05-01

    Resistance to insecticides by modification of their molecular targets is a serious problem in chemical control of many arthropod pests. Neonicotinoids target the nicotinic acetylcholine receptor (nAChR) of arthropods. The spectrum of possible resistance-conferring mutations of this receptor is poorly understood. Prediction of resistance is complicated by the existence of multiple genes encoding the different subunits of this essential component of neurotransmission. We focused on the cluster of three Drosophila melanogaster nAChR subunit genes at cytological region 96A. EMS mutagenesis and selection for resistance to nitenpyram was performed on hybrids carrying a deficiency for this chromosomal region. Two complementation groups were defined for the four strains isolated. Molecular characterisation of the mutations found lesions in two nAChR subunit genes, Dalpha1 (encoding an alpha-type subunit) and Dbeta2 (beta-type). Mutations conferring resistance in beta-type receptors have not previously been reported, but we found several lesions in the Dbeta2 sequence, including locations distant from the predicted neonicotinoid-binding site. This study illustrates that mutations in a single-receptor subunit can confer nitenpyram resistance. Moreover, some of the mutations may protect the insect against nitenpyram by interfering with subunit assembly or channel activation, rather than affecting binding affinities of neonicotinoids to the channel.

  9. Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance

    PubMed Central

    LaMonte, Gregory; Lim, Michelle Yi-Xiu; Wree, Melanie; Reimer, Christin; Nachon, Marie; Corey, Victoria; Gedeck, Peter; Plouffe, David; Du, Alan; Figueroa, Nelissa; Yeung, Bryan; Winzeler, Elizabeth A.

    2016-01-01

    ABSTRACT Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites. PMID:27381290

  10. Frequency of spontaneous mutations that confer antibiotic resistance in Chlamydia spp.

    PubMed

    Binet, Rachel; Maurelli, Anthony T

    2005-07-01

    Mutations in rRNA genes (rrn) that confer resistance to ribosomal inhibitors are typically recessive or weakly codominant and have been mostly reported for clinical strains of pathogens possessing only one or two rrn operons, such as Helicobacter pylori and Mycobacterium spp. An analysis of the genome sequences of several members of the Chlamydiaceae revealed that these obligate intracellular bacteria harbor only one or two sets of rRNA genes. To study the contribution of rRNA mutations to the emergence of drug resistance in the Chlamydiaceae, we used the sensitivities of Chlamydia trachomatis L2 (two rrn operons) and Chlamydophila psittaci 6BC (one rrn operon) to the aminoglycoside spectinomycin as a model. Confluent cell monolayers were infected in a plaque assay with about 10(8) wild-type infectious particles and then treated with the antibiotic. After a 2-week incubation time, plaques formed by spontaneous spectinomycin-resistant (Spc(r)) mutants appeared with a frequency of 5 x 10(-5) for C. psittaci 6BC. No Spc(r) mutants were isolated for C. trachomatis L2, although the frequencies of rifampin resistance were in the same range for both strains (i.e., 10(-7)). The risk of emergence of Chlamydia strains resistant to tetracyclines and macrolides, the ribosomal drugs currently used to treat chlamydial infections, is discussed.

  11. Sdt97: A Point Mutation in the 5′ Untranslated Region Confers Semidwarfism in Rice

    PubMed Central

    Tong, Jiping; Han, Zhengshu; Han, Aonan; Liu, Xuejun; Zhang, Shiyong; Fu, Binying; Hu, Jun; Su, Jingping; Li, Shaoqing; Wang, Shengjun; Zhu, Yingguo

    2016-01-01

    Semidwarfism is an important agronomic trait in rice breeding programs. The semidwarf mutant gene Sdt97 was previously described. However, the molecular mechanism underlying the mutant is yet to be elucidated. In this study, we identified the mutant gene by a map-based cloning method. Using a residual heterozygous line (RHL) population, Sdt97 was mapped to the long arm of chromosome 6 in the interval of nearly 60 kb between STS marker N6 and SNP marker N16 within the PAC clone P0453H04. Sequencing of the candidate genes in the target region revealed that a base transversion from G to C occurred in the 5′ untranslated region of Sdt97. qRT-PCR results confirmed that the transversion induced an obvious change in the expression pattern of Sdt97 at different growth and developmental stages. Plants transgenic for Sdt97 resulted in the restoration of semidwarfism of the mutant phenotype, or displayed a greater dwarf phenotype than the mutant. Our results indicate that a point mutation in the 5′ untranslated region of Sdt97 confers semidwarfism in rice. Functional analysis of Sdt97 will open a new field of study for rice semidwarfism, and also expand our knowledge of the molecular mechanism of semidwarfism in rice. PMID:27172200

  12. DMSO increases mutation-scanning detection sensitivity in clinical samples using high resolution melting

    PubMed Central

    Song, Chen; Castellanos-Rizaldos, Elena; Bejar, Rafael; Ebert, Benjamin L.; Makrigiorgos, G. Mike

    2016-01-01

    BACKGROUND Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. METHODS We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. RESULTS The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. CONCLUSIONS DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM. PMID:26432802

  13. Increasing the imaging depth through computational scattering correction (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Koberstein-Schwarz, Benno; Omlor, Lars; Schmitt-Manderbach, Tobias; Mappes, Timo; Ntziachristos, Vasilis

    2016-03-01

    Imaging depth is one of the most prominent limitations in light microscopy. The depth in which we are still able to resolve biological structures is limited by the scattering of light within the sample. We have developed an algorithm to compensate for the influence of scattering. The potential of algorithm is demonstrated on a 3D image stack of a zebrafish embryo captured with a selective plane illumination microscope (SPIM). With our algorithm we were able shift the point in depth, where scattering starts to blur the imaging and effect the image quality by around 30 µm. For the reconstruction the algorithm only uses information from within the image stack. Therefore the algorithm can be applied on the image data from every SPIM system without further hardware adaption. Also there is no need for multiple scans from different views to perform the reconstruction. The underlying model estimates the recorded image as a convolution between the distribution of fluorophores and a point spread function, which describes the blur due to scattering. Our algorithm performs a space-variant blind deconvolution on the image. To account for the increasing amount of scattering in deeper tissue, we introduce a new regularizer which models the increasing width of the point spread function in order to improve the image quality in the depth of the sample. Since the assumptions the algorithm is based on are not limited to SPIM images the algorithm should also be able to work on other imaging techniques which provide a 3D image volume.

  14. A point mutation in influenza B neuraminidase confers resistance to peramivir and loss of slow binding.

    PubMed

    Baum, Ellen Z; Wagaman, Pamela C; Ly, Linh; Turchi, Ignatius; Le, Jianhua; Bucher, Doris; Bush, Karen

    2003-06-01

    The influenza neuraminidase (NA) inhibitors peramivir, oseltamivir, and zanamivir are potent inhibitors of NAs from both influenza A and B strains. In general, these inhibitors are slow, tight binders of NA, exhibiting time-dependent inhibition. A mutant of influenza virus B/Yamagata/16/88 which was resistant to peramivir was generated by passage of the virus in tissue culture, in the presence of increasing concentrations (0.1-120 microM over 15 passages) of the compound. Whereas the wild type (WT) virus was inhibited by peramivir with an EC(50) value of 0.10 microM, virus isolated at passages 3 and 15 displayed EC(50) values of 10 and >50 microM, respectively. Passage 3 virus contained 3 hemagglutinin (HA) mutations, but no NA mutation. Passage 15 (P15R) virus contained an additional 3 HA mutations, plus the NA mutation His273Tyr. The mechanism of inhibition of WT and P15R NA by peramivir was examined in enzyme assays. The WT and P15R NAs displayed IC(50) values of 8.4+/-0.4 and 127+/-16 nM, respectively, for peramivir. Peramivir inhibited the WT enzyme in a time-dependent fashion, with a K(i) value of 0.066+/-0.002nM. In contrast, the P15R enzyme did not display the property of slow binding and was inhibited competitively with a K(i) value of 4.69+/-0.44nM. Molecular modeling suggested that His273 was relatively distant from peramivir (>5A) in the NA active site, but that Tyr273 introduced a repulsive interaction between the enzyme and inhibitor, which may have been responsible for peramivir resistance.

  15. Fungal Infection Increases the Rate of Somatic Mutation in Scots Pine (Pinus sylvestris L.).

    PubMed

    Ranade, Sonali Sachin; Ganea, Laura-Stefana; Razzak, Abdur M; García Gil, M R

    2015-01-01

    Somatic mutations are transmitted during mitosis in developing somatic tissue. Somatic cells bearing the mutations can develop into reproductive (germ) cells and the somatic mutations are then passed on to the next generation of plants. Somatic mutations are a source of variation essential to evolve new defense strategies and adapt to the environment. Stem rust disease in Scots pine has a negative effect on wood quality, and thus adversely affects the economy. It is caused by the 2 most destructive fungal species in Scandinavia: Peridermium pini and Cronartium flaccidum. We studied nuclear genome stability in Scots pine under biotic stress (fungus-infected, 22 trees) compared to a control population (plantation, 20 trees). Stability was assessed as accumulation of new somatic mutations in 10 microsatellite loci selected for genotyping. Microsatellites are widely used as molecular markers in population genetics studies of plants, and are particularly used for detection of somatic mutations as their rate of mutation is of a much higher magnitude when compared with other DNA markers. We report double the rate of somatic mutation per locus in the fungus-infected trees (4.8×10(-3) mutations per locus), as compared to the controls (2.0×10(-3) mutations per locus) when individual samples were analyzed at 10 different microsatellite markers. Pearson's chi-squared test indicated a significant effect of the fungal infection which increased the number of mutations in the fungus-infected trees (χ(2) = 12.9883, df = 1, P = 0.0003134).

  16. Gestational mutations in radiation carcinogenesis

    NASA Astrophysics Data System (ADS)

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

    Mutations in critical genes during gestation could increase substantially the risk of cancer. We examine the consequences of such mutations using the Luebeck-Moolgavkar model for colorectal cancer and the Lea-Coulson modification of the Luria-Delbruck model for the accumulation of mutations during gestation. When gestational mutation rates are high, such mutations make a significant contribution to cancer risk even for adult tumors. Furthermore, gestational mutations ocurring at distinct times during emryonic developmemt lead to substantially different numbers of mutated cells at birth, with early mutations leading to a large number (jackpots) of mutated cells at birth and mutation occurring late leading to only a few mutated cells. Thus gestational mutations could confer considerable heterogeneity of the risk of cancer. If the fetus is exposed to an environmental mutagen, such as ionizing radiation, the gestational mutation rate would be expected to increase. We examine the consequences of such exposures during gestation on the subsequent development of cancer.

  17. Increased mitochondrial mutation frequency after an island colonization: positive selection or accumulation of slightly deleterious mutations?

    PubMed

    Hardouin, Emilie A; Tautz, Diethard

    2013-04-23

    Island colonizations are excellent models for studying early processes of evolution. We found in a previous study on mice that had colonized the sub-Antarctic Kerguelen Archipelago about 200 years ago that they were derived from a single founder lineage and that this showed an unexpectedly large number of new mutations in the mitochondrial D-loop. To assess whether positive selection has played a role in the emergence of these variants, we have obtained 16 full mitochondrial genome sequences from these mice. For comparison, we have compiled 57 mitochondrial genome sequences from laboratory inbred lines that became established about 100 years ago, also starting from a single founder lineage. We find that the island mice and the laboratory lines show very similar mutation frequencies and patterns. None of the patterns in the Kerguelen mice provides evidence for positive selection. We conclude that nearly neutral evolutionary processes that assume the presence of slightly deleterious variants can fully explain the patterns. This supports the notion of time-dependency of molecular evolution and provides a new calibration point. Based on the observed mutation frequency, we calculate an average evolutionary rate of 0.23 substitutions per site per Myr for the earliest time frame of divergence, which is about six times higher than the long-term rate of 0.037 substitutions per site per Myr.

  18. Using Student Conferences to Increase Participation in the Classroom: A Case Study

    ERIC Educational Resources Information Center

    Arenas, M. G.; Castillo, P. A.; de Vega, F. F.; Merelo, J. J.

    2012-01-01

    This paper describes the use of a student conference as a novel experience aimed at motivating students enrolled in various computer architecture courses, such as Microprocessor Systems. The goal was to increase student engagement, to decrease failure rates, and to introduce students to the world of research. This multidisciplinary experience…

  19. Increasing Participation in Learning. Symposium 19. [Concurrent Symposium Session at AHRD Annual Conference, 2000.

    ERIC Educational Resources Information Center

    2000

    This document contains three papers from a symposium on increasing participation in learning that was conducted as part of a conference on human resource development (HRD). "Factors Influencing Employee Participation in Training: An Empirical Investigation" (Reid A. Bates) reports on a mediated model of employee participation in training…

  20. Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

    PubMed Central

    Melançon, P; Tapprich, W E; Brakier-Gingras, L

    1992-01-01

    Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes. PMID:1281147

  1. In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice.

    PubMed

    Logan, Angela; Shabalina, Irina G; Prime, Tracy A; Rogatti, Sebastian; Kalinovich, Anastasia V; Hartley, Richard C; Budd, Ralph C; Cannon, Barbara; Murphy, Michael P

    2014-08-01

    In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria-targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro-apoptotic and pro-inflammatory redox signaling pathways.

  2. Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas.

    PubMed

    Castro-Vega, Luis Jaime; Buffet, Alexandre; De Cubas, Aguirre A; Cascón, Alberto; Menara, Mélanie; Khalifa, Emmanuel; Amar, Laurence; Azriel, Sharona; Bourdeau, Isabelle; Chabre, Olivier; Currás-Freixes, Maria; Franco-Vidal, Valérie; Guillaud-Bataille, Marine; Simian, Christophe; Morin, Aurélie; Letón, Rocío; Gómez-Graña, Alvaro; Pollard, Patrick J; Rustin, Pierre; Robledo, Mercedes; Favier, Judith; Gimenez-Roqueplo, Anne-Paule

    2014-05-01

    Malignant pheochromocytoma (PCC) and paraganglioma (PGL) are mostly caused by germline mutations of SDHB, encoding a subunit of succinate dehydrogenase. Using whole-exome sequencing, we recently identified a mutation in the FH gene encoding fumarate hydratase, in a PCC with an 'SDH-like' molecular phenotype. Here, we investigated the role of FH in PCC/PGL predisposition, by screening for germline FH mutations in a large international cohort of patients. We screened 598 patients with PCC/PGL without mutations in known PCC/PGL susceptibility genes. We searched for FH germline mutations and large deletions, by direct sequencing and multiplex ligation-dependent probe amplification methods. Global alterations in DNA methylation and protein succination were assessed by immunohistochemical staining for 5-hydroxymethylcytosine (5-hmC) and S-(2-succinyl) cysteine (2SC), respectively. We identified five pathogenic germline FH mutations (four missense and one splice mutation) in five patients. Somatic inactivation of the second allele, resulting in a loss of fumarate hydratase activity, was demonstrated in tumors with FH mutations. Low tumor levels of 5-hmC, resembling those in SDHB-deficient tumors, and positive 2SC staining were detected in tumors with FH mutations. Clinically, metastatic phenotype (P = 0.007) and multiple tumors (P = 0.02) were significantly more frequent in patients with FH mutations than those without such mutations. This study reveals a new role for FH in susceptibility to malignant and/or multiple PCC/PGL. Remarkably, FH-deficient PCC/PGLs display the same pattern of epigenetic deregulation as SDHB-mutated malignant PCC/PGL. Therefore, we propose that mutation screening for FH should be included in PCC/PGL genetic testing, at least for tumors with malignant behavior.

  3. Novel mutations conferring resistance to kanamycin in Mycobacterium tuberculosis clinical isolates from Northern India.

    PubMed

    Kaur, Simerpreet; Rana, Vibhuti; Singh, Pooja; Trivedi, Garima; Anand, Shashi; Kaur, Amanpreet; Gupta, Pawan; Jain, Amita; Sharma, Charu

    2016-01-01

    Twenty-nine Kanamycin resistant clinical isolates of Mycobacterium tuberculosis from Northern India were screened to evaluate genetic mutations in rrs gene, eis gene with its promoter, and whiB7 gene along with its 5'UTR. 14 strains (~48.0%) collectively exhibited mutations in rrs, eis or whiB7 target regions. While the highest frequency of mutations was found in rrs gene, eis and whiB7 loci displayed novel mutations. The novel mutations displayed by eis and whiB7 loci were found to be associated specifically with the Kanamycin resistance as none of the twenty nine Kanamycin sensitive strains harbor them. The inclusion of novel mutations of eis and whiB7 loci will be useful in improving the specificity of future diagnostics.

  4. Somatostatin analogues increase AIP expression in somatotropinomas, irrespective of Gsp mutations.

    PubMed

    Jaffrain-Rea, Marie-Lise; Rotondi, Sandra; Turchi, Annarita; Occhi, Gianluca; Barlier, Anne; Peverelli, Erika; Rostomyan, Lilya; Defilles, Céline; Angelini, Mariolina; Oliva, Maria-Antonietta; Ceccato, Filippo; Maiorani, Orlando; Daly, Adrian F; Esposito, Vincenzo; Buttarelli, Francesca; Figarella-Branger, Dominique; Giangaspero, Felice; Spada, Anna; Scaroni, Carla; Alesse, Edoardo; Beckers, Albert

    2013-10-01

    Germline aryl hydrocarbon receptor interacting protein (AIP) gene mutations confer a predisposition to pituitary adenoma (PA), predominantly GH-secreting (GH-PA). As recent data suggest a role for AIP in the pathogenesis of sporadic GH-PA and their response to somatostatin analogues (SSA), the expression of AIP and its partner, aryl hydrocarbon receptor (AHR), was determined by semiquantitative immunohistochemistry scoring in 62 sporadic GH-PA (37 treated with SSA preoperatively). The influence of Gsp status was studied in a subset of tumours (n=39, 14 Gsp(+)) and six GH-PA were available for primary cultures. AIP and AHR were detected in most cases, with a positive correlation between AIP and cytoplasmic AHR (P=0.012). Low AIP expression was significantly more frequent in untreated vs SSA-treated tumours (44.0 vs 20.5%, P=0.016). AHR expression or localisation did not differ between the two groups. Similarly, in vitro octreotide induced a median twofold increase in AIP expression (range 1.2-13.9, P=0.027) in GH-PA. In SSA-treated tumours, the AIP score was significantly higher in the presence of preoperative IGF1 decrease or tumour shrinkage (P=0.008 and P=0.014 respectively). In untreated tumours, low AIP expression was significantly associated with invasiveness (P=0.028) and suprasellar extension (P=0.019). The only effect of Gsp status was a significantly lower nuclear AHR score in Gsp(+) vs Gsp(-) tumours (P=0.025), irrespective of SSA. In conclusion, AIP is involved in the aggressiveness of sporadic GH-PA, regardless of Gsp status, and AIP up-regulation in SSA-treated tumours is associated with a better preoperative response, with no clear role for AHR.

  5. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  6. A novel insertion mutation on exon 20 of epidermal growth factor receptor, conferring resistance to erlotinib.

    PubMed

    Khan, Nawazish A; Mirshahidi, Saied; Mirshahidi, Hamid R

    2014-05-01

    The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein tyrosine kinase receptor. The small-molecule tyrosine kinase receptor inhibitors (TKIs) are in clinical use to treat non-small cell lung cancer with EGFR mutations. Variable tumor responses to erlotinib and gefitinib have been observed. The response to these TKIs varies by the type of EGFR mutations found in the tumor. The deletion on exon 19 and the L858R substitution on exon 21 constitute the most frequent mutations and are known to show good response to TKIs. However, mutations on exon 20 are less common and seem to respond poorly to TKIs. In clinical settings, the reported response of exon 20 mutations to reversible TKIs (both gefitinib and erlotinib) remains inconstant. The type of coexisting mutation seems to affect the response of these insertions to TKIs. We herein present a case of disease progression despite the use of erlotinib in a female patient who had a novel insertion mutation on exon 20. Our patient was a never-smoker and was identified to have a Pro772_His773insGlnCysPro mutation on exon 20. She had previously been treated with cisplatin and gemcitabine and then with carboplatin and pemetrexed. She was treated with erlotinib upon intolerance to second-line chemotherapy and did not respond. Our patient had a novel insertion mutation on exon 20, which was found to be resistant to erlotinib.

  7. Acquisition of a single EZH2 D1 domain mutation confers acquired resistance to EZH2-targeted inhibitors

    PubMed Central

    Baker, Theresa; Nerle, Sujata; Pritchard, Justin; Zhao, Boyang; Rivera, Victor M.

    2015-01-01

    Although targeted therapies have revolutionized cancer treatment, overcoming acquired resistance remains a major clinical challenge. EZH2 inhibitors (EZH2i), EPZ-6438 and GSK126, are currently in the early stages of clinical evaluation and the first encouraging signs of efficacy have recently emerged in the clinic. To anticipate mechanisms of resistance to EZH2i, we used a forward genetic platform combining a mutagenesis screen with next generation sequencing technology and identified a hotspot of secondary mutations in the EZH2 D1 domain (Y111 and I109). Y111D mutation within the WT or A677G EZH2 allele conferred robust resistance to both EPZ-6438 and GSK126, but it only drove a partial resistance within the Y641F allele. EZH2 mutants required histone methyltransferase (HMT) catalytic activity and the polycomb repressive complex 2 (PRC2) components, SUZ12 and EED, to drive drug resistance. Furthermore, D1 domain mutations not only blocked the ability of EZH2i to bind to WT and A677G mutant, but also abrogated drug binding to the Y641F mutant. These data provide the first cellular validation of the mechanistic model underpinning the oncogenic function of WT and mutant EZH2. Importantly, our findings suggest that acquired-resistance to EZH2i may arise in WT and mutant EZH2 patients through a single mutation that remains targetable by second generation EZH2i. PMID:26360609

  8. Human Slack potassium channel mutations increase positive cooperativity between individual channels

    PubMed Central

    Barcia, Giulia; Quraishi, Imran H.; Martin, Hilary C.; Blair, Edward; Taylor, Jenny C.; Dulac, Olivier; Colleaux, Laurence

    2015-01-01

    Summary Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation or voltage-dependence. We examined nine different mutations of the KCNT1 (Slack) Na+-activated K+ channel that give rise to three distinct forms of epilepsy. All produced many fold-increases in current amplitude over that of the wild type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gives rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of the mutations is to alter channel-channel interactions. PMID:25482562

  9. Human slack potassium channel mutations increase positive cooperativity between individual channels.

    PubMed

    Kim, Grace E; Kronengold, Jack; Barcia, Giulia; Quraishi, Imran H; Martin, Hilary C; Blair, Edward; Taylor, Jenny C; Dulac, Olivier; Colleaux, Laurence; Nabbout, Rima; Kaczmarek, Leonard K

    2014-12-11

    Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation, and voltage dependence. We examined nine different mutations of the KCNT1 (Slack) Na(+)-activated K(+) channel that give rise to three distinct forms of epilepsy. All produced many-fold increases in current amplitude compared to the wild-type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild-type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gave rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of these mutations is to alter channel-channel interactions.

  10. Screening for streptomycin resistance conferring mutations in Mycobacterium tuberculosis isolates from Iran.

    PubMed

    Rezaei, Faranak; Haeili, Mehri; Imani Fooladi, Abbasali; Azari Garmjan, Gholam Ali; Feizabadi, Mohammad Mehdi

    2017-02-01

    Point mutations in the rpsL and rrs genes can lead to development of streptomycin (STR) resistance in Mycobacterium tuberculosis. The aims of this study were to determine the frequency of mutations in STR resistant M. tuberculosis isolates in Iran and to analyze the possible relationship between bacterial genotype and STR resistance. Twenty-three M. tuberculosis samples comprising 9 multidrug-resistant (MDR) and 14 non-MDR isolates, recovered from TB patients in four regions: Tehran (n = 14), Isfahan (n = 2), Zahedan (n = 2), and Khorasan (n = 5), were analysed. Mutational profiling was performed by sequencing of the rrs and rpsL genes and spoligotyping method was used for genotyping. Nineteen isolates were resistant to STR, among them 7 exhibited mutations in the rpsL gene and 7 had mutations in the rrs gene. The remaining 5 STR resistant as well as all susceptible isolates lacked any mutation in both genes. Beijing genotype was associated with both MDR and STR resistance in which all mutations occurred at codon 43 of the rpsL gene. There was an association between mutations in the rpsL and rrs genes and STR resistance. We also found a correlation between Beijing genotype and STR resistance.

  11. Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia

    PubMed Central

    Troup, Camille B.; Gelston, Laura C.; Haliburton, John; Chow, Eric D.; Yu, Kristie B.; Akutagawa, Jon; Taylor-Weiner, Amaro N.; Liu, Y. Lucy; Wang, Yong-Dong; Beckman, Kyle; Emanuel, Peter D.; Braun, Benjamin S.; Abate, Adam; Gerbing, Robert B.; Alonzo, Todd A.; Loh, Mignon L.

    2015-01-01

    Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood associated with a poor prognosis. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders. These lesions were detected in nearly 10% of patients with JMML and have been characterized as secondary events. We hypothesized that rare subclones with SETBP1 mutations are present at diagnosis in a large portion of patients who relapse, but are below the limits of detection for conventional deep sequencing platforms. Using droplet digital polymerase chain reaction, we identified SETBP1 mutations in 17/56 (30%) of patients who were treated in the Children’s Oncology Group sponsored clinical trial, AAML0122. Five-year event-free survival in patients with SETBP1 mutations was 18% ± 9% compared with 51% ± 8% for those without mutations (P = .006). PMID:25395418

  12. BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

    PubMed

    Zabriskie, Matthew S; Eide, Christopher A; Tantravahi, Srinivas K; Vellore, Nadeem A; Estrada, Johanna; Nicolini, Franck E; Khoury, Hanna J; Larson, Richard A; Konopleva, Marina; Cortes, Jorge E; Kantarjian, Hagop; Jabbour, Elias J; Kornblau, Steven M; Lipton, Jeffrey H; Rea, Delphine; Stenke, Leif; Barbany, Gisela; Lange, Thoralf; Hernández-Boluda, Juan-Carlos; Ossenkoppele, Gert J; Press, Richard D; Chuah, Charles; Goldberg, Stuart L; Wetzler, Meir; Mahon, Francois-Xavier; Etienne, Gabriel; Baccarani, Michele; Soverini, Simona; Rosti, Gianantonio; Rousselot, Philippe; Friedman, Ran; Deininger, Marie; Reynolds, Kimberly R; Heaton, William L; Eiring, Anna M; Pomicter, Anthony D; Khorashad, Jamshid S; Kelley, Todd W; Baron, Riccardo; Druker, Brian J; Deininger, Michael W; O'Hare, Thomas

    2014-09-08

    Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

  13. Non-recessive Bt toxin resistance conferred by an intracellular cadherin mutation in field-selected populations of cotton bollworm.

    PubMed

    Zhang, Haonan; Wu, Shuwen; Yang, Yihua; Tabashnik, Bruce E; Wu, Yidong

    2012-01-01

    Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.

  14. Acquired resistance to dasatinib in lung cancer cell lines conferred by DDR2 gatekeeper mutation and NF1 loss.

    PubMed

    Beauchamp, Ellen M; Woods, Brittany A; Dulak, Austin M; Tan, Li; Xu, Chunxiao; Gray, Nathanael S; Bass, Adam J; Wong, Kwok-kin; Meyerson, Matthew; Hammerman, Peter S

    2014-02-01

    The treatment of non-small cell lung cancer has evolved dramatically over the past decade with the adoption of widespread use of effective targeted therapies in patients with distinct molecular alterations. In lung squamous cell carcinoma (lung SqCC), recent studies have suggested that DDR2 mutations are a biomarker for therapeutic response to dasatinib and clinical trials are underway testing this hypothesis. Although targeted therapeutics are typically quite effective as initial therapy for patients with lung cancer, nearly all patients develop resistance with long-term exposure to targeted drugs. Here, we use DDR2-dependent lung cancer cell lines to model acquired resistance to dasatinib therapy. We perform targeted exome sequencing to identify two distinct mechanisms of acquired resistance: acquisition of the T654I gatekeeper mutation in DDR2 and loss of NF1. We show that NF1 loss activates a bypass pathway, which confers ERK dependency downstream of RAS activation. These results indicate that acquired resistance to dasatinib can occur via both second-site mutations in DDR2 and by activation of bypass pathways. These data may help to anticipate mechanisms of resistance that may be identified in upcoming clinical trials of anti-DDR2 therapy in lung cancer and suggest strategies to overcome resistance.

  15. A mutation in Caenorhabditis elegans that increases recombination frequency more than threefold.

    PubMed

    Rose, A M; Baillie, D L

    1979-10-18

    In higher organisms the rate of recombination between genetic loci is presumably responsive to selective pressure. Recently, selective pressures and mutational events that influence recombination have been reviewed. Mutational sites and chromosomal rearrangements that enhance or suppress recombination frequency in specific regions are known, but general mechanisms that enhance recombination have not yet been discovered. We describe here the isolation and characterisation of a strain of the hermaphroditic nematode, Caenorhabditis elegans, that has a recombination frequency at least threefold higher than that found in the wild type. In this strain, rec-1, the number of reciprocal recombination events between linked loci is increased. This is true for all pairs of linked loci studies so far. The high recombination strain behaves as if it carries a classical recessive mutation, although a second mutation exists which can alter the recessive behaviour of rec-1.

  16. Increased in vivo amyloid-β42 production, exchange, and loss in presenilin mutation carriers.

    PubMed

    Potter, Rachel; Patterson, Bruce W; Elbert, Donald L; Ovod, Vitaliy; Kasten, Tom; Sigurdson, Wendy; Mawuenyega, Kwasi; Blazey, Tyler; Goate, Alison; Chott, Robert; Yarasheski, Kevin E; Holtzman, David M; Morris, John C; Benzinger, Tammie L S; Bateman, Randall J

    2013-06-12

    Alzheimer's disease (AD) is hypothesized to be caused by an overproduction or reduced clearance of amyloid-β (Aβ) peptide. Autosomal dominant AD (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aβ42 compared to Aβ40 in the central nervous system (CNS). This has been demonstrated in rodent models of ADAD but not in human mutation carriers. We used compartmental modeling of stable isotope labeling kinetic (SILK) studies in human carriers of PSEN mutations and related noncarriers to evaluate the pathophysiological effects of PSEN1 and PSEN2 mutations on the production and turnover of Aβ isoforms. We compared these findings by mutation status and amount of fibrillar amyloid deposition as measured by positron emission tomography (PET) using the amyloid tracer Pittsburgh compound B (PIB). CNS Aβ42 to Aβ40 production rates were 24% higher in mutation carriers compared to noncarriers, and this was independent of fibrillar amyloid deposits quantified by PET PIB imaging. The fractional turnover rate of soluble Aβ42 relative to Aβ40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aβ42 into plaques, leading to reduced recovery of Aβ42 in cerebrospinal fluid (CSF). Reversible exchange of Aβ42 peptides with preexisting unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aβ42 is overproduced in the CNS of humans with PSEN mutations that cause AD, and demonstrate that soluble Aβ42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aβ42 concentrations in the CSF.

  17. Germline ETV6 Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia

    PubMed Central

    Jacobs, Lauren; Maria, Ann; Villano, Danylo; Gaddam, Pragna; Wu, Gang; McGee, Rose B.; Quinn, Emily; Inaba, Hiroto; Hartford, Christine; Pui, Ching-hon; Pappo, Alberto; Edmonson, Michael; Zhang, Michael Y.; Stepensky, Polina; Steinherz, Peter; Schrader, Kasmintan; Lincoln, Anne; Bussel, James; Lipkin, Steve M.; Goldgur, Yehuda; Harit, Mira; Stadler, Zsofia K.; Mullighan, Charles; Weintraub, Michael; Shimamura, Akiko; Zhang, Jinghui; Downing, James R.; Nichols, Kim E.; Offit, Kenneth

    2015-01-01

    Somatic mutations affecting ETV6 often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline ETV6 p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second ETV6 p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type ETV6 with retention of the ETV6 p. N385fs. Enforced expression of the ETV6 mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of ETV6 target genes. Our findings highlight a novel role for ETV6 in leukemia predisposition. PMID:26102509

  18. Genetic Interactions among Chlamydomonas Reinhardtii Mutations That Confer Resistance to anti-Microtubule Herbicides

    PubMed Central

    James, S. W.; Lefebvre, P. A.

    1992-01-01

    We previously described two types of genetic interactions among recessive mutations in the APM1 and APM2 loci of Chlamydomonas reinhardtii that may reflect a physical association of the gene products or their involvement in a common structure/process: (1) allele-specific synthetic lethality, and (2) unlinked noncomplementation, or dominant enhancement. To further investigate these interactions, we isolated revertants in which the heat sensitivity caused by the apm2-1 mutation is lost. The heat-insensitive revertants were either fully or partially suppressed for the drug-resistance caused by the apm2-1 allele. In recombination tests the revertants behaved as if the suppressing mutation mapped within the APM2 locus; the partial suppressors of apm2-1 herbicide resistance failed to complement apm2-1, leading to the conclusion that they were likely to be intragenic pseudorevertants. The apm2-1 partial suppressor mutations reversed apm1(-)apm2-1 synthetic lethality in an allele-specific manner with respect both to apm1(-) alleles and apm2-1 suppressor mutations. Those apm1(-) apm2-1(rev) strains that regained viability also regained heat sensitivity characteristic of the original apm2-1 mutation, even though the apm2-1 suppressor strains were fully heat-insensitive. The Hs(+) phenotypes of apm2-1 partial suppressors were also reversed by treatment with the microtubule-stabilizing agent deuterium oxide (D(2)O). In addition to the above interactions, we observed interallelic complementation and phenotypic enhancement of temperature conditionality among apm1(-) alleles. Evidence of a role for the products of the two genes in microtubule-based processes was obtained from studying flagellar assembly in apm1(-) and apm2(-) mutants. PMID:1311696

  19. Lamivudine/Adefovir Treatment Increases the Rate of Spontaneous Mutation of Hepatitis B Virus in Patients

    PubMed Central

    Pereira-Gómez, Marianoel; Bou, Juan-Vicente; Andreu, Iván; Sanjuán, Rafael

    2016-01-01

    The high levels of genetic diversity shown by hepatitis B virus (HBV) are commonly attributed to the low fidelity of its polymerase. However, the rate of spontaneous mutation of human HBV in vivo is currently unknown. Here, based on the evolutionary principle that the population frequency of lethal mutations equals the rate at which they are produced, we have estimated the mutation rate of HBV in vivo by scoring premature stop codons in 621 publicly available, full-length, molecular clone sequences derived from patients. This yielded an estimate of 8.7 × 10−5 spontaneous mutations per nucleotide per cell infection in untreated patients, which should be taken as an upper limit estimate because PCR errors and/or lack of effective lethality may inflate observed mutation frequencies. We found that, in patients undergoing lamivudine/adefovir treatment, the HBV mutation rate was elevated by more than sixfold, revealing a mutagenic effect of this treatment. Genome-wide analysis of single-nucleotide polymorphisms indicated that lamivudine/adefovir treatment increases the fraction of A/T-to-G/C base substitutions, consistent with recent work showing similar effects of lamivudine in cellular DNA. Based on these data, the rate at which HBV produces new genetic variants in treated patients is similar to or even higher than in RNA viruses. PMID:27649318

  20. Determination of the primary target of a quinolone drug and the effect of quinolone resistance-conferring mutations by measuring quinolone sensitivity based on its mode of action.

    PubMed

    Pfeiffer, Emily S; Hiasa, Hiroshi

    2007-09-01

    We used an assay to measure quinolone sensitivity as a shift in the position of the cleavage-religation equilibrium. This assay was found to be useful in identifying the primary target of a quinolone drug and assessing the effect of quinolone resistance-conferring mutations.

  1. Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population.

    PubMed

    Chevalier, Frédéric D; Le Clec'h, Winka; Eng, Nina; Rugel, Anastasia R; Assis, Rafael Ramiro de; Oliveira, Guilherme; Holloway, Stephen P; Cao, Xiaohang; Hart, P John; LoVerde, Philip T; Anderson, Timothy J C

    2016-06-01

    Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles.

  2. Genetic Correlations Greatly Increase Mutational Robustness and Can Both Reduce and Enhance Evolvability

    PubMed Central

    Greenbury, Sam F.; Schaper, Steffen; Ahnert, Sebastian E.; Louis, Ard A.

    2016-01-01

    Mutational neighbourhoods in genotype-phenotype (GP) maps are widely believed to be more likely to share characteristics than expected from random chance. Such genetic correlations should strongly influence evolutionary dynamics. We explore and quantify these intuitions by comparing three GP maps—a model for RNA secondary structure, the HP model for protein tertiary structure, and the Polyomino model for protein quaternary structure—to a simple random null model that maintains the number of genotypes mapping to each phenotype, but assigns genotypes randomly. The mutational neighbourhood of a genotype in these GP maps is much more likely to contain genotypes mapping to the same phenotype than in the random null model. Such neutral correlations can be quantified by the robustness to mutations, which can be many orders of magnitude larger than that of the null model, and crucially, above the critical threshold for the formation of large neutral networks of mutationally connected genotypes which enhance the capacity for the exploration of phenotypic novelty. Thus neutral correlations increase evolvability. We also study non-neutral correlations: Compared to the null model, i) If a particular (non-neutral) phenotype is found once in the 1-mutation neighbourhood of a genotype, then the chance of finding that phenotype multiple times in this neighbourhood is larger than expected; ii) If two genotypes are connected by a single neutral mutation, then their respective non-neutral 1-mutation neighbourhoods are more likely to be similar; iii) If a genotype maps to a folding or self-assembling phenotype, then its non-neutral neighbours are less likely to be a potentially deleterious non-folding or non-assembling phenotype. Non-neutral correlations of type i) and ii) reduce the rate at which new phenotypes can be found by neutral exploration, and so may diminish evolvability, while non-neutral correlations of type iii) may instead facilitate evolutionary exploration and so

  3. The Trojan Female Technique for pest control: a candidate mitochondrial mutation confers low male fertility across diverse nuclear backgrounds in Drosophila melanogaster

    PubMed Central

    Dowling, Damian K; Tompkins, Daniel M; Gemmell, Neil J

    2015-01-01

    Pest species represent a major ongoing threat to global biodiversity. Effective management approaches are required that regulate pest numbers, while minimizing collateral damage to nontarget species. The Trojan Female Technique (TFT) was recently proposed as a prospective approach to biological pest control. The TFT draws on the evolutionary hypothesis that maternally inherited mitochondrial genomes are prone to the accumulation of male, but not female, harming mutations. These mutations could be harnessed to provide trans-generational fertility-based control of pest species. A candidate TFT mutation was recently described in the fruit fly, Drosophila melanogaster, which confers male-only sterility in the specific isogenic nuclear background in which it is maintained. However, applicability of the TFT relies on mitochondrial mutations whose male-sterilizing effects are general across nuclear genomic contexts. We test this assumption, expressing the candidate TFT-mutation bearing haplotype alongside a range of nuclear backgrounds and comparing its fertility in males, relative to that of control haplotypes. We document consistently lower fertility for males harbouring the TFT mutation, in both competitive and noncompetitive mating contexts, across all nuclear backgrounds screened. This indicates that TFT mutations conferring reduced male fertility can segregate within populations and could be harnessed to facilitate this novel form of pest control. PMID:26495040

  4. The Trojan Female Technique for pest control: a candidate mitochondrial mutation confers low male fertility across diverse nuclear backgrounds in Drosophila melanogaster.

    PubMed

    Dowling, Damian K; Tompkins, Daniel M; Gemmell, Neil J

    2015-10-01

    Pest species represent a major ongoing threat to global biodiversity. Effective management approaches are required that regulate pest numbers, while minimizing collateral damage to nontarget species. The Trojan Female Technique (TFT) was recently proposed as a prospective approach to biological pest control. The TFT draws on the evolutionary hypothesis that maternally inherited mitochondrial genomes are prone to the accumulation of male, but not female, harming mutations. These mutations could be harnessed to provide trans-generational fertility-based control of pest species. A candidate TFT mutation was recently described in the fruit fly, Drosophila melanogaster, which confers male-only sterility in the specific isogenic nuclear background in which it is maintained. However, applicability of the TFT relies on mitochondrial mutations whose male-sterilizing effects are general across nuclear genomic contexts. We test this assumption, expressing the candidate TFT-mutation bearing haplotype alongside a range of nuclear backgrounds and comparing its fertility in males, relative to that of control haplotypes. We document consistently lower fertility for males harbouring the TFT mutation, in both competitive and noncompetitive mating contexts, across all nuclear backgrounds screened. This indicates that TFT mutations conferring reduced male fertility can segregate within populations and could be harnessed to facilitate this novel form of pest control.

  5. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

    PubMed

    Grassian, Alexandra R; Parker, Seth J; Davidson, Shawn M; Divakaruni, Ajit S; Green, Courtney R; Zhang, Xiamei; Slocum, Kelly L; Pu, Minying; Lin, Fallon; Vickers, Chad; Joud-Caldwell, Carol; Chung, Franklin; Yin, Hong; Handly, Erika D; Straub, Christopher; Growney, Joseph D; Vander Heiden, Matthew G; Murphy, Anne N; Pagliarini, Raymond; Metallo, Christian M

    2014-06-15

    Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

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

    PubMed

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

    2016-09-07

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

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

    PubMed Central

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

    2016-01-01

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

  8. IDH1 Mutations Alter Citric Acid Cycle Metabolism and Increase Dependence on Oxidative Mitochondrial Metabolism

    PubMed Central

    Grassian, Alexandra R.; Parker, Seth J.; Davidson, Shawn M.; Divakarun, Ajit S.; Green, Courtney R.; Zhang, Xiamei; Slocum, Kelly L.; Pu, Minying; Lin, Fallon; Vickers, Chad; Joud-Caldwell, Carol; Chung, Franklin; Yin, Hong; Handly, Erika D.; Straub, Christopher; Growney, Joseph D.; Vander Heiden, Matthew G.; Murphy, Anne N.; Pagliarini, Raymond; Metallo, Christian M.

    2016-01-01

    Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed 13C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation. PMID:24755473

  9. The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

    PubMed Central

    Julias, J G; Kim, T; Arnold, G; Pathak, V K

    1997-01-01

    It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis. PMID:9151812

  10. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma.

    PubMed

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W; Amin, Hesham M; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D; Rosen, Steven T; Pham, Lan V; Ngo, Vu N

    2016-11-08

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5-10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.

  11. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma

    PubMed Central

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W.; Amin, Hesham M.; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D.; Rosen, Steven T.; Pham, Lan V.; Ngo, Vu N.

    2016-01-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5–10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL. PMID:27713153

  12. Free-energy computations identify the mutations required to confer trans-sialidase activity into Trypanosoma rangeli sialidase.

    PubMed

    Pierdominici-Sottile, Gustavo; Palma, Juliana; Roitberg, Adrian E

    2014-03-01

    Trypanosoma rangeli's sialidase (TrSA) and Trypanosoma cruzi's trans-sialidase (TcTS) are members of the glycoside hydrolase family 33 (GH-33). They share 70% of sequence identity and their crystallographic Cα RMSD is 0.59 Å. Despite these similarities they catalyze different reactions. TcTS transfers sialic acid between glycoconjugates while TrSA can only cleave sialic acid from sialyl-glyconjugates. Significant effort has been invested into unraveling the differences between TrSA and TcTS, and into conferring TrSA with trans-sialidase activity through appropriate point mutations. Recently, we calculated the free-energy change for the formation of the covalent intermediate (CI) in TcTS and performed an energy decomposition analysis of that process. In this article we present a similar study for the formation of the CI in TrSA, as well as in a quintuple mutant (TrSA5mut), which has faint trans-sialidase activity. The comparison of these new results with those previously obtained for TcTS allowed identifying five extra mutations to be introduced in TrSA5mut that should create a mutant (TrSA10mut ) with high trans-sialidase activity.

  13. The A395T mutation in ERG11 gene confers fluconazole resistance in Candida tropicalis causing candidemia.

    PubMed

    Tan, Jingwen; Zhang, Jinqing; Chen, Wei; Sun, Yi; Wan, Zhe; Li, Ruoyu; Liu, Wei

    2015-04-01

    The mechanism of fluconazole resistance in Candida tropicalis is still unclear. Recently, we isolated a fluconazole-resistant strain of C. tropicalis from the blood specimen of a patient with candidemia in China. In vitro antifungal susceptibility of the isolate was determined by using CLSI M27-A3 and E-test methods. The sequence of ERG11 gene was then analyzed, and the three-dimensional model of Erg11p encoded by ERG11 gene was also investigated. The sequencing of ERG11 gene revealed the mutation of A395T in this fluconazole-resistant isolate of C. tropicalis, resulting in the Y132F substitution in Erg11p. Sequence alignment and three-dimensional model comparison of Erg11ps showed high similarity between fluconazole-susceptible isolates of C. tropicalis and Candida albicans. The comparison of the three-dimensional models of Erg11ps demonstrated that the position of the Y132F substitution in this isolate of C. tropicalis is identical to the isolate of C. albicans with fluconazole resistance resulting from Y132F substitution in Erg11p. Hence, we ascertain that the Y132F substitution of Erg11p caused by A395T mutation in ERG11 gene confers the fluconazole resistance in C. tropicalis.

  14. Mutation of Rv2887, a marR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

    PubMed Central

    Winglee, Kathryn; Lun, Shichun; Pieroni, Marco; Kozikowski, Alan

    2015-01-01

    Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation. PMID:26303802

  15. Mechanisms by which a CACNA1H mutation in epilepsy patients increases seizure susceptibility

    PubMed Central

    Eckle, Veit-Simon; Shcheglovitov, Aleksandr; Vitko, Iuliia; Dey, Deblina; Yap, Chan Choo; Winckler, Bettina; Perez-Reyes, Edward

    2014-01-01

    T-type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Cav3.2 T-type Ca2+ channels, CACNA1H, have been found in association with various forms of idiopathic generalized epilepsy. We and others have found that these mutations may influence neuronal excitability either by altering the biophysical properties of the channels or by increasing their surface expression. The goals of the present study were to investigate the excitability of neurons expressing Cav3.2 with the epilepsy mutation, C456S, and to elucidate the mechanisms by which it influences neuronal properties. We found that expression of the recombinant C456S channels substantially increased the excitability of cultured neurons by increasing the spontaneous firing rate and reducing the threshold for rebound burst firing. Additionally, we found that molecular determinants in the I–II loop (the region in which most childhood absence epilepsy-associated mutations are found) substantially increase the surface expression of T-channels but do not alter the relative distribution of channels into dendrites of cultured hippocampal neurons. Finally, we discovered that expression of C456S channels promoted dendritic growth and arborization. These effects were reversed to normal by either the absence epilepsy drug ethosuximide or a novel T-channel blocker, TTA-P2. As Ca2+-regulated transcription factors also increase dendritic development, we tested a transactivator trap assay and found that the C456S variant can induce changes in gene transcription. Taken together, our findings suggest that gain-of-function mutations in Cav3.2 T-type Ca2+ channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression. PMID:24277868

  16. Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).

    PubMed

    Roditakis, Emmanouil; Steinbach, Denise; Moritz, Gerald; Vasakis, Emmanouil; Stavrakaki, Marianna; Ilias, Aris; García-Vidal, Lidia; Martínez-Aguirre, María Del Rosario; Bielza, Pablo; Morou, Evangelia; Silva, Jefferson E; Silva, Wellington M; Siqueira, Ηerbert A A; Iqbal, Sofia; Troczka, Bartlomiej J; Williamson, Martin S; Bass, Chris; Tsagkarakou, Anastasia; Vontas, John; Nauen, Ralf

    2017-01-01

    Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance

  17. A CLAG3 mutation in an amphipathic transmembrane domain alters malaria parasite nutrient channels and confers leupeptin resistance.

    PubMed

    Sharma, Paresh; Rayavara, Kempaiah; Ito, Daisuke; Basore, Katherine; Desai, Sanjay A

    2015-06-01

    Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted α-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC.

  18. GATA1 mutations in patients with down syndrome and acute megakaryoblastic leukaemia do not always confer a good prognosis.

    PubMed

    Ariffin, Hany; Garcia, Jaime Castillo; Daud, Siti Sarah; Ibrahim, Kamariah; Aizah, Nik; Ong, Gek-Bee; Chong, Lee-Ai; Mohamad, Zulqarnain

    2009-07-01

    Children with Down syndrome and acute megakaryoblastic leukemia (DS-AMKL) have been shown to have increased sensitivity to cytarabine based chemotherapy. The excellent prognosis in patients with DS-AMKL may be due to mutations in the GATA1 gene leading to reduced expression of the enzyme cytidine deaminase. This leads to a decreased ability to convert cytarabine into its inactive metabolite, resulting in high intracellular concentration of this cytotoxic agent. We report two cases of DS-AMKL with GATA1 mutations who had poor outcome. These patients had high expression levels of cytidine deaminase mRNA transcripts. We speculate that other factors can affect overall outcome in patients with DS-AMKL irrespective of the presence of GATA1 mutations.

  19. LEOPARD syndrome-associated SHP2 mutation confers leanness and protection from diet-induced obesity

    PubMed Central

    Tajan, Mylène; Batut, Aurélie; Cadoudal, Thomas; Deleruyelle, Simon; Le Gonidec, Sophie; Saint Laurent, Céline; Vomscheid, Maëlle; Wanecq, Estelle; Tréguer, Karine; De Rocca Serra-Nédélec, Audrey; Vinel, Claire; Marques, Marie-Adeline; Pozzo, Joffrey; Kunduzova, Oksana; Salles, Jean-Pierre; Tauber, Maithé; Raynal, Patrick; Cavé, Hélène; Edouard, Thomas; Valet, Philippe; Yart, Armelle

    2014-01-01

    LEOPARD syndrome (multiple Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth, sensorineural Deafness; LS), also called Noonan syndrome with multiple lentigines (NSML), is a rare autosomal dominant disorder associating various developmental defects, notably cardiopathies, dysmorphism, and short stature. It is mainly caused by mutations of the PTPN11 gene that catalytically inactivate the tyrosine phosphatase SHP2 (Src-homology 2 domain-containing phosphatase 2). Besides its pleiotropic roles during development, SHP2 plays key functions in energetic metabolism regulation. However, the metabolic outcomes of LS mutations have never been examined. Therefore, we performed an extensive metabolic exploration of an original LS mouse model, expressing the T468M mutation of SHP2, frequently borne by LS patients. Our results reveal that, besides expected symptoms, LS animals display a strong reduction of adiposity and resistance to diet-induced obesity, associated with overall better metabolic profile. We provide evidence that LS mutant expression impairs adipogenesis, triggers energy expenditure, and enhances insulin signaling, three features that can contribute to the lean phenotype of LS mice. Interestingly, chronic treatment of LS mice with low doses of MEK inhibitor, but not rapamycin, resulted in weight and adiposity gains. Importantly, preliminary data in a French cohort of LS patients suggests that most of them have lower-than-average body mass index, associated, for tested patients, with reduced adiposity. Altogether, these findings unravel previously unidentified characteristics for LS, which could represent a metabolic benefit for patients, but may also participate to the development or worsening of some traits of the disease. Beyond LS, they also highlight a protective role of SHP2 global LS-mimicking modulation toward the development of obesity and associated disorders

  20. Functional EGFR germline polymorphisms may confer risk for EGFR somatic mutations in non-small cell lung cancer, with a predominant effect on exon 19 microdeletions

    PubMed Central

    Liu, Wanqing; He, Lijun; Ramírez, Jacqueline; Krishnaswamy, Soundararajan; Kanteti, Rajani; Wang, Yi-Ching; Salgia, Ravi; Ratain, Mark J

    2011-01-01

    Somatic mutations in the EGFR tyrosine kinase (TK) domain play a critical role in the development and treatment of non-small cell lung cancer (NSCLC). Strong genetic influence on susceptibility to these mutations has been suggested. To identify the genetic factors conferring risk for the EGFR TK mutations in NSCLC, a case-control study was conducted in 141 Taiwanese NSCLC patients by focusing on three functional polymorphisms in the EGFR gene [-216G/T, intron 1(CA)n and R497K]. Allelic imbalance (AI) of the EGFR -216G/T polymorphism was also tested in the heterozygous patients as well as in the NCI-60 cancer cell lines to further verify its function. We found that the frequencies of the alleles -216T and CA-19 are significantly higher in the patients with any mutation (p=0.032 and 0.01, respectively), in particular in those with exon 19 microdeletions (p=0.006 and 0.033, respectively), but not in the patients with L858R mutation. The -216T allele is favored to be amplified in both tumor DNA of lung cancer patients and cancer cell lines. We conclude that the local haplotype structures across the EGFR gene may favor the development of cellular malignancies and thus significantly confer risk to the occurrence of EGFR mutations in NSCLC, particularly the exon 19 microdeletions. PMID:21292812

  1. A Mutation in a Saccharomyces Cerevisiae Gene (Rad3) Required for Nucleotide Excision Repair and Transcription Increases the Efficiency of Mismatch Correction

    PubMed Central

    Yang, Y.; Johnson, A. L.; Johnston, L. H.; Siede, W.; Friedberg, E. C.; Ramachandran, K.; Kunz, B. A.

