Science.gov

Sample records for mutation confers increased

  1. Cooperativity of Negative Autoregulation Confers Increased Mutational Robustness.

    PubMed

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

    2016-06-24

    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

    SciTech Connect

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

    1996-04-01

    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{sup 2+})] tested (0.15 mM). Furthermore, these mutants are more sensitive than wild type to application of the potassium channel-blocking drug quinidine: following quinidine 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 behavorial abnormalities and male sterility. 43 refs., 5 figs., 1 tab.

  4. Increased Selectivity towards Cytoplasmic versus Mitochondrial Ribosome Confers Improved Efficiency of Synthetic Aminoglycosides in Fixing Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense Mutations

    PubMed Central

    Kandasamy, Jeyakumar; Atia-Glikin, Dana; Shulman, Eli; Shapira, Katya; Shavit, Michal; Belakhov, Valery; Baasov, Timor

    2012-01-01

    Compelling evidence is now available that gentamicin and geneticin (G418) can induce mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity towards cytoplasmic ribosome correlates with the increased activity, and that the decreased specificity towards mitochondrial ribosome confers to the lowered cytotoxicity. PMID:23148581

  5. A mutation in reverse transcriptase of bis(heteroaryl)piperazine-resistant human immunodeficiency virus type 1 that confers increased sensitivity to other nonnucleoside inhibitors.

    PubMed Central

    Dueweke, T J; Pushkarskaya, T; Poppe, S M; Swaney, S M; Zhao, J Q; Chen, I S; Stevenson, M; Tarpley, W G

    1993-01-01

    Several nonnucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been described, including Nevirapine, thiobenzimidazolone (TIBO) derivatives, pyridinone derivatives such as L-697,661, and the bis(heteroaryl)piperazines (BHAPs). HIV-1 resistant to L-697,661 or Nevirapine emerges rapidly in infected patients treated with these drugs, and the resistance is caused primarily by substitutions at amino acids 181 and 103 of RT that also confer cross resistance to the other nonnucleoside inhibitors. We describe derivation and characterization of two BHAP-resistant HIV-1 variants that differ from this pattern of cross resistance. With both variants, HIV-1 resistance to BHAP RT inhibitors was caused by a RT mutation that results in a proline-to-leucine substitution at amino acid 236 (P236L). Rather than conferring cross resistance to other RT inhibitors, this substitution sensitized RT 7- to 10-fold to Nevirapine, TIBO R82913, and L-697,661 without influencing sensitivity to nucleoside analogue RT inhibitors. This sensitization caused by P236L was also observed in cell culture with BHAP-resistant HIV-1. The effects of the P236L RT substitution suggest that emergence of BHAP-resistant virus in vivo could produce a viral population sensitized to inhibition by these other nonnucleoside RT inhibitors. PMID:7685109

  6. Mutations in the Fusion Protein Cleavage Site of Avian Paramyxovirus Serotype 4 Confer Increased Replication and Syncytium Formation In Vitro but Not Increased Replication and Pathogenicity in Chickens and Ducks

    PubMed Central

    Kim, Shin-Hee; Xiao, Sa; Shive, Heather; Collins, Peter L.; Samal, Siba K.

    2013-01-01

    To evaluate the role of the F protein cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks. PMID:23341874

  7. Mutations in the fusion protein cleavage site of avian paramyxovirus serotype 4 confer increased replication and syncytium formation in vitro but not increased replication and pathogenicity in chickens and ducks.

    PubMed

    Kim, Shin-Hee; Xiao, Sa; Shive, Heather; Collins, Peter L; Samal, Siba K

    2013-01-01

    To evaluate the role of the F protein cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks. PMID:23341874

  8. Mutations of the von Hippel-Lindau gene confer increased susceptibility to natural killer cells of clear-cell renal cell carcinoma.

    PubMed

    Perier, A; Fregni, G; Wittnebel, S; Gad, S; Allard, M; Gervois, N; Escudier, B; Azzarone, B; Caignard, A

    2011-06-01

    The tumor suppressor gene von Hippel-Lindau (VHL) is involved in the development of sporadic clear-cell renal cell carcinoma (RCC). VHL interferes with angiogenesis and also controls cell adhesion and invasion. Therapies that target VHL-controlled genes are currently being evaluated in RCC patients. RCC is a immunogenic tumor and treatment with interleukin-2 (IL2) or interferon (IFN)-α results in regression in some patients. We used two renal tumor cell lines (RCC6 and RCC4) carrying VHL loss-of-function mutations to investigate the role of mutant VHL in susceptibility to natural killer (NK) cell-mediated lysis. The RCC6 and RCC4 cell lines were transfected with the wild-type gene to restore the function of VHL. The presence of the gene in RCC cells downregulated hypoxia-inducible factor (HIF)-1α and subsequently decreased vascular endothelial growth factor (VEGF) production. Relative to control transfectants and parental cells, pVHL-transfected cell lines activated resting and IL2-activated NK cells less strongly, as assessed by IFNγ secretion, NK degranulation and cell lysis. NKG2A, a human leukocyte antigen (HLA)-I-specific inhibitory NK receptor, controls the lysis of tumor targets. We show that HLA-I expression in RCC-pVHL cells is stronger than that in parental and controls cells, although the expression of activating receptor NK ligands remains unchanged. Blocking NKG2A/HLA-I interactions substantially increased lysis of RCC-pVHL, but had little effect on the lysis of VHL-mutated RCC cell lines. In addition, in response to IFNα, the exponential growth of RCC-pVHL was inhibited more than that of RCC-pE cells, indicating that VHL mutations may be involved in IFNα resistance. These results indicate that a decreased expression of HLA-I molecules in mutated VHL renal tumor cells sensitizes them to NK-mediated lysis. These results suggest that combined immunotherapy with anti-angiogenic drugs may be beneficial for patients with mutated VHL. PMID:21258414

  9. Target Enzyme Mutations Confer Differential Echinocandin Susceptibilities in Candida kefyr

    PubMed Central

    Staab, Janet F.; Neofytos, Dionysios; Rhee, Peter; Jiménez-Ortigosa, Cristina; Zhang, Sean X.; Perlin, David S.

    2014-01-01

    Candida kefyr is an increasingly reported pathogen in patients with hematologic malignancies. We studied a series of bloodstream isolates that exhibited reduced echinocandin susceptibilities (RES). Clinical and surveillance isolates were tested for susceptibilities to all three echinocandins, and those isolates displaying RES to one or more echinocandins were selected for molecular and biochemical studies. The isolates were analyzed for genetic similarities, and a subset was analyzed for mutations in the echinocandin target gene FKS1 and glucan synthase echinocandin sensitivities using biochemical methods. The molecular typing did not indicate strong genetic relatedness among the isolates except for a series of strains recovered from a single patient. Two unrelated isolates with RES had previously uncharacterized FKS1 mutations: R647G and deletion of amino acid 641 (F641Δ). Biochemical analysis of the semipurified R647G glucan synthase generated differential echinocandin sensitivity (resistance to micafungin only), while the deletion of F641 resulted in a glucan synthase highly insensitive to all three echinocandins. The consecutive isolates from a single patient with RES all harbored the common S645P mutation, which conferred resistance to all three echinocandins. The MIC values paralleled the glucan synthase inhibition kinetic data, although the S645P isolates displayed relatively higher susceptibility to caspofungin (2 μg/ml) than the other two echinocandins (>8 μg/ml). These findings highlight novel and common FKS1 mutations in C. kefyr isolates. The observation of differential susceptibilities to echinocandins may provide important mechanistic insights for echinocandin antifungals. PMID:24982083

  10. Germline BRCA1 mutations increase prostate cancer risk

    PubMed Central

    Leongamornlert, D; Mahmud, N; Tymrakiewicz, M; Saunders, E; Dadaev, T; Castro, E; Goh, C; Govindasami, K; Guy, M; O'Brien, L; Sawyer, E; Hall, A; Wilkinson, R; Easton, D; Goldgar, D; Eeles, R; Kote-Jarai, Z

    2012-01-01

    Background: Prostate cancer (PrCa) is one of the most common cancers affecting men but its aetiology is poorly understood. Family history of PrCa, particularly at a young age, is a strong risk factor. There have been previous reports of increased PrCa risk in male BRCA1 mutation carriers in female breast cancer families, but there is a controversy as to whether this risk is substantiated. We sought to evaluate the role of germline BRCA1 mutations in PrCa predisposition by performing a candidate gene study in a large UK population sample set. Methods: We screened 913 cases aged 36–86 years for germline BRCA1 mutation, with the study enriched for cases with an early age of onset. We analysed the entire coding region of the BRCA1 gene using Sanger sequencing. Multiplex ligation-dependent probe amplification was also used to assess the frequency of large rearrangements in 460 cases. Results: We identified 4 deleterious mutations and 45 unclassified variants (UV). The frequency of deleterious BRCA1 mutation in this study is 0.45% three of the mutation carriers were affected at age ⩽65 years and one developed PrCa at 69 years. Using previously estimated population carrier frequencies, deleterious BRCA1 mutations confer a relative risk of PrCa of ∼3.75-fold, (95% confidence interval 1.02–9.6) translating to a 8.6% cumulative risk by age 65. Conclusion This study shows evidence for an increased risk of PrCa in men who harbour germline mutations in BRCA1. This could have a significant impact on possible screening strategies and targeted treatments. PMID:22516946

  11. Kinase Impaired BRAF Mutations Confer Lung Cancer Sensitivity to Dasatinib

    PubMed Central

    Sen, Banibrata; Peng, Shaohua; Tang, Ximing; Erickson, Heidi S.; Galindo, Hector; Mazumdar, Tuhina; Stewart, David J.; Wistuba, Ignacio; Johnson, Faye M.