    1996-01-01

    RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMS1, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase δ was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency. PMID:8889512

  2. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  3. Frameshift Mutation Confers Function as Virulence Factor to Leucine-Rich Repeat Protein from Acidovorax avenae

    PubMed Central

    Kondo, Machiko; Hirai, Hiroyuki; Furukawa, Takehito; Yoshida, Yuki; Suzuki, Aika; Kawaguchi, Takemasa; Che, Fang-Sik

    2017-01-01

    Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen Acidovorax avenae causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of A. avenae K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein (Lrp) gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 Lrp gene. An Lrp-deleted K1 strain (KΔLrp) did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 (NΔLrp) did not interfere with infection of finger millet. In addition, NΔLrp retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain α, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain α was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation. PMID:28101092

  4. Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin

    PubMed Central

    Frohloff, Frank; Fichtner, Lars; Jablonowski, Daniel; Breunig, Karin D.; Schaffrath, Raffael

    2001-01-01

    Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G1 cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with Elongator. Intriguingly, strains lacking non-Elongator HATs (gcn5Δ, hat1Δ, hpa3Δ and sas3Δ) or non-Elongator transcription elongation factors TFIIS (dst1Δ) and Spt4p (spt4Δ) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin. PMID:11296232

  5. Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells

    PubMed Central

    Spender, Lindsay C.; Ferguson, G. John; Liu, Sijia; Cui, Chao; Girotti, Maria Romina; Sibbet, Gary; Higgs, Ellen B.; Shuttleworth, Morven K.; Hamilton, Tom; Lorigan, Paul; Weller, Michael; Vincent, David F.; Sansom, Owen J.; Frame, Margaret; Dijke, Peter ten; Marais, Richard; Inman, Gareth J.

    2016-01-01

    Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance. PMID:27835901

  6. Dual E627K and D701N mutations in the PB2 protein of A(H7N9) influenza virus increased its virulence in mammalian models

    PubMed Central

    Zhu, Wenfei; Li, Long; Yan, Zhigang; Gan, Tanhuan; Li, Lifeng; Chen, Rirong; Chen, Ruidong; Zheng, Zuoyi; Hong, Wenshan; Wang, Jia; Smith, David K.; Guan, Yi; Zhu, Huachen; Shu, Yuelong

    2015-01-01

    The ongoing avian H7N9 influenza outbreaks in China have caused significant human fatal cases and the virus is becoming established in poultry. Mutations with potential to increase mammalian adaptation have occurred in the polymerase basic protein 2 (PB2) and other viral genes. Here we found that dual 627K and 701N mutations could readily occur during transmission of the virus among ferrets via direct physical contact, and these mutations conferred higher polymerase activity and improved viral replication in mammalian cells, and enhanced virulence in mice. Special attention needs to be paid to patients with such mutations, as these may serve as an indicator of higher virus replication and increased pathogenicity. PMID:26391278

  7. A novel mutation, D404N, in the connection subdomain of reverse transcriptase of HIV-1 CRF08_BC subtype confers cross-resistance to NNRTIs

    PubMed Central

    Zhang, Xiao-Min; Wu, Hao; Zhang, Qiwei; Lau, Terrence Chi-Kong; Chu, Hin; Chen, Zhi-Wei; Jin, Dong-Yan; Zheng, Bo-Jian

    2015-01-01

    Objectives Growing evidence suggests that mutations in the connection domain of the HIV-1 reverse transcriptase (RT) can contribute to viral resistance to RT inhibitors. This work was designed to determine the effects of a novel mutation, D404N, in the connection subdomain of RT of HIV-1 CRF08_BC subtype on drug resistance, viral replication capacity (RC) and RT activity. Methods Mutation D404N, alone or together with the other reported mutations, was introduced into an HIV-1 CRF08_BC subtype infectious clone by site-directed mutagenesis. Viral susceptibility to nine RT inhibitors, viral RC and the DNA polymerase activity of viral RT of the constructed virus mutants were investigated. A modelling study using the server SWISS-MODEL was conducted to explore the possible structure-related drug resistance mechanism of the mutation D404N. Results Single mutations D404N and H221Y conferred low-level resistance to nevirapine, efavirenz, rilpivirine and zidovudine. Double mutations Y181C/D404N and Y181C/H221Y significantly reduced susceptibility to NNRTIs. The most pronounced resistance to NNRTIs was observed with the triple mutation Y181C/D404N/H221Y. Virus containing D404N as the only mutation displayed ∼50% RC compared with the WT virus. The modelling study suggested that the D404N mutation might abolish the hydrogen bonds between residues 404 and K30 in p51 or K431 in p66, leading to impaired RT subunit structure and enhanced drug resistance. Conclusions These results indicate that D404N is a novel NNRTI-associated mutation in the HIV-1 subtype CRF08_BC and provides information valuable for the monitoring of clinical RTI resistance. PMID:25637519

  8. Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

    PubMed Central

    Sexton, Nicole R.; Smith, Everett Clinton; Blanc, Hervé; Vignuzzi, Marco; Peersen, Olve B.

    2016-01-01

    ABSTRACT Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(−)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex. IMPORTANCE RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity

  9. Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice.

    PubMed

    Morgan, Daniel J; Davis, Brian J; Kearn, Chris S; Marcus, David; Cook, Alex J; Wager-Miller, Jim; Straiker, Alex; Myoga, Michael H; Karduck, Jeffrey; Leishman, Emma; Sim-Selley, Laura J; Czyzyk, Traci A; Bradshaw, Heather B; Selley, Dana E; Mackie, Ken

    2014-04-09

    For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-protein-coupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ(9)-THC), have delayed tolerance to Δ(9)-THC, and showed increased dependence for Δ(9)-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ(9)-THC was absent in S426A/S430A mutants. Δ(9)-THC-induced downregulation of CB1R in the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.

  10. Resistance to Bacillus thuringiensis Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect.

    PubMed

    Xiao, Yutao; Liu, Kaiyu; Zhang, Dandan; Gong, Lingling; He, Fei; Soberón, Mario; Bravo, Alejandra; Tabashnik, Bruce E; Wu, Kongming

    2016-02-01

    Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control.

  11. Resistance to Bacillus thuringiensis Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect

    PubMed Central

    Zhang, Dandan; Gong, Lingling; He, Fei; Soberón, Mario; Bravo, Alejandra; Tabashnik, Bruce E.; Wu, Kongming

    2016-01-01

    Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control. PMID:26872031

  12. Women in Nontraditional Jobs: A Conference Guide. Increasing Job Options for Women.

    ERIC Educational Resources Information Center

    Women's Bureau (DOL), Washington, DC.

    Designed to help organizations interested in expanding job options for women to plan and hold a community-based conference on nontraditional jobs, this guide outlines basic steps in planning, provides information about successful programs, and makes suggestions about how to deal with the mechanics of a conference. Following an introduction which…

  13. Mutations in blaKPC-3 that confer ceftazidime-avibactam resistance encode novel KPC-3 variants that function as extended-spectrum β-lactamases.

    PubMed

    Haidar, Ghady; Clancy, Cornelius J; Shields, Ryan K; Hao, Binghua; Cheng, Shaoji; Nguyen, M Hong

    2017-02-21

    We identified four blaKPC-3 mutations in ceftazidime-avibactam resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli, we conclusively demonstrated that mutant blaKPC-3 encoded enzymes that functioned as extended-spectrum β-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam MICs, and decreased MICs of carbapenems and other β-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop.

  14. Mutations in alternative carbon utilization pathways in Candida albicans attenuate virulence and confer pleiotropic phenotypes.

    PubMed

    Ramírez, Melissa A; Lorenz, Michael C

    2007-02-01

    The interaction between Candida albicans and cells of the innate immune system is a key determinant of disease progression. Transcriptional profiling has revealed that C. albicans has a complex response to phagocytosis, much of which is similar to carbon starvation. This suggests that nutrient limitation is a significant stress in vivo, and we have shown that glyoxylate cycle mutants are less virulent in mice. To examine whether other aspects of carbon metabolism are important in vivo during an infection, we have constructed strains lacking FOX2 and FBP1, which encode key components of fatty acid beta-oxidation and gluconeogenesis, respectively. As expected, fox2Delta mutants failed to utilize several fatty acids as carbon sources. Surprisingly, however, these mutants also failed to grow in the presence of several other carbon sources, whose assimilation is independent of beta-oxidation, including ethanol and citric acid. Mutants lacking the glyoxylate enzyme ICL1 also had more severe carbon utilization phenotypes than were expected. These results suggest that the regulation of alternative carbon metabolism in C. albicans is significantly different from that in other fungi. In vivo, fox2Delta mutants show a moderate but significant reduction in virulence in a mouse model of disseminated candidiasis, while disruption of the glyoxylate cycle or gluconeogenesis confers a severe attenuation in this model. These data indicate that C. albicans often encounters carbon-poor conditions during growth in the host and that the ability to efficiently utilize multiple nonfermentable carbon sources is a virulence determinant. Consistent with this in vivo requirement, C. albicans uniquely regulates carbon metabolism in a more integrated manner than in Saccharomyces cerevisiae, such that defects in one part of the machinery have wider impacts than expected. These aspects of alternative carbon metabolism may then be useful as targets for therapeutic intervention.

  15. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    PubMed Central

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  16. Salmonella recD mutations increase recombination in a short sequence transduction assay.

    PubMed Central

    Miesel, L; Roth, J R

    1994-01-01

    We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene of S. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3- to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease. Images PMID:8021190

  17. Origin and dissemination of the pollen-part mutated SC haplotype which confers self-compatibility in apricot (Prunus armeniaca).

    PubMed

    Halász, Júlia; Pedryc, Andrzej; Hegedus, Attila

    2007-01-01

    In China, its centre of origin, apricot (Prunus armeniaca) is self-incompatible. However, most European cultivars are self-compatible. In most cases, self-compatibility is a result of a loss-of-function mutation within the pollen gene (SFB) in the SC haplotype. Controlled pollinations performed in this work revealed that the cross 'Ceglédi óriás' (S8S9)x'Ceglédi arany' (SCS9) set well, as expected, but the reciprocal cross did not. Apricot S8, S9 and SC haplotypes were analysed using a multilevel approach including fruit set evaluation, pollen tube growth analysis, RNase activity assays, polymerase chain reaction (PCR) analysis and DNA sequencing of the S-RNase and SFB alleles. SFB8 was revealed to be the first known progenitor allele of a naturally occurring self-compatibility allele in Prunus, and consequently SC=The first intron of SC-RNase is a phase one intron, indicating its more recent evolutionary origin compared with the second intron. Sequence analysis of different cultivars revealed that more single nucleotide polymorphisms accumulated in SC-RNase than in SFBC. New methods were designed to allow high-throughput analysis of S genotypes of apricot cultivars and selections. S-RNase sequence data from various sources helped to elucidate the putative origin and dissemination of self-compatibility in apricot conferred by the SC haplotype.

  18. Hungarian surveillance of germinal mutations. Lack of detectable increase in indicator conditions caused by germinal mutations following the Chernobyl accident.

    PubMed

    Czeizel, A

    1989-07-01

    The Hungarian surveillance of germinal mutations is based on three indicator conditions seen in offspring, i.e., 15 sentinel anomalies, Down syndrome and component anomaly pairs of unidentified multiple congenital anomalies. It is an "opportunistic program," because the necessary data are available from the Hungarian Congenital Malformation Registry. This system is described and the criteria of a good registry are summarized. The analysis of indicator conditions caused by germinal mutations did not reveal any measurable mutagenic effects in Hungary following the accident at the Chernobyl nuclear power plant. The pros and cons of germinal mutation surveillance are discussed.

  19. Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL

    PubMed Central

    Zhang, Yixi; Meng, Xiangkun; Yang, Yuanxue; Li, Hong; Wang, Xin; Yang, Baojun; Zhang, Jianhua; Li, Chunrui; Millar, Neil S.; Liu, Zewen

    2016-01-01

    Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance. PMID:27557781

  20. An Arabidopsis soil-salinity-tolerance mutation confers ethylene-mediated enhancement of sodium/potassium homeostasis.

    PubMed

    Jiang, Caifu; Belfield, Eric J; Cao, Yi; Smith, J Andrew C; Harberd, Nicholas P

    2013-09-01

    High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ethylene overproducer1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ethylene resistant1-constitutive triple response1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of respiratory burst oxidase homolog F (RBOHF)-dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated high-affinity K(+) TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation.

  1. Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus

    PubMed Central

    Effner, Emily E.; Iglesias-Sánchez, Maria José; Golden, Susan S.

    2016-01-01

    In natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacterium Synechococcus elongatus PCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678–16683, 2012, http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide of S. elongatus. Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains. PMID:26921432

  2. Novel Timothy Syndrome Mutation Leading to Increase in CACNA1C Window Current

    PubMed Central

    Boczek, Nicole J.; Miller, Erin M.; Ye, Dan; Nesterenko, Vlad V.; Tester, David J.; Antzelevitch, Charles; Czosek, Richard J.; Ackerman, Michael J.; Ware, Stephanie M.

    2016-01-01

    Background Timothy syndrome (TS) is a rare multi-system genetic disorder characterized by a myriad of abnormalities including QT prolongation, syndactyly, and neurological symptoms. The predominant genetic causes are recurrent de novo missense mutations in exon 8/8A of the CACNA1C-encoded L-type calcium channel, however some cases remain genetically elusive. Objective To identify the genetic cause of TS in a case that did not harbor a CACNA1C mutation in exon 8/8A, and was negative for all other plausible genetic substrates. Methods Utilization of diagnostic exome sequencing to identify the genetic substrate responsible for our case of TS. The identified mutation was characterized using whole cell patch-clamp technique and the results of these analyses were modeled using a modified Luo-Rudy dynamic model to determine the effects on the cardiac action potential. Results Whole exome sequencing revealed a novel CACNA1C mutation, p.Ile1166Thr, in a young male with diagnosed TS. Functional electrophysiological analysis identified a novel mechanism of TS-mediated disease, with an overall loss of current density and a gain-of-function shift in activation, leading to an increased window current. Modeling studies of this variant predicted prolongation of the action potential, as well as the development of spontaneous early afterdepolarizations. Conclusion Through expanded whole exome sequencing, we have identified a novel genetic substrate for TS, p.Ile1166Thr-CACNA1C. Electrophysiological experiments combined with modeling studies have identified a novel TS mechanism through increased window current. Therefore, expanded genetic testing in cases of TS to the entire CACNA1C coding region, if initial targeted testing is negative, may be warranted. PMID:25260352

  3. Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage.

    PubMed

    Kennedy, Scott R; Salk, Jesse J; Schmitt, Michael W; Loeb, Lawrence A

    2013-01-01

    Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >10(7) wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ~5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G → T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G → A and T → C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.

  4. Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells.

    PubMed

    Xu, G Wei; Toth, Julia I; da Silva, Sara R; Paiva, Stacey-Lynn; Lukkarila, Julie L; Hurren, Rose; Maclean, Neil; Sukhai, Mahadeo A; Bhattacharjee, Rabindra N; Goard, Carolyn A; Medeiros, Bruno; Gunning, Patrick T; Dhe-Paganon, Sirano; Petroski, Matthew D; Schimmer, Aaron D

    2014-01-01

    The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562(MLN), R-U937(MLN)) were selected. R-K562(MLN) and R-U937(MLN) cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme's affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562(MLN) cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.

  5. Hyperferritinaemia-cataract syndrome: Worldwide mutations and phenotype of an increasingly diagnosed genetic disorder

    PubMed Central

    2010-01-01

    The hereditary hyperferritinaemia-cataract syndrome (HHCS) is characterised by an autosomal dominant cataract and high levels of serum ferritin without iron overload. The cataract develops due to L-ferritin deposits in the lens and its pulverulent aspect is pathognomonic. The syndrome is caused by mutations within the iron-responsive element of L-ferritin. These mutations prevent efficient binding of iron regulatory proteins 1 and 2 to the IRE in L-ferritin mRNA, resulting in an unleashed ferritin translation. This paper reviews all 31 mutations (27 single nucleotide transitions and four deletions) that have been described since 1995. Laboratory test showing hyperferritinaemia, normal serum iron and normal transferrin saturation are indicative for HHCS after exclusion of other causes of increased ferritin levels (inflammation, malignancy, alcoholic liver disease) and should prompt an ophthalmological consultation for diagnostic confirmation. Invasive diagnostics such as liver biopsy are not indicated. HHCS is an important differential diagnosis of hyperferritinaemia. Haematologists, gastroenterologists and ophthalmologists should be aware of this syndrome to spare patients from further invasive diagnosis (liver biopsy), and also from a false diagnosis of hereditary haemochromatosis followed by venesections. Patients diagnosed with HHCS should be counselled regarding the relative harmlessness of this genetic disease, with early cataract surgery as the only clinical consequence. PMID:20511138

  6. Essential Genes Embody Increased Mutational Robustness to Compensate for the Lack of Backup Genetic Redundancy

    PubMed Central

    Cohen, Osher; Oberhardt, Matthew; Yizhak, Keren; Ruppin, Eytan

    2016-01-01

    Genetic robustness is a hallmark of cells, occurring through many mechanisms and at many levels. Essential genes lack the common robustness mechanism of genetic redundancy (i.e., existing alongside other genes with the same function), and thus appear at first glance to leave cells highly vulnerable to genetic or environmental perturbations. Here we explore a hypothesis that cells might protect against essential gene loss through mechanisms that occur at various cellular levels aside from the level of the gene. Using Escherichia coli and Saccharomyces cerevisiae as models, we find that essential genes are enriched over non-essential genes for properties we call “coding efficiency” and “coding robustness”, denoting respectively a gene’s efficiency of translation and robustness to non-synonymous mutations. The coding efficiency levels of essential genes are highly positively correlated with their evolutionary conservation levels, suggesting that this feature plays a key role in protecting conserved, evolutionarily important genes. We then extend our hypothesis into the realm of metabolic networks, showing that essential metabolic reactions are encoded by more “robust” genes than non-essential reactions, and that essential metabolites are produced by more reactions than non-essential metabolites. Taken together, these results testify that robustness at the gene-loss level and at the mutation level (and more generally, at two cellular levels that are usually treated separately) are not decoupled, but rather, that cellular vulnerability exposed due to complete gene loss is compensated by increased mutational robustness. Why some genes are backed up primarily against loss and others against mutations still remains an open question. PMID:27997585

  7. Dependence On Glycolysis Sensitizes BRAF-mutated Melanomas For Increased Response To Targeted BRAF Inhibition

    PubMed Central

    Hardeman, Keisha N.; Peng, Chengwei; Paudel, Bishal B.; Meyer, Christian T.; Luong, Thong; Tyson, Darren R.; Young, Jamey D.; Quaranta, Vito; Fessel, Joshua P.

    2017-01-01

    Dysregulated metabolism can broadly affect therapy resistance by influencing compensatory signaling and expanding proliferation. Given many BRAF-mutated melanoma patients experience disease progression with targeted BRAF inhibitors, we hypothesized therapeutic response is related to tumor metabolic phenotype, and that altering tumor metabolism could change therapeutic outcome. We demonstrated the proliferative kinetics of BRAF-mutated melanoma cells treated with the BRAF inhibitor PLX4720 fall along a spectrum of sensitivity, providing a model system to study the interplay of metabolism and drug sensitivity. We discovered an inverse relationship between glucose availability and sensitivity to BRAF inhibition through characterization of metabolic phenotypes using nearly a dozen metabolic parameters in Principle Component Analysis. Subsequently, we generated rho0 variants that lacked functional mitochondrial respiration and increased glycolytic metabolism. The rho0 cell lines exhibited increased sensitivity to PLX4720 compared to the respiration-competent parental lines. Finally, we utilized the FDA-approved antiretroviral drug zalcitabine to suppress mitochondrial respiration and to force glycolysis in our cell line panel, resulting in increased PLX4720 sensitivity via shifts in EC50 and Hill slope metrics. Our data suggest that forcing tumor glycolysis in melanoma using zalcitabine or other similar approaches may be an adjunct to increase the efficacy of targeted BRAF therapy. PMID:28205616

  8. Estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant A(H1N1)pdm09 influenza viruses.

    PubMed

    Butler, Jeff; Hooper, Kathryn A; Petrie, Stephen; Lee, Raphael; Maurer-Stroh, Sebastian; Reh, Lucia; Guarnaccia, Teagan; Baas, Chantal; Xue, Lumin; Vitesnik, Sophie; Leang, Sook-Kwan; McVernon, Jodie; Kelso, Anne; Barr, Ian G; McCaw, James M; Bloom, Jesse D; Hurt, Aeron C

    2014-04-01

    Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.

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

    PubMed

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

    2013-08-28

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

  10. Increased Use of Twitter at a Medical Conference: A Report and a Review of the Educational Opportunities

    PubMed Central

    Cumming, Grant P; Lee, Amanda J

    2012-01-01

    Background Most consider Twitter as a tool purely for social networking. However, it has been used extensively as a tool for online discussion at nonmedical and medical conferences, and the academic benefits of this tool have been reported. Most anesthetists still have yet to adopt this new educational tool. There is only one previously published report of the use of Twitter by anesthetists at an anesthetic conference. This paper extends that work. Objective We report the uptake and growth in the use of Twitter, a microblogging tool, at an anesthetic conference and review the potential use of Twitter as an educational tool for anesthetists. Methods A unique Twitter hashtag (#WSM12) was created and promoted by the organizers of the Winter Scientific Meeting held by The Association of Anaesthetists of Great Britain and Ireland (AAGBI) in London in January 2012. Twitter activity was compared with Twitter activity previously reported for the AAGBI Annual Conference (September 2011 in Edinburgh). All tweets posted were categorized according to the person making the tweet and the purpose for which they were being used. The categories were determined from a literature review. Results A total of 227 tweets were posted under the #WSM12 hashtag representing a 530% increase over the previously reported anesthetic conference. Sixteen people joined the Twitter stream by using this hashtag (300% increase). Excellent agreement (κ = 0.924) was seen in the classification of tweets across the 11 categories. Delegates primarily tweeted to create and disseminate notes and learning points (55%), describe which session was attended, undertake discussions, encourage speakers, and for social reasons. In addition, the conference organizers, trade exhibitors, speakers, and anesthetists who did not attend the conference all contributed to the Twitter stream. The combined total number of followers of those who actively tweeted represented a potential audience of 3603 people. Conclusions This

  11. A G protein alpha null mutation confers prolificacy potential in maize

    DOE PAGES

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-05-06

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. Lastly, the maize heterotrimeric G protein complex is importantmore » in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.« less

  12. A G protein alpha null mutation confers prolificacy potential in maize

    SciTech Connect

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-05-06

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. Lastly, the maize heterotrimeric G protein complex is important in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.

  13. A G protein alpha null mutation confers prolificacy potential in maize

    PubMed Central

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-01-01

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. The maize heterotrimeric G protein complex is important in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences. PMID:25948706

  14. VEGF Promoter Polymorphism Confers an Increased Risk of Pulmonary Arterial Hypertension in a Chinese Population

    PubMed Central

    Zeng, Qingchun; Zhang, Peng; Li, Guoyang; Xie, Qiang; Cheng, Ying

    2017-01-01

    Purpose Evidence on the contribution of genes to the hereditary predisposition to pulmonary arterial hypertension (PAH) is limited. Materials and Methods In this study, we hypothesized that single nucleotide variants in vascular endothelial growth factor (VEGF) gene may alter gene function and expression and may be associated with PAH risk. Five putatively functional loci (rs699947C>A and rs833061T>C in the promoter, rs3025040C>T, rs10434G>A and rs3025053G>A in the 3'-UTR) in the VEGF gene were genotyped and analyzed in a retrospective study of 587 patients with PAH and 736 healthy subjects from southern China. Results We found that the rs833061T>C polymorphism was significantly associated with PAH risk, while the other single nucleotide polymorphisms were not. Compared to carriers with TT genotype, those with rs833061C variant genotype (CT/CC) had an increased risk of PAH (odds ratio=1.47, 95% confidence interval=1.18–1.83, p=0.001). Functional assays indicated that CT/CC variant genotype had significantly higher mRNA levels of VEGF in peripheral blood mononuclear cells than TT genotype (p=0.021). Luciferase reporter assay indicated that having a C allele conferred a significantly higher transcription activity than that with a T allele. Conclusion Our findings suggest that the functional polymorphism rs833061T>C in VEGF gene promoter modulates VEGF expression and may be a valuable biomarker for predicting PAH susceptibility. PMID:28120560

  15. Dominant mutations causing alterations in acetyl-coenzyme A carboxylase confer tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides in maize.

    PubMed Central

    Parker, W B; Marshall, L C; Burton, J D; Somers, D A; Wyse, D L; Gronwald, J W; Gengenbach, B G

    1990-01-01

    A partially dominant mutation exhibiting increased tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides was isolated by exposing susceptible maize (Zea mays) tissue cultures to increasingly inhibitory concentrations of sethoxydim (a cyclohexanedione). The selected tissue culture (S2) was greater than 40-fold more tolerant to sethoxydim and 20-fold more tolerant to haloxyfop (an aryloxyphenoxypropionate) than the nonselected wild-type tissue culture. Regenerated S2 plants were heterozygous for the mutant allele and exhibited a high-level, but not complete, tolerance to both herbicides. Homozygous mutant families derived by self-pollinating the regenerated S2 plants exhibited no injury after treatment with 0.8 kg of sethoxydim per ha, which was greater than 16-fold the rate lethal to wild-type plants. Acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2) is the target enzyme of cyclohexanedione and aryloxyphenoxypropionate herbicides. ACCase activities of the nonselected wild-type and homozygous mutant seedlings were similar in the absence of herbicide. ACCase activity from homozygous tolerant plants required greater than 100-fold more sethoxydim and 16-fold more haloxyfop for 50% inhibition than ACCase from wild-type plants. These results indicate that tolerance to sethoxydim and haloxyfop is controlled by a partially dominant nuclear mutation encoding a herbicide-insensitive alteration in maize ACCase. Images PMID:1976254

  16. Mutations in Succinate Dehydrogenase Subunit C Increase Radiosensitivity and Bystander Responses

    NASA Astrophysics Data System (ADS)

    Zhou, Hongning; Hei, Tom K.

    Although radiation-induced bystander effect is well studied in the past decade, the precise mech-anisms are still unclear. It is likely that a combination of pathways involving both primary and secondary signaling processes is involved in producing a bystander effect. There is recent evidence that mitochondria play a critical role in bystander responses. Recently studies found that a mutation in succinate dehydrogenese subunit C (SDHC), an integral membrane protein in complex II of the electron transport chain, resulted in increased superoxide, oxidative stress, apoptosis, tumorigenesis, and genomic instability, indicating that SDHC play a critical role in maintaining mitochondrial function. In the present study, using Chinese hamster fibroblasts (B1 cells) and the mutants (B9 cells) containing a single base substitution that produced a premature stop codon resulting in a 33-amino acid COOH-terminal truncation of the SDHC protein, we found that B9 cells had an increase in intracellular superoxide content, nitric oxide species, and mitochondrial membrane potential when compared with wild type cells. After irradiated with a grade of doses of gamma rays, B9 cells show an increased radiosensitivity, especially at high doses. The HPRT- mutant yield after gamma-ray irradiation in B9 cells was significantly higher than that of B1 cells. A single, 3Gy dose of gamma-rays increased the background mutant level by more than 4 fold. In contrast, the mutant induction was less than 2 fold in B1 cells. In addition, B9 cells produced a higher bystander mutagenesis after alpha particle irradiation than the B1 cells. Furthermore, pretreated with carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide scavenger, significantly decreased the bystander effect. Our findings demonstrate that a mutation in SDHC increases radiosensitivity in both directly irradiated cells and in neighboring bystander cells, and mito-chondrial function play an essential role in

  17. Mutations in Nonessential eIF3k and eIF3l Genes Confer Lifespan Extension and Enhanced Resistance to ER Stress in Caenorhabditis elegans

    PubMed Central

    Reddy, Kirthi C.; Droste, Rita; Kim, Dennis H.

    2016-01-01

    The translation initiation factor eIF3 is a multi-subunit protein complex that coordinates the assembly of the 43S pre-initiation complex in eukaryotes. Prior studies have demonstrated that not all subunits of eIF3 are essential for the initiation of translation, suggesting that some subunits may serve regulatory roles. Here, we show that loss-of-function mutations in the genes encoding the conserved eIF3k and eIF3l subunits of the translation initiation complex eIF3 result in a 40% extension in lifespan and enhanced resistance to endoplasmic reticulum (ER) stress in Caenorhabditis elegans. In contrast to previously described mutations in genes encoding translation initiation components that confer lifespan extension in C. elegans, loss-of-function mutations in eif-3.K or eif-3.L are viable, and mutants show normal rates of growth and development, and have wild-type levels of bulk protein synthesis. Lifespan extension resulting from EIF-3.K or EIF-3.L deficiency is suppressed by a mutation in the Forkhead family transcription factor DAF-16. Mutations in eif-3.K or eif-3.L also confer enhanced resistance to ER stress, independent of IRE-1-XBP-1, ATF-6, and PEK-1, and independent of DAF-16. Our data suggest a pivotal functional role for conserved eIF3k and eIF3l accessory subunits of eIF3 in the regulation of cellular and organismal responses to ER stress and aging. PMID:27690135

  18. Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

    PubMed Central

    Berrazeg, M.; Jeannot, K.; Ntsogo Enguéné, Véronique Yvette; Broutin, I.; Loeffert, S.; Fournier, D.

    2015-01-01

    Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting. PMID:26248364

  19. Rhbdf2 mutations increase its protein stability and drive EGFR hyperactivation through enhanced secretion of amphiregulin

    PubMed Central

    Hosur, Vishnu; Johnson, Kenneth R.; Burzenski, Lisa M.; Stearns, Timothy M.; Maser, Richard S.; Shultz, Leonard D.

    2014-01-01

    The rhomboid 5 homolog 2 (Rhbdf2) gene encodes an inactive rhomboid (iRhom) protease, iRhom2, one of a family of enzymes containing a long cytosolic N terminus and a dormant peptidase domain of unknown function. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of epidermal growth factor receptor (EGFR) signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. We show that iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin, independent of metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) activity. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but they do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains. PMID:24825892

  20. Rhbdf2 mutations increase its protein stability and drive EGFR hyperactivation through enhanced secretion of amphiregulin.

    PubMed

    Hosur, Vishnu; Johnson, Kenneth R; Burzenski, Lisa M; Stearns, Timothy M; Maser, Richard S; Shultz, Leonard D

    2014-05-27

    The rhomboid 5 homolog 2 (Rhbdf2) gene encodes an inactive rhomboid (iRhom) protease, iRhom2, one of a family of enzymes containing a long cytosolic N terminus and a dormant peptidase domain of unknown function. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of epidermal growth factor receptor (EGFR) signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. We show that iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin, independent of metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) activity. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but they do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains.

  1. Low pesticide rates may hasten the evolution of resistance by increasing mutation frequencies.

    PubMed

    Gressel, Jonathan

    2011-03-01

    At very low pesticide rates, a certain low proportion of pests may receive a sublethal dose, are highly stressed by the pesticide and yet survive. Stress is a general enhancer of mutation rates. Thus, the survivors are likely to have more than normal mutations, which might include mutations leading to pesticide resistance, both for multifactorial (polygenic, gene amplification, sequential allelic mutations) and for major gene resistance. Management strategies should consider how to eliminate the subpopulation of pests with the high mutation rates, but the best strategy is probably to avoid too low application rates of pesticides from the outset.

  2. A calcium-dependent potassium current is increased by a single-gene mutation in Paramecium.

    PubMed

    Hennessey, T M; Kung, C

    1987-01-01

    The membrane currents of wild type Paramecium tetraurelia and the behavioral mutant teaA were analyzed under voltage clamp. The teaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs+ and external TEA+. This, along with previous work (Satow, Y., Kung, C., 1976, J. Exp. Biol. 65:51-63) identified this as a K+ current. It was further found to be a calcium-activated K+ current since this increased outward K+ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutation pwB, which blocks the inward calcium current, also blocks this increased outward K+ current in teaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K+ current are the same for mutant and wild type, the teaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of the teaA mutant. The teaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of the teaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K+ current which prematurely repolarizes the action potential.

  3. Mutations Conferring Resistance to SCH6, a Novel Hepatitis C Virus NS3/4A Protease Inhibitor: Reduced DNA Replication Fitness and Partial Rescue by Second-Site Mutations

    SciTech Connect

    Yi, MinKyung; Tong, Xiao; Skelton, Angela; Chase, Robert; Chen, Tong; Prongay, Andrew; Bogen, Stephane L.; Saksena, Anil K.; Njoroge, F. George; Veselenak, Ronald L.; Pyles, Richard B.; Bourne, Nigel; Malcolm, Bruce A.; Lemon, Stanley M.

    2008-06-30

    Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P{prime}-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

  4. Long-range DHPS mutations unexpectedly increase Mycobacterium chimaera susceptibility to sulfonamides.

    PubMed

    Gotthard, Guillaume; Muhammed Ameen, Sirwan; Drancourt, Michel; Chabriere, Eric

    2013-12-01

    The two closely related mycobacteria, Mycobacterium intracellulare and Mycobacterium chimaera, exhibit a more than two-fold difference in their in vitro susceptibility to sulfonamides. Sulfonamides are antibiotics targeting the 6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS) enzyme involved in the folate synthesis pathway. Comparing the DHPS gene sequence in six M. intracellulare and M. chimaera types trains and clinical isolates yielded only four amino acid changes. In silico structural modelling surprisingly indicated that these amino acids are not located in the active site of DHPS and do not interact directly with sulfonamides. Unexpectedly, these amino acids in distal positions may play a key role in the increased sulfonamide susceptibility observed in M. chimaera compared with M. intracellulare. This example illustrates how three-dimensional models could help to identify distal mutations capable of modulating enzymatic activity.

  5. Increased Chromosomal Radiosensitivity in Women Carrying BRCA1/BRCA2 Mutations Assessed With the G2 Assay

    SciTech Connect

    Ernestos, Beroukas; Nikolaos, Pandis; Koulis, Giannoukakos; Eleni, Rizou; Konstantinos, Beroukas; Alexandra, Giatromanolaki; Michael, Koukourakis

    2010-03-15

    Purpose: Several in vitro studies suggest that BRCA1 and BRCA2 mutation carriers present increased sensitivity to ionizing radiation. Different assays for the assessment of deoxyribonucleic acid double-strand break repair capacity have been used, but results are rather inconsistent. Given the concerns about the possible risks of breast screening with mammography in mutation carrier women and the potentially damaging effects of radiotherapy, the purpose of this study was to further investigate the radiosensitivity of this population. Methods and Materials: The G2 chromosomal radiosensitivity assay was used to assess chromosomal breaks in lymphocyte cultures after exposure to 1 Gy. A group of familiar breast cancer patients carrying a mutation in the BRCA1 or BRCA2 gene (n = 15) and a group of healthy mutation carriers (n = 5) were investigated and compared with a reference group of healthy women carrying no mutation (n = 21). Results: BRCA1 and BRCA2 mutation carriers had a significantly higher number of mean chromatid breaks per cell (p = 0.006) and a higher maximum number of breaks (p = 0.0001) as compared with their matched controls. Both healthy carriers and carriers with a cancer history were more radiosensitive than controls (p = 0.002 and p = 0.025, respectively). Age was not associated with increased radiosensitivity (p = 0.868). Conclusions: Our results indicate that BRCA1 and BRCA2 mutation carriers show enhanced radiosensitivity, presumably because of the involvement of the BRCA genes in deoxyribonucleic acid repair and cell cycle control mechanisms.

  6. Next-generation sequencing-based method shows increased mutation detection sensitivity in an Indian retinoblastoma cohort

    PubMed Central

    Singh, Jaya; Mishra, Avshesh; Pandian, Arunachalam Jayamuruga; Mallipatna, Ashwin C.; Khetan, Vikas; Sripriya, S.; Kapoor, Suman; Agarwal, Smita; Sankaran, Satish; Katragadda, Shanmukh; Veeramachaneni, Vamsi; Hariharan, Ramesh; Subramanian, Kalyanasundaram

    2016-01-01

    Purpose Retinoblastoma (Rb) is the most common primary intraocular cancer of childhood and one of the major causes of blindness in children. India has the highest number of patients with Rb in the world. Mutations in the RB1 gene are the primary cause of Rb, and heterogeneous mutations are distributed throughout the entire length of the gene. Therefore, genetic testing requires screening of the entire gene, which by conventional sequencing is time consuming and expensive. Methods In this study, we screened the RB1 gene in the DNA isolated from blood or saliva samples of 50 unrelated patients with Rb using the TruSight Cancer panel. Next-generation sequencing (NGS) was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. Results We were able to detect germline pathogenic mutations in 66% (33/50) of the cases, 12 of which were novel. We were able to detect all types of mutations, including missense, nonsense, splice site, indel, and structural variants. When we considered bilateral Rb cases only, the mutation detection rate increased to 100% (22/22). In unilateral Rb cases, the mutation detection rate was 30% (6/20). Conclusions Our study suggests that NGS-based approaches increase the sensitivity of mutation detection in the RB1 gene, making it fast and cost-effective compared to the conventional tests performed in a reflex-testing mode. PMID:27582626

  7. Inhibition of MEK Confers Hypersensitivity to X-radiation in the context of BRAF mutation in a Model of Childhood Astrocytoma

    PubMed Central

    Studebaker, Adam; Bondra, Kathryn; Seum, Star; Shen, Changxian; Phelps, Doris A.; Chronowski, Christopher; Leasure, Justin; Smith, Paul D.; Kurmasheva, Raushan T.; Mo, Xiaokui; Fouladi, Maryam; Houghton, Peter J.

    2015-01-01

    Purpose Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment, however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma. Experimental Design The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. Results Inhibition of MEK signaling by selumetinib, suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity. Conclusions Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae. PMID:25981859

  8. cps1+, a Schizosaccharomyces pombe gene homolog of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclosporin A and papulacandin B.

    PubMed Central

    Ishiguro, J; Saitou, A; Durán, A; Ribas, J C

    1997-01-01

    The Schizosaccharomyces pombe cps1-12 (for chlorpropham supersensitive) mutant strain was originally isolated as hypersensitive to the spindle poison isopropyl N-3-chlorophenyl carbamate (chlorpropham) (J. Ishiguro and Y. Uhara, Jpn. J. Genet. 67:97-109, 1992). We have found that the cps1-12 mutation also confers (i) hypersensitivity to the immunosuppressant cyclosporin A (CsA), (ii) hypersensitivity to the drug papulacandin B, which specifically inhibits 1,3-beta-D-glucan synthesis both in vivo and in vitro, and (iii) thermosensitive growth at 37 degrees C. Under any of these restrictive treatments, cells swell up and finally lyse. With an osmotic stabilizer, cells do not lyse, but at 37 degrees C they become multiseptated and multibranched. The cps1-12 mutant, grown at a restrictive temperature, showed an increase in sensitivity to lysis by enzymatic cell wall degradation, in in vitro 1,3-beta-D-glucan synthase activity (173% in the absence of GTP in the reaction), and in cell wall biosynthesis (130% of the wild-type amount). Addition of Ca2+ suppresses hypersensitivity to papulacandin B and septation and branching phenotypes. All of these data suggest a relationship between the cps1+ gene and cell wall synthesis. A DNA fragment containing the cps1+ gene was cloned, and sequence analysis indicated that it encodes a predicted membrane protein of 1,729 amino acids with 15 to 16 transmembrane domains. S. pombe cps1p has overall 55% sequence identity with Fks1p or Fks2p, proposed to be catalytic or associated subunits of Saccharomyces cerevisiae 1,3-beta-D-glucan synthase. Thus, the cps1+ product might be a catalytic or an associated copurifying subunit of the fission yeast 1,3-beta-D-glucan synthase that plays an essential role in cell wall synthesis. PMID:9401022

  9. A point mutation in transthyretin increases affinity for thyroxine and produces euthyroid hyperthyroxinemia.

    PubMed Central

    Moses, A C; Rosen, H N; Moller, D E; Tsuzaki, S; Haddow, J E; Lawlor, J; Liepnieks, J J; Nichols, W C; Benson, M D

    1990-01-01

    In a family expressing euthyroid hyperthyroxinemia, an increased association of plasma thyroxine (T4) with transthyretin (TTR) is transmitted by autosomal dominant inheritance and is secondary to a mutant TTR molecule with increased affinity for T4. Eight individuals spanning three generations exhibited the abnormality. Although five of eight individuals had elevated total T4 concentrations, all affected individuals were clinically euthyroid and all had normal free T4 levels. Purified TTR from the propositus had an affinity for 125I-T4 three times that of control TTR. Exons 2, 3, and 4 (representing greater than 97% of the coding sequence) of the TTR gene of DNA prepared from the propositus' peripheral blood leukocytes were amplified using the polymerase chain reaction (PCR) and were sequenced after subcloning. Exons 2 and 3 were indistinguishable from normal. In 50% of clones amplified from exon 4, a substitution of adenine (ACC) for guanine (GCC) in codon 109 resulted in the replacement of threonine-for-alanine, a mutation confirmed by amino acid sequencing of tryptic peptides derived from purified plasma TTR. The adenine-for-guanine substitution abolishes one of two Fnu 4H I restriction sites in exon 4. PCR amplification of exon 4 of TTR and restriction digestion with Fnu 4H I confirmed that five affected family members with increased binding of 125I-T4 to TTR are heterozygous for the threonine 109 substitution that increases the affinity of this abnormal TTR for T4. Images PMID:1979335

  10. eis Promoter C14G and C15G Mutations Do Not Confer Kanamycin Resistance in Mycobacterium tuberculosis.

    PubMed

    Pholwat, Suporn; Stroup, Suzanne; Heysell, Scott; Ogarkov, Oleg; Zhdanova, Svetlana; Ramakrishnan, Girija; Houpt, Eric

    2016-12-01

    We studied the significance of particular eis mutations on Mycobacterium tuberculosis drug resistance using a specialized transduction strategy. Recombinant strains harboring eis promoter mutations C14T, C12T, and G10A exhibited kanamycin resistance with MICs of 40, 10, and 20 μg/ml, respectively, while recombinant strains harboring C14G and C15G mutations were kanamycin susceptible (MIC, 2.5 to 5 μg/ml). Each of the eis mutants tested remained amikacin susceptible (MIC, 0.5 to 4 μg/ml). The identification of specific eis mutations is needed for accurate genotypic susceptibility testing for kanamycin.

  11. Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation

    PubMed Central

    Liu, Ta-Ming; Woyach, Jennifer A.; Zhong, Yiming; Lozanski, Arletta; Lozanski, Gerard; Dong, Shuai; Strattan, Ethan; Lehman, Amy; Zhang, Xiaoli; Jones, Jeffrey A.; Flynn, Joseph; Andritsos, Leslie A.; Maddocks, Kami; Jaglowski, Samantha M.; Blum, Kristie A.; Byrd, John C.; Dubovsky, Jason A.

    2015-01-01

    Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib’s capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2R665W confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance. PMID:25972157

  12. Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae

    PubMed Central

    Bommakanti, Ananth S.; Lindahl, Lasse; Zengel, Janice M.

    2008-01-01

    The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both Gram-positive and Gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes. PMID:18218702

  13. A novel nonsense mutation in androgen receptor confers resistance to CYP17 inhibitor treatment in prostate cancer

    PubMed Central

    Han, Dong; Gao, Shuai; Valencia, Kevin; Owiredu, Jude; Han, Wanting; de Waal, Eric; Macoska, Jill A; Cai, Changmeng

    2017-01-01

    The standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that blocks transcriptional activity of androgen receptor (AR). However, ADT invariably leads to the development of castration-resistant PCa (CRPC) with restored activity of AR. CRPC can be further treated with CYP17 inhibitors to block androgen synthesis pathways, but most patients still relapse after a year of such treatment. The mechanisms that drive this progression are not fully understood, but AR activity, at least in a subset of cancers, appears to be restored again. Importantly, AR mutations are more frequently detected in this type of cancer. By analyzing tumor biopsy mRNA from CRPC patients who had developed resistance to CYP17 inhibitor treatment, we have identified a novel nonsense mutation (Q784*) at the ligand binding domain (LBD) of AR, which produces a C-terminal truncated AR protein that lacks intact LBD. This AR-Q784* mutant is transcriptionally inactive, but it is constitutively expressed in the nucleus and can bind to DNA in the absence of androgen. Significantly, our results show that AR-Q784* can heterodimerize with, and enhance the transcriptional activity of, full-length AR. Moreover, expressing AR-Q784* in an AR positive PCa cell line enhances the chromatin binding of endogenous AR and the recruitment of p300 coactivator under the low androgen condition, leading to increased cell growth. This activity of AR-Q784* mimics the function of some AR splice variants, indicating that CYP17 inhibitor treatment in CRPC may select for LBD-truncated forms of AR to restore AR signaling. PMID:28036278

  14. Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity

    PubMed Central

    Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua

    2016-01-01

    ABSTRACT Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo. Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human

  15. Mouse somatic mutation orthologous to MELAS A3302G mutation in the mitochondrial tRNA(Leu(UUR)) gene confers respiration defects.

    PubMed

    Shimizu, Akinori; Enoki, Shunkei; Ishikawa, Kaori; Mito, Takayuki; Obata, Kanae; Nagashima, Ruriko; Yonekawa, Hiromichi; Nakada, Kazuto; Hayashi, Jun-Ichi

    2015-11-27

    We searched for mtDNA harboring somatic mutations in mouse B82 cells, and found an A2748G mutation orthologous to the A3302G mutation in tRNA(Leu(UUR)) gene reported in a patient with MELAS, the most prevalent mitochondrial disease. We isolated subclones of B82 cells until we obtained one subclone harboring >95% A2748G mtDNA. Cytoplasmic transfer of A2748G mtDNA resulted in cotransfer of A2748G mtDNA and respiration defects into mouse ES cells. Thus, A2748G mtDNA is responsible for respiration defects, and the ES cells harboring A2748G mtDNA may be useful for generation of transmitochondrial mice harboring A2748G mtDNA as potential disease models of MELAS.

  16. Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation

    PubMed Central

    Fanning, Sean W; Mayne, Christopher G; Dharmarajan, Venkatasubramanian; Carlson, Kathryn E; Martin, Teresa A; Novick, Scott J; Toy, Weiyi; Green, Bradley; Panchamukhi, Srinivas; Katzenellenbogen, Benita S; Tajkhorshid, Emad; Griffin, Patrick R; Shen, Yang; Chandarlapaty, Sarat; Katzenellenbogen, John A; Greene, Geoffrey L

    2016-01-01

    Somatic mutations in the estrogen receptor alpha (ERα) gene (ESR1), especially Y537S and D538G, have been linked to acquired resistance to endocrine therapies. Cell-based studies demonstrated that these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance. Here, we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα. We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist (estradiol) and antagonist (4-hydroxytamoxifen) are reduced. Further, they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα, which leads to a stabilized agonist state and an altered antagonist state that resists inhibition. DOI: http://dx.doi.org/10.7554/eLife.12792.001 PMID:26836308

  17. Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo

    PubMed Central

    Kuo, Cheng-Ju; Chen, Jenn-Wei; Chiu, Hao-Chieh; Teng, Ching-Hao; Hsu, Tai-I; Lu, Pei-Jung; Syu, Wan-Jr; Wang, Sin-Tian; Chou, Ting-Chen; Chen, Chang-Shi

    2016-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection. PMID:27570746

  18. Detection of JAK2 V617F mutation increases the diagnosis of myeloproliferative neoplasms

    PubMed Central

    ZHANG, SHU-PENG; LI, HUI; LAI, REN-SHENG

    2015-01-01

    The Janus kinase (JAK)2 gene, which is located on chromosome 9p24, is involved in the signaling transduction pathways of the hematopoietic and immune system. Mutations in the JAK2 gene have served as disease markers for myeloproliferative neoplasms (MPNs). The aim of the present study was to investigate the occurrence of the JAK2 gene mutation in 140 clinical samples, and to evaluate its clinical significance in MPNs and other hematological diseases. Genomic DNA was extracted from the peripheral blood leukocytes or bone marrow karyocytes of 140 clinical samples, which included 130 patients with various types of hematological disease and 10 control patients. In addition, exons 12 and 14 of the JAK2 gene were analyzed by direct sequencing and the mutation rates of various MPN subtypes were evaluated. Of the 140 samples, exons 12 and 14 were tested in 74 samples, however, exon 14 only was tested in 66 samples. No mutations were identified in exon 12. The V617F mutation rate in polycythemia vera was 82.1% (23/28), and the mutation rates in essential thrombocythemia histiocytosis, primary myelofibrosis and other MPNs were 53.1% (17/32), 40.0% (4/10) and 60.0% (6/10), respectively. Therefore, the total mutation rate of the JAK2 gene in MPN was 62.5% (50/80). For non-MPN hematological diseases, four V617F mutations were detected in samples of leukocytosis of unknown origin (4/12), however, no JAK2 V617F mutations were identified in the 10 controls. Therefore, JAK2 V617F mutations may present a novel marker for diagnosis of MPNs. Furthermore, the direct sequencing method appeared to be satisfactory for the clinical gene testing of hematological samples. PMID:25624900

  19. The BRAF{sup T1799A} mutation confers sensitivity of thyroid cancer cells to the BRAF{sup V600E} inhibitor PLX4032 (RG7204)

    SciTech Connect

    Xing, Joanna; Liu, Ruixin; Xing, Mingzhao; Trink, Barry

    2011-01-28

    Research highlights: {yields} Exciting therapeutic potential has been recently reported for the BRAF{sup V600E} inhibitor PLX4032 in melanoma. {yields} We tested the effects of PLX4032 on the growth of thyroid cancer cells which often harbor the BRAF{sup V600E} mutation. {yields} We observed a potent BRAF{sup V600E}-dependent inhibition of thyroid cancer cells by PLX4032. {yields} We thus demonstrated an important therapeutic potential of PLX4032 for thyroid cancer. -- Abstract: Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF{sup V600E}, as a result of the BRAF{sup T1799A} mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF{sup V600E}-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF{sup T1799A} mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF{sup T1799A} mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC{sub 50} values (0.115-1.156 {mu}M) in BRAF{sup V600E} mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC{sub 50} values (56.674-1349.788 {mu}M). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF{sup T1799A} mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF{sup T1799A} mutation-selective therapeutic agent for thyroid cancer.