    2013-01-01

    During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non–small cell lung cancer (NSCLC) one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase inactivating BRAF mutation (Y472CBRAF); no inactivating BRAF mutations were found in the non-responding tumors taken from other patients. Cells transfected with Y472CBRAF exhibited CRAF, MEK, and ERK activation – characteristics identical to signaling changes that occur with previously known kinase inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells’ dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by Y472CBRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization resulting in ERK activation in NSCLC cells with kinase inactivating BRAF mutations. The sensitivity of NSCLC with kinase impaired BRAF to dasatinib suggested synthetic lethality of BRAF and a dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells’ dasatinib sensitivity. Thus, the patient’s BRAF mutation was likely responsible for his tumor’s marked response to dasatinib, suggesting that tumors bearing kinase impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF. PMID:22649091

  12. 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

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

    PubMed

    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

    2015-01-23

    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

  14. Mutations in the Pneumocystis jirovecii DHPS Gene Confer Cross-Resistance to Sulfa Drugs

    PubMed Central

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

    2005-01-01

    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 (T517A or P519S) 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. PMID:15673759

  15. Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.

    PubMed

    Deshpande, A; Reddy, M M; Schade, G O M; Ray, A; Chowdary, T K; Griffin, J D; Sattler, M

    2012-04-01

    The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs. PMID:21926964

  16. Negative feedback confers mutational robustness in yeast transcription factor regulation

    PubMed Central

    Denby, Charles M.; Im, Joo Hyun; Yu, Richard C.; Pesce, C. Gustavo; Brem, Rachel B.

    2012-01-01

    Organismal fitness depends on the ability of gene networks to function robustly in the face of environmental and genetic perturbations. Understanding the mechanisms of this stability is one of the key aims of modern systems biology. Dissecting the basis of robustness to mutation has proven a particular challenge, with most experimental models relying on artificial DNA sequence variants engineered in the laboratory. In this work, we hypothesized that negative regulatory feedback could stabilize gene expression against the disruptions that arise from natural genetic variation. We screened yeast transcription factors for feedback and used the results to establish ROX1 (Repressor of hypOXia) as a model system for the study of feedback in circuit behaviors and its impact across genetically heterogeneous populations. Mutagenesis experiments revealed the mechanism of Rox1 as a direct transcriptional repressor at its own gene, enabling a regulatory program of rapid induction during environmental change that reached a plateau of moderate steady-state expression. Additionally, in a given environmental condition, Rox1 levels varied widely across genetically distinct strains; the ROX1 feedback loop regulated this variation, in that the range of expression levels across genetic backgrounds showed greater spread in ROX1 feedback mutants than among strains with the ROX1 feedback loop intact. Our findings indicate that the ROX1 feedback circuit is tuned to respond to perturbations arising from natural genetic variation in addition to its role in induction behavior. We suggest that regulatory feedback may be an important element of the network architectures that confer mutational robustness across biology. PMID:22355134

  17. Dysferlin deficiency confers increased susceptibility to coxsackievirus-induced cardiomyopathy.

    PubMed

    Wang, Chen; Wong, Jerry; Fung, Gabriel; Shi, Junyan; Deng, Haoyu; Zhang, Jingchun; Bernatchez, Pascal; Luo, Honglin

    2015-10-01

    Coxsackievirus infection can lead to viral myocarditis and its sequela, dilated cardiomyopathy, which represent major causes of cardiovascular mortality worldwide in children. Yet, the host genetic susceptible factors and the underlying mechanisms by which viral infection damages cardiac function remain to be fully resolved. Dysferlin is a transmembrane protein highly expressed in skeletal and cardiac muscles. In humans, mutations in the dysferlin gene can cause limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin deficiency has also been linked to cardiomyopathy. Defective muscle membrane repair has been suggested to be an important mechanism responsible for muscle degeneration in dysferlin-deficient patients and animals. Using both naturally occurring and genetically engineered dysferlin-deficient mice, we demonstrated that loss of dysferlin confers increased susceptibility to coxsackievirus infection and myocardial damage. More interestingly, we found that dysferlin is cleaved following coxsackieviral infection through the proteolytic activity of virally encoded proteinases, suggesting an important mechanism underlying virus-induced cardiac dysfunction. Our results in this study not only identify dysferlin deficiency as a novel host risk factor for viral myocarditis but also reveal a key mechanism by which coxsackievirus infection impairs cardiac function, leading to the development of dilated cardiomyopathy. PMID:26073173

  18. 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...