  20. TP53 mutation hits energy metabolism and increases glycolysis in breast cancer

    PubMed Central

    Munkácsy, Gyöngyi; Horváth, Gergő; Nagy, Ádám M.; Ambrus, Attila; Hauser, Péter; Szabó, András; Tretter, László; Győrffy, Balázs

    2016-01-01

    Promising new hallmarks of cancer is alteration of energy metabolism that involves molecular mechanisms shifting cancer cells to aerobe glycolysis. Our goal was to evaluate the correlation between mutation in the commonly mutated tumor suppressor gene TP53 and metabolism. We established a database comprising mutation and RNA-seq expression data of the TCGA repository and performed receiver operating characteristics (ROC) analysis to compare expression of each gene between TP53 mutated and wild type samples. All together 762 breast cancer samples were evaluated of which 215 had TP53 mutation. Top up-regulated metabolic genes include glycolytic enzymes (e.g. HK3, GPI, GAPDH, PGK1, ENO1), glycolysis regulator (PDK1) and pentose phosphate pathway enzymes (PGD, TKT, RPIA). Gluconeogenesis enzymes (G6PC3, FBP1) were down-regulated. Oxygen consumption and extracellular acidification rates were measured in TP53 wild type and mutant breast cell lines with a microfluorimetric analyzer. Applying metabolic inhibitors in the presence and absence of D-glucose and L-glutamine in cell culture experiments resulted in higher glycolytic and mitochondrial activity in TP53 mutant breast cancer cell lines. In summary, TP53 mutation influences energy metabolism at multiple levels. Our results provide evidence for the synergistic activation of multiple hallmarks linking to these the mutation status of a key driver gene. PMID:27582538

  1. E119D Neuraminidase Mutation Conferring Pan-Resistance to Neuraminidase Inhibitors in an A(H1N1)pdm09 Isolate From a Stem-Cell Transplant Recipient

    PubMed Central

    L'Huillier, Arnaud G.; Abed, Yacine; Petty, Tom J.; Cordey, Samuel; Thomas, Yves; Bouhy, Xavier; Schibler, Manuel; Simon, Audrey; Chalandon, Yves; van Delden, Christian; Zdobnov, Evgeny; Boquete-Suter, Patricia; Boivin, Guy; Kaiser, Laurent

    2015-01-01

    Background. An influenza A(H1N1)pdm09 infection was diagnosed in a hematopoietic stem cell transplant recipient during conditioning regimen. He was treated with oral oseltamivir, later combined with intravenous zanamivir. The H275Y neuraminidase (NA) mutation was first detected, and an E119D NA mutation was identified during zanamivir therapy. Methods. Recombinant wild-type (WT) E119D and E119D/H275Y A(H1N1)pdm09 NA variants were generated by reverse genetics. Susceptibility to NA inhibitors (NAIs) was evaluated with a fluorometric assay using the 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) substrate. Susceptibility to favipiravir (T-705) was assessed using plaque reduction assays. The NA affinity and velocity values were determined with NA enzymatic studies. Results. We identified an influenza A(H1N1)pdm09 E119D mutant that exhibited a marked increase in the 50% inhibitory concentrations against all tested NAIs (827-, 25-, 286-, and 702-fold for zanamivir, oseltamivir, peramivir, and laninamivir, respectively). The double E119D/H275Y mutation further increased oseltamivir and peramivir 50% inhibitory concentrations by 790- and >5000-fold, respectively, compared with the WT. The mutant viruses remained susceptible to favipiravir. The NA affinity and velocity values of the E119D variant decreased by 8.1-fold and 4.5-fold, respectively, compared with the WT. Conclusions. The actual emergence of a single NA mutation conferring pan-NAI resistance in the clinical setting reinforces the pressing need to develop new anti-influenza strategies. PMID:25985905

  2. BRCA Mutations Increase Fertility in Families at Hereditary Breast/Ovarian Cancer Risk

    PubMed Central

    Kwiatkowski, Fabrice; Arbre, Marie; Bidet, Yannick; Laquet, Claire; Uhrhammer, Nancy; Bignon, Yves-Jean

    2015-01-01

    Background Deleterious mutations in the BRCA genes are responsible for a small, but significant, proportion of breast and ovarian cancers (5 - 10 %). Proof of de novo mutations in hereditary breast/ovarian cancer (HBOC) families is rare, in contrast to founder mutations, thousands of years old, that may be carried by as much as 1 % of a population. Thus, if mutations favoring cancer survive selection pressure through time, they must provide advantages that compensate for the loss of life expectancy. Method This hypothesis was tested within 2,150 HBOC families encompassing 96,325 individuals. Parameters included counts of breast/ovarian cancer, age at diagnosis, male breast cancer and other cancer locations. As expected, well-known clinical parameters discriminated between BRCA-mutated families and others: young age at breast cancer, ovarian cancer, pancreatic cancer and male breast cancer. The major fertility differences concerned men in BRCA-mutated families: they had lower first and mean age at paternity, and fewer remained childless. For women in BRCA families, the miscarriage rate was lower. In a logistic regression including clinical factors, the different miscarriage rate and men's mean age at paternity remained significant. Results Fertility advantages were confirmed in a subgroup of 746 BRCA mutation carriers and 483 non-carriers from BRCA mutated families. In particular, female carriers were less often nulliparous (9.1 % of carriers versus 16.0 %, p = 0.003) and had more children (1.8 ± 1.4 SD versus 1.5 ± 1.3, p = 0.002) as well as male carriers (1.7 ± 1.3 versus 1.4 ± 1.3, p = 0.024). Conclusion Although BRCA mutations shorten the reproductive period due to cancer mortality, they compensate by improving fertility both in male and female carriers. PMID:26047126

  3. Hypomorphic, homozygous mutations in Phosphoglucomutase 3 impair immunity and increase serum IgE levels

    PubMed Central

    Sassi, Atfa; Lazaroski, Sandra; Wu, Gang; Haslam, Stuart M.; Fliegauf, Manfred; Mellouli, Fethi; Patiroglu, Turkan; Unal, Ekrem; Ozdemir, Mehmet Akif; Jouhadi, Zineb; Khadir, Khadija; Ben-Khemis, Leila; Ben-Ali, Meriem; Ben-Mustapha, Imen; Borchani, Lamia; Pfeifer, Dietmar; Jakob, Thilo; Khemiri, Monia; Asplund, A. Charlotta; Gustafsson, Manuela O.; Lundin, Karin E.; Falk-Sörqvist, Elin; Moens, Lotte N.; Gungor, Hatice Eke; Engelhardt, Karin R.; Dziadzio, Magdalena; Stauss, Hans; Fleckenstein, Bernhard; Meier, Rebecca; Prayitno, Khairunnadiya; Maul-Pavicic, Andrea; Schaffer, Sandra; Rakhmanov, Mirzokhid; Henneke, Philipp; Kraus, Helene; Eibel, Hermann; Kölsch, Uwe; Nadifi, Sellama; Nilsson, Mats; Bejaoui, Mohamed; Schäffer, Alejandro A.; Edvard Smith, C. I.; Dell, Anne; Barbouche, Mohamed-Ridha; Grimbacher, Bodo

    2016-01-01

    Background Recurrent bacterial and fungal infections, eczema and elevated serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in STAT3 and DOCK8, involved in signal transduction pathways. However, glycosylation defects have not been described in HIES. One crucial enzyme in the glycosylation pathway is Phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of UDP-GlcNAc which is required for the biosynthesis of N-glycans. Objective To elucidate the genetic cause in HIES patients who do not carry mutations in STAT3 or DOCK8. Methods After establishing a linkage interval by SNP-chip genotyping and homozygosity mapping in two HIES families from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by Western blotting and glycosylation was profiled by mass spectrometry. Results Mutational analysis of candidate genes in a 11.9 Mb linkage region on chromosome 6 shared by two multiplex families identified two homozygous mutations in PGM3 which segregated with the disease status and followed a recessive inheritance trait. The mutations predict amino acid changes in Phosphoglucomutase-3; PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in two other affected families, respectively. These hypomorphic mutations have impact on the biosynthetic reactions involving UDP-GlcNAc. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-/tetra-antennary N-glycans. T cell proliferation and differentiation was impaired in patients. Most patients showed developmental delay and many had psychomotor retardation. Conclusion Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity, as biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper

  4. fur (-) mutation increases the survival time of Escherichia coli under photooxidative stress in aquatic environments.

    PubMed

    Darcan, C; Aydin, Ebru

    2012-09-01

    We investigated the survival of the wild type Escherichia coli (QC771) and fur- mutant strain (QC1732) under photooxidative stress in different water sources. The survival of fur- mutant and wild type E. coli was seen as a significant decrease in the visible light samples in the presence of methylene blue (MB). The fur-E. coli strain lived longer than the wild type E. coli strain on exposure to MB and visible light, which generates singlet oxygen, in both lake water (48-h) and pure water (16-h). It is interesting to note that the survival of both wild type and the fur- mutant strain was more protected at 24 °C than at other temperatures. The Fur protein does not have any relation to the entry of E. coli into the viable but nonculturable state (VBNC) under photooxidative stress. This is the first study which shows that fur- mutation increases the resistance of E. coli to photooxidative stress in aquatic environments, and the Fur protein does not have any relation to the entry of E. coli into the VBNC state.

  5. Mutation rate is reduced by increased dosage of mutL gene in Escherichia coli K-12.

    PubMed

    Galán, Juan-Carlos; Turrientes, María-Carmen; Baquero, María-Rosario; Rodríguez-Alcayna, Manuel; Martínez-Amado, Jorge; Martínez, José-Luis; Baquero, Fernando

    2007-10-01

    A variable but substantial proportion of wild Escherichia coli isolates present consistently lower mutation frequencies than that found in the ensemble of strains. The genetic mechanisms responsible for the hypo-mutation phenotype are much less known than those involved in hyper-mutation. Changes in E. coli mutation frequencies derived from the gene-copy effect of mutS, mutL, mutH, uvrD, mutT, mutY, mutM, mutA, dnaE, dnaQ, and rpoS are explored. When present in a very high copy number ( approximately 300 copies cell(-1)), mutL, mutH, and mutA gene copies yielded >/=twofold decrease in mutation rates determined by Luria-Delbrück fluctuation tests. Nevertheless, when the copy number was not such high ( approximately 15 copies cell(-1)), only mutL results in a consistent twofold decrease in the mutation rate. This reduction seems to be independent from the RecA background, phase of growth, or from the presence of proficient MutS. An increase in mutL gene copies was also able to partially compensate the hypermutator phenotype of a mutS-defective E. coli derivative.

  6. The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in Saccharomyces cerevisiae under high-pressure and low-temperature conditions.

    PubMed

    Nagayama, Ai; Kato, Chiaki; Abe, Fumiyoshi

    2004-04-01

    Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2(E27F)) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2(D563N)) and HPG2-3 (Tat2(E570K)) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.

  7. Impact of individual mutations on increased fitness in adaptively evolved strains of Escherichia coli.

    PubMed

    Applebee, M Kenyon; Herrgård, Markus J; Palsson, Bernhard Ø

    2008-07-01

    We measured the relative fitness among a set of experimentally evolved Escherichia coli strains differing by a small number of adaptive mutations by directly measuring allelic frequencies in head-to-head competitions using a mass spectrometry-based method. We compared the relative effects of mutations to the same or similar genes acquired in multiple strains when expressed in allele replacement strains. We found that the strongest determinant of fitness among the evolved strains was the impact of beneficial mutations to the RNA polymerase beta and beta' subunit genes. We also identified several examples of epistatic interactions between rpoB/C and glpK mutations and identified two other mutations that are beneficial only in the presence of previously acquired mutations but that have little or no adaptive benefit to the wild-type strain. Allele frequency estimation is shown to be a highly sensitive method for measuring selection rates during competitions between strains differing by as little as a single-nucleotide polymorphism and may be of great use for investigating epistatic interactions.

  8. OPTN 691_692insAG is a founder mutation causing recessive ALS and increased risk in heterozygotes

    PubMed Central

    Goldstein, Orly; Nayshool, Omri; Nefussy, Beatrice; Traynor, Bryan J.; Renton, Alan E.; Gana-Weisz, Mali; Drory, Vivian E.

    2016-01-01

    Objective: To detect genetic variants underlying familial and sporadic amyotrophic lateral sclerosis (ALS). Methods: We analyzed 2 founder Jewish populations of Moroccan and Ashkenazi origins and ethnic matched controls. Exome sequencing of 2 sisters with ALS from Morocco was followed by genotyping the identified causative null mutation in 379 unrelated patients with ALS and 1,000 controls. The shared risk haplotype was characterized using whole-genome single nucleotide polymorphism array. Results: We identified 5 unrelated patients with ALS homozygous for the null 691_692insAG mutation in the optineurin gene (OPTN), accounting for 5.8% of ALS of Moroccan origin and 0.3% of Ashkenazi. We also identified a high frequency of heterozygous carriers among patients with ALS, 8.7% and 2.9%, respectively, compared to 0.75% and 1.0% in controls. The risk of carriers for ALS was significantly increased, with odds ratio of 13.46 and 2.97 in Moroccan and Ashkenazi Jews, respectively. We determined that 691_692insAG is a founder mutation in the tested populations with a minimal risk haplotype of 58.5 Kb, encompassing the entire OPTN gene. Conclusions: Our data show that OPTN 691_692insAG mutation is a founder mutation in Moroccan and Ashkenazi Jews. This mutation causes autosomal recessive ALS and significantly increases the risk to develop the disease in heterozygous carriers, suggesting both a recessive mode of inheritance and a dominant with incomplete penetrance. These data emphasize the important role of OPTN in ALS pathogenesis, and demonstrate the complex genetics of ALS, as the same mutation leads to different phenotypes and appears in 2 patterns of inheritance. PMID:26740678

  9. Digenic inheritance novel mutations in SCN5a and SNTA1 increase late I(Na) contributing to LQT syndrome.

    PubMed

    Hu, Rou-Mu; Tan, Bi-Hua; Orland, Kate M; Valdivia, Carmen R; Peterson, Amber; Pu, Jielin; Makielski, Jonathan C

    2013-04-01

    SCN5A and SNTA1 are reported susceptible genes for long QT syndrome (LQTS). This study was designed to elucidate a plausible pathogenic arrhythmia mechanism for the combined novel mutations R800L-SCN5A and A261V-SNTA1 on cardiac sodium channels. A Caucasian family with syncope and marginally prolonged QT interval was screened for LQTS-susceptibility genes and found to harbor the R800L mutation in SCN5A and A261V mutation in SNTA1, and those with both mutations had the strongest clinical phenotype. The mutations were engineered into the most common splice variant of human SCN5A and SNTA1 cDNA, respectively, and sodium current (INa) was characterized in human embryonic kidney 293 cells cotransfected with neuronal nitric oxide synthase (nNOS) and the cardiac isoform of the plasma membrane Ca-ATPase (PMCA4b). Peak INa densities were unchanged for wild-type (WT) and for mutant channels containing R800L-SCN5A, A261V-SNTA1, or R800L-SCN5A plus A261V-SNTA1. However, late INa for either single mutant was moderately increased two- to threefold compared with WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1 significantly enhanced the INa late/peak ratio by 5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The gain-of-function effect could be blocked by the N(G)-monomethyl-l-arginine, a nNOS inhibitor. We conclude that novel mutations in SCN5A and SNTA1 jointly exert a nNOS-dependent gain-of-function on SCN5A channels, which may consequently prolong the action potential duration and lead to LQTS phenotype.

  10. In Utero Exposure to Benzo[a]Pyrene Increases Mutation Burden in the Soma and Sperm of Adult Mice

    PubMed Central

    Meier, Matthew J.; O’Brien, Jason M.; Beal, Marc A.; Allan, Beverly; Yauk, Carole L.; Marchetti, Francesco

    2016-01-01

    Background: Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged as an important contributor to disease. Mutational events occurring during embryonic development can cause mosaicism in any tissue, but the influence of environmental factors on levels of mosaicism is unclear. Objectives: We investigated whether in utero exposure to the widespread environmental mutagen benzo[a]pyrene (BaP) has an impact on the burden and distribution of mutations in adult mice. Methods: We used the Muta™Mouse transgenic rodent model to quantify and characterize mutations in the offspring of pregnant mice exposed to BaP during postconception days 7 through 16, covering the major period of organogenesis in mice. Next-generation DNA sequencing was then used to determine the spectrum of mutations induced in adult mice that were exposed to BaP during fetal development. Results: Mutation frequency was significantly increased in the bone marrow, liver, brain, and sperm of first filial generation (F1) males. Developing embryos accumulated more mutations and exhibited higher proportions of mosaicism than exposed adults, particularly in the brain. Decreased sperm count and motility revealed additional negative impacts on the reproductive function of F1 males. Conclusion: In utero exposure to environmental mutagens contributes to somatic and germline mosaicism, permanently affecting both the genetic health of the F1 and the population gene pool. Citation: Meier MJ, O’Brien JM, Beal MA, Allan B, Yauk CL, Marchetti F. 2017. In utero exposure to benzo[a]pyrene increases mutation burden in the soma and sperm of adult mice. Environ Health Perspect 125:82–88; http://dx.doi.org/10.1289/EHP211 PMID:27448386

  11. Increased progerin expression associated with unusual LMNA mutations causes severe progeroid syndromes.

    PubMed

    Moulson, Casey L; Fong, Loren G; Gardner, Jennifer M; Farber, Emily A; Go, Gloriosa; Passariello, Annalisa; Grange, Dorothy K; Young, Stephen G; Miner, Jeffrey H

    2007-09-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare precocious aging syndrome caused by mutations in LMNA that lead to synthesis of a mutant form of prelamin A, generally called progerin, that cannot be processed to mature lamin A. Most HGPS patients have a recurrent heterozygous de novo mutation in exon 11 of LMNA, c.1824C>T/p.G608G; this synonymous mutation activates a nearby cryptic splice donor site, resulting in synthesis of the mutant prelamin A, progerin, which lacks 50 amino acids within the carboxyl-terminal domain. Abnormal splicing is incomplete, so the mutant allele produces some normally-spliced transcripts. Nevertheless, the synthesis of progerin is sufficient to cause misshapen nuclei in cultured cells and severe disease phenotypes in affected patients. Here we present two patients with extraordinarily severe forms of progeria caused by unusual mutations in LMNA. One had a splice site mutation (c.1968+1G>A; or IVS11+1G>A), and the other had a novel synonymous coding region mutation (c.1821G>A/p.V607V). Both mutations caused very frequent use of the same exon 11 splice donor site that is activated in typical HGPS patients. As a consequence, the ratios of progerin mRNA and protein to wild-type were higher than in typical HGPS patients. Fibroblasts from both patients exhibited nuclear shape abnormalities typical of HGPS, and cells treated with a protein farnesyltransferase inhibitor exhibited fewer misshapen nuclei. Thus, farnesyltransferase inhibitors may prove to be useful even when progerin expression levels are higher than those in typical HGPS patients.

  12. Increased Artemis levels confer radioresistance to both high and low LET radiation exposures

    PubMed Central

    2012-01-01

    Background Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation. Results Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage. Conclusions These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies. PMID:22713703

  13. Increased calvaria cell differentiation and bone matrix formation induced by fibroblast growth factor receptor 2 mutations in Apert syndrome.

    PubMed

    Lomri, A; Lemonnier, J; Hott, M; de Parseval, N; Lajeunie, E; Munnich, A; Renier, D; Marie, P J

    1998-03-15

    Apert syndrome, associated with fibroblast growth factor receptor (FGFR) 2 mutations, is characterized by premature fusion of cranial sutures. We analyzed proliferation and differentiation of calvaria cells derived from Apert infants and fetuses with FGFR-2 mutations. Histological analysis revealed premature ossification, increased extent of subperiosteal bone formation, and alkaline phosphatase- positive preosteoblastic cells in Apert fetal calvaria compared with age-matched controls. Preosteoblastic calvaria cells isolated from Apert infants and fetuses showed normal cell growth in basal conditions or in response to exogenous FGF-2. In contrast, the number of alkaline phosphatase- positive calvaria cells was fourfold higher than normal in mutant fetal calvaria cells with the most frequent Apert FGFR-2 mutation (Ser252Trp), suggesting increased maturation rate of cells in the osteoblastic lineage. Biochemical and Northern blot analyses also showed that the expression of alkaline phosphatase and type 1 collagen were 2-10-fold greater than normal in mutant fetal calvaria cells. The in vitro production of mineralized matrix formed by immortalized mutant fetal calvaria cells cultured in aggregates was also increased markedly compared with control immortalized fetal calvaria cells. The results show that Apert FGFR-2 mutations lead to an increase in the number of precursor cells that enter the osteogenic pathway, leading ultimately to increased subperiosteal bone matrix formation and premature calvaria ossification during fetal development, which establishes a connection between the altered genotype and cellular phenotype in Apert syndromic craniosynostosis.

  14. Mutation at residue 376 of ALS confers tribenuron-methyl resistance in flixweed (Descurainia sophia) populations from Hebei Province, China.

    PubMed

    Xu, Xian; Liu, Guiqiao; Chen, Silong; Li, Binghua; Liu, Xiaomin; Wang, Xiaoyun; Fan, Cuiqin; Wang, Guiqi; Ni, Hanwen

    2015-11-01

    The acetolactate synthase (ALS) inhibitor tribenuron has been used continuously for approximately twenty years as an herbicide in winter wheat fields in China. Flixweed (Descurainia sophia) has evolved resistance to tribenuron, due to multiple amino acid mutations at the 197th residue of ALS. In this study, the molecular basis of tribenuron resistance was investigated using two resistant populations, Xingtai (XT) and Shijiazhuang (SJ), and two susceptible populations, Cangzhou (CZ) and Handan (HD). Whole-plant tests and ALS activity assays showed that the two resistant populations were highly resistant to tribenuron. Targeted amplification of ALS genes from the four populations showed that there were two ALS genes in each population, and both of them were expressed in flixweed; the full coding lengths of the two ALS genes were 1998bp and 2004bp. Mutations related to tribenuron resistance in flixweed were located in only the 1998bp paralog. An ALS activity assay showed that the resistant population SJ displayed slight cross-resistance to florasulam, with a resistance factor of 4.81, but the resistant population XT did not have cross-resistance to florasulam. The resistant population XT was found to carry the previously reported mutation Pro197Ser, but the resistant population SJ carried a different mutation, Asp376Glu, known from other weeds but novel in flixweed. Our results demonstrated that multiple versions of ALS genes exist in flixweed and that mutations at multiple sites may result in ALS-inhibitor resistance in this weed.

  15. Assessing the risk that Phytophthora melonis can develop a point mutation (V1109L) in CesA3 conferring resistance to carboxylic acid amide fungicides.

    PubMed

    Chen, Lei; Zhu, Shusheng; Lu, Xiaohong; Pang, Zhili; Cai, Meng; Liu, Xili

    2012-01-01

    The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI  =  mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.

  16. Cyclic ichthyosis with epidermolytic hyperkeratosis: A phenotype conferred by mutations in the 2B domain of keratin K1.

    PubMed Central

    Sybert, V P; Francis, J S; Corden, L D; Smith, L T; Weaver, M; Stephens, K; McLean, W H

    1999-01-01

    Bullous congenital ichthyosiform erythroderma (BCIE) is characterized by blistering and erythroderma in infancy and by erythroderma and ichthyosis thereafter. Epidermolytic hyperkeratosis is a hallmark feature of light and electron microscopy. Here we report on four individuals from two families with a unique clinical disorder with histological findings of epidermolytic hyperkeratosis. Manifesting erythema and superficial erosions at birth, which improved during the first few months of life, affected individuals later developed palmoplantar hyperkeratosis with patchy erythema and scale elsewhere on the body. Three affected individuals exhibit dramatic episodic flares of annular, polycyclic erythematous plaques with scale, which coalesce to involve most of the body surface. The flares last weeks to months. In the interim periods the skin may be normal, except for palmoplantar hyperkeratosis. Abnormal keratin-filament aggregates were observed in suprabasal keratinocytes from both probands, suggesting that the causative mutation might reside in keratin K1 or keratin K10. In one proband, sequencing of K1 revealed a heterozygous mutation, 1436T-->C, predicting a change of isoleucine to threonine in the highly conserved helix-termination motif. In the second family, a heterozygous mutation, 1435A-->T, was found in K1, predicting an isoleucine-to-phenylalanine substitution in the same codon. Both mutations were excluded in both a control population and all unaffected family members tested. These findings reveal that a clinical phenotype distinct from classic BCIE but with similar histology can result from K1 mutations and that mutations at this codon give rise to a clinically unique condition. PMID:10053007

  17. Strategic priorities for increasing physical activity among adults age 50 and older: the national blueprint consensus conference summary report.

    PubMed

    Sheppard, Lisa; Senior, Jane; Park, Chae Hee; Mockenhaupt, Robin; Bazzarre, Terry; Chodzko-Zajko, Wojtek

    2003-12-01

    On May 1, 2001, a coalition of national organizations released a major planning document designed to develop a national strategy for the promotion of physically active lifestyles among the mid-life and older adult population. The National Blueprint: Increasing Physical Activity Among Adults Age 50 and Older was developed with input from 46 organizations with expertise in health, medicine, social and behavioral sciences, epidemiology, gerontology/geriatrics, clinical science, public policy, marketing, medical systems, community organization, and environmental issues. The Blueprint notes that, despite a wealth of evidence about the benefits of physical activity for mid-life and older persons, there has been little success in convincing age 50+ Americans to adopt physically active lifestyles. The Blueprint identifies barriers in the areas of research, home and community programs, medical systems, public policy and advocacy, and marketing and communications. In addition to identifying barriers, the Blueprint proposes a number of concrete strategies that could be employed in order to overcome the barriers to physical activity in society at large. This report summarizes the outcome of the National Blueprint Consensus Conference that was held in October 2002. In this conference, representatives of more than 50 national organizations convened in Washington, D.C. with the goal of identifying high priority and high feasibility strategies which would advance the National Blueprint and which could be initiated within the next 12 to 24 months. Participants in the consensus conference were assigned to one of five breakout groups: home and community, marketing, medical systems, public policy, and research. Each breakout group was charged with identifying the three highest priority strategies within their area for effectively increasing physical activity levels in the mid-life and older adult population. In addition to the 15 strategies identified by the breakout groups, three

  18. Increasing Collaborative Efforts in Career Education, K-12. A Series of Mini-Conferences. Phase I. Final Report.

    ERIC Educational Resources Information Center

    Coley, Walton, Ed.

    During 1977-78, a series of Office of Career Education "mini-conferences" was held, each conference devoted to attacking the concept of collaboration in career education from a different perspective. The notes from this set of mini-conferences form the body of this document. (A companion document, CE 020 111, describes the follow-up…

  19. A short insertion mutation disrupts genesis of miR-16 and causes increased body weight in domesticated chicken

    PubMed Central

    Jia, Xinzheng; Lin, Huiran; Nie, Qinghua; Zhang, Xiquan; Lamont, Susan J.

    2016-01-01

    Body weight is one of the most important quantitative traits with high heritability in chicken. We previously mapped a quantitative trait locus (QTL) for body weight by genome-wide association study (GWAS) in an F2 chicken resource population. To identify the causal mutations linked to this QTL, expression profiles were determined on livers of high-weight and low-weight chicken lines by microarray. Combining the expression pattern with SNP effects by GWAS, miR-16 was identified as the most likely potential candidate with a 3.8-fold decrease in high-weight lines. Re-sequencing revealed that a 54-bp insertion mutation in the upstream region of miR-15a-16 displayed high allele frequencies in high-weight commercial broiler line. This mutation resulted in lower miR-16 expression by introducing three novel splicing sites instead of the missing 5′ terminal splicing of mature miR-16. Elevating miR-16 significantly inhibited DF-1 chicken embryo cell proliferation, consistent with a role in suppression of cellular growth. The 54-bp insertion was significantly associated with increased body weight, bone size and muscle mass. Also, the insertion mutation tended towards fixation in commercial broilers (Fst > 0.4). Our findings revealed a novel causative mutation for body weight regulation that aids our basic understanding of growth regulation in birds. PMID:27808177

  20. Possible introgression of the VRTN mutation increasing vertebral number, carcass length and teat number from Chinese pigs into European pigs

    PubMed Central

    Yang, Jie; Huang, Lusheng; Yang, Ming; Fan, Yin; Li, Lin; Fang, Shaoming; Deng, Wenjiang; Cui, Leilei; Zhang, Zhen; Ai, Huashui; Wu, Zhenfang; Gao, Jun; Ren, Jun

    2016-01-01

    Vertnin (VRTN) variants have been associated with the number of thoracic vertebrae in European pigs, but the association has not been evidenced in Chinese indigenous pigs. In this study, we first performed a genome-wide association study in Chinese Erhualian pigs using one VRTN candidate causative mutation and the Illumina Porcine 60K SNP Beadchips. The VRTN mutation is significantly associated with thoracic vertebral number in this population. We further show that the VRTN mutation has pleiotropic and desirable effects on teat number and carcass (body) length across four diverse populations, including Erhualian, White Duroc × Erhualian F2 population, Duroc and Landrace pigs. No association was observed between VRTN genotype and growth and fatness traits in these populations. Therefore, testing for the VRTN mutation in pig breeding schemes would not only increase the number of vertebrae and nipples, but also enlarge body size without undesirable effects on growth and fatness traits, consequently improving pork production. Further, by using whole-genome sequence data, we show that the VRTN mutation was possibly introgressed from Chinese pigs into European pigs. Our results provide another example showing that introgressed Chinese genes greatly contributed to the development and production of modern European pig breeds. PMID:26781738

  1. Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation

    PubMed Central

    Šolman, Maja; Ligabue, Alessio; Blaževitš, Olga; Jaiswal, Alok; Zhou, Yong; Liang, Hong; Lectez, Benoit; Kopra, Kari; Guzmán, Camilo; Härmä, Harri; Hancock, John F; Aittokallio, Tero; Abankwa, Daniel

    2015-01-01

    Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer. DOI: http://dx.doi.org/10.7554/eLife.08905.001 PMID:26274561

  2. IDH1R132H Mutation Increases U87 Glioma Cell Sensitivity to Radiation Therapy in Hypoxia

    PubMed Central

    Wang, Xiao-Wei; Labussière, Marianne; Valable, Samuel; Pérès, Elodie A.; Guillamo, Jean-Sébastien; Sanson, Marc

    2014-01-01

    Objective. IDH1 codon 132 mutation (mostly Arg132His) is frequently found in gliomas and is associated with longer survival. However, it is still unclear whether IDH1 mutation renders the cell more vulnerable to current treatment, radio- and chemotherapy. Materials and Methods. We transduced U87 with wild type IDH1 or IDH1R132H expressing lentivirus and analyzed the radiosensitivity (dose ranging 0 to 10 Gy) under normoxia (20% O2) and moderate hypoxia (1% O2). Results. We observed that IDH1R132H U87 cells grow faster in hypoxia and were more sensitive to radiotherapy (in terms of cell mortality and colony formation assay) compared to nontransduced U87 and IDH1wt cells. This effect was not observed in normoxia. Conclusion. These data suggest that IDH1R132H mutation increases radiosensitivity in mild hypoxic conditions. PMID:24895549

  3. The Y137H mutation of VvCYP51 gene confers the reduced sensitivity to tebuconazole in Villosiclava virens

    PubMed Central

    Wang, Fei; Lin, Yang; Yin, Wei-Xiao; Peng, You-Liang; Schnabel, Guido; Huang, Jun-Bin; Luo, Chao-Xi

    2015-01-01

    Management of rice false smut disease caused by Villosiclava virens is dependent on demethylation inhibitor (DMI) fungicides. Investigation of molecular mechanisms of resistance is therefore of upmost importance. In this study the gene encoding the target protein for DMI fungicides (VvCYP51) was cloned and investigated. The VvCYP51 gene in the resistant mutant revealed both a change from tyrosine to histidine at position 137 (Y137H) and elevated gene expression compared to the parental isolate. In order to determine which of these mechanisms was responsible for the reduced sensitivity to DMI fungicide tebuconazole, transformants expressing the mutated or the wild type VvCYP51 gene were generated. Transformants carrying the mutated gene were more resistant to tebuconazole compared to control transformants lacking the mutation, but the expression of the VvCYP51 gene was not significantly correlated with EC50 values. The wild type VvCYP51 protein exhibited stronger affinity for tebuconazole compared to the VvCYP51/Y137H in both molecular docking analysis and experimental binding assays. The UV-generated mutant as well as transformants expressing the VvCYP51/Y137H did not exhibit significant fitness penalties based on mycelial growth and spore germination, suggesting that isolates resistant to DMI fungicides based on the Y137H mutation may develop and be competitive in the field. PMID:26631591

  4. Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation.

    PubMed

    Zhang, Shirong; Zheng, Xiaoliang; Huang, Haixiu; Wu, Kan; Wang, Bing; Chen, Xufeng; Ma, Shenglin

    2015-03-20

    Afatinib is a second-generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor and has shown a significant clinical benefit in non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. However, the potential therapeutic effects of afatinib combining with other modalities, including ionizing radiation (IR), are not well understood. In this study, we developed a gefitinib-resistant cell subline (PC-9-GR) with a secondary EGFR mutation (T790M) from NSCLC PC-9 cells after chronic exposures to increasing doses of gefitinib. The presence of afatinib significantly increases the cell killing effect of radiation in PC-9-GR cells harboring acquired T790M, but not in H1975 cells with de novo T790M or in H460 cells that express wild-type EGFR. In PC-9-GR cells, afatinib remarkable blocks baseline of EGFR and ERK phosphorylations, and causes delay of IR-induced AKT phosphorylation. Afatinib treatment also leads to increased apoptosis and suppressed DNA damage repair in irradiated PC-9-GR cells, and enhanced tumor growth inhibition when combined with IR in PC-9-GR xenografts. Our findings suggest a potential therapeutic impact of afatinib as a radiation sensitizer in lung cancer cells harboring acquired T790M mutation, providing a rationale for a clinical trial with combination of afatinib and radiation in NSCLCs with EGFR T790M mutation.

  5. Mutations Affecting Starch Synthase III in Arabidopsis Alter Leaf Starch Structure and Increase the Rate of Starch Synthesis1

    PubMed Central

    Zhang, Xiaoli; Myers, Alan M.; James, Martha G.

    2005-01-01

    The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis. PMID:15908598

  6. Somatic Mutation of the SNP rs11614913 and Its Association with Increased MIR 196A2 Expression in Breast Cancer.

    PubMed

    Zhao, Huanhuan; Xu, Jingman; Zhao, Dan; Geng, Meijuan; Ge, Haize; Fu, Li; Zhu, Zhengmao

    2016-02-01

    Common genetic variants (single-nucleotide polymorphisms [SNPs]) in microRNA genes may alter their maturation or expression, resulting in varied functional consequences. Several studies have evaluated the association between the SNP rs11614913 and cancer risk in diverse populations and in a range of cancers, with contradictory outcomes. In this study, we examined 114 paired samples (tumor and normal tissues) from breast cancer patients to study the genotype distribution and somatic mutation of the SNP in MIR 196A2 (rs11614913 C-T). In addition, we evaluated their influence on the mature MIR 196A2 expression. We found that 14% (16/114) of tumors underwent somatic mutation of the SNP rs11614913. Moreover, the CT heterozygous and the CC homozygous states of SNP rs11614913 were more prone to mutation, while the TT homozygous state appeared to be resistant. We further detected a significant increase (p = 0.002) in mature MIR 196A2 expression in breast cancer. In particular, we found a significant association between the occurrence of SNP rs11614913 mutation and high expression (p = 0.0002). In addition, the mature MIR 196A2 expression level was significantly associated with the higher tumor grade (p = 0.004). Taken together, our results seem to demonstrate that somatic mutation of SNP rs11614913 in MIR 196A2 can have an influence on its expression. In addition, it indicated that an unknown mechanism is responsible for both the mutation of SNP rs11614913 and the dysregulation of mature MIR 196A2 expression.

  7. Familial mutations in fibrinogen Aα (FGA) chain identified in renal amyloidosis increase in vitro amyloidogenicity of FGA fragment.

    PubMed

    Sivalingam, Vishwanath; Patel, Basant K

    2016-08-01

    Amyloidoses are clinical disorders where deposition of β-sheet rich, misfolded protein aggregates called amyloid occurs in vital organs like brain, kidney, liver or heart etc. Aggregation of several proteins such as immunoglobulin light chain, fibrinogen Aα chain (FGA) and lysozyme have been found to be associated with renal amyloidosis. Fibrinogen amyloidosis (AFib) is predominantly familial and is associated with the deposition of mutant FGA amyloid, primarily in kidneys. Over ten substitution and frame-shift mutations in FGA have been identified from AFib patients. Whether wild-type FGA is also involved in AFib is yet unknown. The affected tissues from AFib patients usually show ∼10 kDA peptide from C-terminal 80 amino acid residues of mutant FGA. Notably, this region also encompasses all known disease-related mutations. Whether these point mutations increase the amyloidogenicity of FGA leading to disease progression, have not been studied yet. Here, we have investigated the role of two disease-related mutations in affecting amyloidogenic propensity of an FGA(496-581) fragment. We found that at physiological pH, the wild-type FGA(496-581) fragment remains monomeric, whereas its E540V mutant forms amyloid-like fibrils as observed by AFM. Also, FGA(496-581) harbouring another familial mutation, R554L, converts in vitro into globular, β-sheet rich aggregates, showing amyloid-like properties. These findings suggest that familial mutations in FGA may have role in renal amyloidosis via enhanced amyloid formation.

  8. Nickel accumulation in lung tissues is associated with increased risk of p53 mutation in lung cancer patients.

    PubMed

    Chiou, Yu-Hu; Wong, Ruey-Hong; Chao, Mu-Rong; Chen, Chih-Yi; Liou, Saou-Hsing; Lee, Huei

    2014-10-01

    Occupational exposure to nickel compounds has been associated with lung cancer. The correlation between high nickel levels and increased risk of lung cancer has been previously reported in a case-control study. This study assessed whether nickel exposure increased the occurrence of p53 mutations due to DNA repair inhibition by nickel. A total of 189 lung cancer patients were enrolled to determine nickel levels in tumor-adjacent normal lung tissues and p53 mutation status in lung tumors through atomic absorption spectrometry and direct sequencing, respectively. Nickel levels in p53 mutant patients were significantly higher than those in p53 wild-type patients. When patients were divided into high- and low-nickel subgroups by median nickel level, the high-nickel subgroup of patients had an odds ratio (OR) of 3.25 for p53 mutation risk relative to the low-nickel subgroup patients. The OR for p53 mutation risk of lifetime non-smokers, particularly females, in the high-nickel subgroup was greater than that in the low-nickel subgroup. To determine whether nickel affected DNA repair capacity, we conducted the host cell reactivation assay in A549 and H1975 lung cancer cells and showed that the DNA repair activity was reduced by nickel chloride in a dose-dependent manner. This was associated with elevated production of hydrogen peroxide-induced 8-oxo-deoxyguanosine. Therefore, increased risk of p53 mutation due to defective DNA repair caused by high nickel levels in lung tissues may be one mechanism by which nickel exposure contributes to lung cancer development, especially in lifetime female non-smokers.

  9. Treatment-emergent mutations in NAEβ confer resistance to the NEDD8-activating enzyme inhibitor MLN4924.

    PubMed

    Milhollen, Michael A; Thomas, Michael P; Narayanan, Usha; Traore, Tary; Riceberg, Jessica; Amidon, Benjamin S; Bence, Neil F; Bolen, Joseph B; Brownell, James; Dick, Lawrence R; Loke, Huay-Keng; McDonald, Alice A; Ma, Jingya; Manfredi, Mark G; Sells, Todd B; Sintchak, Mike D; Yang, Xiaofeng; Xu, Qing; Koenig, Erik M; Gavin, James M; Smith, Peter G

    2012-03-20

    MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations.

  10. Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (Hordeum vulgare).

    PubMed

    Kreis, M; Shewry, P R; Forde, B G; Rahman, S; Miflin, B J

    1983-08-01

    We have analyzed the molecular nature of the Riso 56 mutation that occurs in barley. This mutation results in a depression of hordein accumulation in the grain and consequently in a higher overall lysine content. In particular, the amount of B hordein, which is encoded by the complex locus Hor-2, is decreased by about 75% because of the absence of the major components. The synthesis of certain minor polypeptides, with properties similar to the major B hordeins, remains unaffected. Analysis of endosperm RNA, by in vitro translation and hybridization to various cloned cDNAs derived from hordein mRNA, shows that mRNA for the major B hordeins is not present in the endosperm. Hybridization of a B hordein cDNA clone to gel-fractionated restriction digests of mutant and wild-type DNA indicates that at least 85 kb of DNA has been deleted from the Hor-2 locus in the high-lysine mutant.

  11. Mutations in H5N1 Influenza Virus Hemagglutinin that Confer Binding to Human Tracheal Airway Epithelium

    PubMed Central

    Scull, Margaret A.; Ren, Junyuan; Jones, Ian M.; Pickles, Raymond J.; Barclay, Wendy S.

    2009-01-01

    The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary “dead end.” We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic. PMID:19924306

  12. I223R Mutation in Influenza A(H1N1)pdm09 Neuraminidase Confers Reduced Susceptibility to Oseltamivir and Zanamivir and Enhanced Resistance with H275Y

    PubMed Central

    Abou-Jaoudé, Georges; Scemla, Anne; Ribaud, Patricia; Mercier-Delarue, Séverine; Caro, Valérie; Enouf, Vincent; Simon, François; Molina, Jean-Michel; van der Werf, Sylvie

    2012-01-01

    Background Resistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility. Methods The NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0–15) and zanamivir (days 15–25 and 70–80). Results Compared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6–69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin. Conclusions Reduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines

  13. Recurrent HOXB13 mutations in the Dutch population do not associate with increased breast cancer risk

    PubMed Central

    Liu, Jingjing; Prager–van der Smissen, Wendy J. C.; Schmidt, Marjanka K.; Collée, J. Margriet; Cornelissen, Sten; Lamping, Roy; Nieuwlaat, Anja; Foekens, John A.; Hooning, Maartje J.; Verhoef, Senno; van den Ouweland, Ans M. W.; Hogervorst, Frans B. L.; Martens, John W. M.; Hollestelle, Antoinette

    2016-01-01

    The HOXB13 p.G84E mutation has been firmly established as a prostate cancer susceptibility allele. Although HOXB13 also plays a role in breast tumor progression, the association of HOXB13 p.G84E with breast cancer risk is less evident. Therefore, we comprehensively interrogated the entire HOXB13 coding sequence for mutations in 1,250 non-BRCA1/2 familial breast cancer cases and 800 controls. We identified two predicted deleterious missense mutations, p.G84E and p.R217C, that were recurrent among breast cancer cases and further evaluated their association with breast cancer risk in a larger study. Taken together, 4,520 familial non-BRCA1/2 breast cancer cases and 3,127 controls were genotyped including the cases and controls of the whole gene screen. The concordance rate for the genotyping assays compared with Sanger sequencing was 100%. The prostate cancer risk allele p.G84E was identified in 18 (0.56%) of 3,187 cases and 16 (0.70%) of 2,300 controls (OR = 0.81, 95% CI = 0.41–1.59, P = 0.54). Additionally, p.R217C was identified in 10 (0.31%) of 3,208 cases and 2 (0.087%) of 2,288 controls (OR = 3.57, 95% CI = 0.76–33.57, P = 0.14). These results imply that none of the recurrent HOXB13 mutations in the Dutch population are associated with breast cancer risk, although it may be worthwhile to evaluate p.R217C in a larger study. PMID:27424772

  14. Fitness Conferred by BCR-ABL Kinase Domain Mutations Determines the Risk of Pre-Existing Resistance in Chronic Myeloid Leukemia

    PubMed Central

    Skaggs, Brian; Gorre, Mercedes; Sawyers, Charles L.; Michor, Franziska

    2011-01-01

    Chronic myeloid leukemia (CML) is the first human malignancy to be successfully treated with a small molecule inhibitor, imatinib, targeting a mutant oncoprotein (BCR-ABL). Despite its successes, acquired resistance to imatinib leads to reduced drug efficacy and frequent progression of disease. Understanding the characteristics of pre-existing resistant cells is important for evaluating the benefits of first-line combination therapy with second generation inhibitors. However, due to limitations of assay sensitivity, determining the existence and characteristics of resistant cell clones at the start of therapy is difficult. Here we combined a mathematical modeling approach using branching processes with experimental data on the fitness changes (i.e., changes in net reproductive rate) conferred by BCR-ABL kinase domain mutations to investigate the likelihood, composition, and diversity of pre-existing resistance. Furthermore, we studied the impact of these factors on the response to tyrosine kinase inhibitors. Our approach predicts that in most patients, there is at most one resistant clone present at the time of diagnosis of their disease. Interestingly, patients are no more likely to harbor the most aggressive, pan-resistant T315I mutation than any other resistance mutation; however, T315I cells on average establish larger-sized clones at the time of diagnosis. We established that for patients diagnosed late, the relative benefit of combination therapy over monotherapy with imatinib is significant, while this benefit is modest for patients with a typically early diagnosis time. These findings, after pre-clinical validation, will have implications for the clinical management of CML: we recommend that patients with advanced-phase disease be treated with combination therapy with at least two tyrosine kinase inhibitors. PMID:22140458

  15. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

  16. CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model

    PubMed Central

    Wert, Katherine J.; Bassuk, Alexander G.; Wu, Wen-Hsuan; Gakhar, Lokesh; Coglan, Diana; Mahajan, MaryAnn; Wu, Shu; Yang, Jing; Lin, Chyuan-Sheng; Tsang, Stephen H.; Mahajan, Vinit B.

    2015-01-01

    A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5R243L only in the retina. The resulting hCAPN5R243L transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5R243L mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5R243L mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients. PMID:25994508

  17. Marked increase in biofilm-derived rough pneumococcal variants and rifampin-resistant strains not due to hex gene mutations.

    PubMed

    McEllistrem, M Catherine; Scott, Jennifer R; Zuniga-Castillo, Jacobo; Khan, Saleem A

    2009-06-01

    Otitis, pneumonia, and meningitis are tissue-based pneumococcal infections that can be associated with biofilms. The emergence of phenotypic rough variants, also known as acapsular small-colony variants, is essential for pneumococcal biofilm formation. These rough variants can increase nearly 100-fold in biofilms over time and can arise through single nucleotide polymorphisms (SNPs), deletions, or tandem duplications in the first gene of the capsular operon, cps3D. We detected a 100-fold increase in rifampin-resistant (Rif(r)) mutants in biofilms compared to planktonic cultures using a nonvaccine serotype 3 strain, which is causing an increasing number of cases of otitis in the 7-valent pneumococcal conjugate vaccine era. Since both rough variants and Rif(r) strains can arise through SNPs, they could emerge due to alteration of the mismatch repair (MMR) system. The Hex system, a pneumococcal MMR system, repairs mismatches during replication and transformation. In this study, no mutations were detected in the hexAB gene sequences among several rough variants with unique mutations in the cps3D gene. Within a hexA null mutant grown in broth, we detected only a 17.5-fold increase in rough variants compared to the wild-type parental strain. Taken together, these data suggest that mutations in the hex genes and modulation of hexA activity are unlikely to account for the generation of biofilm-derived rough variants.

  18. Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis▿

    PubMed Central

    Venkatesan, Ranga N.; Treuting, Piper M.; Fuller, Evan D.; Goldsby, Robert E.; Norwood, Thomas H.; Gooley, Ted A.; Ladiges, Warren C.; Preston, Bradley D.; Loeb, Lawrence A.

    2007-01-01

    Mammalian DNA polymerase δ (Pol δ) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol δ. Homozygous Pold1L604G/L604G and Pold1L604K/L604K mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1+/L604G mice did not differ, while that of Pold1+/L604K mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1+/L604K mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase δ reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer. PMID:17785453

  19. ARID5B polymorphism confers an increased risk to acquire specific MLL rearrangements in early childhood leukemia

    PubMed Central

    2014-01-01

    Background Acute leukemia in early age (EAL) is characterized by acquired genetic alterations such as MLL rearrangements (MLL-r). The aim of this case-controlled study was to investigate whether single nucleotide polymorphisms (SNPs) of IKZF1, ARID5B, and CEBPE could be related to the onset of EAL cases (<24 months-old at diagnosis). Methods The SNPs (IKZF1 rs11978267, ARID5B rs10821936 and rs10994982, CEBPE rs2239633) were genotyped in 265 cases [169 acute lymphoblastic leukemia (ALL) and 96 acute myeloid leukaemia (AML)] and 505 controls by Taqman allelic discrimination assay. Logistic regression was used to evaluate the association between SNPs of cases and controls, adjusted on skin color and/or age. The risk was determined by calculating odds ratios (ORs) with 95% confidence interval (CI). Results Children with the IKZF1 SNP had an increased risk of developing MLL-germline ALL in white children. The heterozygous/mutant genotype in ARID5B rs10994982 significantly increased the risk for MLL-germline leukemia in white and non-white children (OR 2.60, 95% CI: 1.09-6.18 and OR 3.55, 95% CI: 1.57-8.68, respectively). The heterozygous genotype in ARID5B rs10821936 increased the risk for MLL-r leukemia in both white and non-white (OR 2.06, 95% CI: 1.12-3.79 and OR 2.36, 95% CI: 1.09-5.10, respectively). Furthermore, ARID5B rs10821936 conferred increased risk for MLL-MLLT3 positive cases (OR 7.10, 95% CI:1.54-32.68). Our data do not show evidence that CEBPE rs2239633 confers increased genetic susceptibility to EAL. Conclusions IKZF1 and CEBPE variants seem to play a minor role in genetic susceptibility to EAL, while ARID5B rs10821936 increased the risk of MLL-MLLT3. This result shows that genetic susceptibility could be associated with the differences regarding MLL breakpoints and partner genes. PMID:24564228

  20. Hydrogen Peroxide Removes TRPM4 Current Desensitization Conferring Increased Vulnerability to Necrotic Cell Death*

    PubMed Central

    Simon, Felipe; Leiva-Salcedo, Elías; Armisén, Ricardo; Riveros, Ana; Cerda, Oscar; Varela, Diego; Eguiguren, Ana Luisa; Olivero, Pablo; Stutzin, Andrés

    2010-01-01

    Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H2O2 induces a sustained activity of TRPM4, a Ca2+-activated, Ca2+-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H2O2 was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys1093 residue is crucial for the H2O2-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H2O2 elicited necrosis as well as apoptosis. H2O2-mediated necrosis but not apoptosis was abolished by replacement of external Na+ ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H2O2-induced necrotic cell death. In addition, HeLa cells exposed to H2O2 displayed an irreversible loss of membrane potential, which was prevented by TRPM4 knockdown. PMID:20884614

  1. Increased mutant frequency and altered mutation spectrum of the lacI transgene in Wilson disease rats with hepatitis.

    PubMed

    Sone, H; Li, Y J; Ishizuka, M; Aoki, Y; Nagao, M

    2000-09-15

    The mutant strain Long-Evans Cinnamon (LEC) rat, which accumulates copper in the liver because of a mutation in the Atp7b gene, encoding a copper-ATPase, is a model of Wilson disease. It spontaneously develops hepatitis, and subsequently hepatocellular carcinoma and cholangiofibrosis. Excess intracellular copper has been thought to induce DNA damage through reactive oxygen species produced by Cu (II)/Cu (I) redox cycling, and also by direct interaction with DNA. We have developed lacI transgenic Wilson disease (WND-B) rats by mating LEC with Big Blue F344 rats carrying a lambda shuttle vector harboring the lacI gene. lacI mutations of the livers of C-B heterozygous (Atp7b w/m, lacI) and WND-B homozygous (Atp7b m/m, lacI) rats at 6, 24, and 40 weeks of ages were analyzed. Mutant frequencies in the WND-B rats were 2.0 +/- 0.7 x 10(-5), 5.3 +/- 0.9 x 10(-5), and 5.3 +/- 1.0 x 10(-5), respectively, significantly higher than those of C-B rats. Nucleotide sequence analysis revealed that the frequency of deletion mutations of more than two nucleotides were much higher, 15% in WND-B rats, but only 2% in C-B rats. In addition, the average size of deletion was larger in the former. Loss of oligonucleotide-repeat units was specific and relatively frequent in WND-B rats. This type of mutation might be implicated in the induction of DNA strand scissions by reactive oxygen species. These findings suggest that the increase in mutant frequencies and/or the specific type of mutation according to copper accumulation play a crucial role in hepatocarcinogenesis in LEC rats.

  2. JAK3 deregulation by activating mutations confers invasive growth advantage in extranodal nasal-type natural killer cell lymphoma.

    PubMed

    Bouchekioua, A; Scourzic, L; de Wever, O; Zhang, Y; Cervera, P; Aline-Fardin, A; Mercher, T; Gaulard, P; Nyga, R; Jeziorowska, D; Douay, L; Vainchenker, W; Louache, F; Gespach, C; Solary, E; Coppo, P

    2014-02-01

    Extranodal, nasal-type natural killer (NK)/T-cell lymphoma (NKCL) is an aggressive malignancy with poor prognosis in which, usually, signal transducer and activator of transcription 3 (STAT3) is constitutively activated and oncogenic. Here, we demonstrate that STAT3 activation mostly results from constitutive Janus kinase (JAK)3 phosphorylation on tyrosine 980, as observed in three of the four tested NKCL cell lines and in 20 of the 23 NKCL tumor samples under study. In one of the cell lines and in 4 of 19 (21%) NKCL primary tumor samples, constitutive JAK3 activation was related to an acquired mutation (A573V or V722I) in the JAK3 pseudokinase domain. We then show that constitutive activation of the JAK3/STAT3 pathway has a major role in NKCL cell growth and survival and in the invasive phenotype. Indeed, NKCL cell growth was slowed down in vitro by targeting JAK3 with chemical inhibitors or small-interfering RNAs. In a human NKCL xenograft mouse model, tumor growth was significantly delayed by the JAK3 inhibitor CP-690550. Altogether, the constitutive activation of JAK3, which can result from JAK3-activating mutations, is a frequent feature of NKCL that deserves to be tested as a therapeutic target.

  3. Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons.

    PubMed

    Gopal, Pallavi P; Nirschl, Jeffrey J; Klinman, Eva; Holzbaur, Erika L F

    2017-03-21

    Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid-liquid phase separation and liquid-like non-membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 (TDP-43) and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation.

  4. Oxidative Stress Increases the Blood Brain Barrier Permeability Resulting in Increased Incidence of Brain Metastasis in BRCA Mutation Carriers

    DTIC Science & Technology

    2012-02-01

    inhibitors and/or selenium in preventing BBB-induced damage by oxidative stress, and in inhibiting breast cancer metastasis to the brain. Further...since selenium has anti- cancer properties that are linked with protection against oxidative stress and Poly (ADP-ribose) polymerase (PARP) inhibitors...BRCA1 in breast cancer cells resulted in an increase in cell proliferation, anchorage-independent growth, cell migration, invasion and a loss of p21

  5. LacR mutations are frequently observed in Streptococcus intermedius and are responsible for increased intermedilysin production and virulence.

    PubMed

    Tomoyasu, Toshifumi; Imaki, Hidenori; Masuda, Sachiko; Okamoto, Ayumi; Kim, Hyejin; Waite, Richard D; Whiley, Robert A; Kikuchi, Ken; Hiramatsu, Keiichi; Tabata, Atsushi; Nagamune, Hideaki

    2013-09-01

    Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.

  6. The G1138A mutation rate in the fibroblast growth factor receptor 3 (FGFR3) gene is increased in cells carrying the t (4; 14) translocation.

    PubMed

    Reddy, P L; Grewal, R P

    2009-04-22

    Spontaneous mutations are a common phenomenon, occurring in both germ-line and somatic genomes. They may have deleterious consequences including the development of genetic disorders or, when occurring in somatic tissues, may participate in the process of carcinogenesis. Similar to many mutational hotspots, the G1138A mutation in the fibroblast growth factor receptor 3 (FGFR3) gene occurs at a CpG site. In germ-line tissues, the G1138A mutation results in achondroplasia and has one of the highest spontaneous mutation rates in the human genome. Although not at the G1138A site, there are increased rates of other somatic mutations in the FGFR3 gene that have been reported in multiple myeloma cases associated with a translocation, t (4; 14). The chromosome-4 break points in this translocation are clustered in a 70-kb region centromeric to the FGFR3 gene. We hypothesized that this translocation may impact the mutation rate at the G1138A site. We employed a semi-quantitative polymerase chain reaction-based assay to measure the frequency of this mutation in multiple myeloma cell lines carrying t (4; 14) translocation. Analysis of these cell lines varied from no change to a 10-fold increase in the mutation frequency compared with normal controls. In general, there was an increase in the G1138A mutational frequency suggesting that chromosomal rearrangement can affect the stability of the CpG hotspots.

  7. Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis.

    PubMed

    Lall, Rahul K; Syed, Deeba N; Khan, Mohammad Imran; Adhami, Vaqar M; Gong, Yuansheng; Lucey, John A; Mukhtar, Hasan

    2016-09-01

    We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC-ESI-MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa.

  8. Kras mutations increase telomerase activity and targeting telomerase is a promising therapeutic strategy for Kras-mutant NSCLC

    PubMed Central

    Shi, Bowen; Zhang, Lianmin; Qian, Dong; Li, Chenguang; Zhang, Hua; Wang, Shengguang; Zhu, Jinfang; Gao, Liuwei; Zhang, Qiang; Jia, Bin; Hao, Ligang; Wang, Changli; Zhang, Bin

    2017-01-01

    As shortened telomeres inhibit tumor formation and prolong life span in a KrasG12D mouse lung cancer model, we investigated the implications of telomerase in Kras-mutant NSCLC. We found that Kras mutations increased TERT (telomerase reverse transcriptase) mRNA expression and telomerase activity and telomere length in both immortalized bronchial epithelial cells (BEAS-2B) and lung adenocarcinoma cells (Calu-3). MEK inhibition led to reduced TERT expression and telomerase activity. Furthermore, telomerase inhibitor BIBR1532 shortened telomere length and inhibited mutant Kras-induced long-term proliferation, colony formation and migration capabilities of BEAS-2B and Calu-3 cells. Importantly, BIBR1532 sensitized oncogenic Kras expressing Calu-3 cells to chemotherapeutic agents. The Calu-3-KrasG12D xenograft mouse model confirmed that BIBR1532 enhanced the antitumor efficacy of paclitaxel in vivo. In addition, higher TERT expression was seen in Kras-mutant NSCLC than that with wild-type Kras. Our data suggest that Kras mutations increase telomerase activity and telomere length by activating the RAS/MEK pathway, which contributes to an aggressive phenotype of NSCLC. Kras mutations-induced lung tumorigenesis and chemoresistance are attenuated by telomerase inhibition. Targeting telomerase/telomere may be a promising therapeutic strategy for patients with Kras-mutant NSCLC. PMID:27329725

  9. Recent insertion/deletion (reINDEL) mutations: increasing awareness to boost molecular-based research in ecology and evolution

    PubMed Central

    Schlick-Steiner, Birgit C; Arthofer, Wolfgang; Moder, Karl; Steiner, Florian M

    2015-01-01

    Today, the comparative analysis of DNA molecules mainly uses information inferred from nucleotide substitutions. Insertion/deletion (INDEL) mutations, in contrast, are largely considered uninformative and discarded, due to our lacking knowledge on their evolution. However, including rather than discarding INDELs would be relevant to any research area in ecology and evolution that uses molecular data. As a practical approach to better understanding INDEL evolution in general, we propose the study of recent INDEL (reINDEL) mutationsmutations where both ancestral and derived state are seen in the sample. The precondition for reINDEL identification is knowledge about the pedigree of the individuals sampled. Sound reINDEL knowledge will allow the improved modeling needed for including INDELs in the downstream analysis of molecular data. Both microsatellites, currently still the predominant marker system in the analysis of populations, and sequences generated by next-generation sequencing, a promising and rapidly developing range of technologies, offer the opportunity for reINDEL identification. However, a 2013 sample of animal microsatellite studies contained unexpectedly few reINDELs identified. As most likely explanation, we hypothesize that reINDELs are underreported rather than absent and that this underreporting stems from common reINDEL unawareness. If our hypothesis applies, increased reINDEL awareness should allow gathering data rapidly. We recommend the routine reporting of either the absence or presence of reINDELs together with standardized key information on the nature of mutations when they are detected and the use of the keyword “reINDEL” to increase visibility in both instances of successful and unsuccessful search. PMID:25628861

  10. Antibiotic Resistance in Pseudomonas aeruginosa Strains with Increased Mutation Frequency Due to Inactivation of the DNA Oxidative Repair System▿

    PubMed Central

    Mandsberg, L. F.; Ciofu, O.; Kirkby, N.; Christiansen, L. E.; Poulsen, H. E.; Høiby, N.

    2009-01-01

    The chronic Pseudomonas aeruginosa infection of the lungs of cystic fibrosis (CF) patients is characterized by the biofilm mode of growth and chronic inflammation dominated by polymorphonuclear leukocytes (PMNs). A high percentage of P. aeruginosa strains show high frequencies of mutations (hypermutators [HP]). P. aeruginosa is exposed to oxygen radicals, both those generated by its own metabolism and especially those released by a large number of PMNs in response to the chronic CF lung infection. Our work therefore focused on the role of the DNA oxidative repair system in the development of HP and antibiotic resistance. We have constructed and characterized mutT, mutY, and mutM mutants in P. aeruginosa strain PAO1. The mutT and mutY mutants showed 28- and 7.5-fold increases in mutation frequencies, respectively, over that for PAO1. These mutators had more oxidative DNA damage (higher levels of 7,8-dihydro-8-oxodeoxyguanosine) than PAO1 after exposure to PMNs, and they developed resistance to antibiotics more frequently. The mechanisms of resistance were increased β-lactamase production and overexpression of the MexCD-OprJ efflux-pump. Mutations in either the mutT or the mutY gene were found in resistant HP clinical isolates from patients with CF, and complementation with wild-type genes reverted the phenotype. In conclusion, oxidative stress might be involved in the development of resistance to antibiotics. We therefore suggest the possible use of antioxidants for CF patients to prevent the development of antibiotic resistance. PMID:19332676

  11. Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels*

    PubMed Central

    Tang, Qiong-Yao; Larry, Trevor; Hendra, Kalen; Yamamoto, Erica; Bell, Jessica; Cui, Meng; Logothetis, Diomedes E.; Boland, Linda M.

    2015-01-01

    All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81–104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404–404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification. PMID:25957411

  12. Epithelial-to-Mesenchymal Transition of RPE Cells In Vitro Confers Increased β1,6-N-Glycosylation and Increased Susceptibility to Galectin-3 Binding

    PubMed Central

    Priglinger, Claudia S.; Obermann, Jara; Szober, Christoph M.; Merl-Pham, Juliane; Ohmayer, Uli; Behler, Jennifer; Gruhn, Fabian; Kreutzer, Thomas C.; Wertheimer, Christian; Geerlof, Arie; Priglinger, Siegfried G.; Hauck, Stefanie M.

    2016-01-01

    Epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells is a crucial event in the onset of proliferative vitreoretinopathy (PVR), the most common reason for treatment failure in retinal detachment surgery. We studied alterations in the cell surface glycan expression profile upon EMT of RPE cells and focused on its relevance for the interaction with galectin-3 (Gal-3), a carbohydrate binding protein, which can inhibit attachment and spreading of human RPE cells in a dose- and carbohydrate-dependent manner, and thus bares the potential to counteract PVR-associated cellular events. Lectin blot analysis revealed that EMT of RPE cells in vitro confers a glycomic shift towards an abundance of Thomsen-Friedenreich antigen, poly-N-acetyllactosamine chains, and complex-type branched N-glycans. Using inhibitors of glycosylation we found that both, binding of Gal-3 to the RPE cell surface and Gal-3-mediated inhibition of RPE attachment and spreading, strongly depend on the interaction of Gal-3 with tri- or tetra-antennary complex type N-glycans and sialylation of glycans but not on complex-type O-glycans. Importantly, we found that β1,6 N-acetylglucosaminyltransferase V (Mgat5), the key enzyme catalyzing the synthesis of tetra- or tri-antennary complex type N-glycans, is increased upon EMT of RPE cells. Silencing of Mgat5 by siRNA and CRISPR-Cas9 genome editing resulted in reduced Gal-3 binding. We conclude from these data that binding of recombinant Gal-3 to the RPE cell surface and inhibitory effects on RPE attachment and spreading largely dependent on interaction with Mgat5 modified N-glycans, which are more abundant on dedifferentiated than on the healthy, native RPE cells. Based on these findings we hypothesize that EMT of RPE cells in vitro confers glycomic changes, which account for high affinity binding of recombinant Gal-3, particularly to the cell surface of myofibroblastic RPE. From a future perspective recombinant Gal-3 may disclose a

  13. Powdery Mildew Resistance Conferred by Loss of the ENHANCED DISEASE RESISTANCE1 Protein Kinase Is Suppressed by a Missense Mutation in KEEP ON GOING, a Regulator of Abscisic Acid Signaling1[W][OA

    PubMed Central

    Wawrzynska, Anna; Christiansen, Katy M.; Lan, Yinan; Rodibaugh, Natalie L.; Innes, Roger W.

    2008-01-01

    Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to infection by powdery mildew (Golovinomyces cichoracearum). EDR1 encodes a protein kinase, but its substrates and the pathways regulated by EDR1 are unknown. To identify components of the EDR1 signal transduction pathway(s), we conducted a forward genetic screen for mutations that suppressed edr1-mediated disease resistance. Genetic mapping and cloning of one of these suppressor mutations revealed a recessive missense mutation in the KEEP ON GOING gene (KEG; At5g13530), which we designated keg-4. KEG encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like repeats. The KEG protein has previously been shown to have ubiquitin ligase activity and to negatively regulate protein levels of the transcription factor ABCISIC ACID INSENSITIVE5. KEG mRNA levels were found to be 3-fold higher in edr1 mutant plants compared to wild type. Loss-of-function mutations in KEG are seedling lethal and are hypersensitive to glucose and abscisic acid (ABA). The keg-4 mutation, in contrast, conferred resistance to 6% glucose and suppressed edr1-mediated hypersensitivity to ABA, suggesting that the keg-4 mutation suppresses ABA signaling by altering KEG function. Several ABA-responsive genes were found to be further up-regulated in the edr1 mutant following ABA treatment, and this up-regulation was suppressed by the keg-4 mutation. We conclude that edr1-mediated resistance to powdery mildew is mediated, in part, by enhanced ABA signaling. PMID:18815384

  14. Powdery mildew resistance conferred by loss of the ENHANCED DISEASE RESISTANCE1 protein kinase is suppressed by a missense mutation in KEEP ON GOING, a regulator of abscisic acid signaling.

    PubMed

    Wawrzynska, Anna; Christiansen, Katy M; Lan, Yinan; Rodibaugh, Natalie L; Innes, Roger W

    2008-11-01

    Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to infection by powdery mildew (Golovinomyces cichoracearum). EDR1 encodes a protein kinase, but its substrates and the pathways regulated by EDR1 are unknown. To identify components of the EDR1 signal transduction pathway(s), we conducted a forward genetic screen for mutations that suppressed edr1-mediated disease resistance. Genetic mapping and cloning of one of these suppressor mutations revealed a recessive missense mutation in the KEEP ON GOING gene (KEG; At5g13530), which we designated keg-4. KEG encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like repeats. The KEG protein has previously been shown to have ubiquitin ligase activity and to negatively regulate protein levels of the transcription factor ABCISIC ACID INSENSITIVE5. KEG mRNA levels were found to be 3-fold higher in edr1 mutant plants compared to wild type. Loss-of-function mutations in KEG are seedling lethal and are hypersensitive to glucose and abscisic acid (ABA). The keg-4 mutation, in contrast, conferred resistance to 6% glucose and suppressed edr1-mediated hypersensitivity to ABA, suggesting that the keg-4 mutation suppresses ABA signaling by altering KEG function. Several ABA-responsive genes were found to be further up-regulated in the edr1 mutant following ABA treatment, and this up-regulation was suppressed by the keg-4 mutation. We conclude that edr1-mediated resistance to powdery mildew is mediated, in part, by enhanced ABA signaling.

  15. Induced mutations in the starch branching enzyme II (SBEII) genes increase amylose and resistant starch content in durum wheat

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Colasuonno, Pasqualina; Uauy, Cristobal; Beckles, Diane M.; Dubcovsky, Jorge

    2016-01-01

    Starch is the largest component of the wheat (Triticum aestivum L.) grain and consists of approximately 70-80% amylopectin and 20-30% amylose. Amylopectin is a highly-branched, readily digested polysaccharide, whereas amylose has few branches and forms complexes that resist digestion and mimic dietary fiber (resistant starch). Down-regulation of the starch branching enzyme II (SBEII) gene by RNA interference (RNAi) was previously shown to increase amylose content in both hexaploid and tetraploid wheat. We generated ethyl methane sulphonate (EMS) mutants for the SBEIIa-A and SBEIIa-B homoeologs in the tetraploid durum wheat variety Kronos (T. turgidum ssp. durum L.). Single-gene mutants showed non-significant increases in amylose and resistant starch content, but a double mutant combining a SBEIIa-A knock-out mutation with a SBEIIa-B splice-site mutation showed a 22% increase in amylose content (P<0.0001) and a 115% increase in resistant starch content (P<0.0001). In addition, we obtained mutants for the A and B genome copies of the paralogous SBEIIb gene, mapped them 1-2 cM from SBEIIa, and generated double SBEIIa-SBEIIb mutants to study the effect of the SBEIIb gene in the absence of SBEIIa. These mutants are available to those interested in increasing amylose content and resistant starch in durum wheat. PMID:26924849

  16. Increasing prevalence of ciprofloxacin-resistant food-borne Salmonella strains harboring multiple PMQR elements but not target gene mutations

    PubMed Central

    Lin, Dachuan; Chen, Kaichao; Wai-Chi Chan, Edward; Chen, Sheng

    2015-01-01

    Fluoroquinolone resistance in Salmonella has become increasingly prevalent in recent years. To probe the molecular basis of this phenomenon, the genetic and phenotypic features of fluoroquinolone resistant Salmonella strains isolated from food samples were characterized. Among the 82 Salmonella strains tested, resistance rate of the three front line antibiotics of ceftriaxone, ciprofloxacin and azithromycin was 10%, 39% and 25% respectively, which is significantly higher than that reported in other countries. Ciprofloxacin resistant strains typically exhibited cross-resistance to multiple antibiotics including ceftriaxone, primarily due to the presence of multiple PMQR genes and the blaCTX-M-65, blaCTX-M-55 blaCMY-2 and blaCMY-72 elements. The prevalence rate of the oqxAB and aac(6’)-Ib-cr genes were 91% and 75% respectively, followed by qnrS (66%), qnrB (16%) and qnrD (3%). The most common PMQR combination observable was aac(6’)-Ib-cr-oqxAB-qnrS2, which accounted for 50% of the ciprofloxacin resistant strains. Interestingly, such isolates contained either no target mutations or only a single gyrA mutation. Conjugation and hybridization experiments suggested that most PMQR genes were located either in the chromosome or a non-transferrable plasmid. To summarize, findings in this work suggested that PMQRs greatly facilitate development of fluoroquinolone resistance in Salmonella by abolishing the requirement of target gene mutations. PMID:26435519

  17. Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ɛ-cleavage site

    NASA Astrophysics Data System (ADS)

    Chen, Wen; Gamache, Eric; Rosenman, David J.; Xie, Jian; Lopez, Maria M.; Li, Yue-Ming; Wang, Chunyu

    2014-01-01

    The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer’s disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ɛ-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ɛ-cleavage, and consequently shift cleavage preference towards Aβ42.

  18. Microenvironment mediated alterations to metabolic pathways confer increased chemo-resistance in CD133+ tumor initiating cells

    PubMed Central

    Nomura, Alice; Dauer, Patricia; Gupta, Vineet; McGinn, Olivia; Arora, Nivedita; Majumdar, Kaustav; III, Charles Uhlrich; Dalluge, Joseph; Dudeja, Vikas; Saluja, Ashok; Banerjee, Sulagna

    2016-01-01

    Chemoresistance in pancreatic cancer has been attributed to tumor-initiating cells (TICs), a minor sub-population of tumor cells. However, the mechanism of chemo-resistance in these cells is still unclear. In the current study, immunohistochemical analysis of LSL-KrasG12D; LSL-Trp53R172H; PdxCre (KPC) murine tumors indicated that hypoxic regions developed through tumor progression. This hypoxic “niche” correlated with increased CD133+ population that had an increased HIF1A activity. Consistent with this observation, CD133+ cells had increased glucose uptake and activity of glycolytic pathway enzymes compared to CD133− cells. Mass spectrometric analysis (UPLC-TQD) following metabolic labeling of CD133+ cells with [13C]-U6 glucose confirmed this observation. Furthermore, although both populations had functionally active mitochondria, CD133+ cells had low mitochondrial complex I and complex IV activity and lesser accumulation of ROS in response to standard chemotherapeutic compounds like paclitaxel, 5FU and gemcitabine. CD133+ cells also showed increased resistance to all three chemotherapeutic compounds and treatment with Glut1 inhibitor (STF31) reversed this resistance, promoting apoptotic death in these cells similar to CD133− cells. Our study indicates that the altered metabolic profile of CD133+ pancreatic TIC protects them against apoptosis, by reducing accumulation of ROS induced by standard chemotherapeutic agents, thereby confering chemoresistance. Since resistance to existing chemotherapy contributes to the poor prognosis in pancreatic cancer, our study paves the way for identifying novel therapeutic targets for managing chemoresistance and tumor recurrence in pancreatic cancer. PMID:27472388

  19. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia.

    PubMed

    Minson, Katherine A; Smith, Catherine C; DeRyckere, Deborah; Libbrecht, Clara; Lee-Sherick, Alisa B; Huey, Madeline G; Lasater, Elisabeth A; Kirkpatrick, Gregory D; Stashko, Michael A; Zhang, Weihe; Jordan, Craig T; Kireev, Dmitri; Wang, Xiaodong; Frye, Stephen V; Earp, H Shelton; Shah, Neil P; Graham, Douglas K

    2016-03-01

    FMS-like tyrosine kinase 3-targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD-mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML.

  20. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia

    PubMed Central

    Minson, Katherine A.; Smith, Catherine C.; Libbrecht, Clara; Lee-Sherick, Alisa B.; Huey, Madeline G.; Lasater, Elisabeth A.; Kirkpatrick, Gregory D.; Stashko, Michael A.; Zhang, Weihe; Jordan, Craig T.; Kireev, Dmitri; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton; Shah, Neil P.; Graham, Douglas K.

    2016-01-01

    FMS-like tyrosine kinase 3–targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD–mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML. PMID:27158668

  1. Multiple Influenza A (H3N2) Mutations Conferring Resistance to Neuraminidase Inhibitors in a Bone Marrow Transplant Recipient

    PubMed Central

    Eshaghi, Alireza; Shalhoub, Sarah; Rosenfeld, Paul; Li, Aimin; Higgins, Rachel R.; Stogios, Peter J.; Savchenko, Alexei; Bastien, Nathalie; Li, Yan; Rotstein, Coleman

    2014-01-01

    Immunocompromised patients are predisposed to infections caused by influenza virus. Influenza virus may produce considerable morbidity, including protracted illness and prolonged viral shedding in these patients, thus prompting higher doses and prolonged courses of antiviral therapy. This approach may promote the emergence of resistant strains. Characterization of neuraminidase (NA) inhibitor (NAI)-resistant strains of influenza A virus is essential for documenting causes of resistance. In this study, using quantitative real-time PCR along with conventional Sanger sequencing, we identified an NAI-resistant strain of influenza A (H3N2) virus in an immunocompromised patient. In-depth analysis by deep gene sequencing revealed that various known markers of antiviral resistance, including transient R292K and Q136K substitutions and a sustained E119K (N2 numbering) substitution in the NA protein emerged during prolonged antiviral therapy. In addition, a combination of a 4-amino-acid deletion at residues 245 to 248 (Δ245-248) accompanied by the E119V substitution occurred, causing resistance to or reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir). Resistant variants within a pool of viral quasispecies arose during combined antiviral treatment. More research is needed to understand the interplay of drug resistance mutations, viral fitness, and transmission. PMID:25246391

  2. Mutation of cysteine 46 in IKK-beta increases inflammatory responses

    PubMed Central

    Jiang, Zhi Hong; Jiang, Shui Ping; Liu, Yan; Wang, Ting Yu; Yao, Xiao Jun; Su, Xiao Hui; Yan, Feng Gen; Liu, Juan; Leung, Elaine Lai-Han; Yi, Xiao Qin; Wong, Yuen Fan; Zhou, Hua; Liu, Liang

    2015-01-01

    Activation of IκB kinase β (IKK-β) and nuclear factor (NF)-κB signaling contributes to cancer pathogenesis and inflammatory disease; therefore, the IKK-β−NF-κB signaling pathway is a potential therapeutic target. Current drug design strategies focus on blocking NF-κB signaling by binding to specific cysteine residues on IKK-β. However, mutations in IKK-β have been found in patients who may eventually develop drug resistance. For these patients, a new generation of IKK-β inhibitors are required to provide novel treatment options. We demonstrate in vitro that cysteine-46 (Cys-46) is an essential residue for IKK-β kinase activity. We then validate the role of Cys-46 in the pathogenesis of inflammation using delayed-type hypersensitivity (DTH) and an IKK-βC46A transgenic mouse model. We show that a novel IKK-β inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-βC46A transgenic mice. These findings reveal the role of Cys-46 in the promotion of inflammatory responses, and suggest that Cys-46 is a novel drug-binding site for the inhibition of IKK-β. PMID:26378659

  3. Do Mutations Causing Low HDL-C Promote Increased Carotid Intima-Media Thickness?

    PubMed Central

    Miller, Michael; Rhyne, Jeffrey; Hong, Seung Ho; Friel, Gina; Dolinar, Christina; Riley, Ward

    2007-01-01

    Background Although observational data support an inverse relationship between high-density lipoprotein (HDL) cholesterol and coronary heart disease (CHD), genetic HDL deficiency states often do not correlate with premature CHD. Methods Carotid intima-media thickness (cIMT) measurements were obtained in cases comprising 10 different mutations in LCAT, ABCA1 and APOA1 to further evaluate the relationship between low HDL resulting from genetic variation and early atherosclerosis. Results In a 1:2 case-control study of sex and age-related (± 5 y) subjects (n=114), cIMT was nearly identical between cases (0.66 ± 0.17 cm) and controls (0.65 ± 0.18 cm) despite significantly lower HDL cholesterol (0.67 vs 1.58 mmol/l) and apolipoprotein A-I levels (96.7 vs. 151.4 mg/dl) (P < 0.05). Conclusions Genetic variants identified in the present study may be insufficient to promote early carotid atherosclerosis. PMID:17113061

  4. A single mutation within a Ca(2+) binding loop increases proteolytic activity, thermal stability, and surfactant stability.

    PubMed

    Okuda, Mitsuyoshi; Ozawa, Tadahiro; Tohata, Masatoshi; Sato, Tsuyoshi; Saeki, Katsuhisa; Ozaki, Katsuya

    2013-03-01

    We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca(2+) ion. This residue lies 15-20Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate-enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca(2+) and affects the packing of the Ca(2+) binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.

  5. Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance.

    PubMed

    Liu, Simu; Bartnikas, Lisa M; Volko, Sigrid M; Ausubel, Frederick M; Tang, Dingzhong

    2016-01-01

    Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

  6. A Novel Mutation p.A59P in N-Terminal Domain of Methyl-CpG-Binding Protein 2 Confers Phenotypic Variability in 3 Cases of Tunisian Rett Patients: Clinical Evaluations and In Silico Investigations.

    PubMed

    Kharrat, Marwa; Hsairi, Ines; Fendri-Kriaa, Nourhene; Kenoun, Houda; Othmen, Houda Ben; Ben Mahmoud, Afif; Ghorbel, Rania; Abid, Imen; Triki, Chahnez; Fakhfakh, Faiza

    2015-11-01

    Rett syndrome is a monogenic X-linked dominant neurodevelopmental disorder related to mutation in MECP2, which encodes the methyl-CpG-binding protein MeCP2. The aim of this study was to search for mutations of MECP2 gene in Tunisian Rett patients and to evaluate the impact of the found variants on structural and functional features of MeCP2. The result of mutation analysis revealed that 3 Rett patients shared the same novel heterozygous point mutation c.175G>C (p.A59P). The p.A59P mutation was located in a conserved amino acid in the N-terminal segment of MeCP2. This novel mutation confers a phenotypic variability with different clinical severity scores (3, 8, and 9) and predicted by Sift and PolyPhen to be damaging. Modeling results showed that p.A59P adds 2 hydrogen bonds and changes the structural conformation of MeCP2 with a significant root mean square deviation value (9.66 Å), suggesting that this mutation could probably affect the conformation, function and stability of MeCP2.

  7. Mast cells control insulitis and increase Treg cells to confer protection against STZ-induced type 1 diabetes in mice.

    PubMed

    Carlos, Daniela; Yaochite, Juliana N U; Rocha, Fernanda A; Toso, Vanina D; Malmegrim, Kelen C R; Ramos, Simone G; Jamur, Maria C; Oliver, Constance; Camara, Niels O; Andrade, Marcus V M; Cunha, Fernando Q; Silva, João S

    2015-10-01

    Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W(v) or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-β, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.

  8. G206D Mutation of Presenilin-1 Reduces Pen2 Interaction, Increases Aβ42/Aβ40 Ratio and Elevates ER Ca(2+) Accumulation.

    PubMed

    Chen, Wei-Ting; Hsieh, Yi-Fang; Huang, Yan-Jing; Lin, Che-Ching; Lin, Yen-Tung; Liu, Yu-Chao; Lien, Cheng-Chang; Cheng, Irene Han-Juo

    2015-12-01

    Early-onset familial Alzheimer's disease (AD) is most commonly associated with the mutations in presenilin-1 (PS1). PS1 is the catalytic component of the γ-secretase complex, which cleaves amyloid precursor protein to produce amyloid-β (Aβ), the major cause of AD. Presenilin enhancer 2 (Pen2) is critical for activating γ-secretase and exporting PS1 from endoplasmic reticulum (ER). Among all the familial AD-linked PS1 mutations, mutations at the G206 amino acid are the most adjacent position to the Pen2 binding site. Here, we characterized the effect of a familial AD-linked PS1 G206D mutation on the PS1-Pen2 interaction and the accompanied alteration in γ-secretase-dependent and -independent functions. We found that the G206D mutation reduced PS1-Pen2 interaction, but did not abolish γ-secretase formation and PS1 endoproteolysis. For γ-secretase-dependent function, the G206D mutation increased Aβ42 production but not Notch cleavage. For γ-secretase-independent function, this mutation disrupted the ER calcium homeostasis but not lysosomal calcium homeostasis and autophagosome maturation. Impaired ER calcium homeostasis may due to the reduced mutant PS1 level in the ER. Although this mutation did not alter the cell survival under stress, both increased Aβ42 ratio and disturbed ER calcium regulation could be the mechanisms underlying the pathogenesis of the familial AD-linked PS1 G206D mutation.

  9. Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ-transgenic mouse.

    PubMed

    Ono, T; Ikehata, H; Nakamura, S; Saito, Y; Hosoi, Y; Takai, Y; Yamada, S; Onodera, J; Yamamoto, K

    2000-02-14

    Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.

  10. Microbial gut overgrowth guarantees increased spontaneous mutation leading to polyclonality and antibiotic resistance in the critically ill.

    PubMed

    van Saene, H K F; Taylor, N; Damjanovic, V; Sarginson, R E

    2008-05-01

    Polyclonality is defined as the occurrence of different genotypes of a bacterial species. We are of the opinion that these different clones originate within the patient. When infections and outbreaks occur, the terms of polyclonal infections and polyclonal outbreaks have been used, respectively. The origin of polyclonality has never been reported, although some authors suggest the acquisition of different clones from different animate and inanimate sources. We think that the gut of the critically ill patient with microbial overgrowth is the ideal site for the de-novo development of new clones, following increased spontaneous mutation.

  11. Increased rate of missense/in-frame mutations in individuals with NF1-related pulmonary stenosis: a novel genotype-phenotype correlation.

    PubMed

    Ben-Shachar, Shay; Constantini, Shlomi; Hallevi, Hen; Sach, Emma K; Upadhyaya, Meena; Evans, Gareth D; Huson, Susan M

    2013-05-01

    Neurofibromatosis type 1 (NF1) and its related disorders (NF1-Noonan syndrome (NFNS) and Watson syndrome (WS)) are caused by heterozygous mutations in the NF1 gene. Pulmonary stenosis (PS) occurs more commonly in NF1 and its related disorders than in the general population. This study investigated whether PS is associated with specific types of NF1 gene mutations in NF1, NFNS and WS. The frequency of different NF1 mutation types in a cohort of published and unpublished cases with NF1/NFNS/WS and PS was examined. Compared with NF1 in general, NFNS patients had higher rates of PS (9/35=26% vs 25/2322=1.1%, P value<0.001). Stratification according to mutation type showed that the increased PS rate appears to be driven by the NFNS group with non-truncating mutations. Eight of twelve (66.7%) NFNS cases with non-truncating mutations had PS compared with a 1.1% PS frequency in NF1 in general (P<0.001); there was no increase in the frequency of PS in NFNS patients with truncating mutations. Eight out of eleven (73%) individuals with NF1 and PS, were found to have non-truncating mutations, a much higher frequency than the 19% reported in NF1 cohorts (P<0.015). Only three cases of WS have been published with intragenic mutations, two of three had non-truncating mutations. Therefore, PS in NF1 and its related disorders is clearly associated with non-truncating mutations in the NF1 gene providing a new genotype-phenotype correlation. The data indicate a specific role of non-truncating mutations on the NF1 cardiac phenotype.

  12. Point mutations in Escherichia coli DNA pol V that confer resistance to non-cognate DNA damage also alter protein-protein interactions.

    PubMed

    Hawver, Lisa A; Tehrani, Mohammad; Antczak, Nicole M; Kania, Danielle; Muser, Stephanie; Sefcikova, Jana; Beuning, Penny J

    2015-10-01

    Y-family DNA polymerases are important for conferring cellular resistance to DNA damaging agents in part due to their specialized ability to copy damaged DNA. The Escherichia coli Y-family DNA polymerases are encoded by the umuDC and dinB genes. UmuC and the cleaved form of UmuD, UmuD', form UmuD'2C (pol V), which is able to bypass UV photoproducts such as cyclobutane pyrimidine dimers and 6-4 thymine-thymine dimers, whereas DinB is specialized to copy N(2)-dG adducts, such as N(2)-furfuryl-dG. To better understand this inherent specificity, we used hydroxylamine to generate a random library of UmuC variants from which we then selected those with the ability to confer survival to nitrofurazone (NFZ), which is believed to cause N(2)-furfuryl-dG lesions. We tested the ability of three of the selected UmuC variants, A9V, H282P, and T412I, to bypass N(2)-furfuryl-dG in vitro, and discovered that pol V containing UmuC A9V has overall modestly better primer extension activity than WT pol V, whereas the UmuC T412I and UmuC H282P mutations result in much lower primer extension efficiency. Upon further characterization, we found that the ability of the UmuC variant A9V to render cells UV-mutable is dependent on the proper length of the arm of UmuD'. Cells harboring UmuC variants T412I and H282P show enhanced cleavage of UmuD to form UmuD', which, together with our other observations, suggests that this may be due to a disruption of a direct interaction between UmuC and UmuD. Thus, we find that protein interactions as well as protein conformation appear to be crucial for resistance to specific types of DNA damage.

  13. Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-Cardiac Myosin.

    PubMed

    Adhikari, Arjun S; Kooiker, Kristina B; Sarkar, Saswata S; Liu, Chao; Bernstein, Daniel; Spudich, James A; Ruppel, Kathleen M

    2016-12-13

    Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%-90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

  14. A Single Mutation in the Gene Responsible for the Mucoid Phenotype of Bifidobacterium animalis subsp. lactis Confers Surface and Functional Characteristics

    PubMed Central

    Hidalgo-Cantabrana, Claudio; Sánchez, Borja; Álvarez-Martín, Pablo; López, Patricia; Martínez-Álvarez, Noelia; Delley, Michele; Martí, Marc; Varela, Encarna; Suárez, Ana; Antolín, María; Guarner, Francisco; Berger, Bernard

    2015-01-01

    Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells. PMID:26362981

  15. A novel ENU-induced ankyrin-1 mutation impairs parasite invasion and increases erythrocyte clearance during malaria infection in mice

    PubMed Central

    Huang, Hong Ming; Bauer, Denis C.; Lelliott, Patrick M.; Greth, Andreas; McMorran, Brendan J.; Foote, Simon J.; Burgio, Gaetan

    2016-01-01

    Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1(MRI61689/+) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1(MRI61689/+) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1(MRI61689/+) erythrocytes were also more likely to be cleared from circulation during infection; the “bystander effect”. This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1(MRI61689/+) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance. PMID:27848995

  16. A rare mutation in UNC5C predisposes to Alzheimer’s disease and increases neuronal cell death

    PubMed Central

    Wetzel-Smith, MK; Hunkapiller, J; Bhangale, TR; Srinivasan, K; Maloney, JA; Atwal, JK; Sa, SM; Yaylaoglu, MB; Foreman, O; Ortmann, W; Rathore, N; Hansen, DV; Tessier-Lavigne, M; Mayeux, R; Pericak-Vance, M; Haines, J; Farrer, LA; Schellenberg, GD; Goate, A; Behrens, TW

    2015-01-01

    We have identified a rare coding mutation, T835M (rs137875858), in the Netrin receptor UNC5C that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer’s disease (LOAD), and was associated with disease across four large case/control cohorts (OR = 2.15, Pmeta= 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and in vitro studies demonstrate that this mutation leads to increased cell death in several cell types, including neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to multiple neurodegenerative stimuli, including β-Amyloid (Aβ). Based on these data and the enriched hippocampal expression of UNC5C in the adult nervous system, we propose one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer’s disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer’s brain. PMID:25419706

  17. PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers.

    PubMed

    Alquézar, Carolina; Esteras, Noemí; de la Encarnación, Ana; Alzualde, Ainhoa; Moreno, Fermín; López de Munain, Adolfo; Martín-Requero, Angeles

    2014-04-01

    Loss-of-function progranulin (PGRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Previously, we reported cell cycle-related alterations in lymphoblasts from FTLD-TDP patients, carrying the c.709-1G>A null PGRN mutation, suggesting aberrant cell cycle activation in affected neurons. Here we report that PGRN haploinsufficiency activates the extracellular signal-regulated protein kinases 1 and 2 pathway in a Ca(2+), protein kinase C-dependent, and pertussis toxin-sensitive manner. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that noncanonical Wnt5a signaling might be overactivated by PGRN deficiency. We detected increased cellular and secreted levels of Wnt5a in PGRN-deficient lymphoblasts associated with enhanced phosphorylated calmodulin kinase II. Moreover, treatment of control cells with exogenous Wingless-type 5a (Wnt5a)-activated Ca(2+)/calmodulin kinase II (CaMKII), increased extracellular signal-regulated protein kinases 1 and 2 activity and cell proliferation up to the levels found in c.709-1G>A carrier cells. PGRN knockdown SH-SY5Y neuroblastoma cells also show enhanced Wnt5a content and signaling. Taken together, our results revealed an important role of Wnt signaling in FTLD-TDP pathology and suggest a novel target for therapeutic intervention.

  18. Polymorphism of FGFR4 Gly388Arg does not confer an increased risk to breast cancer development.

    PubMed

    Naidu, R; Har, Y C; Taib, N A

    2009-01-01

    The genotype analysis of the Gly and Arg allele at codon 388 of fibroblast growth factor receptor-4 (FGFR4) gene was evaluated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in a hospital-based Malaysian population. Peripheral blood samples were collected from 387 breast cancer patients and 252 normal and healthy women who had no history of any malignancy. The aim of the present study was to evaluate the association between the FGFR4 Gly388Arg polymorphism and breast cancer risk as well as clinicopathological parameters of the patients. The Gly/Gly, Gly/Arg, Arg/Arg, and Arg allele genotypes were detected in 46.3%, 44.4%, 9.3%, and 53.7% of breast cancer cases, respectively. The distribution of genotype (p = 0.204) and allele (p = 0.086) frequencies of FGFR4 polymorphism were not significantly different between the breast cancer cases and normal individuals. Women who were Arg/ Arg homozygotes (OR = 1.714, 95% CI 0.896-3.278), Gly/Arg heterozygotes (OR = 1.205, 95% CI 0.863-1.683), carriers of Arg allele genotype (OR = 1.269, 95% CI 0.921-1.750), or Arg allele (OR = 1.246, 95% CI 0.970-1.602) were not associated with breast cancer risk. The Arg allele genotype was significantly associated with lymph node metastases (p = 0.001) but not with other clinicopathological parameters. Our findings suggest that the polymorphic variant at codon 388 of FGFR4 gene does not confer increased risk to breast cancer development but it may be a potential genetic marker for tumor prognosis.

  19. Using polarization-sensitive optical coherence tomography to identify tumor stromal fibrosis and increase tumor biopsy yield (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hariri, Lida P.; Adams, David C.; Miller, Alyssa J.; Mino-Kenudson, Mari; Suter, Melissa J.

    2016-03-01

    Tissue biopsy is the principal method used to diagnose tumors in a variety of organ systems. It is essential to maximize tumor yield in biopsy specimens for both clinical diagnostic and research purposes. This is particularly important in tumors where additional tissue is needed for molecular analysis to identify patients who would benefit from mutation-specific targeted therapy, such as in lung carcinomas. Inadvertent sampling of fibrotic stroma within tumor nodules contaminates biopsies, decreases tumor yield, and can impede diagnosis. The ability to assess tumor composition and guide biopsy site selection in real time is likely to improve diagnostic yield. Polarization sensitive OCT (PS-OCT) measures birefringence in organized tissues, such as collagen, and could be used to distinguish tumor from fibrosis. In this study, PS-OCT was obtained in 65 lung nodule samples from surgical resection specimens containing varying ratios of tumor and fibrosis. PS-OCT was obtained with either a custom-built helical scanning catheter (0.8 or 1.6mm in diameter) or a dual-axis bench top scanner. Strong birefringence was observed in nodules containing dense fibrosis, with no birefringence in adjacent regions of tumor. Tumors admixed with early, loosely-organized collagen demonstrated mild-to-moderate birefringence, and tumors with little collagen content showed little to no birefringent signal. PS-OCT provides significant insights into tumor nodule composition, and has potential to differentiate tumor from stromal fibrosis during biopsy site selection to increase diagnostic tumor yield.

  20. Increased uracil insertion in DNA is cytotoxic and increases the frequency of mutation, double strand break formation and VSG switching in Trypanosoma brucei.

    PubMed

    Castillo-Acosta, Víctor M; Aguilar-Pereyra, Fernando; Bart, Jean-Mathieu; Navarro, Miguel; Ruiz-Pérez, Luis M; Vidal, Antonio E; González-Pacanowska, Dolores

    2012-12-01

    Deoxyuridine 5'-triphosphate pyrophosphatase (dUTPase) and uracil-DNA glycosylase (UNG) are key enzymes involved in the control of the presence of uracil in DNA. While dUTPase prevents uracil misincorporation by removing dUTP from the deoxynucleotide pool, UNG excises uracil from DNA as a first step of the base excision repair pathway (BER). Here, we report that strong down-regulation of dUTPase in UNG-deficient Trypanosoma brucei cells greatly impairs cell viability in both bloodstream and procyclic forms, underscoring the extreme sensitivity of trypanosomes to uracil in DNA. Depletion of dUTPase activity in the absence of UNG provoked cell cycle alterations, massive dUTP misincorporation into DNA and chromosomal fragmentation. Overall, trypanosomatid cells that lack dUTPase and UNG activities exhibited greater proliferation defects and DNA damage than cells deficient in only one of these activities. To determine the mutagenic consequences of uracil in DNA, mutation rates and spectra were analyzed in dUTPase-depleted cells in the presence of UNG activity. These cells displayed a spontaneous mutation rate 9-fold higher than the parental cell line. Base substitutions at A:T base pairs and deletion frequencies were both significantly enhanced which is consistent with the generation of mutagenic AP sites and DNA strand breaks. The increase in strand breaks conveyed a concomitant increase in VSG switching in vitro. The low tolerance of T. brucei to uracil in DNA emphasizes the importance of uracil removal and regulation of intracellular dUTP pool levels in cell viability and genetic stability and suggests potential strategies to compromise parasite survival.

  1. A protective antigen mutation increases the pH threshold of anthrax toxin receptor 2-mediated pore formation.

    PubMed

    Dennis, Melissa K; Mogridge, Jeremy

    2014-04-08

    Anthrax toxin protective antigen (PA) binds cellular receptors and self-assembles into oligomeric prepores. A prepore converts to a protein translocating pore after it has been transported to an endosome where the low pH triggers formation of a membrane-spanning β-barrel channel. Formation of this channel occurs after some PA-receptor contacts are broken to allow pore formation, while others are retained to preserve receptor association. The interaction between PA and anthrax toxin receptor 1 (ANTXR1) is weaker than its interaction with ANTXR2 such that the pH threshold of ANTXR1-mediated pore formation is higher by 1 pH unit. Here we examine receptor-specific differences in toxin binding and pore formation by mutating PA residue G342 that selectively abuts ANTXR2. Mutation of G342 to valine, leucine, isoleucine, or tryptophan increased the amount of PA bound to ANTXR1-expressing cells and decreased the amount of PA bound to ANTXR2-expressing cells. The more conservative G342A mutation did not affect the level of binding to ANTXR2, but ANTXR2-bound PA-G342A prepores exhibited a pH threshold higher than that of wild-type prepores. Mixtures of wild-type PA and PA-G342A were functional in toxicity assays, and the pH threshold of ANTXR2-mediated pore formation was dictated by the relative amounts of the two proteins in the hetero-oligomers. These results suggest that PA subunits within an oligomer do not have to be triggered simultaneously for a productive membrane insertion event to occur.

  2. Reversal of clavulanate resistance conferred by a Ser-244 mutant of TEM-1 beta-lactamase as a result of a second mutation (Arg to Ser at position 164) that enhances activity against ceftazidime.

    PubMed Central

    Imtiaz, U; Manavathu, E K; Mobashery, S; Lerner, S A

    1994-01-01

    The mutation of Arg-244 to Ser (Arg-244-->Ser mutation) in the TEM-1 beta-lactamase has been shown to produce resistance to inactivation by clavulanate in the mutant enzyme and resistance to ampicillin plus clavulanate in a strain of Escherichia coli producing this enzyme. The Arg-164-->Ser mutation in the TEM-1 beta-lactamase (TEM-12 enzyme) is known to enhance the activity of the enzyme against ceftazidime, resulting in resistance to the drug in a strain producing the mutant enzyme (D. A. Weber, C. C. Sanders, J. S. Bakken, and J. P. Quinn, J. Infect. Dis. 162:460-465, 1990). The doubly mutated derivative of the TEM-1 enzyme (Ser-164/Ser-244) retains the characteristics of the Ser-164 mutant enzyme, i.e., enhanced activity against ceftazidime and sensitivity to inactivation by clavulanate. It also confers the same phenotype as the Ser-164 mutant enzyme, i.e., resistance to ceftazidime and ampicillin, with reversal of this resistance in the presence of clavulanate. Thus, the Arg-164-->Ser mutation in the TEM-1 beta-lactamase suppresses the effect of the Arg-244-->Ser mutation which, by itself, reduces the sensitivity of the enzyme to inactivation by clavulanate. PMID:8067751

  3. Mutations in the Polybasic Juxtamembrane Sequence of Both Plasma Membrane- and Endoplasmic Reticulum-localized Epidermal Growth Factor Receptors Confer Ligand-independent Cell Transformation*

    PubMed Central

    Bryant, Kirsten L.; Antonyak, Marc A.; Cerione, Richard A.; Baird, Barbara; Holowka, David

    2013-01-01

    Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling. PMID:24142702

  4. Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

    PubMed

    Dunn, Cory D

    2011-10-01

    Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan.

  5. MDE heteroduplex analysis of PCR products spanning each exon of the fibrillin (FBN1) gene greatly increases the efficiency of mutation detection in the Marfan syndrome

    SciTech Connect

    Nijbroek, G.; Dietz, H.C.; Pereira, L.; Ramirz, F.

    1994-09-01

    Defects in fibrillin (FNB1) cause the Marfan syndrome (MFS). Classic Marfan phenotype cosegregates with intragenic and/or flanking marker alleles in all families tested and a significant number of FBN1 mutations have been identified in affected individuals. Using a standard method of mutation detection, SSCP analysis of overlapping RT-PCR amplimers that span the entire coding sequence, the general experience has been a low yield of identifiable mutations, ranging from 10-20%. Possible explanations included low sensitivity of mutation screening procedures, under-representation of mutant transcript in patient samples either due to deletions or mutant alleles containing premature termination codons, clustering of mutations in yet uncharacterized regions of the gene, including regulatory elements, or genetic heterogeneity. In order to compensate for a potential reduced mutant transcript stability, we have devised a method to screen directly from genomic DNA. The intronic boundaries flanking each of the 65 FBN1 exons were characterized and primer pairs were fashioned such that all splice junctions would be included in the resultant amplimers. The entire gene was screened for a panel of 9 probands with classic Marfan syndrome using mutation detection enhancement (MDE) gel heteroduplex analysis. A mutation was identified in 5/9 (55%) of patient samples. All were either missense mutations involving a cysteine residue or small deletions that did not create a frame shift. In addition, 10 novel polymorphisms were found. We conclude that the majority of mutations causing Marfan syndrome reside in the FBN1 gene and that mutations creating premature termination codons are not the predominant cause of inefficient mutation detection using RT-PCR. We are currently modifying screening methods to increase sensitivity and targeting putative FBN1 gene promoter sequences for study.

  6. Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%.

    PubMed Central

    Macek, M; Mackova, A; Hamosh, A; Hilman, B C; Selden, R F; Lucotte, G; Friedman, K J; Knowles, M R; Rosenstein, B J; Cutting, G R

    1997-01-01

    Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans. PMID:9150159

  7. Chronic Ethanol Consumption Increases Myocardial Mitochondrial DNA Mutations: A Potential Contribution by Mitochondrial Topoisomerases

    PubMed Central

    Laurent, D.; Mathew, J.E.; Mitry, M.; Taft, M.; Force, A.; Edwards, J.G.

    2014-01-01

    Aims: Alcoholic cardiomyopathy (ACM) presents as decreased myocardial contractility, arrhythmias and secondary non-ischemic dilated cardiomyopathy leading to heart failure. Mitochondrial dysfunction is known to have a significant role in the development and complications of ACM. This study investigated if chronic ethanol feeding promoted myocardial mitochondrial topoisomerase dysfunction as one underlying cause of mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in ACM. Methods: The impact of chronic ethanol exposure on the myocardial mitochondria was examined in both neonatal cardiomyocytes using 50 mM ethanol for 6 days and in rats assigned to control or ethanol feeding groups for 4 months. Results: Chronic ethanol feeding led to significant (P < 0.05) decreases in M-mode Fractional Shortening, ejection fraction, and the cardiac output index as well as increases in Tau. Ethanol feeding promoted mitochondrial dysfunction as evidenced by significantly decreased left ventricle cytochrome oxidase activity and decreases in mitochondrial protein content. Both in rats and in cultured cardiomyocytes, chronic ethanol presentation significantly increased mtDNA damage. Using isolated myocardial mitochondria, both mitochondrial topoisomerase-dependent DNA cleavage and DNA relaxation were significantly altered by ethanol feeding. Conclusion: Chronic ethanol feeding compromised cardiovascular and mitochondrial function as a result of a decline in mtDNA integrity that was in part the consequence of mitochondrial topoisomerase dysfunction. Understanding the regulation of the mitochondrial topoisomerases is critical for protection of mtDNA, not only for the management of alcoholic cardiomyopathy, but also for the many other clinical treatments that targets the topoisomerases in the alcoholic patient. PMID:24852753

  8. Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans.

    PubMed

    Jakubowski, Hieronim; Boers, Godfried H J; Strauss, Kevin A

    2008-12-01

    Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also show that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients.