  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. 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. PMID:26464158

  1. Complex long-distance effects of mutations that confer linezolid resistance in the large ribosomal subunit

    PubMed Central

    Fulle, Simone; Saini, Jagmohan S.; Homeyer, Nadine; Gohlke, Holger

    2015-01-01

    The emergence of multidrug-resistant pathogens will make current antibiotics ineffective. For linezolid, a member of the novel oxazolidinone class of antibiotics, 10 nucleotide mutations in the ribosome have been described conferring resistance. Hypotheses for how these mutations affect antibiotics binding have been derived based on comparative crystallographic studies. However, a detailed description at the atomistic level of how remote mutations exert long-distance effects has remained elusive. Here, we show that the G2032A-C2499A double mutation, located > 10 Å away from the antibiotic, confers linezolid resistance by a complex set of effects that percolate to the binding site. By molecular dynamics simulations and free energy calculations, we identify U2504 and C2452 as spearheads among binding site nucleotides that exert the most immediate effect on linezolid binding. Structural reorganizations within the ribosomal subunit due to the mutations are likely associated with mutually compensating changes in the effective energy. Furthermore, we suggest two main routes of information transfer from the mutation sites to U2504 and C2452. Between these, we observe cross-talk, which suggests that synergistic effects observed for the two mutations arise in an indirect manner. These results should be relevant for the development of oxazolidinone derivatives that are active against linezolid-resistant strains. PMID:26202966

  2. 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. PMID:25441450

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

    PubMed

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

    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

  4. 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

  5. Glucocerebrosidase L444P mutation confers genetic risk for Parkinson’s disease in central China

    PubMed Central

    2012-01-01

    Background Mutations of the glucocerebrosidase (GBA) gene have reportedly been associated with Parkinson disease (PD) in various ethnic populations such as Singaporean, Japanese, Formosan, Canadian, American, Portuguese, Greek, Brazilian, British, Italian, Ashkenazi Jewish, southern and southwestern Chinese. The purpose of this study is to determine in central China whether or not the reported GBA mutations remain associated with PD. Methods In this project, we conducted a controlled study in a cohort of 208 central Chinese PD patients and 298 controls for three known GBA mutations (L444P, N370S and R120W). Results Our data reveals a significantly higher frequency of L444P mutation in GBA gene of PD cases (3.4%) compared with the controls (0.3%) (P = 0.007, OR = 10.34, 95% CI = 1.26 - 84.71). Specifically, the frequency of L444P mutation was higher in the late onset PD (LOPD) cases compared with that in control subjects. The N370S and R120W mutations were detected in neither the PD group nor the control subjects. Conclusions Our observations demonstrated that the GBA L444P mutation confers genetic risk for PD, especially LOPD, among the population in the central China area. PMID:23227814

  6. 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. PMID:27185800

  7. Identification and Characterization of Mutations Conferring Resistance to d-Amino Acids in Bacillus subtilis

    PubMed Central

    Leiman, Sara A.; Richardson, Charles; Foulston, Lucy; Elsholz, Alexander K. W.; First, Eric A.

    2015-01-01

    ABSTRACT Bacteria produce d-amino acids for incorporation into the peptidoglycan and certain nonribosomally produced peptides. However, d-amino acids are toxic if mischarged on tRNAs or misincorporated into protein. Common strains of the Gram-positive bacterium Bacillus subtilis are particularly sensitive to the growth-inhibitory effects of d-tyrosine due to the absence of d-aminoacyl-tRNA deacylase, an enzyme that prevents misincorporation of d-tyrosine and other d-amino acids into nascent proteins. We isolated spontaneous mutants of B. subtilis that survive in the presence of a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine. Whole-genome sequencing revealed that these strains harbored mutations affecting tRNATyr charging. Three of the most potent mutations enhanced the expression of the gene (tyrS) for tyrosyl-tRNA synthetase. In particular, resistance was conferred by mutations that destabilized the terminator hairpin of the tyrS riboswitch, as well as by a mutation that transformed a tRNAPhe into a tyrS riboswitch ligand. The most potent mutation, a substitution near the tyrosine recognition site of tyrosyl-tRNA synthetase, improved enzyme stereoselectivity. We conclude that these mutations promote the proper charging of tRNATyr, thus facilitating the exclusion of d-tyrosine from protein biosynthesis in cells that lack d-aminoacyl-tRNA deacylase. IMPORTANCE Proteins are composed of l-amino acids. Mischarging of tRNAs with d-amino acids or the misincorporation of d-amino acids into proteins causes toxicity. This work reports on mutations that confer resistance to d-amino acids and their mechanisms of action. PMID:25733611

  8. 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.