  9. Identifying potential functional impact of mutations and polymorphisms: linking heart failure, increased risk of arrhythmias and sudden cardiac death

    PubMed Central

    Jagu, Benoît; Charpentier, Flavien; Toumaniantz, Gilles

    2013-01-01

    Researchers and clinicians have discovered several important concepts regarding the mechanisms responsible for increased risk of arrhythmias, heart failure, and sudden cardiac death. One major step in defining the molecular basis of normal and abnormal cardiac electrical behavior has been the identification of single mutations that greatly increase the risk for arrhythmias and sudden cardiac death by changing channel-gating characteristics. Indeed, mutations in several genes encoding ion channels, such as SCN5A, which encodes the major cardiac Na+ channel, have emerged as the basis for a variety of inherited cardiac arrhythmias such as long QT syndrome, Brugada syndrome, progressive cardiac conduction disorder, sinus node dysfunction, or sudden infant death syndrome. In addition, genes encoding ion channel accessory proteins, like anchoring or chaperone proteins, which modify the expression, the regulation of endocytosis, and the degradation of ion channel a-subunits have also been reported as susceptibility genes for arrhythmic syndromes. The regulation of ion channel protein expression also depends on a fine-tuned balance among different other mechanisms, such as gene transcription, RNA processing, post-transcriptional control of gene expression by miRNA, protein synthesis, assembly and post-translational modification and trafficking. The aim of this review is to inventory, through the description of few representative examples, the role of these different biogenic mechanisms in arrhythmogenesis, HF and SCD in order to help the researcher to identify all the processes that could lead to arrhythmias. Identification of novel targets for drug intervention should result from further understanding of these fundamental mechanisms. PMID:24065925

  10. Deletion of p66Shc in mice increases the frequency of size-change mutations in the lacZ transgene.

    PubMed

    Beltrami, Elena; Ruggiero, Antonella; Busuttil, Rita; Migliaccio, Enrica; Pelicci, Pier Giuseppe; Vijg, Jan; Giorgio, Marco

    2013-04-01

    Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest, or eliminate the damaged cell. Thus, reactive oxygen species (ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knockout (p66KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from organs into Escherichia coli to measure mutation rate. Liver and small intestine from 2- to 24-month-old, lacZ (p66Shc+/+) and lacZp66KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase in the frequency of size-change mutations in the intestine of lacZp66KO mice. This difference was further increased upon irradiation of mice with X-ray. In addition, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66Shc, increased the size-change mutation frequency in small intestine. Size-change mutations also accumulated in death-resistant embryonic fibroblasts from lacZp66KO mice treated with H2 O2 . These results indicate that p66Shc plays a role in the accumulation of DNA rearrangements and suggest that p66Shc functions to clear damaged cells rather than affect DNA metabolism.

  11. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts

    PubMed Central

    Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A.; Verma, Amit; Boultwood, Jacqueline

    2015-01-01

    Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival. PMID:26623729

  12. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

    PubMed

    Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A; Verma, Amit; Boultwood, Jacqueline

    2015-12-29

    Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

  13. Amyloid-beta (Aβ) D7H mutation increases oligomeric Aβ42 and alters properties of Aβ-zinc/copper assemblies.

    PubMed

    Chen, Wei-Ting; Hong, Chen-Jee; Lin, Ya-Tzu; Chang, Wen-Han; Huang, He-Ting; Liao, Jhih-Ying; Chang, Yu-Jen; Hsieh, Yi-Fang; Cheng, Chih-Ya; Liu, Hsiu-Chih; Chen, Yun-Ru; Cheng, Irene H

    2012-01-01

    Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid β-protein (Aβ) levels or aggregation. Here, we identified a novel APP mutation, located within the Aβ sequence (Aβ(D7H)), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Aβ production, Aβ42/40 ratio and prolonged Aβ42 oligomer state with higher neurotoxicity. Because the D7H mutant Aβ has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Aβ to ion. When co-incubated with Zn(2+) or Cu(2+), Aβ(D7H) aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a "double punch" effect on elevating both Aβ production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aβ N-terminal region potentially modulates APP processing and Aβ aggregation, and further provides a genetic indication of the importance of Zn(2+) and Cu(2+) in the etiology of AD.

  14. Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies.

    PubMed

    Liu, Xia; Zheng, Hong; Li, Xiaobo; Wang, Siying; Meyerson, Howard J; Yang, Wentian; Neel, Benjamin G; Qu, Cheng-Kui

    2016-01-26

    Gain-of-function (GOF) mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling pathways, are associated with childhood leukemias and solid tumors. The underlying mechanisms are not fully understood. Here, we report that Ptpn11 GOF mutations disturb mitosis and cytokinesis, causing chromosomal instability and greatly increased susceptibility to DNA damage-induced malignancies. We find that Shp2 is distributed to the kinetochore, centrosome, spindle midzone, and midbody, all of which are known to play critical roles in chromosome segregation and cytokinesis. Mouse embryonic fibroblasts with Ptpn11 GOF mutations show a compromised mitotic checkpoint. Centrosome amplification and aberrant mitosis with misaligned or lagging chromosomes are significantly increased in Ptpn11-mutated mouse and patient cells. Abnormal cytokinesis is also markedly increased in these cells. Further mechanistic analyses reveal that GOF mutant Shp2 hyperactivates the Polo-like kinase 1 (Plk1) kinase by enhancing c-Src kinase-mediated tyrosine phosphorylation of Plk1. This study provides novel insights into the tumorigenesis associated with Ptpn11 GOF mutations and cautions that DNA-damaging treatments in Noonan syndrome patients with germ-line Ptpn11 GOF mutations could increase the risk of therapy-induced malignancies.

  15. Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius.

    PubMed

    Weyda, István; Lübeck, Mette; Ahring, Birgitte K; Lübeck, Peter S

    2014-04-01

    Aspergillus carbonarius accumulates xylitol when it grows on D-xylose. In fungi, D-xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR). Xylitol is then further oxidized by the NAD(+)-dependent xylitol dehydrogenase (XDH). The cofactor impairment between the XR and XDH can lead to the accumulation of xylitol under oxygen-limiting conditions. Most of the XRs are NADPH dependent and contain a conserved Ile-Pro-Lys-Ser motif. The only known naturally occurring NADH-dependent XR (from Candida parapsilosis) carries an arginine residue instead of the lysine in this motif. In order to overcome xylitol accumulation in A. carbonarius a Lys-274 to Arg point mutation was introduced into the XR with the aim of changing the specificity toward NADH. The effect of the genetic engineering was examined in fermentation for citric acid production and xylitol accumulation by using D-xylose as the sole carbon source. Fermentation with the mutant strain showed a 2.8-fold reduction in xylitol accumulation and 4.5-fold increase in citric acid production compared to the wild-type strain. The fact that the mutant strain shows decreased xylitol levels is assumed to be associated with the capability of the mutated XR to use the NADH generated by the XDH, thus preventing the inhibition of XDH by the high levels of NADH and ensuring the flux of xylose through the pathway. This work shows that enhanced production of citric acid can be achieved using xylose as the sole carbon source by reducing accumulation of other by-products, such as xylitol.

  16. The Single T65S Mutation Generates Brighter Cyan Fluorescent Proteins with Increased Photostability and pH Insensitivity

    PubMed Central

    Fredj, Asma; Pasquier, Hélène; Demachy, Isabelle; Jonasson, Gabriella; Levy, Bernard; Derrien, Valérie; Bousmah, Yasmina; Manoussaris, Gallia; Wien, Frank; Ridard, Jacqueline; Erard, Marie; Merola, Fabienne

    2012-01-01

    Cyan fluorescent proteins (CFP) derived from Aequorea victoria GFP, carrying a tryptophan-based chromophore, are widely used as FRET donors in live cell fluorescence imaging experiments. Recently, several CFP variants with near-ultimate photophysical performances were obtained through a mix of site-directed and large scale random mutagenesis. To understand the structural bases of these improvements, we have studied more specifically the consequences of the single-site T65S mutation. We find that all CFP variants carrying the T65S mutation not only display an increased fluorescence quantum yield and a simpler fluorescence emission decay, but also show an improved pH stability and strongly reduced reversible photoswitching reactions. Most prominently, the Cerulean-T65S variant reaches performances nearly equivalent to those of mTurquoise, with QY  = 0.84, an almost pure single exponential fluorescence decay and an outstanding stability in the acid pH range (pK1/2 = 3.6). From the detailed examination of crystallographic structures of different CFPs and GFPs, we conclude that these improvements stem from a shift in the thermodynamic balance between two well defined configurations of the residue 65 hydroxyl. These two configurations differ in their relative stabilization of a rigid chromophore, as well as in relaying the effects of Glu222 protonation at acid pHs. Our results suggest a simple method to greatly improve numerous FRET reporters used in cell imaging, and bring novel insights into the general structure-photophysics relationships of fluorescent proteins. PMID:23133673

  17. Gender and plasma iron biomarkers, but not HFE gene mutations, increase the risk of colorectal cancer and polyps.

    PubMed

    Castiella, Agustin; Múgica, Fernando; Zapata, Eva; Zubiaurre, Leire; Iribarren, Arantxa; de Juan, M Dolores; Alzate, Luis; Gil, Ines; Urdapilleta, Gregorio; Otazua, Pedro; Emparanza, José Ignacio

    2015-09-01

    A cohort study of patients included in the Basque Country colorectal cancer (CRC) screening programme was carried out to assess the risk of adenomatous polyps and CRC (P-CRC) associated with HFE gene mutations, with gender and with iron biomarkers (serum ferritin (SF), iron (Fe) and transferrin saturation index (TSI)). Among 432 included patients (mean age 59.8 years), 263 were men (60.9 %) and 169 women (39.1 %). P-CRC were identified in 221 patients (51.2 %) and no polyps (NP) in 211 patients (48.8 %). HFE mutations were identified in 43.8 % of the patients. C282Y/wt genotypic frequency was 6.8 % in the P-CRC group and 1.4 % in the NP group (p < 0.05). The allelic frequency was 3.8 versus 1.2 % (p < 0.05). For laboratory, all three iron biomarkers showed a statistically significant difference: mean Fe, 91.29 ± 34 for P-CRC and 80.81 ± 30.59 for NP group. Mean TSI for P-CRC was 24.95 ± 8.90 and 22.74 ± 8.79 for NP group. Mean SF 308.09 ± 536.32 for P-CRC and 177.55 ± 159.95 for NP group. In a multivariate logistic regression analysis, only male gender (odds ratio (OR) = 2.04, 1.29-3.22), SF (OR = 1.001, 1.0004-1.003) and Fe (OR = 1.01, 1.004-1.02) were related with the presence of CRC and adenoma. Men gender and raised serum iron biomarkers increase the risk of P-CRC.

  18. The fitness costs of antibiotic resistance mutations

    PubMed Central

    Melnyk, Anita H; Wong, Alex; Kassen, Rees

    2015-01-01

    Antibiotic resistance is increasing in pathogenic microbial populations and is thus a major threat to public health. The fate of a resistance mutation in pathogen populations is determined in part by its fitness. Mutations that suffer little or no fitness cost are more likely to persist in the absence of antibiotic treatment. In this review, we performed a meta-analysis to investigate the fitness costs associated with single mutational events that confer resistance. Generally, these mutations were costly, although several drug classes and species of bacteria on average did not show a cost. Further investigations into the rate and fitness values of compensatory mutations that alleviate the costs of resistance will help us to better understand both the emergence and management of antibiotic resistance in clinical settings. PMID:25861385

  19. Follow #eHealth2011: Measuring the Role and Effectiveness of Online and Social Media in Increasing the Outreach of a Scientific Conference

    PubMed Central

    Winandy, Marcel; St Louis, Connie; Szomszor, Martin

    2016-01-01

    Background Social media promotion is increasingly adopted by organizers of industry and academic events; however, the success of social media strategies is rarely questioned or the real impact scientifically analyzed. Objective We propose a framework that defines and analyses the impact, outreach, and effectiveness of social media for event promotion and research dissemination to participants of a scientific event as well as to the virtual audience through the Web. Methods Online communication channels Twitter, Facebook, Flickr, and a Liveblog were trialed and their impact measured on outreach during five phases of an eHealth conference: the setup, active and last-minute promotion phases before the conference, the actual event, and after the conference. Results Planned outreach through online channels and social media before and during the event reached an audience several magnitudes larger in size than would have been possible using traditional means. In the particular case of eHealth 2011, the outreach using traditional means would have been 74 attendees plus 23 extra as sold proceedings and the number of downloaded articles from the online proceedings (4107 until October 2013). The audience for the conference reached via online channels and social media was estimated at more than 5300 in total during the event. The role of Twitter for promotion before the event was complemented by an increased usage of the website and Facebook during the event followed by a sharp increase of views of posters on Flickr after the event. Conclusions Although our case study is focused on a particular audience around eHealth 2011, our framework provides a template for redefining “audience” and outreach of events, merging traditional physical and virtual communities and providing an outline on how these could be successfully reached in clearly defined event phases. PMID:27436012

  20. Altering a gene involved in nuclear distribution increases the repeat-induced point mutation process in the fungus Podospora anserina.

    PubMed Central

    Bouhouche, Khaled; Zickler, Denise; Debuchy, Robert; Arnaise, Sylvie

    2004-01-01

    Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of ami1, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that ami1 is involved in nuclear distribution during the sexual cycle and that alteration of ami1 delays the fruiting-body development. We also demonstrate that ami1 alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an ami1 mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the ami1 mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR. PMID:15166143

  1. Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue.

    PubMed

    Lebel, Michel

    2002-01-01

    Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.

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

    PubMed

    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.

  3. Full Genome Characterization of Human Influenza A/H3N2 Isolates from Asian Countries Reveals a Rare Amantadine Resistance-Conferring Mutation and Novel PB1-F2 Polymorphisms.

    PubMed

    Zaraket, Hassan; Kondo, Hiroki; Hibino, Akinobu; Yagami, Ren; Odagiri, Takashi; Takemae, Nobuhiro; Tsunekuni, Ryota; Saito, Takehiko; Myint, Yi Yi; Kyaw, Yadanar; Oo, Khin Yi; Tin, Htay Htay; Lin, Nay; Anh, Nguyen Phuong; Hang, Nguyen Le Khanh; Mai, Le Quynh; Hassan, Mohd R; Shobugawa, Yugo; Tang, Julian; Dbaibo, Ghassan; Saito, Reiko

    2016-01-01

    Influenza A viruses evolve at a high rate requiring continuous monitoring to maintain the efficacy of vaccines and antiviral drugs. We performed next generation sequencing analysis of 100 influenza A/H3N2 isolates collected in four Asian countries (Japan, Lebanon, Myanmar, and Vietnam) during 2012-2015. Phylogenetic analysis revealed several reassortment events leading to the circulation of multiple clades within the same season. This was particularly evident during the 2013 and 2013/2014 seasons. Importantly, our data showed that certain lineages appeared to be fitter and were able to persist into the following season. The majority of A/H3N2 viruses continued to harbor the M2-S31N mutation conferring amantadine-resistance. In addition, an S31D mutation in the M2-protein, conferring a similar level of resistance as the S31N mutation, was detected in three isolates obtained in Japan during the 2014/2015 season. None of the isolates possessed the NA-H274Y mutation conferring oseltamivir-resistance, though a few isolates were found to contain mutations at the catalytic residue 151 (D151A/G/N or V) of the NA protein. These variations did not alter the susceptibility to neuraminidase inhibitors and were not detected in the original clinical specimens, suggesting that they had been acquired during their passage in MDCK cells. Novel polymorphisms were detected in the PB1-F2 open-reading frame resulting in truncations in the protein of 24-34 aminoacids in length. Thus, this study has demonstrated the utility of monitoring the full genome of influenza viruses to allow the detection of the potentially fittest lineages. This enhances our ability to predict the strain(s) most likely to persist into the following seasons and predict the potential degree of vaccine match or mismatch with the seasonal influenza season for that year. This will enable the public health and clinical teams to prepare for any related healthcare burden, depending on whether the vaccine match is

  4. Evidence for increased prevalence of SRY mutations in XY females with complete rather than partial gonadal dysgenesis

    SciTech Connect

    Hawkins, J.R.; Taylor, A.; Goodfellow, P.N. ); Migeon, C.J.; Smith, K.D.; Berkovitz, G.D. )

    1992-11-01

    The Y chromosome gene SRY (sex-determining region, Y gene) has been equated with the mammalian testis-determining factor. The SRY gene of five subjects with 46,XY complete gonadal dysgenesis (46,XY karyotype, completely female external genitalia, normal Muellerian ducts, and streak gonads) was evaluated for possible mutations in the coding region by using both single-strand conformation polymorphism (SSCP) assay and DNA sequencing. Mutations were identified in three subjects, of which two gave altered SSCP patterns. Two of them were point mutations causing amino acid substitutions, and the third was a single-base deletion causing a frameshift. All three mutations caused alterations in the putative DNA-binding region of the SRY protein. Genomic DNA was obtained from the fathers of two of the three mutant patients: one mutation was demonstrated to be de novo, and the other was inherited. The presence of SRY mutations in three of five patients suggest that the frequency of SRY mutations in XY females is higher than current estimates. 25 refs., 2 figs.

  5. Faster cross-bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation

    PubMed Central

    Witjas-Paalberends, E Rosalie; Ferrara, Claudia; Scellini, Beatrice; Piroddi, Nicoletta; Montag, Judith; Tesi, Chiara; Stienen, Ger J M; Michels, Michelle; Ho, Carolyn Y; Kraft, Theresia; Poggesi, Corrado; van der Velden, Jolanda

    2014-01-01

    The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s−1, respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l–1 s−1 kN−1 m−2, respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation. PMID:24928957

  6. Mutational analysis in the glycone binding pocket of Dalbergia cochinchinensis β-glucosidase to increase catalytic efficiency toward mannosides.

    PubMed

    Ratananikom, Khakhanang; Choengpanya, Khuanjarat; Tongtubtim, Nusra; Charoenrat, Theppanya; Withers, Stephen G; Kongsaeree, Prachumporn T

    2013-05-24

    Dalcochinase and Abg are glycoside hydrolase family 1 β-glucosidases from Dalbergia cochinchinensis Pierre and Agrobacterium sp., respectively, with 35% sequence identity. However, Abg shows much higher catalytic efficiencies toward a broad range of glycone substrates than dalcochinase does, possibly due to the difference in amino acid residues around their glycone binding pockets. Site-directed mutagenesis was used to replace the amino acid residues of dalcochinase with the corresponding residues of Abg, generating three single mutants, F196H, S251V, and M369E, as well as the corresponding three double mutants and one triple mutant. Among these, the F196H mutant showed increases in catalytic efficiency toward almost all glycoside substrates tested, with the most improved catalytic efficiency being a 3-fold increase for hydrolysis of p-nitrophenyl β-D-mannoside, suggesting a preferred polar residue at this position and consistent with the presence of histidine at this position in two other GH1 glycosidases from barley and rice that prefer β-mannosides. In addition, the M369E mutation resulted in a small increase in catalytic efficiency for cleavage of p-nitrophenyl β-D-galactoside. By contrast, the multiple mutants were up to 8-fold less efficient than the recombinant wild-type dalcochinase, and displayed primarily antagonistic interactions between these residues. Thus, differences in catalytic efficiency between dalcochinase and Abg are therefore not primarily due to differences in the residues that directly contact the substrate, but derive largely from contributions from more remote residues and the overall architecture of the active site.

  7. Stochastic epigenetic mutations (DNA methylation) increase exponentially in human aging and correlate with X chromosome inactivation skewing in females.

    PubMed

    Gentilini, Davide; Garagnani, Paolo; Pisoni, Serena; Bacalini, Maria Giulia; Calzari, Luciano; Mari, Daniela; Vitale, Giovanni; Franceschi, Claudio; Di Blasio, Anna Maria

    2015-08-01

    In this study we applied a new analytical strategy to investigate the relations between stochastic epigenetic mutations (SEMs) and aging. We analysed methylation levels through the Infinium HumanMethylation27 and HumanMethylation450 BeadChips in a population of 178 subjects ranging from 3 to 106 years. For each CpG probe, epimutated subjects were identified as the extreme outliers with methylation level exceeding three times interquartile ranges the first quartile (Q1-(3 x IQR)) or the third quartile (Q3+(3 x IQR)). We demonstrated that the number of SEMs was low in childhood and increased exponentially during aging. Using the HUMARA method, skewing of X chromosome inactivation (XCI) was evaluated in heterozygotes women. Multivariate analysis indicated a significant correlation between log(SEMs) and degree of XCI skewing after adjustment for age (β = 0.41; confidence interval: 0.14, 0.68; p-value = 0.0053). The PATH analysis tested the complete model containing the variables: skewing of XCI, age, log(SEMs) and overall CpG methylation. After adjusting for the number of epimutations we failed to confirm the well reported correlation between skewing of XCI and aging. This evidence might suggest that the known correlation between XCI skewing and aging could not be a direct association but mediated by the number of SEMs.

  8. SCN4A pore mutation pathogenetically contributes to autosomal dominant essential tremor and may increase susceptibility to epilepsy

    PubMed Central

    Bergareche, Alberto; Bednarz, Marcin; Sánchez, Elena; Krebs, Catharine E.; Ruiz-Martinez, Javier; De La Riva, Patricia; Makarov, Vladimir; Gorostidi, Ana; Jurkat-Rott, Karin; Marti-Masso, Jose Felix; Paisán-Ruiz, Coro

    2015-01-01

    Essential tremor (ET) is the most prevalent movement disorder, affecting millions of people in the USA. Although a positive family history is one of the most important risk factors for ET, the genetic causes of ET remain unknown. In an attempt to identify genetic causes for ET, we performed whole-exome sequencing analyses in a large Spanish family with ET, in which two patients also developed epilepsy. To further assess pathogenicity, site-directed mutagenesis, mouse and human brain expression analyses, and patch clamp techniques were performed. A disease-segregating mutation (p.Gly1537Ser) in the SCN4A gene was identified. Posterior functional analyses demonstrated that more rapid kinetics at near-threshold potentials altered ion selectivity and facilitated the conductance of both potassium and ammonium ions, which could contribute to tremor and increase susceptibility to epilepsy, respectively. In this report, for the first time, we associated the genetic variability of SCN4A with the development of essential tremor, which adds ET to the growing list of neurological channelopathies. PMID:26427606

  9. SCN4A pore mutation pathogenetically contributes to autosomal dominant essential tremor and may increase susceptibility to epilepsy.

    PubMed

    Bergareche, Alberto; Bednarz, Marcin; Sánchez, Elena; Krebs, Catharine E; Ruiz-Martinez, Javier; De La Riva, Patricia; Makarov, Vladimir; Gorostidi, Ana; Jurkat-Rott, Karin; Marti-Masso, Jose Felix; Paisán-Ruiz, Coro

    2015-12-15

    Essential tremor (ET) is the most prevalent movement disorder, affecting millions of people in the USA. Although a positive family history is one of the most important risk factors for ET, the genetic causes of ET remain unknown. In an attempt to identify genetic causes for ET, we performed whole-exome sequencing analyses in a large Spanish family with ET, in which two patients also developed epilepsy. To further assess pathogenicity, site-directed mutagenesis, mouse and human brain expression analyses, and patch clamp techniques were performed. A disease-segregating mutation (p.Gly1537Ser) in the SCN4A gene was identified. Posterior functional analyses demonstrated that more rapid kinetics at near-threshold potentials altered ion selectivity and facilitated the conductance of both potassium and ammonium ions, which could contribute to tremor and increase susceptibility to epilepsy, respectively. In this report, for the first time, we associated the genetic variability of SCN4A with the development of essential tremor, which adds ET to the growing list of neurological channelopathies.

  10. Environmentally Endemic Pseudomonas aeruginosa Strains with Mutations in lasR Are Associated with Increased Disease Severity in Corneal Ulcers

    PubMed Central

    Hammond, John H.; Hebert, Wesley P.; Naimie, Amanda; Ray, Kathryn; Van Gelder, Rachel D.; DiGiandomenico, Antonio; Lalitha, Prajna; Srinivasan, Muthiah; Acharya, Nisha R.; Lietman, Thomas; Hogan, Deborah A.

    2016-01-01

    ABSTRACT The Steroids for Corneal Ulcers Trial (SCUT) was a multicenter, international study of bacterial keratitis in which 101 Pseudomonas aeruginosa infections were treated. Twenty-two of 101 P. aeruginosa isolates collected had a colony morphology characteristic of a loss-of-function mutation in lasR, the gene encoding a quorum-sensing master regulator. Ulcers caused by these 22 strains were associated with larger areas of corneal opacification, worse vision, and a lower rate of vision recovery in response to treatment than ulcers caused by the other isolates. The lasR sequences from these isolates each contained one of three nonsynonymous substitutions, and these strains were deficient in production of LasR-regulated protease and rhamnolipids. Replacement of lasR with either of the two most common lasR alleles from the SCUT isolates was sufficient to decrease protease and rhamnolipid production in PA14. Loss of LasR function is associated with increased production of CupA fimbriae, and the LasR-defective isolates exhibited higher production of CupA fimbriae than LasR-intact isolates. Strains with the same lasR mutation were of the same multilocus sequence type, suggesting that LasR-deficient, environmental P. aeruginosa strains were endemic to the area, and infections caused by these strains were associated with worse patient outcomes in the SCUT study. (This study has been registered at ClinicalTrials.gov under registration no. NCT00324168.) IMPORTANCE The LasR transcription factor is an important regulator of quorum sensing in P. aeruginosa and positively controls multiple virulence-associated pathways. The emergence of strains with lasR loss-of-function alleles in chronic disease is well described and is thought to represent a specific adaptation to the host environment. However, the prevalence and virulence of these strains in acute infections remain unclear. This report describes observations revealing that lasR mutants were common among isolates from

  11. Six Novel Missense Mutations in the LDL Receptor-Related Protein 5 (LRP5) Gene in Different Conditions with an Increased Bone Density

    PubMed Central

    Van Wesenbeeck, Liesbeth; Cleiren, Erna; Gram, Jeppe; Beals, Rodney K.; Bénichou, Olivier; Scopelliti, Domenico; Key, Lyndon; Renton, Tara; Bartels, Cindy; Gong, Yaoqin; Warman, Matthew L.; de Vernejoul, Marie-Christine; Bollerslev, Jens; Van Hul, Wim

    2003-01-01

    Bone is a dynamic tissue that is subject to the balanced processes of bone formation and bone resorption. Imbalance can give rise to skeletal pathologies with increased bone density. In recent years, several genes underlying such sclerosing bone disorders have been identified. The LDL receptor-related protein 5 (LRP5) gene has been shown to be involved in both osteoporosis-pseudoglioma syndrome and the high–bone-mass phenotype and turned out to be an important regulator of peak bone mass in vertebrates. We performed mutation analysis of the LRP5 gene in 10 families or isolated patients with different conditions with an increased bone density, including endosteal hyperostosis, Van Buchem disease, autosomal dominant osteosclerosis, and osteopetrosis type I. Direct sequencing of the LRP5 gene revealed 19 sequence variants. Thirteen of these were confirmed as polymorphisms, but six novel missense mutations (D111Y, G171R, A214T, A214V, A242T, and T253I) are most likely disease causing. Like the previously reported mutation (G171V) that causes the high–bone-mass phenotype, all mutations are located in the aminoterminal part of the gene, before the first epidermal growth factor–like domain. These results indicate that, despite the different diagnoses that can be made, conditions with an increased bone density affecting mainly the cortices of the long bones and the skull are often caused by mutations in the LRP5 gene. Functional analysis of the effects of the various mutations will be of interest, to evaluate whether all the mutations give rise to the same pathogenic mechanism. PMID:12579474

  12. Cellular mechanisms underlying the increased disease severity seen for patients with long QT syndrome caused by compound mutations in KCNQ1.

    PubMed

    Harmer, Stephen C; Mohal, Jagdeep S; Royal, Alice A; McKenna, William J; Lambiase, Pier D; Tinker, Andrew

    2014-08-15

    The KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1) gene encodes the Kv7.1 potassium channel which forms a complex with KCNE1 (potassium voltage-gated channel Isk-related family member 1) in the human heart to produce the repolarizing IKs (slow delayed rectifier potassium current). Mutations in KCNQ1 can perturb IKs function and cause LQT1 (long QT syndrome type 1). In LQT1, compound mutations are relatively common and are associated with increased disease severity. LQT1 compound mutations have been shown to increase channel dysfunction, but whether other disease mechanisms, such as defective channel trafficking, contribute to the increase in arrhythmic risk has not been determined. Using an imaging-based assay we investigated the effects of four compound heterozygous mutations (V310I/R594Q, A341V/P127T, T391I/Q530X and A525T/R518X), one homozygous mutation (W248F) and one novel compound heterozygous mutation (A178T/K422fs39X) (where fs denotes frameshift) on channel trafficking. By analysing the effects in the equivalent of a homozygous, heterozygous and compound heterozygous condition, we identify three different types of behaviour. A341V/P127T and W248F/W248F had no effect, whereas V310I/R594Q had a moderate, but not compound, effect on channel trafficking. In contrast, T391I/Q530X, A525T/R518X and A178T/K422fs39X severely disrupted channel trafficking when expressed in compound form. In conclusion, we have characterized the disease mechanisms for six LQT1 compound mutations and report that, for four of these, defective channel trafficking underlies the severe clinical phenotype.

  13. Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation.

    PubMed

    Sant'Anna, Ricardo; Almeida, Maria Rosário; Varejāo, Nathalia; Gallego, Pablo; Esperante, Sebastian; Ferreira, Priscila; Pereira-Henriques, Alda; Palhano, Fernando L; de Carvalho, Mamede; Foguel, Debora; Reverter, David; Saraiva, Maria João; Ventura, Salvador

    2017-03-24

    More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies.

  14. Naturally occurring basal core promoter A1762T/G1764A dual mutations increase the risk of HBV-related hepatocellular carcinoma: a meta-analysis

    PubMed Central

    Lu, Yunfei; Xu, Qingnian; Tang, Bozong; Chen, Xiaorong

    2016-01-01

    Basal core promoter (BCP) A1762T/G1764A dual mutations in hepatocarcinogenesis remain controversial. Published studies up to June 1, 2015 investigating the frequency of A1762T/G1764A dual mutations from chronic hepatitis B virus (HBV) infection, including hepatocellular carcinoma (HCC), were systematically identified. A total of 10,240 patients with chronic HBV infection, including 3729 HCC cases, were included in 52 identified studies. HCC patients had a higher frequency of BCP A1762T/G1764A dual mutations compared with asymptomatic HBsAg carriers (ASC) and patients with chronic hepatitis B (CHB) and liver cirrhosis (LC) (OR = 5.59, P < 0.00001; OR = 2.87, P < 0.00001; OR = 1.55, P = 0.02, respectively). No statistically significant difference was observed in the frequency of A1762T/G1764A dual mutations in cirrhotic HCC versus non-cirrhotic HCC patients (OR = 2.06, P = 0.05). Chronic HBV-infected patients and HCC patients with genotype B had a significantly lower risk of A1762T/G1764A dual mutations compared with patients with genotype C (OR = 0.30, P < 0.0001 and OR = 0.34, P = 0.04, respectively). In HBV genotype C subjects, A1762T/G1764A dual mutations contributed to significantly higher risk for HCC developing compared with non-mutation ones (OR = 3.47, P < 0.00001). In conclusion, A1762T/G1764A dual mutations increase the risk of HBV-related hepatocellular carcinoma, particularly in an HBV genotype C population, even without progression to cirrhosis. PMID:26848866

  15. A naturally occurring mutation in the human androgen receptor of a subject with complete androgen insensitivity confers binding and transactivation by estradiol.

    PubMed

    Bonagura, Thomas W; Deng, Min; Brown, Terry R

    2007-01-15

    The clinical phenotype of complete androgen insensitivity (CAIS) was associated with a mutation in the human androgen receptor (hAR) gene encoding the amino acid substitution, M745I, in the hAR protein. Transcriptional activation of hAR(M745I) by the synthetic androgen, methyltrienolone (R1881), was reduced compared to wild-type (wt) hAR. The transcriptional co-activator, androgen receptor associated protein 70 (ARA70), failed to enhance transactivation of hAR(M745I) at lower concentrations of R1881 (0.01-0.1 nM), whereas the p160 co-activators, SRC-1 and TIF2, stimulated activity. Transcriptional activity of hAR(M745I) was stimulated by 1 or 10 nM R1881 and activity was further enhanced by co-expression of ARA70 similar to that of the hAR(wt). Transcriptional activity of hAR(wt) was minimally stimulated by estradiol (E2) without or with co-expression of ARA70, whereas 10 or 100 nM E2 increased transactivation by hAR(M745I) of the androgen-responsive MMTV-luciferase reporter gene by 10-fold and activity was further enhanced by ARA70. Increasing concentrations of E2 competed more effectively for binding of R1881 to hAR(M745I) than to hAR(wt), indicative of the preferential binding of E2 to the mutant hAR. Partial tryptic digestion of hAR wt and M745I revealed that activation of the mutant protein was reduced in the presence of R1881. By contrast, tryptic digestion showed that the mutant hAR was activated by the binding of E2. In conclusion, the clinical phenotype of CAIS resulted from a hAR gene mutation encoding hAR(M745I) with reduced binding and transactivation by androgens, but the novel properties of enhanced affinity for and increased transactivation by estradiol.

  16. Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

    PubMed Central

    Shima, J; Hesketh, A; Okamoto, S; Kawamoto, S; Ochi, K

    1996-01-01

    A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery. PMID:8955413

  17. Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli

    PubMed Central

    Lee, Jae Taek; Lee, Seung Sik; Mondal, Suvendu; Tripathi, Bhumi Nath; Kim, Siu; Lee, Keun Woo; Hong, Sung Hyun; Bai, Hyoung-Woo; Cho, Jae-Young; Chung, Byung Yeoup

    2016-01-01

    Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of Ser78 to Cys78 resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of Cys78 in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone. PMID:27457208

  18. Cross-resistance patterns to acetolactate synthase (ALS)-inhibiting herbicides of flixweed (Descurainia sophia L.) conferred by different combinations of ALS isozymes with a Pro-197-Thr mutation or a novel Trp-574-Leu mutation.

    PubMed

    Deng, Wei; Yang, Qian; Zhang, Yongzhi; Jiao, Hongtao; Mei, Yu; Li, Xuefeng; Zheng, Mingqi

    2017-03-01

    Acetolactate synthase (ALS) is the common target of ALS-inhibiting herbicides, and target-site ALS mutations are the main mechanism of resistance to ALS-inhibiting herbicides. In this study, ALS1 and ALS2 genes with full lengths of 2004bp and 1998bp respectively were cloned in individual plants of susceptible (S) or resistant (R) flixweed (Descurainia sophia L.) populations. Two ALS mutations of Pro-197-Thr and/or Trp-574-Leu were identified in plants of three R biotypes (HB24, HB30 and HB42). In order to investigate the function of ALS isozymes in ALS-inhibiting herbicide resistance, pHB24 (a Pro-197-Thr mutation in ALS1 and a wild type ALS2), pHB42 (a Trp-574-Leu mutation in ALS1 and a wild type ALS2) and pHB30 (a Trp-574-Leu mutation in ALS1 and a Pro-197-Thr mutation in ALS2) subpopulations individually homozygous for different ALS mutations were generated. Individuals of pHB30 had mutations in each isozyme of ALS and had higher resistance than pHB24 and pHB42 populations containing mutations in only one ALS isozyme. Moreover, the pHB24 had resistance to SU, TP and SCT herbicides, whereas pHB24 and pHB42 had resistance to these classes of herbicides as well as IMI and PTB herbicides. The sensitivity of isolated ALS enzyme to inhibition by herbicides in these populations correlated with whole plant resistance levels. Therefore, reduced ALS sensitivity resulting from the mutations in ALS was responsible for resistance to ALS-inhibiting herbicides in flixweed.

  19. Mutational signatures associated with tobacco smoking in human cancer.

    PubMed

    Alexandrov, Ludmil B; Ju, Young Seok; Haase, Kerstin; Van Loo, Peter; Martincorena, Iñigo; Nik-Zainal, Serena; Totoki, Yasushi; Fujimoto, Akihiro; Nakagawa, Hidewaki; Shibata, Tatsuhiro; Campbell, Peter J; Vineis, Paolo; Phillips, David H; Stratton, Michael R

    2016-11-04

    Tobacco smoking increases the risk of at least 17 classes of human cancer. We analyzed somatic mutations and DNA methylation in 5243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA editing by APOBEC cytidine deaminases and of an endogenous clocklike mutational process. Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.

  20. Mutational signatures associated with tobacco smoking in human cancer

    DOE PAGES

    Alexandrov, Ludmil B.; Ju, Young Seok; Haase, Kerstin; ...

    2016-11-04

    Tobacco smoking increases the risk of at least 17 classes of cancer. Here, we analyzed somatic mutations and DNA methylation in 5,243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA edi ting by APOBEC cytidine deaminases and of an endogenous clock-like mutational process.more » Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.« less

  1. Mutational signatures associated with tobacco smoking in human cancer

    SciTech Connect

    Alexandrov, Ludmil B.; Ju, Young Seok; Haase, Kerstin; Van Loo, Peter; Martincorena, Inigo; Nik-Zainal, Serena; Totoki, Yasushi; Fujimoto, Akihiro; Nakagawa, Hidewaki; Shibata, Tatsuhiro; Campbell, Peter J.; Vineis, Paolo; Phillips, David H.; Stratton, Michael R.

    2016-11-04

    Tobacco smoking increases the risk of at least 17 classes of cancer. Here, we analyzed somatic mutations and DNA methylation in 5,243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA edi ting by APOBEC cytidine deaminases and of an endogenous clock-like mutational process. Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.

  2. A Point Mutation in DNA Polymerase β (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer

    PubMed Central

    Tan, Xiaohui; Wu, Xiaoling; Ren, Shuyang; Wang, Hongyi; Li, Zhongwu; Alshenawy, Weaam; Li, Wenmei; Cui, Jiantao; Luo, Guangbin; Siegel, Robert S.; Fu, Sidney W.; Lu, Youyong

    2016-01-01

    Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC. PMID:27471563

  3. Structural insight with mutational impact on tyrosinase and PKC-β interaction from Homo sapiens: Molecular modeling and docking studies for melanogenesis, albinism and increased risk for melanoma.

    PubMed

    Banerjee, Arundhati; Ray, Sujay

    2016-10-30

    Human tyrosinase, is an important protein for biosynthetic pathway of melanin. It was studied to be phosphorylated and activated by protein kinase-C, β-subunit (PKC-β) through earlier experimentations with in vivo evidences. Documentation documents that mutation in two essentially vital serine residues in C-terminal end of tyrosinase leads to albinism. Due to the deficiency of protective shield like enzyme; melanin, albinos are at an increased peril for melanoma and other skin cancers. So, computational and residue-level insight including a mutational exploration with evolutionary importance into this mechanism lies obligatory for future pathological and therapeutic developments. Therefore, functional tertiary models of the relevant proteins were analyzed after satisfying their stereo-chemical features. Evolutionarily paramount residues for the activation of tyrosinase were perceived via multiple sequence alignment phenomena. Mutant-type tyrosinase protein (S98A and S102A) was thereby modeled, maintaining the wild-type proteins' functionality. Furthermore, this present comparative study discloses the variation in the stable residual participation (for mutant-type and wild-type tyrosinase-PKCβ complex). Mainly, an increased number of polar negatively charged residues from the wild-type tyrosinase participated with PKC-β, predominantly. Fascinatingly supported by evaluation of statistical significances, mutation even led to a destabilizing impact in tyrosinase accompanied by conformational switches with a helix-to-coil transition in the mutated protein. Even the allosteric sites in the protein got poorly hampered upon mutation leading to weaker tendency for binding partners to interact.

  4. A Single Point Mutation within the Coding Sequence of Cholera Toxin B Subunit Will Increase Its Expression Yield

    PubMed Central

    Bakhshi, Bita; Boustanshenas, Mina; Ghorbani, Masoud

    2014-01-01

    Background: Cholera toxin B subunit (CTB) has been extensively considered as an immunogenic and adjuvant protein, but its yield of expression is not satisfactory in many studies. The aim of this study was to compare the expression of native and mutant recombinant CTB (rCTB) in pQE vector. Methods: ctxB fragment from Vibrio cholerae O1 ATCC14035 containing the substitution of mutant ctxB for amino acid S128T was amplified by PCR and cloned in pGETM-T easy vector. It was then transformed to E. coli Top 10F' and cultured on LB agar plate containing ampicillin. Sequence analysis confirmed the mature ctxB gene sequence and the mutant one in both constructs which were further subcloned to pQE-30 vector. Both constructs were subsequently transformed to E. coli M15 (pREP4) for expression of mature and mutant rCTB. Results: SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 hours after induction and Western-blot analysis confirmed the presence of rCTB in blotting membranes. The expression of mutant rCTB was much higher than mature rCTB, which may be the result of serine-to-threonine substitution at position 128 of mature rCTB amino acid sequence created by PCR mutagenesis. The mutant rCTB retained pentameric stability and its ability to bind to anti- cholera toxin IgG antibodies. Conclusion: Point mutation in ctxB sequence resulted in over-expression of rCTB, probably due to the increase of solubility of produced rCTB. Consequently, this expression system can be used to produce rCTB in high yield. PMID:24842138

  5. Use of a LightCycler gyrA Mutation Assay for Rapid Identification of Mutations Conferring Decreased Susceptibility to Ciprofloxacin in Multiresistant Salmonella enterica Serotype Typhimurium DT104 Isolates

    PubMed Central

    Walker, R. A.; Saunders, N.; Lawson, A. J.; Lindsay, E. A.; Dassama, M.; Ward, L. R.; Woodward, M. J.; Davies, R. H.; Liebana, E.; Threlfall, E. J.

    2001-01-01

    A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC→AAC) mutation, an Asp-87-to-Gly (GAC→GGC) mutation, and a Ser-83-to-Phe (TCC→TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC→TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC→TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104. PMID:11283069

  6. Multiple mutations and increased RNA expression in tetracycline-resistant Streptococcus pneumoniae as determined by genome-wide DNA and mRNA sequencing

    PubMed Central

    Lupien, Andréanne; Gingras, Hélène; Bergeron, Michel G.; Leprohon, Philippe; Ouellette, Marc

    2015-01-01

    Objectives The objective of this study was to characterize chromosomal mutations associated with resistance to tetracycline in Streptococcus pneumoniae. Methods Chronological appearance of mutations in two S. pneumoniae R6 mutants (R6M1TC-5 and R6M2TC-4) selected for resistance to tetracycline was determined by next-generation sequencing. A role for the mutations identified was confirmed by reconstructing resistance to tetracycline in a S. pneumoniae R6 WT background. RNA sequencing was performed on R6M1TC-5 and R6M2TC-4 and the relative expression of genes was reported according to R6. Differentially expressed genes were classified according to their ontology. Results WGS of R6M1TC-5 and R6M2TC-4 revealed mutations in the gene rpsJ coding for the ribosomal protein S10 and in the promoter region and coding sequences of the ABC genes patA and patB. These cells were cross-resistant to ciprofloxacin. Resistance reconstruction confirmed a role in resistance for the mutations in rpsJ and patA. Overexpression of the ABC transporter PatA/PatB or mutations in the coding sequence of patA contributed to resistance to tetracycline, ciprofloxacin and ethidium bromide, and was associated with a decreased accumulation of [3H]tetracycline. Comparative transcriptome profiling of the resistant mutants further revealed that, in addition to the overexpression of patA and patB, several genes of the thiamine biosynthesis and salvage pathway were increased in the two mutants, but also in clinical isolates resistant to tetracycline. This overexpression most likely contributes to the tetracycline resistance phenotype. Conclusions The combination of genomic and transcriptomic analysis coupled to functional studies has allowed the discovery of novel tetracycline resistance mutations in S. pneumoniae. PMID:25862682

  7. BRAF Mutation Correlates With High-Risk Langerhans Cell Histiocytosis and Increased Resistance to First-Line Therapy

    PubMed Central

    Emile, Jean-François; Barkaoui, Mohamed-Aziz; Thomas, Caroline; Fraitag, Sylvie; Boudjemaa, Sabah; Renaud, Florence; Moreau, Anne; Peuchmaur, Michel; Chassagne-Clément, Catherine; Dijoud, Frédérique; Rigau, Valérie; Moshous, Despina; Lambilliotte, Anne; Mazingue, Françoise; Kebaili, Kamila; Miron, Jean; Jeziorski, Eric; Plat, Geneviève; Aladjidi, Nathalie; Ferster, Alina; Pacquement, Hélène; Galambrun, Claire; Brugières, Laurence; Leverger, Guy; Mansuy, Ludovic; Paillard, Catherine; Deville, Anne; Armari-Alla, Corinne; Lutun, Anne; Gillibert-Yvert, Marion; Stephan, Jean-Louis; Cohen-Aubart, Fleur; Haroche, Julien; Pellier, Isabelle; Millot, Frédéric; Lescoeur, Brigitte; Gandemer, Virginie; Bodemer, Christine; Lacave, Roger; Hélias-Rodzewicz, Zofia; Taly, Valérie; Geissmann, Frédéric; Donadieu, Jean

    2016-01-01

    Purpose Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia with a broad spectrum of clinical manifestations and outcomes in children. The somatic BRAFV600E mutation occurs frequently, but clinical significance remains to be determined. Patients and Methods BRAFV600E mutation was investigated in a French LCH cohort. We analyzed associations between mutation status and clinical presentation, extent of disease, reactivation rate, response to therapy, and long-term permanent sequelae. Results Among 315 patients with successfully determined BRAF status, 173 (54.6%) carried a BRAFV600E mutation. Patients with BRAFV600E manifested more severe disease than did those with wild-type BRAF. Patients with BRAFV600E comprised 87.8% of patients (43 of 49) with multisystem LCH with risk organ involvement (liver, spleen, hematology), 68.6% of patients (35 of 51) with multisystem LCH without risk organ involvement, 43.9% of patients (86 of 196) with single-system LCH, and 42.1% of patients (8 of 19) with lung-involved LCH (P < .001). BRAFV600E mutation was also associated with organ involvement that could lead to permanent, irreversible damage, such as neurologic (75%) and pituitary (72.9%) injuries. Compared with patients with wild-type BRAF, patients with BRAFV600E more commonly displayed resistance to combined vinblastine and corticosteroid therapy (21.9% v 3.3%; P = .001), showed a higher reactivation rate (5-year reactivation rate, 42.8% v 28.1%; P = .006), and had more permanent, long-term consequences from disease or treatment (27.9% v 12.6%; P = .001). Conclusion In children with LCH, BRAFV600E mutation was associated with high-risk features, permanent injury, and poor short-term response to chemotherapy. Further population-based studies should be undertaken to confirm our observations and to assess the impact of BRAF inhibitors for this subgroup of patients who may benefit from targeted therapy. PMID:27382093

  8. Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

    PubMed Central

    Deuzing, Ilona P.; Charpentier, Charlotte; Wright, David W.; Matheron, Sophie; Paton, Jack; Frentz, Dineke; van de Vijver, David A.; Coveney, Peter V.; Descamps, Diane; Boucher, Charles A. B.

    2014-01-01

    ABSTRACT Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity. IMPORTANCE Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug

  9. Increased yield of actionable mutations using multi-gene panels to assess hereditary cancer susceptibility in an ethnically diverse clinical cohort.

    PubMed

    Ricker, Charité; Culver, Julie O; Lowstuter, Katrina; Sturgeon, Duveen; Sturgeon, Julia D; Chanock, Christopher R; Gauderman, William J; McDonnell, Kevin J; Idos, Gregory E; Gruber, Stephen B

    2016-04-01

    This study aims to assess multi-gene panel testing in an ethnically diverse clinical cancer genetics practice. We conducted a retrospective study of individuals with a personal or family history of cancer undergoing clinically indicated multi-gene panel tests of 6-110 genes, from six commercial laboratories. The 475 patients in the study included 228 Hispanics (47.6%), 166 non-Hispanic Whites (35.4%), 55 Asians (11.6%), 19 Blacks (4.0%), and seven others (1.5%). Panel testing found that 15.6% (74/475) of patients carried deleterious mutations for a total of 79 mutations identified. This included 7.4% (35/475) of patients who had a mutation identified that would not have been tested with a gene-by-gene approach. The identification of a panel-added mutation impacted clinical management for most of cases (69%, 24/35), and genetic testing was recommended for the first degree relatives of nearly all of them (91%, 32/35). Variants of uncertain significance (VUSs) were identified in a higher proportion of tests performed in ethnic minorities. Multi-gene panel testing increases the yield of mutations detected and adds to the capability of providing individualized cancer risk assessment. VUSs represent an interpretive challenge due to less data available outside of White, non-Hispanic populations. Further studies are necessary to expand understanding of the implementation and utilization of panels across broad clinical settings and patient populations.

  10. Heterozygous carriers of the I171V mutation of the NBS1 gene have a significantly increased risk of solid malignant tumours.

    PubMed

    Nowak, Jerzy; Mosor, Maria; Ziółkowska, Iwona; Wierzbicka, Malgorzta; Pernak-Schwarz, Monika; Przyborska, Marta; Roznowski, Krzysztof; Pławski, Andrzej; Słomski, Ryszard; Januszkiewicz, Danuta

    2008-03-01

    Homozygous mutation 657del5 within the NBS1 gene is responsible for the majority of Nijmegen breakage syndrome (NBS) cases. NBS patients are characterised by increased susceptibility to malignancies mainly of lymphoid origin. Recently it has been postulated that heterozygous carriers of 657del5 NBS1 mutation are at higher risk of cancer development. The aim of the study was to analyse the frequency of I171V mutation in NBS1 gene in 270 women with breast cancer, 176 patients with larynx cancer, 81 with second primary tumours of head and neck, 131 with colorectal carcinoma and 600 healthy individuals. I171V mutation was present in 17 cancer patients compared with only one in healthy individuals. This constitutes 2.58% in studied patients with malignancies and 0.17% in the control group (P=0.0002; relative risk 1.827; odds ratio 15.886; 95% confidence interval 2.107-119.8). Since DNA was isolated from non malignant cells, all mutations found in cancer patients appeared to be of germinal origin. It can be concluded that NBS1 allele I171V may be a general susceptibility gene in solid tumours.

  11. Phenocopy breast cancer rates in Israeli BRCA1 BRCA2 mutation carrier families: is the risk increased in non-carriers?

    PubMed

    Bernholtz, Shiri; Laitman, Yael; Kaufman, Bella; Shimon-Paluch, Shnai; Friedman, Eitan

    2012-04-01

    BRCA1 and BRCA2 mutation carriers have an increased risk for developing breast (and ovarian) cancer. Non-carriers from within such families (=true negatives) are counseled that their risk for developing breast cancer is similar to that of the average-risk population. Breast cancer diagnosed in a non-carrier from a family with a known mutation is coined phenocopy. The rate of breast cancer phenocopy and the risk for breast cancer in true negatives are unsettled. The rate of phenocopy breast cancer was assessed in non-carriers from Jewish families with a BRCA1 or BRCA2 mutation, identified at the Sheba medical center. Analysis was performed by t test for comparison of mean age at counseling or breast cancer diagnosis, and by calculating a standardized incidence ratio (SIR). Overall, 1318 females from 884 mutation carrying families (620 with BRCA1 264 with BRCA2 mutations) were genotyped, of whom 307 women from 245 families were assigned a true negative status (mean age at counseling 43.01 ± 13.03 years (range 19.7-92.8 years). Of these true negatives, 20 women (6.51-2.26% of families) developed breast cancer at a mean age of 54.1 ± 12.9 years (range 48.1 -60.1 years). The SIR for breast cancer in true negatives was not significantly different than the expected in the average-risk Israeli population [observed 20-expected 23.8 cases SIR = 0.84, 95% CI (0.51, 1.30)]. The rate of phenocopy breast cancer in non-carriers from Israeli BRCA1 BRCA2 mutation carrier families is 2.26% with no increased breast cancer risk over the average-risk population.

  12. Overexpression of the yeast transcription activator Msn2 confers furfural resistance and increases the initial fermentation rate in ethanol production.