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

    PubMed

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

    2014-06-01

    MEX3C is an RNA-binding protein with unknown physiological function. We have recently reported that a Mex3c mutation in mice causes growth retardation and reduced adiposity, but how adiposity is reduced remains unclear. Herein, we show that homozygous Mex3c gene trap mice have increased physical activity. The Mex3c mutation consistently conferred full protection from diet-induced obesity, hyperglycemia, insulin resistance, hyperlipidemia, and hepatic steatosis. In ob/ob mice with leptin deficiency, the Mex3c mutation also increased physical activity and improved glucose and lipid profiles. Expressing cre in the neurons of Mex3c gene trap mice, an attempt to partially restoring neuronal Mex3c expression, significantly increased white adipose tissue deposition, but had no effects on body length. Our data suggest that one way in which Mex3c regulates adiposity is through controlling physical activity, and that neuronal Mex3c expression could play an important role in this process. PMID:24741071

  10. Superroot, a recessive mutation in Arabidopsis, confers auxin overproduction.

    PubMed Central

    Boerjan, W; Cervera, M T; Delarue, M; Beeckman, T; Dewitte, W; Bellini, C; Caboche, M; Van Onckelen, H; Van Montagu, M; Inzé, D

    1995-01-01

    We have isolated seven allelic recessive Arabidopsis mutants, designated superroot (sur1-1 to sur1-7), displaying several abnormalities reminiscent of auxin effects. These characteristics include small and epinastic cotyledons, an elongated hypocotyl in which the connection between the stele and cortical and epidermal cells disintegrates, the development of excess adventitious and lateral roots, a reduced number of leaves, and the absence of an inflorescence. When germinated in the dark, sur1 mutants did not develop the apical hook characteristic of etiolated seedlings. We were able to phenocopy the Sur1- phenotype by supplying auxin to wild-type seedlings, to propagate sur1 explants on phytohormone-deficient medium, and to regenerate shoots from these explants by the addition of cytokinins alone to the culture medium. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of both free and conjugated indole-3-acetic acid. sur1 was crossed to the mutant axr2 and the altered-auxin response mutant ctr1. The phenotype of both double mutants was additive. The sur1 gene was mapped on chromosome 2 at 0.5 centimorgans from the gene encoding phytochrome B. PMID:8589625

  11. The penC mutation conferring antibiotic resistance in Neisseria gonorrhoeae arises from a mutation in the PilQ secretin that interferes with multimer stability

    PubMed Central

    Zhao, Shuqing; Tobiason, Deborah M.; Hu, Mei; Seifert, H. Steven; Nicholas, Robert A.

    2008-01-01

    The penC resistance gene was previously characterized in a FA19 penA mtrR penB gonococcal strain (PR100) as a spontaneous mutation that increased resistance to penicillin and tetracycline. We show here that antibiotic resistance mediated by penC is the result of a Glu-666 to Lys missense mutation in the pilQ gene that interferes with the formation of the SDS-resistant high-molecular-mass PilQ secretin complex, disrupts piliation, and decreases transformation frequency by 50-fold. Deletion of pilQ in PR100 confers the same level of antibiotic resistance as the penC mutation, but increased resistance was observed only in strains containing the mtrR and penB resistance determinants. Site-saturation mutagenesis of Glu-666 revealed that only acidic or amidated amino acids at this position preserved PilQ function. Consistent with early studies suggesting the importance of cysteine residues on stability of the PilQ multimer, mutation of either of the two cysteine residues in FA19 PilQ led to a similar phenotype as penC: increased antibiotic resistance, loss of piliation, intermediate levels of transformation competence, and absence of SDS-resistant PilQ oligomers. These data show that a functional secretin complex can enhance the entry of antibiotics into the cell and suggest that the PilQ oligomer forms a pore in the outer membrane through which antibiotics diffuse into the periplasm. PMID:16101998

  12. 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.

  13. Rapid Detection of rpoB Gene Mutations Conferring Rifampin Resistance in Mycobacterium tuberculosis

    PubMed Central

    Ao, Wanyuan; Aldous, Stephen; Woodruff, Evelyn; Hicke, Brian; Rea, Larry; Kreiswirth, Barry

    2012-01-01

    Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute. PMID:22518852

  14. Next-generation sequencing reveals somatic mutations that confer exceptional response to everolimus

    PubMed Central

    Kim, Sangwoo; Kim, Sora; Ali, Siraj M.; Greenbowe, Joel R.; Yang, In Seok; Kwon, Nak-Jung; Lee, Jae Lyun; Ryu, Min-Hee; Ahn, Jin-Hee; Lee, Jeeyun; Lee, Min Goo; Kim, Hyo Song; Kim, Hyunki; Kim, Hye Ryun; Moon, Yong Wha; Chung, Hyun Cheol; Kim, Joo-Hang; Kang, Yoon-Koo; Cho, Byoung Chul