    PubMed

    Sasano, Yu; Watanabe, Daisuke; Ukibe, Ken; Inai, Tomomi; Ohtsu, Iwao; Shimoi, Hitoshi; Takagi, Hiroshi

    2012-04-01

    Lignocellulosic biomass is a promising source for bioethanol production, because it is abundant worldwide and has few competing uses. However, the treatment of lignocelllulosic biomass with weak acid to release cellulose and hemicellulose generates many kinds of byproducts including furfural and 5-hydroxymethylfurfural, which inhibit fermentation by yeast, because they generate reactive oxygen species (ROS) in cells. In order to acquire high tolerance to oxidative stress in bioethanol yeast strains, we focused on the transcription activator Msn2 of Saccharomyces cerevisiae, which regulates numerous genes involved in antioxidative stress responses, and constructed bioethanol yeast strains that overexpress Msn2 constitutively. The Msn2-overexpressing bioethanol strains showed tolerance to oxidative stress, probably due to the high-level expression of various antioxidant enzyme genes. Unexpectedly, these strains showed ethanol sensitivity compared with the control strain, probably due to imbalance of the expression level between Msn2 and Msn4. In the presence of furfural, the engineered strains exhibited reduced intracellular ROS levels, and showed rapid growth compared with the control strain. The fermentation test in the presence of furfural revealed that the Msn2-overexpressing strains showed improvement of the initial rate of fermentation. Our results indicate that overexpression of the transcription activator Msn2 in bioethanol yeast strains confers furfural tolerance by reducing the intracellular ROS levels and enhances the initial rate of fermentation in the presence of furfural, suggesting that these strains are capable of adapting rapidly to various compounds that inhibit fermentation by inducing ROS accumulation. Our results not only promise to improve bioethanol production from lignocellulosic biomass, but also provide novel insights for molecular breeding of industrial yeast strains.

  13. Disrupted nitric oxide signaling due to GUCY1A3 mutations increases risk for moyamoya disease, achalasia and hypertension.

    PubMed

    Wallace, S; Guo, D-C; Regalado, E; Mellor-Crummey, L; Bamshad, M; Nickerson, D A; Dauser, R; Hanchard, N; Marom, R; Martin, E; Berka, V; Sharina, I; Ganesan, V; Saunders, D; Morris, S A; Milewicz, D M

    2016-10-01

    Moyamoya disease (MMD) is a progressive vasculopathy characterized by occlusion of the terminal portion of the internal carotid arteries and its branches, and the formation of compensatory moyamoya collateral vessels. Homozygous mutations in GUCY1A3 have been reported as a cause of MMD and achalasia. Probands (n = 96) from unrelated families underwent sequencing of GUCY1A3. Functional studies were performed to confirm the pathogenicity of identified GUCY1A3 variants. Two affected individuals from the unrelated families were found to have compound heterozygous mutations in GUCY1A3. MM041 was diagnosed with achalasia at 4 years of age, hypertension and MMD at 18 years of age. MM149 was diagnosed with MMD and hypertension at the age of 20 months. Both individuals carry one allele that is predicted to lead to haploinsufficiency and a second allele that is predicted to produce a mutated protein. Biochemical studies of one of these alleles, GUCY1A3 Cys517Tyr, showed that the mutant protein (a subunit of soluble guanylate cyclase) has a significantly blunted signaling response with exposure to nitric oxide (NO). GUCY1A3 missense and haploinsufficiency mutations disrupt NO signaling leading to MMD and hypertension, with or without achalasia.

  14. Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation

    PubMed Central

    Sant’Anna, Ricardo; Almeida, Maria Rosário; Varejāo, Nathalia; Gallego, Pablo; Esperante, Sebastian; Ferreira, Priscila; Pereira-Henriques, Alda; Palhano, Fernando L.; de Carvalho, Mamede; Foguel, Debora; Reverter, David; Saraiva, Maria João; Ventura, Salvador

    2017-01-01

    More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies. PMID:28338000

  15. Mutations in PIK3CD Can Cause Hyper IgM Syndrome (HIGM) Associated with Increased Cancer Susceptibility

    PubMed Central

    Crank, M. C.; Grossman, J. K.; Moir, S.; Pittaluga, S.; Buckner, C. M.; Kardava, L.; Agharahimi, A.; Meuwissen, H.; Stoddard, J.; Niemela, J.; Kuehn, H.

    2014-01-01

    Autosomal dominant gain of function mutations in the gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, CMV and/or EBV viremia and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. Here we describe three patients in two families who were diagnosed with HIGM at a young age and were recently found to carry heterozygous mutations in PIK3CD. These patients had an abnormal circulating B cell distribution featuring a preponderance of early transitional (T1) B cells and plasmablasts. When stimulated in vitro, PIK3CD mutated B cells were able to secrete class-switched immunoglobulins. This finding implies that the patients’ elevated serum IgM levels were unlikely a product of an intrinsic B cell functional inability to class switch. All three patients developed malignant lymphoproliferative syndromes that were not associated with EBV. Thus, we identified a novel subset of patients with PIK3CD mutations associated with HIGM, despite indications of preserved in vitro B cell class switch recombination, as well as susceptibility to non-EBV-associated malignancies. PMID:24610295

  16. The (95)(Δ)G mutation in the 5'untranslated region of the norA gene increases efflux activity in Staphylococcus epidermidis isolates.

    PubMed

    García-Gómez, Elizabeth; Jaso-Vera, Marcos E; Juárez-Verdayes, Marco A; Alcántar-Curiel, María D; Zenteno, Juan C; Betanzos-Cabrera, Gabriel; Peralta, Humberto; Rodríguez-Martínez, Sandra; Cancino-Díaz, Mario E; Jan-Roblero, Janet; Cancino-Diaz, Juan C

    2017-02-01

    In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 μg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR (95)(Δ)G mutation. Isolates carrying the (95)(Δ)G mutation had higher levels of norA expression compared with those that did not. To corroborate that the (95)(Δ)G mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro. This strain also presented the (95)(Δ)G mutation and had a high level of norA expression and EF, indicating that the (95)(Δ)G mutation is important for the HEA phenotype. The (95)(Δ)G mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5'UTR (95)(Δ)G mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene.

  17. [Afatinib as first-line therapy in mutation-positive EGFR. Results by type of mutation].

    PubMed

    Vidal, Óscar Juan

    2016-04-01

    The discovery of endothelial growth factor receptor (EGFR) mutations has laid the foundations for personalized medicine in non-small cell lung carcinoma (NSCLC). In phase III trials, the first-generation tyrosine kinase inhibitors (TKI), gefitinib and erlotinib, demonstrated greater efficacy compared with chemotherapy in patients with EGFR mutations, achieving progression-free survival of 8-13.5 months. Afatinib, a second-generation irreversible pan-ErbB inhibitor, is the first TKI that has shown a benefit in overall survival (OS) compared with chemotherapy in EGFR mutation-positive NSCLC when used as first-line treatment. Exon 19 deletion (Del19) and the single-point substitution mutation (L858R) in exon 21, called activating mutations due to their ability to confer sensitivity to TKI, represent approximately 90% of the EGFR mutations in NSCLC. Distinct sensitivity to TKI has been observed depending on the type of mutation, with greater progression-free survival in patients with the Del19 mutation. The analysis of OS in the LUX-Lung 3 and LUX-Lung 6 trials showed a statistically significant increase in survival in afatinib-treated patients with the Del 19 mutation, but no significant increase in that of patients with the L858R mutation. Direct comparison of afatinib and gefitinib as first-line therapy (LUX-Lung 7 trial) showed a statistically-significant increase in progression-free survival (hazard ratio: 0.73; 95% confidence interval, 0.57-0.95; p=0.0165) with afatinib. In the analysis by type of mutation, this benefit was observed for both the Del19 and the L858R mutations.

  18. PARP inhibitor increases chemosensitivity by upregulating miR-664b-5p in BRCA1-mutated triple-negative breast cancer

    PubMed Central

    Song, Wei; Tang, Lin; Xu, Yumei; Xu, Jing; Zhang, Wenwen; Xie, Hui; Wang, Shui; Guan, Xiaoxiang

    2017-01-01

    Emerging evidence has shown that adding poly(ADP-ribose) polymerase (PARP) inhibitors to chemotherapy regimens is superior to the control regimens alone in BRCA1-mutated triple-negative breast cancer (TNBC) patients, but their underlying mechanisms have not been fully elucidated. In this study, using miRNA microarray analysis of two BRCA1-mutated TNBC cell lines, we found that miR-664b-5p expression was increased after adding a PARP inhibitor, olaparib, to a carboplatin (CBP) plus gemcitabine (GEM) therapy regimen. Functional assays showed miR-664b-5p overexpression inhibited proliferation, migration and invasion in BRCA1-mutated TNBC cells. CCNE2 was identified as a novel functional target of miR-664b-5p, and CCNE2 knockdown revealed effects similar to those observed with miR-664b-5p overexpression. Both CCNE2 knockdown and miR-664b-5p overexpression significantly increased the chemosensitivity of BRCA1-mutated TNBC cells. In addition, in vivo studies indicated that miR-664b-5p inhibited tumour growth compared with the control in tumour xenograft models, and we also found that CCNE2 expression was inversely correlated with miR-664b-5p expression in 90 TNBC patient samples. In conclusion, miR-664b-5p functions as a tumour suppressor and has an important role in the regulation of PARP inhibitors to increase chemosensitivity by targeting CCNE2. This may be one of the possible mechanisms by which PARP inhibitors increase chemosensitivity in BRCA1-mutated TNBC. PMID:28176879

  19. PARP inhibitor increases chemosensitivity by upregulating miR-664b-5p in BRCA1-mutated triple-negative breast cancer.

    PubMed

    Song, Wei; Tang, Lin; Xu, Yumei; Xu, Jing; Zhang, Wenwen; Xie, Hui; Wang, Shui; Guan, Xiaoxiang

    2017-02-08

    Emerging evidence has shown that adding poly(ADP-ribose) polymerase (PARP) inhibitors to chemotherapy regimens is superior to the control regimens alone in BRCA1-mutated triple-negative breast cancer (TNBC) patients, but their underlying mechanisms have not been fully elucidated. In this study, using miRNA microarray analysis of two BRCA1-mutated TNBC cell lines, we found that miR-664b-5p expression was increased after adding a PARP inhibitor, olaparib, to a carboplatin (CBP) plus gemcitabine (GEM) therapy regimen. Functional assays showed miR-664b-5p overexpression inhibited proliferation, migration and invasion in BRCA1-mutated TNBC cells. CCNE2 was identified as a novel functional target of miR-664b-5p, and CCNE2 knockdown revealed effects similar to those observed with miR-664b-5p overexpression. Both CCNE2 knockdown and miR-664b-5p overexpression significantly increased the chemosensitivity of BRCA1-mutated TNBC cells. In addition, in vivo studies indicated that miR-664b-5p inhibited tumour growth compared with the control in tumour xenograft models, and we also found that CCNE2 expression was inversely correlated with miR-664b-5p expression in 90 TNBC patient samples. In conclusion, miR-664b-5p functions as a tumour suppressor and has an important role in the regulation of PARP inhibitors to increase chemosensitivity by targeting CCNE2. This may be one of the possible mechanisms by which PARP inhibitors increase chemosensitivity in BRCA1-mutated TNBC.

  20. Myh11(R247C/R247C) mutations increase thoracic aorta vulnerability to intramural damage despite a general biomechanical adaptivity.

    PubMed

    Bellini, Chiara; Wang, Shanzhi; Milewicz, Dianna M; Humphrey, Jay D

    2015-01-02

    Genetic studies in patients reveal that mutations to genes that encode contractile proteins in medial smooth muscle cells can cause thoracic aortic aneurysms and dissections. Mouse models of such mutations, including Acta2(-/-) and Myh11(R247C/R247C), surprisingly do not present with any severe vascular phenotype under normal conditions. This observation raises the question whether these mutations nevertheless render the thoracic aorta increasingly vulnerable to aneurysms or dissections in the presence of additional, epigenetic, factors such as hypertension, a known risk factor for thoracic aortic disease. Accordingly, we compared the structure and biaxial mechanical properties of the ascending and descending thoracic aorta from male wild-type and Myh11(R247C/R247C) mice under normotension and induced hypertension. On average, the mutant aortas exhibited near normal biomechanics under normotensive hemodynamics and near normal adaptations to hypertensive hemodynamics, yet the latter led to intramural delaminations or premature deaths in over 20% of these mice. Moreover, the delaminated vessels exhibited localized pools of mucoid material, similar to the common histopathologic characteristic observed in aortas from humans affected by thoracic aortic aneurysms and dissections. The present findings suggest, therefore, that mutations to smooth muscle cell contractile proteins may place the thoracic aorta at increased risk to epigenetic factors and that there is a need to focus on focal, not global, changes in aortic structure and properties, including the pooling of glycosaminoglycans/proteoglycans that may lead to thoracic aortic dissection.

  1. Myh11R247C/R247C Mutations Increase Thoracic Aorta Vulnerability to Intramural Damage Despite a General Biomechanical Adaptivity

    PubMed Central

    Bellini, Chiara; Wang, Shanzhi; Milewicz, Dianna M.; Humphrey, Jay D.

    2014-01-01

    Genetic studies in patients reveal that mutations to genes that encode contractile proteins in medial smooth muscle cells can cause thoracic aortic aneurysms and dissections. Mouse models of such mutations, including Acta2−/− and Myh11 R247C/R247C, surprisingly do not present with any severe vascular phenotype under normal conditions. This observation raises the question whether these mutations nevertheless render the thoracic aorta increasingly vulnerable to aneurysms or dissections in the presence of additional, epigenetic, factors such as hypertension, a known risk factor for thoracic aortic disease. Accordingly, we compared the structure and biaxial mechanical properties of the ascending and descending thoracic aorta from male wild-type and Myh11 R247C/R247C mice under normotension and induced hypertension. On average, the mutant aortas exhibited near normal biomechanics under normotensive hemodynamics and near normal adaptations to hypertensive hemodynamics, yet the latter led to intramural delaminations or premature deaths in over 20 percent of these mice. Moreover, the delaminated vessels exhibited localized pools of mucoid material, similar to the common histopathologic characteristic observed in aortas from humans affected by thoracic aortic aneurysms and dissections. The present findings suggest, therefore, that mutations to smooth muscle cell contractile proteins may place the thoracic aorta at increased risk to epigenetic factors and that there is a need to focus on focal, not global, changes in aortic structure and properties, including the pooling of glycosaminoglycans / proteoglycans that may lead to thoracic aortic dissection. PMID:25433566

  2. T394A Mutation at the μ Opioid Receptor Blocks Opioid Tolerance and Increases Vulnerability to Heroin Self-Administration in Mice.

    PubMed

    Wang, Xiao-Fei; Barbier, Elisabeth; Chiu, Yi-Ting; He, Yi; Zhan, Jia; Bi, Guo-Hua; Zhang, Hai-Ying; Feng, Bo; Liu-Chen, Lee-Yuan; Wang, Jia Bei; Xi, Zheng-Xiong

    2016-10-05

    The etiology and pathophysiology underlying opioid tolerance and dependence are still unknown. Because mu opioid receptor (MOR) plays an essential role in opioid action, many vulnerability-related studies have focused on single nucleotide polymorphisms of MOR, particularly on A118G. In this study, we found that a single-point mutation at the MOR T394 phosphorylation site could be another important susceptive factor in the development of opioid tolerance and dependence in mice. T394A mutation, in which a threonine at 394 was replaced by an alanine, did not alter agonist binding to MOR and opioid analgesia, but resulted in loss of etorphine-induced MOR internalization in spinal dorsal horn neurons and opioid analgesic tolerance induced by either morphine or etorphine. In addition, this mutation also caused an increase in intravenous heroin self-administration and in nucleus accumbens dopamine response to heroin. These findings suggest that T394 phosphorylation following MOR activation causes MOR internalization and desensitization, which subsequently contributes to the development of tolerance in both opioid analgesia and opioid reward. Accordingly, T394A mutation blocks opioid tolerance and leads to an increase in brain dopamine response to opioids and in opioid-taking behavior. Thus, the T394 may serve as a new drug target for modulating opioid tolerance and the development of opioid abuse and addiction.

  3. SMARCA4-inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers.

    PubMed

    Tagal, Vural; Wei, Shuguang; Zhang, Wei; Brekken, Rolf A; Posner, Bruce A; Peyton, Michael; Girard, Luc; Hwang, TaeHyun; Wheeler, David A; Minna, John D; White, Michael A; Gazdar, Adi F; Roth, Michael G

    2017-01-19

    Mutations in the SMARCA4/BRG1 gene resulting in complete loss of its protein (BRG1) occur frequently in non-small cell lung cancer (NSCLC) cells. Currently, no single therapeutic agent has been identified as synthetically lethal with SMARCA4/BRG1 loss. We identify AURKA activity as essential in NSCLC cells lacking SMARCA4/BRG1. In these cells, RNAi-mediated depletion or chemical inhibition of AURKA induces apoptosis and cell death in vitro and in xenograft mouse models. Disc large homologue-associated protein 5 (HURP/DLGAP5), required for AURKA-dependent, centrosome-independent mitotic spindle assembly is essential for the survival and proliferation of SMARCA4/BRG1 mutant but not of SMARCA4/BRG1 wild-type cells. AURKA inhibitors may provide a therapeutic strategy for biomarker-driven clinical studies to treat the NSCLCs harbouring SMARCA4/BRG1-inactivating mutations.

  4. SMARCA4-inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers

    PubMed Central

    Tagal, Vural; Wei, Shuguang; Zhang, Wei; Brekken, Rolf A.; Posner, Bruce A.; Peyton, Michael; Girard, Luc; Hwang, TaeHyun; Wheeler, David A.; Minna, John D.; White, Michael A.; Gazdar, Adi F.; Roth, Michael G.

    2017-01-01

    Mutations in the SMARCA4/BRG1 gene resulting in complete loss of its protein (BRG1) occur frequently in non-small cell lung cancer (NSCLC) cells. Currently, no single therapeutic agent has been identified as synthetically lethal with SMARCA4/BRG1 loss. We identify AURKA activity as essential in NSCLC cells lacking SMARCA4/BRG1. In these cells, RNAi-mediated depletion or chemical inhibition of AURKA induces apoptosis and cell death in vitro and in xenograft mouse models. Disc large homologue-associated protein 5 (HURP/DLGAP5), required for AURKA-dependent, centrosome-independent mitotic spindle assembly is essential for the survival and proliferation of SMARCA4/BRG1 mutant but not of SMARCA4/BRG1 wild-type cells. AURKA inhibitors may provide a therapeutic strategy for biomarker-driven clinical studies to treat the NSCLCs harbouring SMARCA4/BRG1-inactivating mutations. PMID:28102363

  5. Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

    PubMed Central

    Yen, H.-L.; McKimm-Breschkin, J. L.; Choy, K.-T.; Wong, D. D. Y.; Cheung, P. P. H.; Zhou, J.; Ng, I. H.; Zhu, H.; Webby, R. J.; Guan, Y.; Webster, R. G.; Peiris, J. S. M.

    2013-01-01

    ABSTRACT We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2′-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. PMID:23860768

  6. Mutations in the transmembrane helix S6 of domain IV confer cockroach sodium channel resistance to sodium channel blocker insecticides and local anesthetics.

    PubMed

    Jiang, Dingxin; Du, Yuzhe; Nomura, Yoshiko; Wang, Xingliang; Wu, Yidong; Zhorov, Boris S; Dong, Ke

    2015-11-01

    Indoxacarb and metaflumizone are two sodium channel blocker insecticides (SCBIs). They preferably bind to and trap sodium channels in the slow-inactivated non-conducting state, a mode of action similar to that of local anesthetics (LAs). Recently, two sodium channel mutations, F1845Y (F(4i15)Y) and V1848I (V(4i18)I), in the transmembrane segment 6 of domain IV (IVS6), were identified to be associated with indoxacarb resistance in Plutella xylostella. F(4i15) is known to be critical for the action of LAs on mammalian sodium channels. Previously, mutation F(4i15)A in a cockroach sodium channel, BgNav1-1a, has been shown to reduce the action of lidocaine, a LA, but not the action of SCBIs. In this study, we introduced mutations F(4i15)Y and V(4i18)A/I individually into the cockroach sodium channel, BgNav1-1a, and conducted functional analysis of the three mutants in Xenopus oocytes. We found that both the F(4i15)Y and V(4i18)I mutations reduced the inhibition of sodium current by indoxacarb, DCJW (an active metabolite of indoxacarb) and metaflumizone. F(4i15)Y and V(4i18)I mutations also reduced the use-dependent block of sodium current by lidocaine. In contrast, substitution V(4i18)A enhanced the action metaflumizone and lidocaine. These results show that both F(4i15)Y and V(4i18)I mutations may contribute to target-site resistance to SCBIs, and provide the first molecular evidence for common amino acid determinants on insect sodium channels involved in action of SCBIs and LA.

  7. Metformin treatment of diabetes mellitus increases the risk for pancreatitis in patients bearing the CFTR-mutation S573C.

    PubMed

    Kongsuphol, Patthara; Cassidy, Diane; Romeiras, Francisco; Schreiber, Rainer; Mehta, Anil; Kunzelmann, Karl

    2010-01-01

    Metformin use in diabetes can cause acidosis and might be linked to pancreatitis. Here, we mechanistically focus on this relationship via a point mutation in the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7). CFTR is an ATP-hydrolyzing, cAMP/PKA-activated anion channel regulating pancreatic bicarbonate/chloride secretion across duct-facing apical membranes in epithelia. CFTR has two nucleotide binding domains (NBD1/2) which clamp two ATP molecules across their opposed, inverted interfacial surfaces which generates anion-conductance after ATP hydrolysis. Notably, CFTR mutations not causal for classical cystic fibrosis segregate with unexplained pancreatitis and one of these lies in NBD1 near its ATP-clamp (S573C; close to the Walker B aspartate D572). We recently showed that after raising [cAMP], wt-CFTR chloride-conductance, when expressed in Xenopus oocytes, remains elevated despite the presence of metformin. Yet here, we find that S573C-CFTR manifests a metformin-inhibitable whole cell chloride-conductance after cAMP elevation. In the absence of metformin, cAMP-activated S573C-CFTR also displays a reduced anion-conductance relative to wt-CFTR. Furthermore, intra-oocyte acidification inhibited wt-CFTR and abolished S573C-CFTR conductance. We conclude that defective S573C-CFTR remains both poorly conducting and inhibited by metformin and intracellular acidosis. This might explain the propensity to pancreatitis with this rare CF mutation.

  8. Increased reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorders patients with JAK2 (V617F) mutation

    PubMed Central

    Hurtado-Nedelec, Margarita; Csillag-Grange, Marie-José; Boussetta, Tarek; Belambri, Sahra Amel; Fay, Michèle; Cassinat, Bruno; Gougerot-Pocidalo, Marie-Anne; Dang, Pham My-Chan; El-Benna, Jamel

    2013-01-01

    Myeloproliferative disorders are associated with increased risk of thrombosis and vascular complications. The pathogenesis of these complications is not completely known. Reactive oxygen species produced by the neutrophil NADPH oxidase could have a role in this process. The aim of this study was to evaluate reactive oxygen species production by neutrophils of myeloproliferative disorder patients. Patients with or without the JAK2 V617F mutation were characterized. Reactive oxygen species production was assessed by chemiluminescence, and phosphorylation of the NADPH oxidase subunit p47phox was analyzed by Western blots. In a comparison of controls and myeloproliferative disorder patients without the JAK2 V617F mutation, reactive oxygen species production by neutrophils from patients with the JAK2 V617F mutation was dramatically increased in non-stimulated and in stimulated conditions. This increase was associated with increased phosphorylation of the p47phox on Ser345 and of the uspstream kinase ERK1/2. In neutrophils from healthy donors, JAK2 can be activated by GM-CSF. GM-CSF-induced p47phox phosphorylation and priming of reactive oxygen species production are inhibited by the selective JAK2 inhibitors AG490 and lestaurtinib (CEP-701), supporting a role for JAK2 in the upregulation of NADPH oxidase activation. These findings show an increase in reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorder patients with the JAK2 V617F mutation, and demonstrate that JAK2 is involved in GM-CSF-induced NADPH oxidase hyperactivation. As neutrophil hyperactivation could be implicated in the thrombophilic status of patients with myeloproliferative disorders, aberrant activation of JAK2 V617F, leading to excessive neutrophil reactive oxygen species production might play a role in this setting. PMID:23975181

  9. Investigating Mutations to Reduce Huntingtin Aggregation by Increasing Htt-N-Terminal Stability and Weakening Interactions with PolyQ Domain

    PubMed Central

    Mazza-Anthony, Cody; Waldispühl, Jérôme

    2016-01-01

    Huntington's disease is a fatal autosomal genetic disorder characterized by an expanded glutamine-coding CAG repeat sequence in the huntingtin (Htt) exon 1 gene. The Htt protein associated with the disease misfolds into toxic oligomers and aggregate fibril structures. Competing models for the misfolding and aggregation phenomena have suggested the role of the Htt-N-terminal region and the CAG trinucleotide repeats (polyQ domain) in affecting aggregation propensities and misfolding. In particular, one model suggests a correlation between structural stability and the emergence of toxic oligomers, whereas a second model proposes that molecular interactions with the extended polyQ domain increase aggregation propensity. In this paper, we computationally explore the potential to reduce Htt aggregation by addressing the aggregation causes outlined in both models. We investigate the mutation landscape of the Htt-N-terminal region and explore amino acid residue mutations that affect its structural stability and hydrophobic interactions with the polyQ domain. Out of the millions of 3-point mutation combinations that we explored, the (L4K E12K K15E) was the most promising mutation combination that addressed aggregation causes in both models. The mutant structure exhibited extreme alpha-helical stability, low amyloidogenicity potential, a hydrophobic residue replacement, and removal of a solvent-inaccessible intermolecular side chain that assists oligomerization. PMID:28096892

  10. A missense mutation in the transcription factor ETV5 leads to sterility, increased embryonic and perinatal death, postnatal growth restriction, renal asymmetry and polydactyly in the mouse.

    PubMed

    Jamsai, Duangporn; Clark, Brett J; Smith, Stephanie J; Whittle, Belinda; Goodnow, Christopher C; Ormandy, Christopher J; O'Bryan, Moira K

    2013-01-01

    ETV5 (Ets variant gene 5) is a transcription factor that is required for fertility. In this study, we demonstrate that ETV5 plays additional roles in embryonic and postnatal developmental processes in the mouse. Through a genome-wide mouse mutagenesis approach, we generated a sterile mouse line that carried a nonsense mutation in exon 12 of the Etv5 gene. The mutation led to the conversion of lysine at position 412 into a premature termination codon (PTC) within the ETS DNA binding domain of the protein. We showed that the PTC-containing allele produced a highly unstable mRNA, which in turn resulted in an undetectable level of ETV5 protein. The Etv5 mutation resulted in male and female sterility as determined by breeding experiments. Mutant males were sterile due to a progressive loss of spermatogonia, which ultimately resulted in a Sertoli cell only phenotype by 8 week-of-age. Further, the ETV5 target genes Cxcr4 and Ccl9 were significantly down-regulated in mutant neonate testes. CXCR4 and CCL9 have been implicated in the maintenance and migration of spermatogonia, respectively. Moreover, the Etv5 mutation resulted in several developmental abnormalities including an increased incidence of embryonic and perinatal lethality, postnatal growth restriction, polydactyly and renal asymmetry. Thus, our data define a physiological role for ETV5 in many aspects of development including embryonic and perinatal survival, postnatal growth, limb patterning, kidney development and fertility.

  11. Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes

    PubMed Central

    Salzer, Ulrich; Bacchelli, Chiara; Buckridge, Sylvie; Pan-Hammarström, Qiang; Jennings, Stephanie; Lougaris, Vassilis; Bergbreiter, Astrid; Hagena, Tina; Birmelin, Jennifer; Plebani, Alessandro; Webster, A. David B.; Peter, Hans-Hartmut; Suez, Daniel; Chapel, Helen; McLean-Tooke, Andrew; Spickett, Gavin P.; Anover-Sombke, Stephanie; Ochs, Hans D.; Urschel, Simon; Belohradsky, Bernd H.; Ugrinovic, Sanja; Kumararatne, Dinakantha S.; Lawrence, Tatiana C.; Holm, Are M.; Franco, Jose L.; Schulze, Ilka; Schneider, Pascal; Gertz, E. Michael; Schäffer, Alejandro A.; Hammarström, Lennart; Thrasher, Adrian J.; Gaspar, H. Bobby

    2009-01-01

    TNFRSF13B encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), a B cell– specific tumor necrosis factor (TNF) receptor superfamily member. Both biallelic and monoallelic TNFRSF13B mutations were identified in patients with common variable immunodeficiency disorders. The genetic complexity and variable clinical presentation of TACI deficiency prompted us to evaluate the genetic, immunologic, and clinical condition in 50 individuals with TNFRSF13B alterations, following screening of 564 unrelated patients with hypogammaglobulinemia. We identified 13 new sequence variants. The most frequent TNFRSF13B variants (C104R and A181E; n = 39; 6.9%) were also present in a heterozygous state in 2% of 675 controls. All patients with biallelic mutations had hypogammaglobulinemia and nearly all showed impaired binding to a proliferation-inducing ligand (APRIL). However, the majority (n = 41; 82%) of the pa-tients carried monoallelic changes in TNFRSF13B. Presence of a heterozygous mutation was associated with antibody deficiency (P <.001, relative risk 3.6). Heterozygosity for the most common mutation, C104R, was associated with disease (P < .001, relative risk 4.2). Furthermore, heterozygosity for C104R was associated with low numbers of IgD−CD27+ B cells (P = .019), benign lymphoproliferation (P < .001), and autoimmune complications (P = .001). These associations indicate that C104R heterozygosity increases the risk for common variable immunodeficiency disorders and influences clinical presentation. PMID:18981294

  12. Yeast NDI1 Improve Oxidative Phosphorylation Capacity and Increases Protection Against Oxidative Stress and Cell Death in Cells Carrying a Leber’s Hereditary Optic Neuropathy Mutation

    PubMed Central

    Park, Jeong Soon; Li, You-fen; Bai, Yidong

    2007-01-01

    G11778A in the subunit ND4 gene of NADH dehydrogenase complex is the most common primary mutation found in Leber’s hereditary optic neuropathy (LHON) patients. The NDI1 gene, which encodes the internal NADH -quinone oxidoreductase in Saccharomyces cerevisiae, was introduced into the nuclear genome of a mitochondrial defective human cell line, Le1.3.1, carrying the G11778A mutation. In transformant cell lines, LeNDI1-1 and -2, total and complex I-dependent respiration were fully restored and largely resistant to complex I inhibitor, rotenone, indicating a dominant role of NDI1 in the transfer of electrons in the host cells. Whereas the original mutant Le1.3.1 cell grows poorly in medium containing galactose, the transformants have a fully restored growth capacity in galactose medium, although the ATP production was not totally recovered. Furthermore, the increased oxidative stress in the cells carrying the G11778A mutation was alleviated in transformants, demonstrated by a decreased reactive oxygen species (ROS) level. Finally, transformants were also shown to be desensitized to induction to apoptosis and also exhibit greater resistance to paraquat-induced cell death. It is concluded that the yeast ND11 enzyme can improve the oxidative phosphorylation capacity in cells carrying the G11778A mutation and protect the cells from oxidative stress and cell death. PMID:17320357

  13. Yeast NDI1 improves oxidative phosphorylation capacity and increases protection against oxidative stress and cell death in cells carrying a Leber's hereditary optic neuropathy mutation.

    PubMed

    Park, Jeong Soon; Li, You-Fen; Bai, Yidong

    2007-05-01

    G11778A in the subunit ND4 gene of NADH dehydrogenase complex is the most common primary mutation found in Leber's hereditary optic neuropathy (LHON) patients. The NDI1 gene, which encodes the internal NADH-quinone oxidoreductase in Saccharomyces cerevisiae, was introduced into the nuclear genome of a mitochondrial defective human cell line, Le1.3.1, carrying the G11778A mutation. In transformant cell lines, LeNDI1-1 and -2, total and complex I-dependent respiration were fully restored and largely resistant to complex I inhibitor, rotenone, indicating a dominant role of NDI1 in the transfer of electrons in the host cells. Whereas the original mutant Le1.3.1 cell grows poorly in medium containing galactose, the transformants have a fully restored growth capacity in galactose medium, although the ATP production was not totally recovered. Furthermore, the increased oxidative stress in the cells carrying the G11778A mutation was alleviated in transformants, demonstrated by a decreased reactive oxygen species (ROS) level. Finally, transformants were also shown to be desensitized to induction to apoptosis and also exhibit greater resistance to paraquat-induced cell death. It is concluded that the yeast NDI1 enzyme can improve the oxidative phosphorylation capacity in cells carrying the G11778A mutation and protect the cells from oxidative stress and cell death.

  14. Mutation R273H confers p53 a stimulating effect on the IGF-1R-AKT pathway via miR-30a suppression in breast cancer.

    PubMed

    Guo, Fangdong; Chen, Hongshen; Chang, Jian; Zhang, Lin

    2016-03-01

    p53 is the most highly mutated tumor suppressor in human malignancies. A wide array of p53 mutations has been revealed to play pivotal roles during cancer progression, which abolish anti-tumor functions of wild type p53 but also elicit tumorigenic effects by activating a diverse subset of downstream molecules. R273H mutation of p53 has been closely implicated in human cancer. Here we report miR-30a as a novel downstream target of p53 R273H mutant, which binds to the promoter region to repress miR-30a expression. Consequently, p53 R273H mutant enhances the migratory capabilities of tumor cells that are compromised by exogenous miR-30a over-expression. Our further investigation indicates that p53 R273H mutation unleashes the inhibition effect of miR-30a on IGF-1R expression, thus leading to elevated activation of IGF-1R-AKT signaling cascade in tumor cells.

  15. Mutation in Torenia fournieri Lind. UFO homolog confers loss of TfLFY interaction and results in a petal to sepal transformation.

    PubMed

    Sasaki, Katsutomo; Yamaguchi, Hiroyasu; Aida, Ryutaro; Shikata, Masahito; Abe, Tomoko; Ohtsubo, Norihiro

    2012-09-01

    We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY.

  16. Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins.

    PubMed

    Fleet, Christine M; Yamaguchi, Shinjiro; Hanada, Atsushi; Kawaide, Hiroshi; David, Charles J; Kamiya, Yuji; Sun, Tai-Ping

    2003-06-01

    The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.

  17. The HOXB13 G84E Mutation is Associated with an Increased Risk for Prostate Cancer and other Malignancies

    PubMed Central

    Beebe-Dimmer, Jennifer L; Hathcock, Matthew; Yee, Cecilia; Okoth, Linda A.; Ewing, Charles M; Isaacs, William B.; Cooney, Kathleen A.; Thibodeau, Stephen N.

    2015-01-01

    Background A rare non-conservative substitution (G84E) in the HOXB13 gene has been shown to be associated with risk of prostate cancer. DNA samples from male patients included in the Mayo Clinic Biobank (MCB) were genotyped to determine the frequency of the G84E mutation and its association with various cancers. Methods Subjects were genotyped using a custom TaqMan (Applied Biosystems) assay for G84E (rs138213197). In addition to donating a blood specimen, all MCB participants completed a baseline questionnaire to collect information on medical history and family history of cancer. Results Forty-nine of 9,012 male patients were carriers of G84E (0.5%). Thirty-one percent (n=2,595) of participants had been diagnosed with cancer, including 51.1% of G84E carriers compared to just 30.6% of non-carriers (p=0.004). G84E was most frequently observed among men with prostate cancer compared to men without cancer (p<0.0001). However, the mutation was also more commonly observed in men with bladder cancer (p=0.06) and leukemia (p=0.01). G84E carriers were more likely to have a positive family history of prostate cancer in a first degree relative compared to non-carriers (36.2% v. 16.0%, p=0.0003). Conclusions Our study confirms the association between the HOXB13 G84E variant and prostate cancer and suggests a novel association between G84E and leukemia and a suggestive association with bladder cancer. Future investigation is warranted to confirm these associations in order to improve our understanding of the role of germline HOXB13 mutations in human cancer. Impact The associations between HOXB13 and prostate, leukemia and bladder suggest that this gene is important in carcinogenesis. PMID:26108461

  18. Step-Wise Increase in Tigecycline Resistance in Klebsiella pneumoniae Associated with Mutations in ramR, lon and rpsJ

    PubMed Central

    Fang, Li; Chen, Qiong; Shi, Keren; Li, Xi; Shi, Qiucheng; He, Fang; Zhou, Jiancang; Yu, Yunsong

    2016-01-01

    Klebsiella pneumoniae is a gram-negative bacterium that causes numerous diseases, including pneumonia and urinary tract infections. An increase in multidrug resistance has complicated the treatment of these bacterial infections, and although tigecycline shows activity against a broad spectrum of bacteria, resistant strains have emerged. In this study, the whole genomes of two clinical and six laboratory-evolved strains were sequenced to identify putative mutations related to tigecycline resistance. Of seven tigecycline-resistant strains, seven (100%) had ramR mutations, five (71.4%) had lon mutations, one (14.2%) had a ramA mutation, and one (14.2%) had an rpsJ mutation. A higher fitness cost was observed in the laboratory-evolved strains but not in the clinical strains. A transcriptome analysis demonstrated high expression of the ramR operon and acrA in all tigecycline-resistant strains. Genes involved in nitrogen metabolism were induced in the laboratory-evolved strains compared with the wild-type and clinical strains, and this difference in nitrogen metabolism reflected the variation between the laboratory-evolved and the clinical strains. Complementation experiments showed that both the wild-type ramR and the lon genes could partially restore the tigecycline sensitivity of K. pneumoniae. We believe that this manuscript describes the first construct of a lon mutant in K. pneumoniae, which allowed confirmation of its association with tigecycline resistance. Our findings illustrate the importance of the ramR operon and the lon and rpsJ genes in K. pneumoniae resistance to tigecycline. PMID:27764207

  19. Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield.

    PubMed

    Tata, Sandeep Kumar; Jung, Jihye; Kim, Yoon-Ha; Choi, Jun Young; Jung, Ji-Yul; Lee, In-Jung; Shin, Jeong Sheop; Ryu, Stephen Beungtae

    2016-01-01

    Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.

  20. Mutation increasing β-carotene concentrations does not adversely affect concentrations of essential mineral elements in pepper fruit

    PubMed Central

    Thompson, Jacqueline A.; Penchev, Emil A.; Nielen, Stephan

    2017-01-01

    Vitamin and mineral deficiencies are prevalent in human populations throughout the world. Vitamin A deficiency affects hundreds of millions of pre-school age children in low income countries. Fruits of pepper (Capsicum annuum L.) can be a major dietary source of precursors to Vitamin A biosynthesis, such as β-carotene. Recently, pepper breeding programs have introduced the orange-fruited (of) trait of the mutant variety Oranzheva kapiya, which is associated with high fruit β-carotene concentrations, to the mutant variety Albena. In this manuscript, concentrations of β-carotene and mineral elements (magnesium, phosphorus, sulphur, potassium, zinc, calcium, manganese, iron and copper) were compared in fruit from P31, a red-fruited genotype derived from the variety Albena, and M38, a genotype developed by transferring the orange-fruited mutation (of) into Albena. It was observed that fruit from M38 plants had greater β-carotene concentration at both commercial and botanical maturity (4.9 and 52.7 mg / kg fresh weight, respectively) than fruit from P31 plants (2.3 and 30.1 mg / kg fresh weight, respectively). The mutation producing high β-carotene concentrations in pepper fruits had no detrimental effect on the concentrations of mineral elements required for human nutrition. PMID:28207797

  1. Mutation increasing β-carotene concentrations does not adversely affect concentrations of essential mineral elements in pepper fruit.

    PubMed

    Tomlekova, Nasya B; White, Philip J; Thompson, Jacqueline A; Penchev, Emil A; Nielen, Stephan

    2017-01-01

    Vitamin and mineral deficiencies are prevalent in human populations throughout the world. Vitamin A deficiency affects hundreds of millions of pre-school age children in low income countries. Fruits of pepper (Capsicum annuum L.) can be a major dietary source of precursors to Vitamin A biosynthesis, such as β-carotene. Recently, pepper breeding programs have introduced the orange-fruited (of) trait of the mutant variety Oranzheva kapiya, which is associated with high fruit β-carotene concentrations, to the mutant variety Albena. In this manuscript, concentrations of β-carotene and mineral elements (magnesium, phosphorus, sulphur, potassium, zinc, calcium, manganese, iron and copper) were compared in fruit from P31, a red-fruited genotype derived from the variety Albena, and M38, a genotype developed by transferring the orange-fruited mutation (of) into Albena. It was observed that fruit from M38 plants had greater β-carotene concentration at both commercial and botanical maturity (4.9 and 52.7 mg / kg fresh weight, respectively) than fruit from P31 plants (2.3 and 30.1 mg / kg fresh weight, respectively). The mutation producing high β-carotene concentrations in pepper fruits had no detrimental effect on the concentrations of mineral elements required for human nutrition.

  2. Mouse model of OPRM1 (A118G) polymorphism increases sociability and dominance and confers resilience to social defeat.

    PubMed

    Briand, Lisa A; Hilario, Monica; Dow, Holly C; Brodkin, Edward S; Blendy, Julie A; Berton, Olivier

    2015-02-25

    A single nucleotide polymorphism (SNP) in the human μ-opioid receptor gene (OPRM1 A118G) has been widely studied for its association in drug addiction, pain sensitivity, and, more recently, social behavior. The endogenous opioid system has been shown to regulate social distress and reward in a variety of animal models. However, mechanisms underlying the associations between the OPRM1 A118G SNP and these behaviors have not been clarified. We used a mouse model possessing the human equivalent nucleotide/amino acid substitution to study social affiliation and social defeat behaviors. In mice with the Oprm1 A112G SNP, we demonstrate that the G allele is associated with an increase in home-cage dominance and increased motivation for nonaggressive social interactions, similar to what is reported in human populations. When challenged by a resident aggressor, G-allele carriers expressed less submissive behavior and exhibited resilience to social defeat, demonstrated by a lack of subsequent social avoidance and reductions in anhedonia as measured by intracranial self-stimulation. Protection from social defeat in G-allele carriers was associated with a greater induction of c-fos in a resilience circuit comprising the nucleus accumbens and periaqueductal gray. These findings led us to test the role of endogenous opioids in the A112G mice. We demonstrate that the increase in social affiliation in G carriers is blocked by pretreatment with naloxone. Together, these data suggest a mechanism involving altered hedonic state and neural activation as well as altered endogenous opioid tone in the differential response to aversive and rewarding social stimuli in G-allele carriers.

  3. Mouse Model of OPRM1 (A118G) Polymorphism Increases Sociability and Dominance and Confers Resilience to Social Defeat

    PubMed Central

    Briand, Lisa A.; Hilario, Monica; Dow, Holly C.; Brodkin, Edward S.; Berton, Olivier

    2015-01-01

    A single nucleotide polymorphism (SNP) in the human μ-opioid receptor gene (OPRM1 A118G) has been widely studied for its association in drug addiction, pain sensitivity, and, more recently, social behavior. The endogenous opioid system has been shown to regulate social distress and reward in a variety of animal models. However, mechanisms underlying the associations between the OPRM1 A118G SNP and these behaviors have not been clarified. We used a mouse model possessing the human equivalent nucleotide/amino acid substitution to study social affiliation and social defeat behaviors. In mice with the Oprm1 A112G SNP, we demonstrate that the G allele is associated with an increase in home-cage dominance and increased motivation for nonaggressive social interactions, similar to what is reported in human populations. When challenged by a resident aggressor, G-allele carriers expressed less submissive behavior and exhibited resilience to social defeat, demonstrated by a lack of subsequent social avoidance and reductions in anhedonia as measured by intracranial self-stimulation. Protection from social defeat in G-allele carriers was associated with a greater induction of c-fos in a resilience circuit comprising the nucleus accumbens and periaqueductal gray. These findings led us to test the role of endogenous opioids in the A112G mice. We demonstrate that the increase in social affiliation in G carriers is blocked by pretreatment with naloxone. Together, these data suggest a mechanism involving altered hedonic state and neural activation as well as altered endogenous opioid tone in the differential response to aversive and rewarding social stimuli in G-allele carriers. PMID:25716856

  4. Inhibition of TRPV1 confers neuroprotection, reduces TNF-α and increases Il-10 in a rat stroke model.

    PubMed

    Hakimizadeh, Elham; Shamsizadeh, Ali; Roohbakhsh, Ali; Arababadi, Mohammad Kazemi; Hajizadeh, Mohammad Reza; Shariati, Mehdi; Rahmani, Mohammad Reza; Allahtavakoli, Mohammad

    2017-02-15

    Stroke is a major cause of mortality and long-term disability in adults. TRPV1 plays a crucial role in neuroinflammation. In the current study, the effects of TRPV1 agonist (capsaicin) and antagonist (AMG9810) on cerebral ischemia were investigated. Forty male Wistar rats were assigned to the following experimental groups: sham, vehicle) ischemic), AMG9810 (selective TRPV1 antagonist, 0.5 mg/kg; 3 h after stroke), and capsaicin (1 mg/kg; 3 h after stroke). Stroke was induced by permanent middle cerebral artery occlusion and neurological deficits were evaluated 1, 3, and 7 days after stroke. Then, infarct volume, brain edema, body temperature, mRNA expression of TRPV1 and serum concentrations of TNF-α and IL-10 were measured. Compared to the vehicle group, AMG9810 significantly decreased the infarct volume (P< 0.01). Latency for the removal of sticky labels from the forepaw and the hanging time were significantly decreased and increased respectively following administration of AMG9810 (P< 0.01 and P< 0.001 versus vehicle) 3 and 7 days after stroke. Compared to the sham group, the mRNA expression of TRPV1 was significantly increased in vehicle group (P< 0.01). Administration of AMG9810 significantly increased the anti-inflammatory cytokine IL-10 and decreased the inflammatory cytokine TNF-α (P< 0.05). Moreover, our results indicate that AMG9810 might a promising candidate for the hypothermic treatment of stroke. The findings also suggest a key role for AMG9810 in reducing inflammation after stroke and imply that TRPV1 could be a potential target for the treatment of ischemic stroke. This article is protected by copyright. All rights reserved.

  5. Neomorphic Mutations in PIK3R1 Confer Sensitivity to MAPK Inhibitors due to Activation of ERK and JNK Pathways | Office of Cancer Genomics

    Cancer.gov

    In a recent publication in Cancer Cell, CTD2 investigators discovered that a known cancer-associated gain-of-function alteration in phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) results in novel protein activity that confers sensitivity to mitogen-activated protein kinase (MAPK) inhibitors. The PIK3R1 gene encodes the p85α regulatory subunit of PIK3. Under normal conditions, p85α suppresses PIK3 mediated activation of downstream pathways that promote cell growth and survival.

  6. Overexpression of an Apocynum venetum DEAD-Box Helicase Gene (AvDH1) in Cotton Confers Salinity Tolerance and Increases Yield in a Saline Field

    PubMed Central

    Chen, Jie; Wan, Sibao; Liu, Huaihua; Fan, Shuli; Zhang, Yujuan; Wang, Wei; Xia, Minxuan; Yuan, Rui; Deng, Fenni; Shen, Fafu

    2016-01-01

    Soil salinity is a major environmental stress limiting plant growth and productivity. We have reported previously the isolation of an Apocynum venetum DEAD-box helicase 1 (AvDH1) that is expressed in response to salt exposure. Here, we report that the overexpression of AvDH1 driven by a constitutive cauliflower mosaic virus-35S promoter in cotton plants confers salinity tolerance. Southern and Northern blotting analyses showed that the AvDH1 gene was integrated into the cotton genome and expressed. In this study, the growth of transgenic cotton expressing AvDH1 was evaluated under saline conditions in a growth chamber and in a saline field trial. Transgenic cotton overexpressing AvDH1 was much more resistant to salt than the wild-type plants when grown in a growth chamber. The lower membrane ion leakage, along with increased activity of superoxide dismutase, in AvDH1 transgenic lines suggested that these characteristics may prevent membrane damage, which increases plant survival rates. In a saline field, the transgenic cotton lines expressing AvDH1 showed increased boll numbers, boll weights and seed cotton yields compared with wild-type plants, especially at high soil salinity levels. This study indicates that transgenic cotton expressing AvDH1 is a promising option for increasing crop productivity in saline fields. PMID:26779246

  7. Characterization of the effects of an rpoC mutation that confers resistance to the Fst peptide toxin-antitoxin system toxin.

    PubMed

    Brinkman, Cassandra L; Bumgarner, Roger; Kittichotirat, Weerayuth; Dunman, Paul M; Kuechenmeister, Lisa J; Weaver, Keith E

    2013-01-01

    Overexpression of the Fst toxin in Enterococcus faecalis strain OG1X leads to defects in chromosome segregation, cell division and, eventually, membrane integrity. The M7 mutant derivative of OG1X is resistant to most of these effects but shows a slight growth defect in the absence of Fst. Full-genome sequencing revealed two differences between M7 and its OG1X parent. First, OG1X contains a frameshift mutation that inactivates the etaR response regulator gene, while M7 is a wild-type revertant for etaR. Second, the M7 mutant contains a missense mutation in the rpoC gene, which encodes the β' subunit of RNA polymerase. Mutagenesis experiments revealed that the rpoC mutation was primarily responsible for the resistance phenotype. Microarray analysis revealed that a number of transporters were induced in OG1X when Fst was overexpressed. These transporters were not induced in M7 in response to Fst, and further experiments indicated that this had a direct protective effect on the mutant cells. Therefore, exposure of cells to Fst appears to have a cascading effect, first causing membrane stress and then potentiation of these effects by overexpression of certain transporters.

  8. Overexpression of the PP2A-C5 gene confers increased salt tolerance in Arabidopsis thaliana

    PubMed Central

    Hu, Rongbin; Zhu, Yinfeng; Shen, Guoxin; Zhang, Hong

    2017-01-01

    ABSTRACT Protein phosphatase 2A (PP2A) was shown to play important roles in biotic and abiotic stress signaling pathways in plants. PP2A is made of 3 subunits: a scaffolding subunit A, a regulatory subunit B, and a catalytic subunit C. It is believed that the B subunit recognizes specific substrates and the C subunit directly acts on the selected substrates, whereas the A subunit brings a B subunit and a C subunit together to form a specific PP2A holoenzyme. Because there are multiple isoforms for each PP2A subunit, there could be hundreds of novel PP2A holoenzymes in plants. For an example, there are 3 A subunits, 17 B subunits, and 5 C subunits in Arabidopsis, which could form 255 different PP2A holoenzymes. Understanding the roles of these PP2A holoenzymes in various signaling pathways is a challenging task. In a recent study,1 we discovered that PP2A-C5, the catalytic subunit 5 of PP2A, plays an important role in salt tolerance in Arabidopsis. We found that a knockout mutant of PP2A-C5 (i.e. pp2a-c5–1) was very sensitive to salt treatments, whereas PP2A-C5-overexpressing plants were more tolerant to salt stresses. Genetic analyses between pp2a-c5–1 and Salt-Overly-Sensitive (SOS) mutants indicated that PP2A-C5 does not function in the same pathway as SOS genes. Using yeast 2-hybrid analysis, we found that PP2A-C5 interacts with several vacuolar membrane bound chloride channel proteins. We hypothesize that these vacuolar chloride channel proteins might be PP2A-C5's substrates in vivo, and the action of PP2A-C5 on these channel proteins could increase or activate their activities, thereby result in accumulation of the chloride and sodium contents in vacuoles, leading to increased salt tolerance in plants. PMID:28045581

  9. Elevated salicylic acid levels conferred by increased expression of ISOCHORISMATE SYNTHASE 1 contribute to hyperaccumulation of SUMO1 conjugates in the Arabidopsis mutant early in short days 4.

    PubMed

    Villajuana-Bonequi, Mitzi; Elrouby, Nabil; Nordström, Karl; Griebel, Thomas; Bachmair, Andreas; Coupland, George

    2014-07-01

    Post-translational modification of proteins by attachment of small ubiquitin-like modifier (SUMO) is essential for plant growth and development. Mutations in the SUMO protease early in short days 4 (ESD4) cause hyperaccumulation of conjugates formed between SUMO and its substrates, and phenotypically are associated with extreme early flowering and impaired growth. We performed a suppressor mutagenesis screen of esd4 and identified a series of mutants called suppressor of esd4 (sed), which delay flowering, enhance growth and reduce hyperaccumulation of SUMO conjugates. Genetic mapping and genome sequencing indicated that one of these mutations (sed111) is in the gene salicylic acid induction-deficient 2 (SID2), which encodes ISOCHORISMATE SYNTHASE I, an enzyme required for biosynthesis of salicylic acid (SA). Analyses showed that compared with wild-type plants, esd4 contains higher levels of SID2 mRNA and about threefold more SA, whereas sed111 contains lower SA levels. Other sed mutants also contain lower SA levels but are not mutant for SID2, although most reduce SID2 mRNA levels. Therefore, higher SA levels contribute to the small size, early flowering and elevated SUMO conjugate levels of esd4. Our results support previous data indicating that SUMO homeostasis influences SA biosynthesis in wild-type plants, and also demonstrate that elevated levels of SA strongly increase the abundance of SUMO conjugates.