    2016-01-01

    Background Given the modest responses to everolimus, a mTOR inhibitor, in multiple tumor types, there is a pressing need to identify predictive biomarkers for this drug. Using targeted ultra-deep sequencing, we aimed to explore genomic alterations that confer extreme sensitivity to everolimus. Results We collected formalin-fixed paraffin-embedded tumor/normal pairs from 39 patients (22 with exceptional clinical benefit, 17 with no clinical benefit) who were treated with everolimus across various tumor types (13 gastric cancers, 15 renal cell carcinomas, 2 thyroid cancers, 2 head and neck cancer, and 7 sarcomas). Ion AmpliSeqTM Comprehensive Cancer Panel was used to identify alterations across all exons of 409 target genes. Tumors were sequenced to a median coverage of 552x. Cancer genomes are characterized by 219 somatic single-nucleotide variants (181 missense, 9 nonsense, 7 splice-site) and 22 frameshift insertions/deletions, with a median of 2.1 mutations per Mb (0 to 12.4 mutations per Mb). Overall, genomic alterations with activating effect on mTOR signaling were identified in 10 of 22 (45%) patients with clinical benefit and these include MTOR, TSC1, TSC2, NF1, PIK3CA and PIK3CG mutations. Recurrently mutated genes in chromatin remodeling genes (BAP1; n = 2, 12%) and receptor tyrosine kinase signaling (FGFR4; n = 2, 12%) were noted only in patients without clinical benefit. Conclusions Regardless of different cancer types, mTOR-pathway-activating mutations confer sensitivity to everolimus. Targeted sequencing of mTOR pathway genes facilitates identification of potential candidates for mTOR inhibitors. PMID:26859683

  15. An Agrobacterium VirB10 Mutation Conferring a Type IV Secretion System Gating Defect▿

    PubMed Central

    Banta, Lois M.; Kerr, Jennifer E.; Cascales, Eric; Giuliano, Meghan E.; Bailey, Megan E.; McKay, Cedar; Chandran, Vidya; Waksman, Gabriel; Christie, Peter J.

    2011-01-01

    Agrobacterium VirB7, VirB9, and VirB10 form a “core complex” during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra+) but did block pilus production (Pil−). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra+ Pil− phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway. PMID:21421757

  16. 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.

  17. JAK-2 V617F mutation increases heparanase procoagulant activity.

    PubMed

    Kogan, Inna; Chap, Dafna; Hoffman, Ron; Axelman, Elena; Brenner, Benjamin; Nadir, Yona

    2016-01-01

    Patients with polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are at increased risk of arterial and venous thrombosis. In patients with ET a positive correlation was observed between JAK-2 V617F mutation, that facilitates erythropoietin receptor signalling, and thrombotic events, although the mechanism involved is not clear. We previously demonstrated that heparanase protein forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF) which leads to increased factor Xa production and subsequent activation of the coagulation system. The present study was aimed to evaluate heparanase procoagulant activity in myeloproliferative neoplasms. Forty bone marrow biopsies of patients with ET, PV, PMF and chronic myelogenous leukaemia (CML) were immunostained to heparanase, TF and TF pathway inhibitor (TFPI). Erythropoietin receptor positive cell lines U87 human glioma and MCF-7 human breast carcinoma were studied. Heparanase and TFPI staining were more prominent in ET, PV and PMF compared to CML. The strongest staining was in JAK-2 positive ET biopsies. Heparanase level and procoagulant activity were higher in U87 cells transfected to over express JAK-2 V617F mutation compared to control and the effect was reversed using JAK-2 inhibitors (Ruxolitinib, VZ3) and hydroxyurea, although the latter drug did not inhibit JAK-2 phosphorylation. Erythropoietin increased while JAK-2 inhibitors decreased the heparanase level and procoagulant activity in U87 and MCF-7 parental cells. In conclusion, JAK-2 is involved in heparanase up-regulation via the erythropoietin receptor. The present findings may potentially point to a new mechanism of thrombosis in JAK-2 positive ET patients. PMID:26489695

  18. 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. PMID:27402614

  19. Physiological, biochemical and molecular characterization of an induced mutation conferring imidazolinone resistance in wheat.

    PubMed

    Jimenez, Francisco; Rojano-Delgado, Antonia M; Fernández, Pablo Tomas; Rodríguez-Suárez, Cristina; Atienza, Sergio G; De Prado, Rafael

    2016-09-01

    The Clearfield(®) wheat cultivars possessing imidazolinone (IMI)-resistant traits provide an efficient option for controlling weeds. The imazamox-resistant cultivar Pantera (Clearfield(®) ) was compared with a susceptible cultivar (Gazul). Target and non-target mechanisms of resistance were studied to characterize the resistance of Pantera and to identify the importance of each mechanism involved in this resistance. Pantera is resistant to imazamox as was determined in previous experiments. The molecular study confirmed that it carries a mutation Ser-Asn627 conferring resistance to imazamox in two out of three acetolactate synthase (ALS) genes (imi1 and imi2), located in wheat on chromosomes 6B and 6D, respectively. However, the last gene (imi3) located on chromosome 6A does not carry any mutation conferring resistance. As a result, photosynthetic activity and chlorophyll content were reduced after imazamox treatment. Detoxification was higher in the resistant biotype as shown by metabolomic study while imazamox translocation was higher in the susceptible cultivar. Interestingly, imazamox metabolism was higher at higher doses of herbicide, which suggests that the detoxification process is an inducible mechanism in which the upregulation of key gene coding for detoxification enzymes could play an important role. Thus, the identification of cultivars with a higher detoxification potential would allow the development of more resistant varieties. PMID:26991509

  20. ERK Mutations Confer Resistance to Mitogen-Activated Protein Kinase Pathway Inhibitors

    PubMed Central

    Goetz, Eva M.; Ghandi, Mahmoud; Treacy, Daniel J.; Wagle, Nikhil; Garraway, Levi A.

    2015-01-01

    The use of targeted therapeutics directed against BRAFV600-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAFV600-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor–resistant alleles were sensitive to RAF/ MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents. PMID:25320010

  1. Mutation Leading to Increased Sensitivity to Chromium in Salmonella typhimurium

    PubMed Central

    Corwin, L. M.; Fanning, G. R.; Feldman, F.; Margolin, P.

    1966-01-01

    Corwin, L. M. (Walter Reed Army Institute of Research, Washington, D.C.), G. R. Fanning, F. Feldman, and P. Margolin. Mutation leading to increased sensitivity to chromium in Salmonella typhimurium. J. Bacteriol. 91:1509–1515. 1966.—Certain deletion mutants including the tryptophan operon in Salmonella typhimurium are unable to utilize several sugars as carbon sources in solid media, although they are able to grow in liquid media with these sugars. The addition of citrate or washing the agar with ethylenediaminetetraacetic acid permits growth on solid media. Analysis of the agar revealed that Fe3+ and Cr3+ were present at concentrations of 22 and 75 μm, respectively. The addition of Fe3+ to liquid media in 0.5 mm concentrations did not inhibit the wild type or the mutants. A similar concentration of Cr3+ did not inhibit the wild type, but concentrations as low as 0.01 to 0.05 mm inhibited the deletion mutants. Other metals were inhibitory at various concentrations, but none showed any significant differential effects on the mutants and the wild type. The increased sensitivity of the mutants to chromium may be due either to an increased permeability to Cr3+, resulting in higher effective intracellular concentrations and inhibition of one or more metabolic functions, or to a binding of Cr3+ to an altered cell wall, resulting in decreased permeability of required substrates. PMID:4956341

  2. Mutation in promoter region of a serine protease inhibitor confers Perkinsus marinus resistance in the eastern oyster (Crassostrea virginica).

    PubMed

    He, Yan; Yu, Haiyang; Bao, Zhenmin; Zhang, Quanqi; Guo, Ximing

    2012-08-01

    Protease inhibitors from the host may inhibit proteases from invading pathogens and confer resistance. We have previously shown that a single-nucleotide polymorphism (SNP198C) in a serine protease inhibitor gene (cvSI-1) is associated with Perkinsus marinus resistance in the eastern oyster. As SNP198 is synonymous, we studied whether its linkage to polymorphism at the promoter region could explain the resistance. A 631 bp fragment of the promoter region was cloned by genome-walking and resequenced, revealing 22 SNPs and 3 insertion/deletions (indels). A 25 bp indel at position -404 was genotyped along with SNP198 for association analysis using before- and after-mortality samples. After mortalities that were primarily caused by P. marinus, the frequency of deletion allele at -404indel increased by 15.6% (p = 0.0437), while that of SNP198C increased by only 3.4% (p = 0.5756). The resistance alleles at the two loci were coupled in 79.6% of the oysters. Oysters with the deletion allele at -404indel showed significant (p = 0.0189) up-regulation of cvSI-1 expression under P. marinus challenge. Our results suggest that mutation at the promoter region causes increased transcription of cvSI-1, which in turn confers P. marinus resistance in the eastern oyster likely through inhibiting pathogenic proteases from the parasite. PMID:22683517