  10. Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome.

    PubMed

    Bernabéu-Herrero, Maria E; Jiménez-Alcázar, Miguel; Anter, Jaouad; Pinto, Sheila; Sánchez Chinchilla, Daniel; Garrido, Sofía; López-Trascasa, Margarita; Rodríguez de Córdoba, Santiago; Sánchez-Corral, Pilar

    2015-10-01

    Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy affecting the renal microvasculature and is associated with complement dysregulation caused by mutations or autoantibodies. Disease penetrance and severity is modulated by inheritance of "risk" polymorphisms in the complement genes MCP, CFH and CFHR1. We describe the prevalence of mutations, the frequency of risk polymorphisms and the occurrence of anti-FH autoantibodies in a Spanish aHUS cohort (n=367). We also report the identification of a polymorphism in CFHR3 (c.721C>T; rs379370) that is associated with increased risk of aHUS (OR=1.78; CI 1.22-2.59; p=0.002), and is most frequently included in an extended risk haplotype spanning the CFH-CFHR3-CFHR1 genes. This extended haplotype integrates polymorphisms in the promoter region of CFH and CFHR3, and is associated with poorer evolution of renal function and decreased FH levels. The CFH-CFHR3-CFHR1 aHUS-risk haplotype seems to be the same as was previously associated with protection against meningococcal infections, suggesting that the genetic variability in this region is limited to a few extended haplotypes, each with opposite effects in various human diseases. These results suggest that the combination of quantitative and qualitative variations in the complement proteins encoded by CFH, CFHR3 and CFHR1 genes is key for the association of these haplotypes with disease.

  11. B7-H1 Blockade Increases Survival of Dysfunctional CD8+ T Cells and Confers Protection against Leishmania donovani Infections

    PubMed Central

    Joshi, Trupti; Rodriguez, Susana; Perovic, Vladimir; Cockburn, Ian A.; Stäger, Simona

    2009-01-01

    Experimental visceral leishmaniasis (VL) represents an exquisite model to study CD8+ T cell responses in a context of chronic inflammation and antigen persistence, since it is characterized by chronic infection in the spleen and CD8+ T cells are required for the development of protective immunity. However, antigen-specific CD8+ T cell responses in VL have so far not been studied, due to the absence of any defined Leishmania-specific CD8+ T cell epitopes. In this study, transgenic Leishmania donovani parasites expressing ovalbumin were used to characterize the development, function, and fate of Leishmania-specific CD8+ T cell responses. Here we show that L. donovani parasites evade CD8+ T cell responses by limiting their expansion and inducing functional exhaustion and cell death. Dysfunctional CD8+ T cells could be partially rescued by in vivo B7-H1 blockade, which increased CD8+ T cell survival but failed to restore cytokine production. Nevertheless, B7-H1 blockade significantly reduced the splenic parasite burden. These findings could be exploited for the design of new strategies for immunotherapeutic interventions against VL. PMID:19436710

  12. Increasing Understanding of Public Problems and Policies, 1994. [National Public Policy Education Conference (44th, Boise, Idaho, September 18-21, 1994).

    ERIC Educational Resources Information Center

    Halbrook, Steve A., Ed.; Grace, Teddee E., Ed.

    The National Public Policy Education Conference is held annually to improve the policy education efforts of extension workers responsible for public affairs programs. The 1994 conference addressed the following topics: (1) ethical perspectives in public policy education; (2) transition of food and agricultural policy; (3) building human…

  13. Overexpression of rice serotonin N-acetyltransferase 1 in transgenic rice plants confers resistance to cadmium and senescence and increases grain yield.

    PubMed

    Lee, Kyungjin; Back, Kyoungwhan

    2017-04-01

    While ectopic overexpression of serotonin N-acetyltransferase (SNAT) in plants has been accomplished using animal SNAT genes, ectopic overexpression of plant SNAT genes in plants has not been investigated. Because the plant SNAT protein differs from that of animals in its subcellular localization and enzyme kinetics, its ectopic overexpression in plants would be expected to give outcomes distinct from those observed from overexpression of animal SNAT genes in transgenic plants. Consistent with our expectations, we found that transgenic rice plants overexpressing rice (Oryza sativa) SNAT1 (OsSNAT1) did not show enhanced seedling growth like that observed in ovine SNAT-overexpressing transgenic rice plants, although both types of plants exhibited increased melatonin levels. OsSNAT1-overexpressing rice plants did show significant resistance to cadmium and senescence stresses relative to wild-type controls. In contrast to tomato, melatonin synthesis in rice seedlings was not induced by selenium and OsSNAT1 transgenic rice plants did not show tolerance to selenium. T2 homozygous OsSNAT1 transgenic rice plants exhibited increased grain yield due to increased panicle number per plant under paddy field conditions. These benefits conferred by ectopic overexpression of OsSNAT1 had not been observed in transgenic rice plants overexpressing ovine SNAT, suggesting that plant SNAT functions differently from animal SNAT in plants.

  14. Accelerated mutation accumulation in asexual lineages of a freshwater snail.

    PubMed

    Neiman, Maurine; Hehman, Gery; Miller, Joseph T; Logsdon, John M; Taylor, Douglas R

    2010-04-01

    Sexual reproduction is both extremely costly and widespread relative to asexual reproduction, meaning that it must also confer profound advantages in order to persist. One theorized benefit of sex is that it facilitates the clearance of harmful mutations, which would accumulate more rapidly in the absence of recombination. The extent to which ineffective purifying selection and mutation accumulation are direct consequences of asexuality and whether the accelerated buildup of harmful mutations in asexuals can occur rapidly enough to maintain sex within natural populations, however, remain as open questions. We addressed key components of these questions by estimating the rate of mutation accumulation in the mitochondrial genomes of multiple sexual and asexual representatives of Potamopyrgus antipodarum, a New Zealand snail characterized by mixed sexual/asexual populations. We found that increased mutation accumulation is associated with asexuality and occurs rapidly enough to be detected in recently derived asexual lineages of P. antipodarum. Our results demonstrate that increased mutation accumulation in asexuals can differentially affect coexisting and ecologically similar sexual and asexual lineages. The accelerated rate of mutation accumulation observed in asexual P. antipodarum provides some of the most direct evidence to date for a link between asexuality and mutation accumulation and implies that mutational buildup could be rapid enough to contribute to the short-term evolutionary mechanisms that favor sexual reproduction.

  15. A Missense Mutation of the Gene Encoding Synaptic Vesicle Glycoprotein 2A (SV2A) Confers Seizure Susceptibility by Disrupting Amygdalar Synaptic GABA Release

    PubMed Central

    Tokudome, Kentaro; Okumura, Takahiro; Terada, Ryo; Shimizu, Saki; Kunisawa, Naofumi; Mashimo, Tomoji; Serikawa, Tadao; Sasa, Masashi; Ohno, Yukihiro

    2016-01-01

    Synaptic vesicle glycoprotein 2A (SV2A) is specifically expressed in the membranes of synaptic vesicles and modulates action potential-dependent neurotransmitter release. To explore the role of SV2A in the pathogenesis of epileptic disorders, we recently generated a novel rat model (Sv2aL174Q rat) carrying a missense mutation of the Sv2a gene and showed that the Sv2aL174Q rats were hypersensitive to kindling development (Tokudome et al., 2016). Here, we further conducted behavioral and neurochemical studies to clarify the pathophysiological mechanisms underlying the seizure vulnerability in Sv2aL174Q rats. Sv2aL174Q rats were highly susceptible to pentylenetetrazole (PTZ)-induced seizures, yielding a significantly higher seizure scores and seizure incidence than the control animals. Brain mapping analysis of Fos expression, a biological marker of neural excitation, revealed that the seizure threshold level of PTZ region-specifically elevated Fos expression in the amygdala in Sv2aL174Q rats. In vivo microdialysis study showed that the Sv2aL174Q mutation preferentially reduced high K+ (depolarization)-evoked GABA release, but not glutamate release, in the amygdala. In addition, specific control of GABA release by SV2A was supported by its predominant expression in GABAergic neurons, which were co-stained with antibodies against SV2A and glutamate decarboxylase 1. The present results suggest that dysfunction of SV2A by the missense mutation elevates seizure susceptibility in rats by preferentially disrupting synaptic GABA release in the amygdala, illustrating the crucial role of amygdalar SV2A-GABAergic system in epileptogenesis. PMID:27471467

  16. The 677C>T mutation of the MTHFR gene increases the risk of venous thromboembolism in Koreans and a meta-analysis from Asian population.

    PubMed

    Jang, Moon Ju; Jeon, Young Joo; Choi, Won-Il; Choi, Yi Seul; Kim, Su Yeoun; Chong, So Young; Oh, Doyeun; Kim, Nam Keun

    2013-06-01

    The frequency of methylenetetrahydrofolate reductase (MTHFR) mutations varies between racial and ethnic groups, and there are also conflicting data regarding MTHFR gene mutations in Asian patients with venous thromboembolism (VTE). The aim of this study was to examine the association between common MTHFR gene mutations (677C>T and 1298A>C) and risk of VTE in Koreans. This study was a retrospective case-control study. We enrolled 203 patients with VTE and 403 controls. For the 677C>T polymorphism, there was no difference in the frequency of the CT genotype and TT genotype between the patients with VTE and the controls. However, in the recessive analysis (CC + CT vs TT), the frequency of the TT genotype was significantly higher in VTE than in controls (odds ratio = 1.700; 95% confidence interval = 1.108-2.607, P = .015). In conclusion, the TT genotype of MTHFR 677C>T increases the risk of VTE in Koreans. This finding was supported by meta-analysis of previous Asian studies.

  17. Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

    NASA Astrophysics Data System (ADS)

    Tomobe, Katsufumi; Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

    2016-05-01

    The conformational change from the cellular prion protein (PrPc) to scrapie prion protein (PrPsc) is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT) PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

  18. 9. international mouse genome conference

    SciTech Connect

    1995-12-31

    This conference was held November 12--16, 1995 in Ann Arbor, Michigan. The purpose of this conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on genetic mapping in mice. This report contains abstracts of presentations, focusing on the following areas: mutation identification; comparative mapping; informatics and complex traits; mutagenesis; gene identification and new technology; and genetic and physical mapping.

  19. Crystal Structure and Biochemical Characterization of Chlamydomonas FDX2 Reveal Two Residues that, When Mutated, Partially Confer FDX2 the Redox Potential and Catalytic Properties of FDX1

    SciTech Connect

    Boehm, Marko; Alahuhta, Markus; Mulder, David W.; Peden, Erin A.; Long, Hai; Brunecky, Roman; Lunin, Vladimir V.; King, Paul W.; Ghirardi, Maria L.; Dubini, Alexandra

    2015-11-03

    The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2 protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.

  20. Crystal Structure and Biochemical Characterization of Chlamydomonas FDX2 Reveal Two Residues that, When Mutated, Partially Confer FDX2 the Redox Potential and Catalytic Properties of FDX1

    DOE PAGES

    Boehm, Marko; Alahuhta, Markus; Mulder, David W.; ...

    2015-11-03

    The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2more » protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.« less

  1. Mutations in the β-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding.

    PubMed

    Begaye, Adrian; Trostel, Shana; Zhao, Zhiming; Taylor, Richard E; Schriemer, David C; Sackett, Dan L

    2011-10-01

    Peloruside A is a microtubule-stabilizing macrolide that binds to beta tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I β-tubulin that result in the following substitutions: R306H, Y340S, N337D, and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10-15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G2/M arrest in the Pel cell lines at concentrations 10-15 times that required in the parental line. The cells show notable changes in morphology compared to the parental line.

  2. NL-103, a novel dual-targeted inhibitor of histone deacetylases and hedgehog pathway, effectively overcomes vismodegib resistance conferred by Smo mutations.

    PubMed

    Zhao, Jie; Quan, Haitian; Xie, Chengying; Lou, Liguang

    2014-06-01

    Misregulation of hedgehog (Hh) signaling has been implicated in the pathogenesis of basal cell carcinoma (BCC) and medulloblastoma. Vismodegib, an orally bioavailable Hh signal pathway inhibitor targeting Smo, has been approved for the treatment of advanced BCC. However, acquired drug resistance to vismodegib induced by point mutation in Smo is emerging as a major problem to vismodegib treatment. In this study, we designed and synthesized a novel chimeric compound NL-103, which comprises structural elements of Hh pathway inhibitor vismodegib, and histone deacetylase (HDAC) inhibitor vorinostat. NL-103 simultaneously and significantly inhibited both HDACs and Hh pathway. Importantly, NL-103, as well as vorinostat, effectively overcame vismodegib resistance induced by Smoothened point mutations. Moreover, NL-103 and vorinostat, but not vismodegib, significantly downregulated the expression of Gli2 which plays an important role in Hh pathway. These results indicate that HDAC inhibitory activity is essential for NL-103 to overcome vismodegib resistance and that dual inhibition of HDAC and Hh signaling pathway may be a rational strategy for overcoming vismodegib resistance. Our findings suggest that NL-103 may be a promising compound for clinical development as a more effective Hh pathway inhibitor.

  3. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.

    PubMed

    Ye, Lin; Wang, Jiaming; Beyer, Ashley I; Teque, Fernando; Cradick, Thomas J; Qi, Zhongxia; Chang, Judy C; Bao, Gang; Muench, Marcus O; Yu, Jingwei; Levy, Jay A; Kan, Yuet Wai

    2014-07-01

    Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

  4. A rare SNP mutation in Brachytic2 moderately reduces plant height and increases yield potential in maize.

    PubMed

    Xing, Anqi; Gao, Yufeng; Ye, Lingfeng; Zhang, Weiping; Cai, Lichun; Ching, Ada; Llaca, Victor; Johnson, Blaine; Liu, Lin; Yang, Xiaohong; Kang, Dingming; Yan, Jianbing; Li, Jiansheng

    2015-07-01

    Plant height has long been an important agronomic trait in maize breeding. Many plant height QTLs have been reported, but few of these have been cloned. In this study, a major plant height QTL, qph1, was mapped to a 1.6kb interval in Brachytic2 (Br2) coding sequence on maize chromosome 1. A naturally occurring rare SNP in qph1, which resulted in an amino acid substitution, was validated as the causative mutation. QPH1 protein is located in the plasma membrane and polar auxin transport is impaired in the short near-isogenic line RIL88(qph1). Allelism testing showed that the SNP variant in qph1 reduces longitudinal cell number and decreases plant height by 20% in RIL88(qph1) compared to RIL88(QPH1), and is milder than known br2 mutant alleles. The effect of qph1 on plant height is significant and has no or a slight influence on yield in four F2 backgrounds and in six pairs of single-cross hybrids. Moreover, qph1 could reduce plant height when heterozygous, allowing it to be easily employed in maize breeding. Thus, a less-severe allele of a known dwarf mutant explains part of the quantitative variation for plant height and has great potential in maize improvement.

  5. A rare SNP mutation in Brachytic2 moderately reduces plant height and increases yield potential in maize

    PubMed Central

    Xing, Anqi; Gao, Yufeng; Ye, Lingfeng; Zhang, Weiping; Cai, Lichun; Ching, Ada; Llaca, Victor; Johnson, Blaine; Liu, Lin; Yang, Xiaohong; Kang, Dingming; Yan, Jianbing; Li, Jiansheng

    2015-01-01

    Plant height has long been an important agronomic trait in maize breeding. Many plant height QTLs have been reported, but few of these have been cloned. In this study, a major plant height QTL, qph1, was mapped to a 1.6kb interval in Brachytic2 (Br2) coding sequence on maize chromosome 1. A naturally occurring rare SNP in qph1, which resulted in an amino acid substitution, was validated as the causative mutation. QPH1 protein is located in the plasma membrane and polar auxin transport is impaired in the short near-isogenic line RIL88(qph1). Allelism testing showed that the SNP variant in qph1 reduces longitudinal cell number and decreases plant height by 20% in RIL88 (qph1) compared to RIL88 (QPH1), and is milder than known br2 mutant alleles. The effect of qph1 on plant height is significant and has no or a slight influence on yield in four F2 backgrounds and in six pairs of single-cross hybrids. Moreover, qph1 could reduce plant height when heterozygous, allowing it to be easily employed in maize breeding. Thus, a less-severe allele of a known dwarf mutant explains part of the quantitative variation for plant height and has great potential in maize improvement. PMID:25922491

  6. Increased co-expression of genes harboring the damaging de novo mutations in Chinese schizophrenic patients during prenatal development.

    PubMed

    Wang, Qiang; Li, Miaoxin; Yang, Zhenxing; Hu, Xun; Wu, Hei-Man; Ni, Peiyan; Ren, Hongyan; Deng, Wei; Li, Mingli; Ma, Xiaohong; Guo, Wanjun; Zhao, Liansheng; Wang, Yingcheng; Xiang, Bo; Lei, Wei; Sham, Pak C; Li, Tao

    2015-12-15

    Schizophrenia is a heritable, heterogeneous common psychiatric disorder. In this study, we evaluated the hypothesis that de novo variants (DNVs) contribute to the pathogenesis of schizophrenia. We performed exome sequencing in Chinese patients (N = 45) with schizophrenia and their unaffected parents (N = 90). Forty genes were found to contain DNVs. These genes had enriched transcriptional co-expression profile in prenatal frontal cortex (Bonferroni corrected p < 9.1 × 10(-3)), and in prenatal temporal and parietal regions (Bonferroni corrected p < 0.03). Also, four prenatal anatomical subregions (VCF, MFC, OFC and ITC) have shown significant enrichment of connectedness in co-expression networks. Moreover, four genes (LRP1, MACF1, DICER1 and ABCA2) harboring the damaging de novo mutations are strongly prioritized as susceptibility genes by multiple evidences. Our findings in Chinese schizophrenic patients indicate the pathogenic role of DNVs, supporting the hypothesis that schizophrenia is a neurodevelopmental disease.

  7. Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

    SciTech Connect

    Hoetzel, Isidro . E-mail: ihotzel@gene.com; Cheevers, William P.

    2005-09-01

    The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains of CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain {beta}-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding.

  8. P53 mutations in triple negative breast cancer upregulate endosomal recycling of epidermal growth factor receptor (EGFR) increasing its oncogenic potency.

    PubMed

    Shapira, Iuliana; Lee, Annette; Vora, Reena; Budman, Daniel R

    2013-11-01

    There is no available targeted therapy for triple-negative or its more aggressive subtype, basal-like breast cancer. Multiple therapeutic strategies based on translational knowledge have not improved the treatment options for triple negative patients. As understanding of molecular pathways that drive tumor development is rapidly increasing, it is imperative to adapt our treatment strategies to perturbations in molecular pathways driving the malignant process. Basal-like breast cancers over-express EGFR (without mutations or EGFR gene amplifications) and have p53 mutations. While EGFR drives the malignant behavior in triple negative breast cancer (TNBC), anti-EGFR therapies have fallen short of the expected results in clinical trials. Here we bring evidence that the less than optimal results of the anti-EGFR therapies may be explained in part by the increased potency of the EGFR signaling due to increased endosomal recycling. The functional connection between EGFR and endosomal trafficking in TNBC is mutant p53 found in the most aggressive forms of TNBC. Mutant p53 acquires oncogenic functions and binds p63 protein, a member of p53 family with tumor suppressor activities. In the absence of functional p63 there is an upregulation of endosomal recycling EGFR and integrin to the membrane with increased proinvasive abilities of cancer cells. Blocking endosomal trafficking combined with anti-EGFR treatments may result in better clinical outcomes in TNBC.

  9. Error-prone initiation factor 2 mutations reduce the fitness cost of antibiotic resistance

    PubMed Central

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

    2010-01-01

    Mutations in the fmt gene (encoding formyl methionine transferase) that eliminate formylation of initiator tRNA (Met-tRNAi) confer resistance to the novel antibiotic class of peptide deformylase inhibitors (PDFIs) while concomitantly reducing bacterial fitness. Here we show in Salmonella typhimurium that novel mutations in initiation factor 2 (IF2) located outside the initiator tRNA binding domain can partly restore fitness of fmt mutants without loss of antibiotic resistance. Analysis of initiation of protein synthesis in vitro showed that with non-formylated Met-tRNAi IF2 mutants initiated much faster than wild-type IF2, whereas with formylated fMet-tRNAi the initiation rates were similar. Moreover, the increase in initiation rates with Met-tRNAi conferred by IF2 mutations in vitro correlated well with the increase in growth rate conferred by the same mutations in vivo, suggesting that the mutations in IF2 compensate formylation deficiency by increasing the rate of in vivo initiation with Met-tRNAi. IF2 mutants had also a high propensity for erroneous initiation with elongator tRNAs in vitro, which could account for their reduced fitness in vivo in a formylation-proficient strain. More generally, our results suggest that bacterial protein synthesis is mRNA-limited and that compensatory mutations in IF2 could increase the persistence of PDFI-resistant bacteria in clinical settings. PMID:20132454

  10. A Gain-of-Function Mutation in Tnni2 Impeded Bone Development through Increasing Hif3a Expression in DA2B Mice

    PubMed Central

    Zhao, Yanyang; Yang, Peng; Chen, Jun; Sun, Hanzi; Liu, Lei; Li, Wenjun; Pan, Lin; Guo, Yanru; Kou, Zhaohui; Zhang, Yu; Zhou, Cheng; He, Jiang; Zhang, Xue; Li, Jianxin; Han, Weitian; Li, Jian; Liu, Guanghui; Gao, Shaorong; Yang, Ze

    2014-01-01

    Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder. PMID:25340332

  11. Mutation of Candida tropicalis by Irradiation with a He-Ne Laser To Increase Its Ability To Degrade Phenol▿

    PubMed Central

    Jiang, Yan; Wen, Jianping; Jia, Xiaoqiang; Caiyin, Qinggele; Hu, Zongding

    2007-01-01

    Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 × 107 cells ml−1 were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter−1 phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter−1. The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did. PMID:17085704

  12. Heterozygous Mutations of FREM1 Are Associated with an Increased Risk of Isolated Metopic Craniosynostosis in Humans and Mice

    PubMed Central

    Short, Kieran M.; Wiradjaja, Fenny; Janssen, Irene M.; Jehee, Fernanda; Bertola, Debora; Liu, Jia; Yagnik, Garima; Sekiguchi, Kiyotoshi; Kiyozumi, Daiji; van Bokhoven, Hans; Marcelis, Carlo; Cunningham, Michael L.; Anderson, Peter J.; Boyadjiev, Simeon A.; Passos-Bueno, Maria Rita; Veltman, Joris A.; Smyth, Ian; Buckley, Michael F.; Roscioli, Tony

    2011-01-01

    The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia. PMID:21931569

  13. Increased Prevalence of the pfdhfr/phdhps Quintuple Mutant and Rapid Emergence of pfdhps Eesistance Mutations at Codons 581 and 613 in Kisumu, Kenya

    DTIC Science & Technology

    2010-11-24

    pfdhps mutations S436A, A581G, and A613S/T combined with A437G increase the IC50 of sulfa drugs in in vitro culture relative to A437G alone [20-21...14. ABSTRACT Background Anti-malarial drug resistance in Kenya prompted two drug policy changes within a decade sulphadoxine-pyrimethamine (SP...conducted to monitor changes in the prevalence of molecular markers of drug resistance over the period in which SP was used as the first-line anti

  14. Mutations alter the sodium versus proton use of a Bacillus clausii flagellar motor and confer dual ion use on Bacillus subtilis motors.

    PubMed

    Terahara, Naoya; Krulwich, Terry A; Ito, Masahiro

    2008-09-23

    Bacterial flagella contain membrane-embedded stators, Mot complexes, that harness the energy of either transmembrane proton or sodium ion gradients to power motility. Use of sodium ion gradients is associated with elevated pH and sodium concentrations. The Mot complexes studied to date contain channels that use either protons or sodium ions, with some bacteria having only one type and others having two distinct Mot types with different ion-coupling. Here, alkaliphilic Bacillus clausii KSM-K16 was shown to be motile in a pH range from 7 to 11 although its genome encodes only one Mot (BCl-MotAB). Assays of swimming as a function of pH, sodium concentration, and ion-selective motility inhibitors showed that BCl-MotAB couples motility to sodium at the high end of its pH range but uses protons at lower pH. This pattern was confirmed in swimming assays of a statorless Bacillus subtilis mutant expressing either BCl-MotAB or one of the two B. subtilis stators, sodium-coupled Bs-MotPS or proton-coupled Bs-MotAB. Pairs of mutations in BCl-MotB were identified that converted the naturally bifunctional BCl-MotAB to stators that preferentially use either protons or sodium ions across the full pH range. We then identified trios of mutations that added a capacity for dual-ion coupling on the distinct B. subtilis Bs-MotAB and Bs-MotPS motors. Determinants that alter the specificity of bifunctional and single-coupled flagellar stators add to insights from studies of other ion-translocating transporters that use both protons and sodium ions.

  15. A novel pyrosequencing assay for the detection of neuraminidase inhibitor resistance-conferring mutations among clinical isolates of avian H7N9 influenza virus.

    PubMed

    Qi, Yuhua; Fan, Huan; Qi, Xian; Zhu, Zheng; Guo, Xiling; Chen, Yin; Ge, Yiyue; Zhao, Kangchen; Wu, Tao; Li, Yan; Shan, Yunfeng; Zhou, Minghao; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao

    2014-01-22

    A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.

  16. Arabidopsis actin capping protein (AtCP) subunits have different expression patterns, and downregulation of AtCPB confers increased thermotolerance of Arabidopsis after heat shock stress.

    PubMed

    Wang, Jue; Qian, Dong; Fan, Tingting; Jia, Honglei; An, Lizhe; Xiang, Yun

    2012-09-01

    As a heterodimer actin-binding protein, capping protein is composed of α and β subunits, and can stabilize the actin filament cytoskeleton by binding to F-actin ends to inhibit G-actin addition or loss from that end. Until now, studies on plant capping protein have focused on biochemical functions in vitro, and so the expression patterns and physiological functions of actin capping protein in Arabidopsis (AtCP) are poorly understood. In the present study, real-time quantitative PCR and Western blot analysis showed that although AtCP α and β subunits (i.e. AtCPA and AtCPB) were expressed in various tissues, their expression patterns were significantly different. GUS staining further indicated they were present in different parts of the same organs. We also demonstrated that the expression levels of both subunits were induced by heat shock stress. However, only the atcpβ-mutant showed enhanced thermotolerance, and confocal microscopy showed that the actin filaments of the atcpβ-mutant were much more complete than that in the wild-type and the atcpα-mutant after heat treatment at 45 °C for 40 and 45 min. In conclusion, these results demonstrated that AtCPA and AtCPB showed distinct expression patterns in vivo, and that downregulation of AtCPB conferred increased plant thermotolerance after heat shock stress.

  17. High-resolution fine mapping of ps-2, a mutated gene conferring functional male sterility in tomato due to non-dehiscent anthers.

    PubMed

    Gorguet, Benoit; Schipper, Danny; van Heusden, Adriaan W; Lindhout, Pim

    2006-11-01

    Functional male sterility is an important trait for the production of hybrid seeds. Among the genes coding for functional male sterility in tomato is the positional sterility gene ps-2. ps-2 is monogenic recessive, confers non-dehiscent anthers and is the most suitable for practical uses. In order to have tools for molecular-assisted selection (MAS) we fine mapped the ps-2 locus. This was done in an F(2) segregating population derived from the interspecific cross between a functionally male sterile line (ps-2/ps-2; Solanum lycopersicum) and a functionally male fertile line (S. pimpinellifolium). Here we report the procedure that has led to the high-resolution fine mapping of the ps-2 locus in a 1.65 cM interval delimited by markers T0958 and T0635 on the short arm of Chromosome 4. The presence of many COS markers in the local high-resolution map allowed us to study the synteny between tomato and Arabidopsis at the ps-2 locus region. No obvious candidate gene for ps-2 was identified among the known functional male sterility genes in Arabidopsis.

  18. The variant allele of the rs188140481 polymorphism confers a moderate increase in the risk of prostate cancer in Polish men.

    PubMed

    Antczak, Andrzej; Wokołorczyk, Dominika; Kluźniak, Wojciech; Kashyap, Aniruddh; Jakubowska, Anna; Gronwald, Jacek; Huzarski, Tomasz; Byrski, Tomasz; Dębniak, Tadeusz; Masojć, Bartłomiej; Górski, Bohdan; Gromowski, Tomasz; Gołąb, Adam; Sikorski, Andrzej; Słojewski, Marcin; Gliniewicz, Bartłomiej; Borkowski, Tomasz; Borkowski, Andrzej; Przybyła, Jacek; Sosnowski, Marek; Małkiewicz, Bartosz; Zdrojowy, Romuald; Sikorska-Radek, Paulina; Matych, Józef; Wilkosz, Jacek; Różański, Waldemar; Kiś, Jacek; Bar, Krzysztof; Janiszewska, Hanna; Stawicka, Małgorzata; Milecki, Piotr; Lubiński, Jan; Narod, Steven A; Cybulski, Cezary

    2015-03-01

    A number of single nucleotide polymorphisms (SNPs) in the human genome have been associated with increased risk of prostate cancer. Recently, a single SNP in the region of chromosome 8q24 (rs188140481) has been associated with a three-fold increased risk of prostate cancer in Europe and North America. To establish whether rs188140481 is associated with the risk of prostate cancer in Poland, we genotyped 3467 men with prostate cancer and 1958 controls. The A allele of rs188140481 was detected in 44 of 3467 (1.3%) men with prostate cancer and in seven of 1958 (0.4%) controls (odds ratio=3.6; 95% confidence interval 1.6-7.9; P=0.0006). The allele was present in eight of 390 (2.1%) men with familial prostate cancer (odds ratio=5.8; 95% confidence interval 2.1-16.2; P=0.001). A positive family history of cancers at sites other than the prostate was observed in 27% of men who carried the rs188140481 risk allele and in 44% of noncarriers (P=0.04). No cancer at a site other than the prostate was more common in first-degree or second-degree relatives of carriers of the rs188140481 risk allele than relatives of noncarriers. The rs188140481 polymorphism in the 8q24 region confers a moderate increase in the risk of prostate cancer in Polish men. The SNP does not appear to be associated with susceptibility to cancers of other types.

  19. D-Alanylation of Lipoteichoic Acids Confers Resistance to Cationic Peptides in Group B Streptococcus by Increasing the Cell Wall Density

    PubMed Central

    Saar-Dover, Ron; Bitler, Arkadi; Nezer, Ravit; Shmuel-Galia, Liraz; Firon, Arnaud; Shimoni, Eyal; Trieu-Cuot, Patrick; Shai, Yechiel

    2012-01-01

    Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance. PMID:22969424

  20. Obesogenic memory can confer long-term increases in adipose tissue but not liver inflammation and insulin resistance after weight loss

    PubMed Central

    Schmitz, J.; Evers, N.; Awazawa, M.; Nicholls, H.T.; Brönneke, H.S.; Dietrich, A.; Mauer, J.; Blüher, M.; Brüning, J.C.

    2016-01-01

    Objective Obesity represents a major risk factor for the development of type 2 diabetes mellitus, atherosclerosis and certain cancer entities. Treatment of obesity is hindered by the long-term maintenance of initially reduced body weight, and it remains unclear whether all pathologies associated with obesity are fully reversible even upon successfully maintained weight loss. Methods We compared high fat diet-fed, weight reduced and lean mice in terms of body weight development, adipose tissue and liver insulin sensitivity as well as inflammatory gene expression. Moreover, we assessed similar parameters in a human cohort before and after bariatric surgery. Results Compared to lean animals, mice that demonstrated successful weight reduction showed increased weight gain following exposure to ad libitum control diet. However, pair-feeding weight-reduced mice with lean controls efficiently stabilized body weight, indicating that hyperphagia was the predominant cause for the observed weight regain. Additionally, whereas glucose tolerance improved rapidly after weight loss, systemic insulin resistance was retained and ameliorated only upon prolonged pair-feeding. Weight loss enhanced insulin action and resolved pro-inflammatory gene expression exclusively in the liver, whereas visceral adipose tissue displayed no significant improvement of metabolic and inflammatory parameters compared to obese mice. Similarly, bariatric surgery in humans (n = 55) resulted in massive weight reduction, improved hepatic inflammation and systemic glucose homeostasis, while adipose tissue inflammation remained unaffected and adipocyte-autonomous insulin action only exhibit minor improvements in a subgroup of patients (42%). Conclusions These results demonstrate that although sustained weight loss improves systemic glucose homeostasis, primarily through improved inflammation and insulin action in liver, a remarkable obesogenic memory can confer long-term increases in adipose tissue

  1. miR-335 Targets SIAH2 and Confers Sensitivity to Anti-Cancer Drugs by Increasing the Expression of HDAC3

    PubMed Central

    Kim, Youngmi; Kim, Hyuna; Park, Deokbum; Jeoung, Dooil

    2015-01-01

    We previously reported the role of histone deacetylase 3 (HDAC3) in response to anti-cancer drugs. The decreased expression of HDAC3 in anti-cancer drug-resistant cancer cell line is responsible for the resistance to anti-cancer drugs. In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression. MG132, an inhibitor of proteasomal degradation, induced the expression of HDAC3 in various anti-cancer drug-resistant cancer cell lines. Ubiquitination of HDAC3 was observed in various anti-cancer drug-resistant cancer cell lines. HDAC3 showed an interaction with SIAH2, an ubiquitin E3 ligase, that has increased expression in various anti-cancer drug-resistant cancer cell lines. miRNA array analysis showed the decreased expression of miR-335 in these cells. Targetscan analysis predicted the binding of miR-335 to the 3′-UTR of SIAH2. miR-335-mediated increased sensitivity to anti-cancer drugs was associated with its effect on HDAC3 and SIAH2 expression. miR-335 exerted apoptotic effects and inhibited ubiquitination of HDAC3 in anti-cancer drug-resistant cancer cell lines. miR-335 negatively regulated the invasion, migration, and growth rate of cancer cells. The mouse xenograft model showed that miR-335 negatively regulated the tumorigenic potential of cancer cells. The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs. The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs. PMID:25997740

  2. Recurrent BRCA1 and BRCA2 mutations in Mexican women with breast cancer

    PubMed Central

    Torres-Mejía, Gabriela; Royer, Robert; Llacuachaqui, Marcia; Akbari, Mohammad R.; Giuliano, Anna R.; Martínez-Matsushita, Louis; Angeles-Llerenas, Angélica; Ortega-Olvera, Carolina; Ziv, Elad; Lazcano-Ponce, Eduardo; Phelan, Catherine M.; Narod, Steven A.

    2015-01-01

    Background Germline mutations in the BRCA1 and BRCA2 genes confer an estimated 58–80% lifetime risk of breast cancer. In general, screening is done for cancer patients if a relative has been diagnosed with breast or ovarian cancer. There are few data on the prevalence of mutations in these genes in Mexican women with breast cancer and this hampers efforts to develop screening policies in Mexico. Methods We screened 810 unselected women with breast cancer from three cities in Mexico (Mexico City, Veracruz and Monterrey) for mutations in BRCA1 and BRCA2, including a panel of 26 previously reported mutations. Results Thirty-five mutations were identified in 34 women (4.3% of total) including 20 BRCA1 mutations and 15 BRCA2 mutations. Twenty-two of the 35 mutations were recurrent mutations (62.8%). Only five of the 34 mutation carriers had a first-degree relative with breast cancer (three with BRCA1 and two with BRCA2 mutations). Conclusion These results support the rationale for a strategy of screening for recurrent mutations in all women with breast cancer in Mexico, as opposed to restricting screening to those with a sister or mother with breast or ovarian cancer. Impact These results will impact cancer genetic testing in Mexico and the identification of at-risk individuals who will benefit from increased surveillance. PMID:25371446

  3. Mutation of the endogenous p53 gene in cells transformed by HPV-16 E7 and EJ c-ras confers a growth advantage involving an autocrine mechanism.

    PubMed Central

    Peacock, J W; Benchimol, S

    1994-01-01

    Rat embryo fibroblasts transformed with the HPV-16 E7 gene and the activated c-H-ras gene fall into two distinct phenotypic classes. At high cell density, clones of one class form colonies in methylcellulose supplemented with low serum; at low cell density, these cells display responsiveness to mitogenic factors present in serum-free conditioned medium from rat embryo fibroblasts. In contrast, clones of the second class exhibit an absolute dependency on growth factors present in serum at all cell densities in the methylcellulose colony assay and fail to respond to conditioned medium. We find that the status of the endogenous p53 gene is tightly correlated with these two classes of clones. Clones of the first class contain missense mutations in the p53 gene and have lost the wild-type allele. Clones of the second class express wild-type p53 protein. The importance of mutant p53 expression in reducing the growth factor dependency of transformed clones was confirmed in a separate series of experiments in which rat embryo fibroblasts were transformed with three genes, E7 + ras + mutant p53. The growth behaviour of these triply transfected clones was similar to that of the E7 + ras clones expressing endogenous mutant p53. We demonstrate that the enhanced proliferation of E7 + ras clones expressing mutant p53 protein involves an autocrine mechanism. Images PMID:8131742

  4. Ectopic Overexpression of SlHsfA3, a Heat Stress Transcription Factor from Tomato, Confers Increased Thermotolerance and Salt Hypersensitivity in Germination in Transgenic Arabidopsis

    PubMed Central

    Li, Zhenjun; Zhang, Lili; Wang, Aoxue; Xu, Xiangyang; Li, Jingfu

    2013-01-01

    Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions. PMID:23349984

  5. Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios+ T cells and autoantibodies

    PubMed Central

    Myers, Darienne R; Polakos, Noelle K; Enders, Anselm; Roots, Carla; Balakishnan, Bhavani; Miosge, Lisa A; Sjollema, Geoff; Bertram, Edward M; Field, Matthew A; Shao, Yunli; Andrews, T Daniel; Whittle, Belinda; Barnes, S Whitney; Walker, John R; Cyster, Jason G

    2013-01-01

    Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1Anaef, with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1Anaef mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44hi Helios+ PD-1+ CD4+ T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1Anaef is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1Anaef naïve CD4+ T cells. CD44 expression, CD4+ T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1AnaefMtorchino double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1Anaef T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001 PMID:24336796

  6. Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement.

    PubMed

    Huang, Sheng-Wen; Wang, Ya-Fang; Yu, Chun-Keung; Su, Ih-Jen; Wang, Jen-Ren

    2012-01-05

    Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2(149) (VP2(149M)) or glutamine to glutamic acid substitution at VP1(145) (VP1(145E)) showed greater viral titers and apoptosis. Synergistic effect of VP2(149M) and VP1(145E) double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2(149M) and VP1(145E) mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.

  7. Mutation in the ntrR gene, a member of the vap gene family, increases the symbiotic efficiency of Sinorhizobium meliloti.

    PubMed

    Oláh, B; Kiss, E; Györgypál, Z; Borzi, J; Cinege, G; Csanádi, G; Batut, J; Kondorosi, A; Dusha, I

    2001-07-01

    In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the "ntrPR operon," which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

  8. Mutations in ABO1/ELO2, a Subunit of Holo-Elongator, Increase Abscisic Acid Sensitivity and Drought Tolerance in Arabidopsis thaliana

    PubMed Central

    Chen, Zhizhong; Zhang, Hairong; Jablonowski, Daniel; Zhou, Xiaofeng; Ren, Xiaozhi; Hong, Xuhui; Schaffrath, Raffael; Zhu, Jian-Kang; Gong, Zhizhong

    2006-01-01

    The phytohormone abscisic acid (ABA) plays an important role in modulating plant growth, development, and stress responses. In a genetic screen for mutants with altered drought stress responses, we identified an ABA-overly sensitive mutant, the abo1 mutant, which showed a drought-resistant phenotype. The abo1 mutation enhances ABA-induced stomatal closing and increases ABA sensitivity in inhibiting seedling growth. abo1 mutants are more resistant to oxidative stress than the wild type and show reduced levels of transcripts of several stress- or ABA-responsive genes. Interestingly, the mutation also differentially modulates the development and growth of adjacent guard cells. Map-based cloning identified ABO1 as a new allele of ELO2, which encodes a homolog of Saccharomyces cerevisiae Iki3/Elp1/Tot1 and human IκB kinase-associated protein. Iki3/Elp1/Tot1 is the largest subunit of Elongator, a multifunctional complex with roles in transcription elongation, secretion, and tRNA modification. Ecotopic expression of plant ABO1/ELO2 in a tot1/elp1Δ yeast Elongator mutant complements resistance to zymocin, a yeast killer toxin complex, indicating that ABO1/ELO2 substitutes for the toxin-relevant function of yeast Elongator subunit Tot1/Elp1. Our results uncover crucial roles for ABO1/ELO2 in modulating ABA and drought responses in Arabidopsis thaliana. PMID:16943431

  9. Increased Permeability of the Aquaporin SoPIP2;1 by Mercury and Mutations in Loop A

    PubMed Central

    Kirscht, Andreas; Survery, Sabeen; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) also referred to as Major intrinsic proteins, regulate permeability of biological membranes for water and other uncharged small polar molecules. Plants encode more AQPs than other organisms and just one of the four AQP subfamilies in Arabidopsis thaliana, the water specific plasma membrane intrinsic proteins (PIPs), has 13 isoforms, the same number as the total AQPs encoded by the entire human genome. The PIPs are more conserved than other plant AQPs and here we demonstrate that a cysteine residue, in loop A of SoPIP2;1 from Spinacia oleracea, is forming disulfide bridges. This is in agreement with studies on maize PIPs, but in contrast we also show an increased permeability of mutants with a substitution at this position. In accordance with earlier findings, we confirm that mercury increases water permeability of both wild type and mutant proteins. We report on the slow kinetics and reversibility of the activation, and on quenching of intrinsic tryptophan fluorescence as a potential reporter of conformational changes associated with activation. Hence, previous studies in plants based on the assumption of mercury as a general AQP blocker have to be reevaluated, whereas mercury and fluorescence studies of isolated PIPs provide new means to follow structural changes dynamically. PMID:27625657

  10. Gene-environment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function.

    PubMed

    Burren, Katie A; Savery, Dawn; Massa, Valentina; Kok, Robert M; Scott, John M; Blom, Henk J; Copp, Andrew J; Greene, Nicholas D E

    2008-12-01

    Risk of neural tube defects (NTDs) is determined by genetic and environmental factors, among which folate status appears to play a key role. However, the precise nature of the link between low folate status and NTDs is poorly understood, and it remains unclear how folic acid prevents NTDs. We investigated the effect of folate level on risk of NTDs in splotch (Sp(2)(H)) mice, which carry a mutation in Pax3. Dietary folate restriction results in reduced maternal blood folate, elevated plasma homocysteine and reduced embryonic folate content. Folate deficiency does not cause NTDs in wild-type mice, but causes a significant increase in cranial NTDs among Sp(2)(H) embryos, demonstrating a gene-environment interaction. Control treatments, in which intermediate levels of folate are supplied, suggest that NTD risk is related to embryonic folate concentration, not maternal blood folate concentration. Notably, the effect of folate deficiency appears more deleterious in female embryos than males, since defects are not prevented by exogenous folic acid. Folate-deficient embryos exhibit developmental delay and growth retardation. However, folate content normalized to protein content is appropriate for developmental stage, suggesting that folate availability places a tight limit on growth and development. Folate-deficient embryos also exhibit a reduced ratio of s-adenosylmethionine (SAM) to s-adenosylhomocysteine (SAH). This could indicate inhibition of the methylation cycle, but we did not detect any diminution in global DNA methylation, in contrast to embryos in which the methylation cycle was specifically inhibited. Hence, folate deficiency increases the risk of NTDs in genetically predisposed splotch embryos, probably via embryonic growth retardation.

  11. The Gastrodia anti-fungal protein confers increased resistance to Phytophthora root rot and the root-knot nematode in a fruit tree species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Gastrodia Anti-Fungal Protein (GAFP) is a monocot mannose-binding lectin isolated from the Asiatic orchid Gastrodia elata. This protein, among others, enables the orchid to live parasitically off the basidiomycete pathogen Armillaria mellea. GAFP has been shown to confer resistance to transgenic...

  12. Proceedings of the Conference on Increasing Opportunities for Mexican American Students in Higher Education (Los Angeles Harbor College, California, May 15-17, 1969).

    ERIC Educational Resources Information Center

    Franklin, Mayer J., Ed.; And Others

    The conference was conducted in order to propose solutions to the problems that confront the Mexican American student in higher education in areas of recruitment, retention, and financial assistance. Colleges and universities from 5 Southwestern states sent teams (student, faculty member, administrator, junior college representative, high school…

  13. ConfChem Conference on Flipped Classroom: Reclaiming Face Time--How an Organic Chemistry Flipped Classroom Provided Access to Increased Guided Engagement

    ERIC Educational Resources Information Center

    Trogden, Bridget G.

    2015-01-01

    Students' active engagement is one of the most critical challenges to any successful learning environment. The blending of active engagement along with rich, meaningful content is necessary for chemical educators to re-examine the purpose of the chemistry classroom. The Spring 2014 ConfChem conference, Flipped Classroom, was held from May 9 to…

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

    PubMed Central

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

    1990-01-01

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

  15. In Egyptians, a mutation in the lymphotoxin-alpha gene may increase susceptibility to hepatitis C virus but not that to schistosomal infection.

    PubMed

    Elsammak, M Y; Al-Sharkaweey, R M; Ragab, M S; Amin, G M; Kandil, M H

    2008-12-01

    In Egypt, human schistosomiasis is a chronic endemic disease that can produce portal hypertension and occasionally death. Curiously, most Egyptian cases of the disease are complicated by co-infection with hepatitis C virus (HCV), the co-infection generally resulting in more severe liver disease than seen in those only infected with HCV. The high frequency of co-infection may be the result of transmission of the virus during parental schistosomal therapy or schistosomiasis-related surgery but it also seems possible that certain individuals are particularly susceptible to both schistosome and HCV infection. Lymphotoxin-alpha (LTalpha) participates in inflammatory responses, and single-nucleotide polymorphisms (SNP) in the human LTalpha gene have recently been found to have profound effects on individual susceptibility to various diseases, including some of those caused by parasitic infection. The possibility that the SNP that create an NcoI restriction site in the gene are associated with increased susceptibility to schistosomal and/or HCV infection has now been investigated in the Egyptian city of Alexandria. The subjects investigated were 22 patients infected only with HCV, 44 cases of schistosomal hepatic fibrosis (SHF) who were either co-infected with HCV (22) or HCV-free (22), and 22 apparently healthy, schistosome-free and HCV-free controls. When each of these subjects was tested for the NcoI polymorphism in their LTalpha gene, by PCR-RFLP, those with isolated HCV infection and those co-infected with Schistosoma and HCV (but not those infected with Schistosoma alone) were found significantly more likely to carry the mutation than the control subjects (P<0.05). When the cases of SHF were pooled together (irrespective of HCV-infection status), they were not found significantly more likely to have the mutation than the controls. At least in Egypt, therefore, the LTalpha mutation may have a role in susceptibility to HCV infection (and the subsequent development of

  16. A single intramuscular vaccination of mice with the HSV-1 VC2 virus with mutations in the glycoprotein K and the membrane protein UL20 confers full protection against lethal intravaginal challenge with virulent HSV-1 and HSV-2 strains.

    PubMed

    Stanfield, Brent A; Stahl, Jacque; Chouljenko, Vladimir N; Subramanian, Ramesh; Charles, Anu-Susan; Saied, Ahmad A; Walker, Jason D; Kousoulas, Konstantin G

    2014-01-01

    Herpes Simplex Virus type-1 (HSV-1) and type-2 (HSV-2) establish life-long infections and cause significant orofacial and genital infections in humans. HSV-1 is the leading cause of infectious blindness in the western world. Currently, there are no available vaccines to protect against herpes simplex infections. Recently, we showed that a single intramuscular immunization with an HSV-1(F) mutant virus lacking expression of the viral glycoprotein K (gK), which prevents the virus from entering into distal axons of ganglionic neurons, conferred significant protection against either virulent HSV-1(McKrae) or HSV-2(G) intravaginal challenge in mice. Specifically, 90% of the mice were protected against HSV-1(McKrae) challenge, while 70% of the mice were protected against HSV-2(G) challenge. We constructed the recombinant virus VC2 that contains specific mutations in gK and the membrane protein UL20 preventing virus entry into axonal compartments of neurons, while allowing efficient replication in cell culture, unlike the gK-null virus, which has a major defect in virus replication and spread. Intramuscular injection of mice with 107 VC2 plaque forming units did not cause any significant clinical disease in mice. A single intramuscular immunization with the VC2 virus protected 100% of mice against lethal intravaginal challenge with either HSV-1(McKrae) or HSV-2(G) viruses. Importantly, vaccination with VC2 produced robust cross protective humoral and cellular immunity that fully protected vaccinated mice against lethal disease. Quantitative PCR did not detect any viral DNA in ganglionic tissues of vaccinated mice, while unvaccinated mice contained high levels of viral DNA. The VC2 virus may serve as an efficient vaccine against both HSV-1 and HSV-2 infections, as well as a safe vector for the production of vaccines against other viral and bacterial pathogens.

  17. Estimation of the HIV-1 backward mutation rate from transmitted drug-resistant strains.

    PubMed

    Kitayimbwa, J M; Mugisha, J Y T; Saenz, R A

    2016-12-01

    One of the serious threats facing the administration of antiretroviral therapy to human immunodeficiency virus (HIV-1) infected patients is the reported increasing prevalence of transmitted drug resistance. However, given that HIV-1 drug-resistant strains are often less fit than the wild-type strains, it is expected that drug-resistant strains that are present during the primary phase of the HIV-1 infection are replaced by the fitter wild-type strains. This replacement of HIV-1 resistant mutations involves the emergence of wild-type strains by a process of backward mutation. How quickly the replacement happens is dependent on the class of HIV-1 mutation group. We estimate the backward mutation rates and relative fitness of various mutational groups known to confer HIV-1 drug resistance. We do this by fitting a stochastic model to data for individuals who were originally infected by an HIV-1 strain carrying any one of the known drug resistance-conferring mutations and observed over a period of time to see whether the resistant strain is replaced. To do this, we seek a distribution, generated from simulations of the stochastic model, that best describes the observed (clinical data) replacement times of a given mutation. We found that Lamivudine/Emtricitabine-associated mutations have a distinctly higher, backward mutation rate and low relative fitness compared to the other classes (as has been reported before) while protease inhibitors-associated mutations have a slower backward mutation rate and high relative fitness. For the other mutation classes, we found more uncertainty in their estimates.

  18. Conference Resolution

    NASA Astrophysics Data System (ADS)

    2009-04-01

    Since the first IUPAP International Conference on Women in Physics (Paris, March 2002) and the Second Conference (Rio de Janeiro, May 2005), progress has continued in most countries and world regions to attract girls to physics and advance women into leadership roles, and many working groups have formed. The Third Conference (Seoul, October 2008), with 283 attendees from 57 countries, was dedicated to celebrating the physics achievements of women throughout the world, networking toward new international collaborations, building each participant's capacity for career success, and aiding the formation of active regional working groups to advance women in physics. Despite the progress, women remain a small minority of the physics community in most countries.

  19. Compound Mutations Cause Increased Cardiac Events in Children with Long QT Syndrome: Can the Sequence Homology-Based Tools be Applied for Prediction of Phenotypic Severity?

    PubMed

    Izumi, Gaku; Hayama, Emiko; Yamazawa, Hirokuni; Inai, Kei; Shimada, Mitsuyo; Furutani, Michiko; Nishizawa, Tsutomu; Furutani, Yoshiyuki; Matsuoka, Rumiko; Nakanishi, Toshio

    2016-06-01

    Long QT syndrome (LQTS) can cause syncope, ventricular fibrillation, and death. Recently, several disease-causing mutations in ion channel genes have been identified, and compound mutations have also been detected. It is unclear whether children who are carriers of compound mutations exhibit a more severe phenotype than those with single mutations. Although predicting phenotypic severity is clinically important, the availability of prediction tools for LQTS is unknown. To determine whether the severity of the LQTS phenotype can be predicted by the presence of compound mutations in children is needed. We detected 97 single mutations (Group S) and 13 compound mutations (Group C) between 1998 and 2012, age at diagnosis ranging 0-19 years old (median age is 9.0) and 18.0 years of follow-up period. The phenotypes and Kaplan-Meier event-free rates of the two groups were compared for cardiac events. This study investigated phenotypic severity in relation to the location of mutations in the protein sequence, which was analyzed using two sequence homology-based tools. In results, compound mutations in children were associated with a high incidence of syncope within the first decade (Group S: 32 % vs. Group C: 61 %), requiring an ICD in the second decade (Group S: 3 % vs. Group C: 56 %). Mortality in these patients was high within 5 years of birth (23 %). Phenotypic prediction tools correctly predicted the phenotypic severity in both Groups S and C, especially by using their coupling method. The coupling prediction method is useful in the initial evaluation of phenotypes both with single and compound mutations of LQTS patients. However, it should be noted that the compound mutation makes more severe phenotype.

  20. Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.

    PubMed Central

    Manak, J R; Prywes, R

    1991-01-01

    Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation. Images PMID:2046671

  1. Increased outer arm and core fucose residues on the N-glycans of mutated alpha-1 antitrypsin protein from alpha-1 antitrypsin deficient individuals.

    PubMed

    McCarthy, Cormac; Saldova, Radka; O'Brien, M Emmet; Bergin, David A; Carroll, Tomás P; Keenan, Joanne; Meleady, Paula; Henry, Michael; Clynes, Martin; Rudd, Pauline M; Reeves, Emer P; McElvaney, Noel G

    2014-02-07

    Alpha-1 antitrypsin (AAT) is the major physiological inhibitor of a range of serine proteases, and in the lung, it maintains a protease-antiprotease balance. AAT deficiency (AATD) is an autosomal co-dominant condition with the Z mutation being the most common cause. Individuals homozygous for Z (PiZZ) have low levels of circulating mutant Z-AAT protein leading to premature emphysematous lung disease. Extensive glycoanalysis has been performed on normal AAT (M-AAT) from healthy individuals and the importance of glycosylation in affecting the immune modulatory roles of AAT is documented. However, no glycoanalysis has been carried out on Z-AAT from deficient individuals to date. In this study, we investigate whether the glycans present on Z-AAT differ to those found on M-AAT from healthy controls. Plasma AAT was purified from 10 individuals: 5 AATD donors with the PiZZ phenotype and 5 PiMM healthy controls. Glycoanalysis was performed employing N-glycan release, exoglycosidase digestion and UPLC analysis. No difference in branched glycans was identified between AATD and healthy controls. However, a significant increase in both outer arm (α1-3) (p = 0.04) and core (α1-6) fucosylated glycans (p < 0.0001) was found on Z-AAT compared to M-AAT. This study has identified increased fucosylation on N-glycans of Z-AAT indicative of ongoing inflammation in AATD individuals with implications for early therapeutic intervention.

  2. Mutations in Acetylcholinesterase2 (ace2) increase the insensitivity of acetylcholinesterase to fosthiazate in the root-knot nematode Meloidogyne incognita.

    PubMed

    Huang, Wen-Kun; Wu, Qin-Song; Peng, Huan; Kong, Ling-An; Liu, Shi-Ming; Yin, Hua-Qun; Cui, Ru-Qiang; Zhan, Li-Ping; Cui, Jiang-Kuan; Peng, De-Liang

    2016-11-29

    The root-knot nematode Meloidogyne incognita causes severe damage to continuously cropping vegetables. The control of this nematode relies heavily on organophosphate nematicides in China. Here, we described resistance to the organophosphate nematicide fosthiazate in a greenhouse-collected resistant population (RP) and a laboratory susceptible population (SP) of M. incognita. Fosthiazate was 2.74-fold less toxic to nematodes from RP than that from SP. Quantitative real-time PCR revealed that the acetylcholinesterase2 (ace2) transcription level in the RP was significantly higher than that in the SP. Eighteen nonsynonymous amino acid differences in ace2 were observed between the cDNA fragments of the RP and SP. The acetylcholinesterase (AChE) protein activity in the RP was significantly reduced compared with that in the SP. After knocking down the ace2 gene, the ace2 transcription level was significantly decreased, but no negative impact on the infection of juveniles was observed. The 50% lethal concentration of the RNAi RP population decreased 40%, but the inhibition rate of fosthiazate against AChE activity was significantly increased in RP population. Thus, the increased fosthiazate insensitivity in the M. incognita resistant population was strongly associated with mutations in ace2. These results provide valuable insights into the resistance mechanism of root-knot nematode to organophosphate nematicides.

  3. Polyamine Resistance Is Increased by Mutations in a Nitrate Transporter Gene NRT1.3 (AtNPF6.4) in Arabidopsis thaliana

    PubMed Central

    Tong, Wurina; Imai, Akihiro; Tabata, Ryo; Shigenobu, Shuji; Yamaguchi, Katsushi; Yamada, Masashi; Hasebe, Mitsuyasu; Sawa, Shinichiro; Motose, Hiroyasu; Takahashi, Taku

    2016-01-01

    Polyamines are small basic compounds present in all living organisms and act in a variety of biological processes. However, the mechanism of polyamine sensing, signaling and response in relation to other metabolic pathways remains to be fully addressed in plant cells. As one approach, we isolated Arabidopsis mutants that show increased resistance to spermine in terms of chlorosis. We show here that two of the mutants have a point mutation in a nitrate transporter gene of the NRT1/PTR family (NPF), NRT1.3 (AtNPF6.4). These mutants also exhibit increased resistance to putrescine and spermidine while loss-of-function mutants of the two closest homologs of NRT1.3, root-specific NRT1.1 (AtNPF6.3) and petiole-specific NRT1.4 (AtNPF6.2), were shown to have a normal sensitivity to polyamines. When the GUS reporter gene was expressed under the control of the NRT1.3 promoter, GUS staining was observed in leaf mesophyll cells and stem cortex cells but not in the epidermis, suggesting that NRT1.3 specifically functions in parenchymal tissues. We further found that the aerial part of the mutant seedling has normal levels of polyamines but shows reduced uptake of norspermidine compared with the wild type. These results suggest that polyamine transport or metabolism is associated with nitrate transport in the parenchymal tissue of the shoot. PMID:27379127

  4. Mutations in Acetylcholinesterase2 (ace2) increase the insensitivity of acetylcholinesterase to fosthiazate in the root-knot nematode Meloidogyne incognita

    PubMed Central

    Huang, Wen-Kun; Wu, Qin-Song; Peng, Huan; Kong, Ling-An; Liu, Shi-Ming; Yin, Hua-Qun; Cui, Ru-Qiang; Zhan, Li-Ping; Cui, Jiang-Kuan; Peng, De-Liang

    2016-01-01

    The root-knot nematode Meloidogyne incognita causes severe damage to continuously cropping vegetables. The control of this nematode relies heavily on organophosphate nematicides in China. Here, we described resistance to the organophosphate nematicide fosthiazate in a greenhouse-collected resistant population (RP) and a laboratory susceptible population (SP) of M. incognita. Fosthiazate was 2.74-fold less toxic to nematodes from RP than that from SP. Quantitative real-time PCR revealed that the acetylcholinesterase2 (ace2) transcription level in the RP was significantly higher than that in the SP. Eighteen nonsynonymous amino acid differences in ace2 were observed between the cDNA fragments of the RP and SP. The acetylcholinesterase (AChE) protein activity in the RP was significantly reduced compared with that in the SP. After knocking down the ace2 gene, the ace2 transcription level was significantly decreased, but no negative impact on the infection of juveniles was observed. The 50% lethal concentration of the RNAi RP population decreased 40%, but the inhibition rate of fosthiazate against AChE activity was significantly increased in RP population. Thus, the increased fosthiazate insensitivity in the M. incognita resistant population was strongly associated with mutations in ace2. These results provide valuable insights into the resistance mechanism of root-knot nematode to organophosphate nematicides. PMID:27897265

  5. ENU-induced mutation in USP18 causes hyperactivation of IFN-αβ signalling and suppresses STAT4-induced IFN-γ production resulting in increased susceptibility to Salmonella Typhimurium

    PubMed Central

    Richer, Etienne; Prendergast, Caitlin; Zhang, Dong-Er; Qureshi, Salman T; Vidal, Silvia M; Malo, Danielle

    2014-01-01

    In order to deepen our knowledge of the natural host response to pathogens our team undertook an in vivo screen of mutagenized 129S1 mice with Salmonella Typhimurium. One mutation affecting Salmonella susceptibility was mapped to a region of 1.3Mb on chromosome 6 that contains 15 protein-coding genes. A missense mutation was identified in the Usp18 (Ubiquitin specific peptidase 18) gene. This mutation results in an increased inflammatory response (IL-6, type 1 IFN) to Salmonella and LPS challenge while paradoxically reducing IFN-γ production during bacterial infection. Increased STAT1 phophorylation correlated with impaired STAT4 phosphorylation resulting in overwhelming IL-6 secretion but reduced IFN-γ production during infection. The reduced IFN-γ levels along with the increased inflammation rationalize the Salmonella Typhimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death. PMID:20693420

  6. Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries).

    PubMed

    Hanrahan, James P; Gregan, Scott M; Mulsant, Philippe; Mullen, Michael; Davis, George H; Powell, Richard; Galloway, Susan M

    2004-04-01

    Belclare and Cambridge are prolific sheep breeds, the origins of which involved selecting ewes with exceptionally high litter size records from commercial flocks. The variation in ovulation rate in both breeds is consistent with segregation of a gene (or genes) with a large effect on this trait. Sterile ewes, due to a failure of normal ovarian follicle development, occur in both breeds. New naturally occurring mutations in genes for the oocyte-derived growth factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are described. These mutations are associated with increased ovulation rate in heterozygous carriers and sterility in homozygous carriers in both breeds. This is the first time that a mutation in the gene for GDF9 has been found that causes increased ovulation rate and infertility in a manner similar to inactivating mutations in BMP15, and shows that GDF9 is essential for normal folliculogenesis in sheep. Furthermore, it is shown, for the first time in any species, that individuals with mutations in both GDF9 and BMP15 have a greater ovulation rate than sheep with either of the mutations separately.

  7. Smoking correlates with increased cytoskeletal protein-related coding region mutations in the lung and head and neck datasets of the cancer genome atlas.

    PubMed

    Yavorski, John M; Blanck, George

    2016-12-01

    Cancer from smoking tobacco is considered dependent on mutagens, but significant molecular aspects of smoking-specific, cancer development remain unknown. We defined sets of coding regions for oncoproteins, tumor suppressor proteins, and cytoskeletal-related proteins that were compared between nonsmokers and smokers, for mutation occurrences, in the lung adenocarcinoma (LUAD), head and neck squamous carcinoma (HNSC), bladder carcinoma (BLCA), and pancreatic adenocarcinoma ( PAAD) datasets from the cancer genome atlas (TCGA). We uncovered significant differences in overall mutation rates, and in mutation rates in cytoskeletal protein-related coding regions (CPCRs, including extracellular matrix protein coding regions), between nonsmokers and smokers in LUAD and HNSC (P < 0.001), raising the question of whether the CPCR mutation differences lead to different clinical courses for nonsmoker and smoker cancers. Another important question inspired by these results is, whether high smoker cancer mutation rates would facilitate genotoxicity or neoantigen-based therapies. No significant, mutation-based differences were found in the BLCA or PAAD datasets, between nonsmokers and smokers. However, a significant difference was uncovered for the average number of overall cancer mutations, in LUAD, for persons who stopped smoking more than 15 years ago, compared with more recent smokers (P < 0.032).

  8. Drug response to HER2 gatekeeper T798M mutation in HER2-positive breast cancer.

    PubMed

    Meng, Xuli; Li, Yongfeng; Tang, Hongchao; Mao, Weimin; Yang, Hongjian; Wang, Xiaojia; Ding, Xianfeng; Xie, Shangnao

    2016-02-01

    The gatekeeper T798M mutation in HER2 kinase domain has been observed to considerably shift drug sensitivity to HER2 in breast cancer therapy. Here, drug response of clinical tyrosine kinase inhibitors (TKIs) to the mutation was profiled using a synthetic biology protocol. It was found that TKIs can be grouped into three classes in terms of their response behavior to T798M mutation: class I inhibitors exhibit drug resistance upon the mutation, such as lapatinib, TAK-285 and AEE788; class II inhibitors are insensitive to the mutation, such as erlotinib and gefitinib; and class III inhibitors can be sensitized by the mutation, such as staurosporine. However, kinetic study indicated that the mutation has only a modest effect on the binding of substrate ATP to HER2. Binding free energy analysis revealed that the drug response is primarily determined by direct interaction between the kinase and inhibitors, but not by indirect kinase interaction with competitive ATP. This is different to the molecular mechanism of "generic" drug resistance conferring from EGFR gatekeeper T790M mutation, which is caused by increased ATP affinity upon the mutation. Structural analysis of kinase-inhibitor complexes unraveled that HER2 T798M mutation induces significant steric hindrance to class I inhibitors, but can establish additional nonbonded interactions for class III inhibitors.

  9. Recombinant human parainfluenza virus type 2 vaccine candidates containing a 3′ genomic promoter mutation and L polymerase mutations are attenuated and protective in non-human primates

    PubMed Central

    Nolan, Sheila M.; Skiadopoulos, Mario H.; Bradley, Konrad; Kim, Olivia S.; Bier, Stacia; Amaro-Carambot, Emerito; Surman, Sonja R.; Davis, Stephanie; St. Claire, Marisa; Elkins, Randy; Collins, Peter L.; Murphy, Brian R.; Schaap-Nutt, Anne

    2007-01-01

    Previously, we identified several attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and genetically stabilized those mutations using reverse genetics (Nolan et al., 2005). Here we describe the discovery of an attenuating mutation at nucleotide 15 (15T→C) in the 3′ genomic promoter that was also present in the previously characterized mutants. We evaluated the properties of this promoter mutation alone and in various combinations with the L polymerase mutations. Amino acid substitutions at L protein positions 460 (460A or 460P) or 948 (948L), or deletion of amino acids 1724 and 1725 (Δ1724), each conferred a temperature sensitivity (ts) phenotype whereas the 15T→C mutation did not. The 460A and 948L mutations each contributed to restricted replication in the lower respiratory tract of African green monkeys, but the Δ1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)/460A/948L and rV94(15C)/948L/Δ1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans. PMID:17658669

  10. Increased incidence of cycloguanil resistance in malaria cases entering France from Africa, determined as point mutations in the parasites' dihydrofolate-reductase genes.

    PubMed

    Durand, R; di Piazza, J P; Longuet, C; Sécardin, Y; Clain, J; le Bras, J

    1999-01-01

    The incidence of cycloguanil resistance in 501 Plasmodium falciparum isolates from individuals entering France from Africa was estimated by a method based on PCR-restriction-fragment-length polymorphisms. None of the subjects had taken antifol prophylaxis. Annual incidence of the resistance, detected as a point mutation at codon 108 in the parasite's dihydrofolate-reductase gene, increased from 19.8% in 1995 to 43.6% in 1997 (P < 0.001). The proportion of isolates found to be susceptible (i.e. wild-type) among travellers returning from the African countries known as Group 2 in France (i.e. Burkina Faso, Côte d'Ivoire, Gambia, Ghana, Guinea, Liberia, Madagascar, Mali, Mauritania, Niger, Senegal, Sierra Leone, Tchad and Togo) was reasonably high (62.9%) and much higher than in the other subjects returning from other identifiable countries in Africa (35.3%). The antimalarial prophylaxis recommended in France to those travelling to Group-2 countries, chloroquine-proguanil, therefore still seems reasonable, although cycloguanil resistance may seriously undermine the efficacy of this drug combination in the future.

  11. Loss-of-Function Mutation of REDUCED WALL ACETYLATION2 in Arabidopsis Leads to Reduced Cell Wall Acetylation and Increased Resistance to Botrytis cinerea1[W][OA

    PubMed Central

    Manabe, Yuzuki; Nafisi, Majse; Verhertbruggen, Yves; Orfila, Caroline; Gille, Sascha; Rautengarten, Carsten; Cherk, Candice; Marcus, Susan E.; Somerville, Shauna; Pauly, Markus; Knox, J. Paul; Sakuragi, Yumiko; Scheller, Henrik Vibe

    2011-01-01

    Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea. PMID:21212300

  12. ROAM mutations causing increased expression of yeast genes: their activation by signals directed toward conjugation functions and their formation by insertion of tyl repetitive elements

    SciTech Connect

    Errede, B.; Cardillo, T.S.; Wever, G.; Sherman, F.

    1980-01-01

    Mechanisms available to eukaryotic organisms for the coordinate regulation of gene expression are being examined by genetic and biochemical characterization of an unusual mutation, CYC7-H2, which causes overproduction of iso-2-cytochrome c in the yeast Saccharomyces cerevisiae. The CYC7-H2 mutation causes approximately a twenty fold overproduction of iso-2-cytochrome c in haploid strains but only a one to four fold overproduction in MATa/MAT..cap alpha.. diploid strains. This regulation of overproduction has been characterized as a response to signals controlling conjugation in yeast. The CYC7-H2 mutation is closely related to other regulatory mutations occurring at the cargA, cargB and DUR1,2 loci which are the structural genes for arginase, ornithine transaminase and urea amidolyase, respectively. Similar to the CYC7-H2 mutation, the mutations designated cargA/sup +/O/sup h/, cargB/sup +/O/sup h/ and durO/sup h/ cause constitutive production of their respective gene products at much lower levels in MATa/MAT..cap alpha.. diploid strains than in the corresponding haploid strains. Observations characterizing the regulation of overproduction in the CYC7-H2 mutant are presented with the additional and parallel observations for the O/sup h/ mutants.

  13. Conference Connections: Rewiring the Circuit

    ERIC Educational Resources Information Center

    Siemens, George; Tittenberger, Peter; Anderson, Terry

    2008-01-01

    Increased openness, two-way dialogue, and blurred distinctions between experts and amateurs have combined with numerous technology tools for dialogue, personal expression, networking, and community formation to "remake" conferences, influencing not only how attendees participate in but also how organizers host conferences today. (Contains 31…

  14. Alzheimer’s disease-associated mutations increase amyloid precursor protein resistance to γ-secretase cleavage and the Aβ42/Aβ40 ratio

    PubMed Central

    Xu, Ting-Hai; Yan, Yan; Kang, Yanyong; Jiang, Yi; Melcher, Karsten; Xu, H Eric

    2016-01-01

    Mutations in the amyloid precursor protein (APP) gene and the aberrant cleavage of APP by γ-secretase are associated with Alzheimer’s disease (AD). Here we have developed a simple and sensitive cell-based assay to detect APP cleavage by γ-secretase. Unexpectedly, most familial AD (FAD)-linked APP mutations make APP partially resistant to γ-secretase. Mutations that alter residues N terminal to the γ-secretase cleavage site Aβ42 have subtle effects on cleavage efficiency and cleavage-site selectivity. In contrast, mutations that alter residues C terminal to the Aβ42 site reduce cleavage efficiency and dramatically shift cleavage-site specificity toward the aggregation-prone Aβ42. Moreover, mutations that remove positive charge at residue 53 greatly reduce the APP cleavage by γ-secretase. These results suggest a model of γ-secretase substrate recognition, in which the APP region C terminal to the Aβ42 site and the positively charged residue at position 53 are the primary determinants for substrate binding and cleavage-site selectivity. We further demonstrate that this model can be extended to γ-secretase processing of notch receptors, a family of highly conserved cell-surface signaling proteins. PMID:27625790

  15. Conference Space

    ERIC Educational Resources Information Center

    Tillett, Wade

    2016-01-01

    The following is an exploration of the spatial configurations (and their implications) within a typical panel session at an academic conference. The presenter initially takes up different roles and hyperbolically describes some possible messages that the spatial arrangement sends. Eventually, the presenter engages the audience members in atypical…

  16. Cancer cell mitochondria confer apoptosis resistance and promote metastasis.

    PubMed

    Kulawiec, Mariola; Owens, Kjerstin M; Singh, Keshav K

    2009-07-01

    Mutations in mtDNA are found in most cancers. In this study, we studied the role of cancer cell mutant mtDNA in tumorigenesis. We sequenced the entire mitochondrial genome of three different breast cancer cell lines and found that all three, MCF7, MDA-MB-231 and MDA-MB-435, contained mutations in mtDNA. MDA-MB-435 cells contained a mutation in the tRNA(Leu(CUN)) gene known to be involved in pathogenesis of mitochondrial diseases. We generated a mutant cybrid (cytoplasmic hybrid) by repopulating the recipient rho(0) (completely devoid of mtDNA) cells with donor mtDNA derived from an enucleated MDA-MB-435 breast cancer cell line. An isogenic wild-type cybrid was produced by transfer of normal mtDNA from a healthy donor. When compared to the wild type, we found that mutant mtDNA increases mitochondrial membrane potential. However, this increase in mitochondrial membrane potential was not associated with increase in reactive oxygen species (ROS) production. MtDNA mutations conferred resistance to apoptosis triggered by etoposide. Our study also revealed that mutations in mtDNA increase metastatic potential. Using a tail-vein model of metastasis in a mouse model, we show that the mutant cybrid metastatizes to the lungs and forms macrometastic foci. Additionally we found that mutations in mtDNA constitutively activate the PI3/Akt pathway that contributes to increased metastatis. Together our study demonstrates that mutant mtDNA promotes apoptotic resistance and metastasis in a mouse model.

  17. Correlation between penicillin-binding protein 2 mutations and carbapenem resistance in Escherichia coli.

    PubMed

    Yamachika, Shinichiro; Sugihara, Chika; Kamai, Yasuki; Yamashita, Makoto

    2013-03-01

    It is well known that carbapenem-resistant mutations in penicillin-binding proteins (PBPs) are not observed in most Gram-negative bacteria under either clinical or experimental conditions. To understand the mechanisms involved in carbapenem resistance, this study constructed a mutS- and tolC-deficient Escherichia coli strain, which was expected to have elevated mutation frequencies and to lack drug efflux. Using this mutant, carbapenem-resistant strains with target mutations were successfully and efficiently isolated. The mutations T547I/A, M574I and G601D were identified in the PBP2 gene. Meropenem (MEPM)-resistant strains with the PBP2 T547I mutation showed fourfold increased resistance to 1-β-methyl-substituted carbapenems, such as doripenem, MEPM and biapenem, but not to non-substituted carbapenems such as imipenem and panipenem and other β-lactams. In addition, resistance resulting from the G601D mutation was limited to MEPM, whilst the M574I mutation conferred resistance to MEPM, imipenem and panipenem. This is the first report, to the best of our knowledge, that E. coli also has a carbapenem-resistance mechanism as a result of PBP2 mutations, and it provides insight into the resistance profiles of PBP2 mutations to carbapenems with and without the 1-β-methyl group.

  18. The conferences for undergraduate women in physics

    NASA Astrophysics Data System (ADS)

    Blessing, Susan K.

    2015-12-01

    The American Physical Society Conferences for Undergraduate Women in Physics are the continuation of a grassroots collaborative effort that began in 2006. The goals of the conferences are to increase retention and improve career outcomes of undergraduate women in physics. I describe the conferences, including organization and participant response, and encourage other countries to host similar programs for their undergraduate women.

  19. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

    PubMed Central

    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

    2012-01-01

    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 (RAC1P29S) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1P29S 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. PMID:22842228

  20. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

    SciTech Connect

    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

    2012-10-11

    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.

  1. Evidence of increasing L1014F kdr mutation frequency in Anopheles gambiae s.l. pyrethroid resistant following a nationwide distribution of LLINs by the Beninese National Malaria Control Programme

    PubMed Central

    Aïzoun, Nazaire; Aïkpon, Rock; Akogbéto, Martin

    2014-01-01

    Objective To determine the susceptibility status to pyrethroid in Anopheles gambiae s.l. (An. gambiae), the distribution of kdr “Leu-Phe” mutation in malaria vectors in Benin and to compare the current frequency of kdr “Leu-Phe” mutation to the previous frequency after long-lasting insecticide treated nets implementation. Methods Larvae and pupae of An. gambiae s.l. mosquitoes were collected from the breeding sites in Littoral, Zou, Borgou and Alibori provinces. CDC susceptibility tests were conducted on unfed females mosquitoes aged 2-5 d old. An. gambiae mosquitoes were identified to species using PCR techniques. Molecular assays were also carried out to identify kdr mutations in individual mosquitoes. Results The results showed that An. gambiae Malanville and Suru-lere populations were resistant to deltamethrin. Regarding An. gambiae Parakou and Bohicon populations, they were resistant to permethrin. PCR revealed 100% of mosquitoes tested were An. gambiae s.s. The L1014F kdr mutation was found in An. gambiae s.s. Malanville and Parakou at various allelic frequencies. The increase of kdr allelic frequency was positively correlated with CDC bioassays data. Conclusions : Pyrethroid resistance is widespread in malaria vector in Benin and kdr mutation is the main resistance mechanism involved. More attention may be paid for the future success of malaria control programmes based on LLINs with pyrethroids in the country. PMID:25182444

  2. Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

    SciTech Connect

    Lee, Yoon Jae; Jang, Yo Han; Kim, Paul; Lee, Yun Ha; Lee, Young Jae; Byun, Young Ho; Lee, Kwang-Hee; Kim, Kyusik; Seong, Baik Lin

    2016-04-15

    In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. - Highlights: • Cold-adaptation process induced four amino acid mutations in the HA of X-31 virus. • The four mutations in the HA also contributed to attenuation of the X-31ca virus • N81K mutation was the most significant marker for the attenuation of X-31ca virus. • Introduction of N81K mutation into H3N2 LAIV further attenuated the vaccine. • This approach provides a useful guideline for enhancing the safety of the LAIVs.

  3. Effective Temperature of Mutations

    NASA Astrophysics Data System (ADS)

    Derényi, Imre; Szöllősi, Gergely J.

    2015-02-01

    Biological macromolecules experience two seemingly very different types of noise acting on different time scales: (i) point mutations corresponding to changes in molecular sequence and (ii) thermal fluctuations. Examining the secondary structures of a large number of microRNA precursor sequences and model lattice proteins, we show that the effects of single point mutations are statistically indistinguishable from those of an increase in temperature by a few tens of kelvins. The existence of such an effective mutational temperature establishes a quantitative connection between robustness to genetic (mutational) and environmental (thermal) perturbations.

  4. Large Deletions in the pAtC58 Megaplasmid of Agrobacterium tumefaciens Can Confer Reduced Carriage Cost and Increased Expression of Virulence Genes

    PubMed Central

    Morton, Elise R.; Merritt, Peter M.; Bever, James D.; Fuqua, Clay

    2013-01-01

    The accessory plasmid pAtC58 of the common laboratory strain of Agrobacterium tumefaciens confers numerous catabolic functions and has been proposed to play a role in virulence. Genomic sequencing of evolved laboratory strains of A. tumefaciens revealed the presence of multiple deletion events in the At plasmid, with reductions in plasmid size ranging from 25% to 30% (115–194 kb). Flanking both ends of the sites of these deletions is a short-nucleotide repeat sequence that is in a single copy in the deleted plasmids, characteristic of a phage- or transposon-mediated deletion event. This repeat sequence is widespread throughout the C58 genome, but concentrated on the At plasmid, suggesting its frequency to be nonrandom. In this study, we assess the prevalence of the larger of these deletions in multiple C58 derivatives and characterize its functional significance. We find that in addition to elevating virulence gene expression, this deletion is associated with a significantly reduced carriage cost to the cell. These observations are a clear demonstration of the dynamic nature of the bacterial genome and suggest a mechanism for genetic plasticity of these costly but otherwise stable plasmids. Additionally, this phenomenon could be the basis for some of the dramatic recombination events so ubiquitous within and among megaplasmids. PMID:23783172

  5. Overexpression of a bHLH1 Transcription Factor of Pyrus ussuriensis Confers Enhanced Cold Tolerance and Increases Expression of Stress-Responsive Genes

    PubMed Central

    Jin, Cong; Huang, Xiao-San; Li, Kong-Qing; Yin, Hao; Li, Lei-Ting; Yao, Zheng-Hong; Zhang, Shao-Ling

    2016-01-01

    The basic helix-loop-helix (bHLH) transcription factors are involved in arrays of physiological and biochemical processes. However, knowledge concerning the functions of bHLHs in cold tolerance remains poorly understood. In this study, a PubHLH1 gene isolated from Pyrus ussuriensis was characterized for its function in cold tolerance. PubHLH1 was upregulated by cold, salt, and dehydration, with the greatest induction under cold conditions. PubHLH1 had the transactivational activity and localized in the nucleus. Ectopic expression of PubHLH1 in transgenic tobacco conferred enhanced tolerance to cold stress. The transgenic lines had higher survival rates, higher chlorophyll, higher proline contents, lower electrolyte leakages and MDA when compared with wild type (WT). In addition, transcript levels of eight genes associated with ROS scavenging, regulation, and stress defense were higher in the transgenic plants relative to the WT under the chilling stress. Taken together, these results demonstrated that PubHLH1 played a key role in cold tolerance and, at least in part, contributed to activation of stress-responsive genes. PMID:27092159

  6. Commentary on the role of treatment-related HIV compensatory mutations on increasing virulence: new discoveries twenty years since the clinical testing of protease inhibitors to block HIV-1 replication.

    PubMed

    Arts, Eric J

    2012-10-03

    Approximately 20 years has passed since the first human trial with HIV-1 protease inhibitors. Protease inhibitors set the stage for combination therapy in the mid-1990s but are now rarely used in first-line combination therapy and reserved for salvage therapy. Initially, resistance to protease inhibitors was deemed unlikely due to the small enzymatic target with limited genetic diversity, the extended drug binding site in protease, and the need to cleave multiple sites in the HIV-1 precursor proteins. However, a highly protease inhibitor-resistant virus can emerge during treatment and is found to harbor a collection of primary drug-resistant mutations near the drug and/or substrate binding site as well as secondary mutations that compensate for fitness loss. For years, the research field has debated the impact of these secondary mutations on the emergence rates of high-level protease inhibitor resistance. A recent study poses a more pertinent question, related to disease progression in patients newly infected with a virus harboring secondary protease inhibitor-associated polymorphisms. The authors of that study show that increased rates of disease progression, inferred by increased viral loads and decreased CD4 cell counts, correlate with a fitness score of the infecting virus. The modeled fitness scores increased with an accumulation of these secondary protease inhibitors mutations, and not because of any one specific polymorphism.

  7. Traceback: A Proposed Framework to Increase Identification and Genetic Counseling of BRCA1 and BRCA2 Mutation Carriers Through Family-Based Outreach.

    PubMed

    Samimi, Goli; Bernardini, Marcus Q; Brody, Lawrence C; Caga-Anan, Charlisse F; Campbell, Ian G; Chenevix-Trench, Georgia; Couch, Fergus J; Dean, Michael; de Hullu, Joanne A; Domchek, Susan M; Drapkin, Ronny; Spencer Feigelson, Heather; Friedlander, Michael; Gaudet, Mia M; Harmsen, Marline G; Hurley, Karen; James, Paul A; Kwon, Janice S; Lacbawan, Felicitas; Lheureux, Stephanie; Mai, Phuong L; Mechanic, Leah E; Minasian, Lori M; Myers, Evan R; Robson, Mark E; Ramus, Susan J; Rezende, Lisa F; Shaw, Patricia A; Slavin, Thomas P; Swisher, Elizabeth M; Takenaka, Masataka; Bowtell, David D; Sherman, Mark E

    2017-04-11

    In May 2016, the Division of Cancer Prevention and the Division of Cancer Control and Population Sciences, National Cancer Institute, convened a workshop to discuss a conceptual framework for identifying and genetically testing previously diagnosed but unreferred patients with ovarian cancer and other unrecognized BRCA1 or BRCA2 mutation carriers to improve the detection of families at risk for breast or ovarian cancer. The concept, designated Traceback, was prompted by the recognition that although BRCA1 and BRCA2 mutations are frequent in women with ovarian cancer, many such women have not been tested, especially if their diagnosis predated changes in testing guidelines. The failure to identify mutation carriers among probands represents a lost opportunity to prevent cancer in unsuspecting relatives through risk-reduction intervention in mutation carriers and to provide appropriate reassurances to noncarriers. The Traceback program could provide an important opportunity to reach families from racial, ethnic, and socioeconomic groups who historically have not sought or been offered genetic counseling and testing and thereby contribute to a reduction in health disparities in women with germline BRCA mutations. To achieve an interdisciplinary perspective, the workshop assembled international experts in genetics, medical and gynecologic oncology, clinical psychology, epidemiology, genomics, cost-effectiveness modeling, pathology, bioethics, and patient advocacy to identify factors to consider when undertaking a Traceback program. This report highlights the workshop deliberations with the goal of stimulating research and providing a framework for pilot studies to assess the feasibility and ethical and logistical considerations related to the development of best practices for implementation of Traceback studies.

  8. Functional impact of HIV coreceptor-binding site mutations

    SciTech Connect

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Reeves, Jacqueline D. . E-mail: jreeves@MonogramBio.com

    2006-07-20

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

  9. Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality

    PubMed Central

    Coleman, Bradley M.; Harrison, Christopher F.; Guo, Belinda; Masters, Colin L.; Barnham, Kevin J.; Lawson, Victoria A.

    2014-01-01

    ABSTRACT Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrPC, into the disease-associated isoform, PrPSc. Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrPC share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrPSc are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrPSc, giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion. IMPORTANCE Prion diseases are transmissible neurodegenerative diseases associated with an infectious agent called a prion. Prions are comprised of an abnormally folded form of the prion protein (PrP) that is normally resistant to enzymes called proteases. In humans, prion disease can occur in individuals who inherited mutations in the prion protein gene. Here we have studied the effects of two of these mutations and show that they influence the properties of the prions that can be formed. We show that

  10. Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

    PubMed Central

    Morsomme, P; de Kerchove d'Exaerde, A; De Meester, S; Thinès, D; Goffeau, A; Boutry, M

    1996-01-01

    In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway. Images PMID:8896445

  11. Concurrent porcine circovirus type 2a (PCV2a) or PCV2b infection increases the rate of amino acid mutations of porcine reproductive and respiratory syndrome virus (PRRSV) during serial passages in pigs.

    PubMed

    Yin, Shuang-Hui; Xiao, Chao-Ting; Gerber, Priscilla F; Beach, Nathan M; Meng, Xiang-Jin; Halbur, Patrick G; Opriessnig, Tanja

    2013-12-26

    Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic and antigenic variability. The purpose of this study was to determine if porcine circovirus type 2 (PCV2) infection increases genetic variability of PRRSV during serial passages in pigs and to determine if there is a difference in the PRRSV mutation rate between pigs concurrently infected with PCV2a or PCV2b. After 8 consecutive passages of PRRSV alone (group 1), PRRSV with PCV2a (group 2), or PCV2b (group 3) in pigs, the sequences of PRRSV structural genes for open reading frame (ORF) 5, ORF6, ORF7 and the partial non-structural protein gene (Nsp) 2 were determined. The total number of identified amino acid mutations in ORF5, ORF6, ORF7 and Nsp2 sequences was 30 for PRRSV infection only, 63 for PRRSV/PCV2a concurrent infection, and 77 for PRRSV/PCV2b concurrent infection when compared with the original VR2385 virus used to infect the passage 1 pigs. Compared to what occurred in pigs infected with PRRSV only, the mutation rates in ORF5 and ORF6 were significantly higher for concurrent PRRSV/PCV2b infected pigs. The PRRSV/PCV2a pigs had a significantly higher mutation rate in ORF7. The results from this study indicated that, besides ORF5 and Nsp2, the PRRSV structural genes ORF6 and ORF7 were shown to mutate at various degrees when the PRRSV was passaged over time in vivo. Furthermore, a significantly higher mutation rate of PRRSV was observed when pigs were co-infected with PCV2 highlighting the importance of concurrent infections on PRRSV evolution and control.

  12. Naturally Occurring Mutations in the PA Gene Are Key Contributors to Increased Virulence of Pandemic H1N1/09 Influenza Virus in Mice

    PubMed Central

    Sun, Yipeng; Xu, Qi; Shen, Ye; Liu, Linqing; Wei, Kai; Sun, Honglei; Pu, Juan; Chang, Kin-Chow

    2014-01-01

    We examined the molecular basis of virulence of pandemic H1N1/09 influenza viruses by reverse genetics based on two H1N1/09 virus isolates (A/California/04/2009 [CA04] and A/swine/Shandong/731/2009 [SD731]) with contrasting pathogenicities in mice. We found that four amino acid mutations (P224S in the PA protein [PA-P224S], PB2-T588I, NA-V106I, and NS1-I123V) contributed to the lethal phenotype of SD731. In particular, the PA-P224S mutation when combined with PA-A70V in CA04 drastically reduced the virus's 50% mouse lethal dose (LD50), by almost 1,000-fold. PMID:24522908

  13. SMARCA4-inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers. | Office of Cancer Genomics

    Cancer.gov

    Mutations in the SMARCA4/BRG1 gene resulting in complete loss of its protein (BRG1) occur frequently in non-small cell lung cancer (NSCLC) cells. Currently, no single therapeutic agent has been identified as synthetically lethal with SMARCA4/BRG1 loss. We identify AURKA activity as essential in NSCLC cells lacking SMARCA4/BRG1. In these cells, RNAi-mediated depletion or chemical inhibition of AURKA induces apoptosis and cell death in vitro and in xenograft mouse models.

  14. Concomitance of oncogenic HPV types, CHEK2 gene mutations, and CYP1B1 gene polymorphism as an increased risk factor for malignancy

    PubMed Central

    Constantinou, Maria; Pietrusiński, Michał; Kępczyński, Łukasz; Jędrzejczyk, Adam; Rożniecki, Marek; Marks, Piotr; Kałużewski, Bogdan

    2013-01-01

    Introduction Urinary bladder carcinoma ranks the fourth position in malignancy incidence rates in men (6.1%) and the 17th position in women (1.6%). In general, neoplastic diseases should be approached from two perspectives: prevention with implementation of prophylactic measures and early diagnostics. Prophylactics is possible in the preclinical phase of neoplasm, being both justified and plausible in patients from high–risk groups. Thus, it is particularly important to select such groups, not only by referring to environmental carcinogenic factors (occupational and extra–occupational) but also from genetic predisposition, which may be conductive for neoplasm formation. The mutations / polymorphisms of CHEK2 and CYP1B1 genes predispose to neoplasm via multiorgan mechanisms, while the human papilloma virus (HPV) may participate in the neoplastic transformation as an environmental factor. Material and methods 131 patients with diagnosed urinary bladder cancer were qualified to the study. Mutations/polymorphisms of CHEK2 (IVS2 + 1G > A gene, 1100delC, del5395, I157T) and CYP1B1– 355T/T were identified by the PCR in DNA isolated directly from the tumor and from peripheral blood. The ELISA test was used for the studies of 37 HPV genotypes in DNA, isolated tumour tissue. Results 11 mutations of CHEK2 gene were found, 355T/T polymorphism if CYP1B1 gene occurred in 18 patients (12.9%). Oncogenic HPV was found in 36 (29.3%), out of 123 examined patients. Conclusions The concomitance of CHEK2 gene mutations or 355T/T polymorphism of CYP1B1 gene and the presence of oncogenic HPV types statistically significantly correlates with histological malignancy grades of urinary bladder carcinoma. PMID:24578981

  15. The relationship between glucocerebrosidase mutations and Parkinson disease.

    PubMed

    Migdalska-Richards, Anna; Schapira, Anthony H V

    2016-10-01

    Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease, whereas Gaucher disease (GD) is the most frequent lysosomal storage disorder caused by homozygous mutations in the glucocerebrosidase (GBA1) gene. Increased risk of developing PD has been observed in both GD patients and carriers. It has been estimated that GBA1 mutations confer a 20- to 30-fold increased risk for the development of PD, and that at least 7-10% of PD patients have a GBA1 mutation. To date, mutations in the GBA1 gene constitute numerically the most important risk factor for PD. The type of PD associated with GBA1 mutations (PD-GBA1) is almost identical to idiopathic PD, except for a slightly younger age of onset and a tendency to more cognitive impairment. Importantly, the pathology of PD-GBA1 is identical to idiopathic PD, with nigral dopamine cell loss, Lewy bodies, and neurites containing alpha-synuclein. The mechanism by which GBA1 mutations increase the risk for PD is still unknown. However, given that clinical manifestation and pathological findings in PD-GBA1 patients are almost identical to those in idiopathic PD individuals, it is likely that, as in idiopathic PD, alpha-synuclein accumulation, mitochondrial dysfunction, autophagic impairment, oxidative and endoplasmic reticulum stress may contribute to the development and progression of PD-GBA1. Here, we review the GBA1 gene, its role in GD, and its link with PD. The impact of glucocerebrosidase 1 (GBA1) mutations on functioning of endoplasmic reticulum (ER), lysosomes, and mitochondria. GBA1 mutations resulting in production of misfolded glucocerebrosidase (GCase) significantly affect the ER functioning. Misfolded GCase trapped in the ER leads to both an increase in the ubiquitin-proteasome system (UPS) and the ER stress. The presence of ER stress triggers the unfolded protein response (UPR) and/or endoplasmic reticulum-associated degradation (ERAD). The prolonged activation of UPR and ERAD

  16. Mutations in LZTR1 add to the complex heterogeneity of schwannomatosis

    PubMed Central

    Smith, Miriam J.; Isidor, Bertand; Beetz, Christian; Williams, Simon G.; Bhaskar, Sanjeev S.; Richer, Wilfrid; O'Sullivan, James; Anderson, Beverly; Daly, Sarah B.; Urquhart, Jill E.; Fryer, Alan; Rustad, Cecilie F.; Mills, Samantha J.; Samii, Amir; du Plessis, Daniel; Halliday, Dorothy; Barbarot, Sebastien; Bourdeaut, Franck

    2015-01-01

    Objectives: We aimed to determine the proportion of individuals in our schwannomatosis cohort whose disease is associated with an LZTR1 mutation. Methods: We used exome sequencing, Sanger sequencing, and copy number analysis to screen 65 unrelated individuals with schwannomatosis who were negative for a germline NF2 or SMARCB1 mutation. We also screened samples from 39 patients with a unilateral vestibular schwannoma (UVS), plus at least one other schwannoma, but who did not have an identifiable germline or mosaic NF2 mutation. Results: We identified germline LZTR1 mutations in 6 of 16 patients (37.5%) with schwannomatosis who had at least one affected relative, 11 of 49 (22%) sporadic patients, and 2 of 39 patients with UVS in our cohort. Three germline mutation–positive patients in total had developed a UVS. Mosaicism was excluded in 3 patients without germline mutation in NF2, SMARCB1, or LZTR1 by mutation screening in 2 tumors from each. Conclusions: Our data confirm the relationship between mutations in LZTR1 and schwannomatosis. They indicate that germline mutations in LZTR1 confer an increased risk of vestibular schwannoma, providing further overlap with NF2, and that further causative genes for schwannomatosis remain to be identified. PMID:25480913

  17. Accumulation of Deleterious Passenger Mutations Is Associated with the Progression of Hepatocellular Carcinoma

    PubMed Central

    d’Avigdor, William M. H.; McCaughan, Geoffrey W.; Luciani, Fabio; Shackel, Nicholas A.

    2016-01-01

    In hepatocellular carcinoma (HCC), somatic genome-wide DNA mutations are numerous, universal and heterogeneous. Some of these somatic mutations are drivers of the malignant process but the vast majority are passenger mutations. These passenger mutations can be deleterious to individual protein function but are tolerated by the cell or are offset by a survival advantage conferred by driver mutations. It is unknown if these somatic deleterious passenger mutations (DPMs) develop in the precancerous state of cirrhosis or if it is confined to HCC. Therefore, we studied four whole-exome sequencing datasets, including patients with non-cirrhotic liver (n = 12), cirrhosis without HCC (n = 6) and paired HCC with surrounding non-HCC liver (n = 74 paired samples), to identify DPMs. After filtering out putative germline mutations, we identified 187±22 DPMs per non-diseased tissue. DPMs number was associated with liver disease progressing to HCC, independent of the number of exonic mutations. Tumours contained significantly more DPMs compared to paired non-tumour tissue (258–293 per HCC exome). Cirrhosis- and HCC-associated DPMs do not occur predominantly in specific genes, chromosomes or biological pathways and the effect on tumour biology is presently unknown. Importantly, for the first time we have shown a significant increase in DPMs with HCC. PMID:27631787

  18. Conferences revisited

    NASA Astrophysics Data System (ADS)

    Radcliffe, Jonathan

    2008-08-01

    Way back in the mid-1990s, as a young PhD student, I wrote a Lateral Thoughts article about my first experience of an academic conference (Physics World 1994 October p80). It was a peach of a trip - most of the lab decamped to Grenoble for a week of great weather, beautiful scenery and, of course, the physics. A whole new community was there for me to see in action, and the internationality of it all helped us to forget about England's non-appearance in the 1994 World Cup finals.

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

    PubMed Central

    Gargouri, Ali; Macadré, Catherine; Lazowska, Jaga

    2015-01-01

    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. PMID:28357310

  20. Mutations in the interdomain loop region of the tetA(A) tetracycline resistance gene increase efflux of minocycline and glycylcyclines.

    PubMed

    Tuckman, M; Petersen, P J; Projan, S J

    2000-01-01

    A novel class of tetracyclines, the glycylcyclines, have been shown to be active against bacterial strains harboring genes encoding tetracycline efflux pumps. However, two veterinary Salmonella isolates that carried tetracycline resistance determinants of the tetA(A) class were found to have reduced susceptibility to glycylcyclines, especially two early investigational glycylcyclines, DMG-MINO and DMG-DMDOT. These isolates were also quite resistant to tetracycline and minocycline. The isolates, one a strain of S. cholerasuis and the other, S. typhimurium, both carried the same novel tetA(A) variant, based on DNA sequencing, with one determinant plasmid encoded and the other located on the chromosome. This tetA(A) variant was cloned and shown to provide reduced susceptibility to the glycylcycline class although GAR-936, a glycylcycline currently in clinical development, was the least affected. The novel tetA(A) gene carries two mutations in the largest cytoplasmic loop of the efflux pump, which causes a double frameshift in codons 201, 202, and 203. This "interdomain region" of the efflux pump has generally been regarded as having no functional role in the efflux of tetracycline but the double frameshift is most likely responsible for the enhanced resistance observed and points to an interaction that was previously unrecognized. Mutants of the tetA(B) class with decreased susceptibility to the glycylcyclines were also generated in vitro. These all carried mutations in the portion of the tetA(B) gene encoding a transmembrane spanning region of the efflux pump. The laboratory-generated mutants point to the tight constraints in substrate recognition of the transmembrane-spanning region and may suggest that it will be the interdomain region of the pump that is likely to be the locus of future glycylcycline resistance mutations as these compounds enter clinical use.

  1. Accumulation of Phosphatidic Acid Increases Vancomycin Resistance in Escherichia coli

    PubMed Central

    Sutterlin, Holly A.; Zhang, Sisi

    2014-01-01

    In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane, preventing entry of toxic molecules such as antibiotics. Mutations in lptD, the beta-barrel component of the LPS transport and assembly machinery, compromise LPS assembly and result in increased antibiotic sensitivity. Here, we report rare vancomycin-resistant suppressors that improve barrier function of a subset of lptD mutations. We find that all seven suppressors analyzed mapped to the essential gene cdsA, which is responsible for the conversion of phosphatidic acid to CDP-diacylglycerol in phospholipid biosynthesis. These cdsA mutations cause a partial loss of function and, as expected, accumulate phosphatidic acid. We show that this suppression is not confined to mutations that cause defects in outer membrane biogenesis but rather that these cdsA mutations confer a general increase in vancomycin resistance, even in a wild-type cell. We use genetics and quadrupole time of flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS) to show that accumulation of phosphatidic acid by means other than cdsA mutations also increases resistance to vancomycin. We suggest that increased levels of phosphatidic acid change the physical properties of the outer membrane to impede entry of vancomycin into the periplasm, hindering access to its target, an intermediate required for the synthesis of the peptidoglycan cell wall. PMID:24957626

  2. A Single Mutation in the VP1 of Enterovirus 71 Is Responsible for Increased Virulence and Neurotropism in Adult Interferon-Deficient Mice

    PubMed Central

    Caine, Elizabeth A.; Moncla, Louise H.; Ronderos, Monica D.; Friedrich, Thomas C.

    2016-01-01

    ABSTRACT Hand, foot, and mouth disease (HFMD) has spread throughout the Asia-Pacific region, affecting millions of young children, who develop symptoms ranging from painful blisters around their mouths and hands to neurological complications. Many members of the genus Enterovirus (family Picornaviridae) cause HFMD. Enterovirus 71 (EV71) is one of the primary causative agents and has been linked to severe disease. Vaccine efficacy and pathogenesis studies for EV71 have been limited because there is a lack of suitable animal models. Previously, we generated a mouse-adapted EV71 (mEV71) capable of infecting 12-week-old interferon receptor-deficient AG129 mice and used the model to evaluate the efficacy of candidate HFMD vaccines. Here, we present data investigating the genetic correlates of EV71 adaptation and characterize the virus's tissue tropism in mice. Using reverse genetics, a VP1 mutation (K244E) was shown to be necessary for mEV71 virulence in adult mice. Another VP1 mutation (H37R) was required for mEV71 recovery on rhabdomyosarcoma (RD) cells. Viral loads determined by real-time reverse transcription (RT)-PCR confirmed the presence of mEV71 in the sera and multiple organs of mice. Histological analysis revealed signs of meningitis and encephalitis, characteristic of severe human disease. The further description of this model has provided insight into EV71 pathogenesis and demonstrates the importance of the VP1 region in facilitating mEV71 adaptation. IMPORTANCE EV71 is a reemerging pathogen, and little is known about the genetic determinants involved in its pathogenesis. The absence of animal models has contributed to this lack of knowledge. The data presented here improve upon the existing animal models by characterizing a mouse-adapted strain of EV71. We determined that a VP1 mutation (K244E) was needed for EV71 virulence in adult AG129 mice. While this mutation was found previously for EV71 adaptation in 5-day-old BALB/c mice, neurotropic disease did not

  3. Activating Somatic FGFR2 Mutations in Breast Cancer

    PubMed Central

    Reintjes, Nadine; Li, Yun; Becker, Alexandra; Rohmann, Edyta; Schmutzler, Rita; Wollnik, Bernd

    2013-01-01

    It is known that FGFR2 gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5–10% of breast tumors. In our study, we sequenced the most important coding regions of FGFR2 in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in FGFR2 and FGF10. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer. PMID:23527311

  4. A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation.

    PubMed

    Wong, Hao Yun; Hoogerbrugge, Jos W; Pang, Kar Lok; van Leeuwen, Marije; van Royen, Martin E; Molier, Michel; Berrevoets, Cor A; Dooijes, Dennis; Dubbink, Hendrikus Jan; van de Wijngaart, Dennis J; Wolffenbuttel, Katja P; Trapman, Jan; Kleijer, Wim J; Drop, Stenvert L S; Grootegoed, J Anton; Brinkmann, Albert O

    2008-09-24

    A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.

  5. Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining.

    PubMed

    Beckta, Jason M; Dever, Seth M; Gnawali, Nisha; Khalil, Ashraf; Sule, Amrita; Golding, Sarah E; Rosenberg, Elizabeth; Narayanan, Aarthi; Kehn-Hall, Kylene; Xu, Bo; Povirk, Lawrence F; Valerie, Kristoffer

    2015-09-29

    Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

  6. Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining

    PubMed Central

    Gnawali, Nisha; Khalil, Ashraf; Sule, Amrita; Golding, Sarah E.; Rosenberg, Elizabeth; Narayanan, Aarthi; Kehn-Hall, Kylene; Xu, Bo; Povirk, Lawrence F.; Valerie, Kristoffer

    2015-01-01

    Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations. PMID:26320175