  3. Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma

    PubMed Central

    Villanueva, Jessie; Infante, Jeffrey R.; Krepler, Clemens; Reyes-Uribe, Patricia; Samanta, Minu; Chen, Hsin-Yi; Li, Bin; Swoboda, Rolf K.; Wilson, Melissa; Vultur, Adina; Fukunaba-Kalabis, Mizuho; Wubbenhorst, Bradley; Chen, Thomas Y.; Liu, Qin; Sproesser, Katrin; DeMarini, Douglas J.; Gilmer, Tona M.; Martin, Anne-Marie; Marmorstein, Ronen; Schultz, David C.; Speicher, David W.; Karakousis, Giorgos C.; Xu, Wei; Amaravadi, Ravi K.; Xu, Xiaowe; Schuchter, Lynn M.; Herlyn, Meenhard; Nathanson, Katherine L.

    2014-01-01

    Summary Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it. PMID:24055054

  4. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia

    PubMed Central

    Lee, Benjamin H.; Tothova, Zuzana; Levine, Ross L.; Anderson, Kristina; Buza-Vidas, Natalija; Cullen, Dana E.; McDowell, Elizabeth P.; Adelsperger, Jennifer; Fröhling, Stefan; Huntly, Brian J.P.; Beran, Miloslav; Jacobsen, Sten Eirik; Gilliland, D. Gary

    2007-01-01

    SUMMARY Despite their known transforming properties, the effects of leukemogenic FLT3-ITD mutations on hematopoietic stem and multipotent progenitor cells and on hematopoietic differentiation are not well understood. We report a mouse model harboring an ITD in the murine Flt3 locus that develops myeloproliferative disease resembling CMML and further identified FLT3-ITD mutations in a subset of human CMML. These findings correlated with an increase in number, cell cycling and survival of multipotent stem and progenitor cells in an ITD dose-dependent manner in animals that exhibited alterations within their myeloid progenitor compartments and a block in normal B-cell development. This model provides insights into the consequences of constitutive signaling by an oncogenic tyrosine kinase on hematopoietic progenitor quiescence, function, and cell fate. SIGNIFICANCE Activating FLT3 mutations are among the most common genetic events in AML and confer a poor clinical prognosis. Essential to our understanding of how these lesions contribute to myeloid leukemia is the development of a Flt3-ITD ‘knock-in’ murine model that has allowed examination of the consequences of constitutive FLT3 signaling on primitive hematopoietic progenitors when expressed at appropriate physiologic levels. These animals informed us to the existence of FLT3-ITD-positive human CMML, which has clinical importance given the availability of FLT3 small molecule inhibitors. This model will not only serve as a powerful biological tool to identify mutations that cooperate with FLT3 in leukemogenesis, but also to assess molecular therapies that target either FLT3 or components of its signaling pathways. PMID:17936561

  5. 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

  6. Increased mutation in crosses between geographically separated strains of Drosophila melanogaster.

    PubMed Central

    Thompson, J N; Woodruff, R C

    1980-01-01

    Mutator activity associated with the common male recombination (MR) chromosomes in Drosophila melanogaster appears to be suppressed in natural populations. Crosses between geographically separated populations, however, lead to the release of mutator activity as measured by a significant increase in visible mutations. Such an increase in mutation in hybrid individuals may be a powerful factor in inducing or releasing variation in nature, and in more extreme instances may contribute to the separation of microdifferentiated populations. PMID:6767240

  7. The SKG Mutation in ZAP-70 also Confers Arthritis Susceptibility in C57 Black Mouse Strains.

    PubMed

    Guerard, S; Boieri, M; Hultqvist, M; Holmdahl, R; Wing, K

    2016-07-01

    Various rodent models of arthritis are essential to dissect the full complexity of human rheumatoid arthritis (RA), a common autoimmune disease affecting joints. The SKG model of arthritis originates from a spontaneous mutation in ZAP-70 found in a BALB/c colony. This mutation affects T cell selection due to reduced TCR signalling, which allows leakage of self-reactive T cells from the thymus. To further expand the practical applicability of this unique model in arthritis research, we investigated the arthritogenicity of the SKG mutation in two common black mouse strains C57BL/6.Q and C57BL/10.Q and compared to BALB/c.Q. Mice retained the reduced TCR signalling characteristic of SKG.BALB/c mice, which leads to similar alteration in thymic selection. Importantly, mice also retained susceptibility to chronic arthritis after a single injection of mannan from Saccharomyces cerevisiae, with comparable prevalence and severity regardless of the genetic background. Further characterization of CD4(+) T cells revealed a similar bias towards IL-17 production and activated T cell phenotype in all SKG strains compared to respective wild type controls. Finally, transfer of SKG thymocytes conferred susceptibility to recipients, which confirm the intrinsic defect and pathogenicity of T cells. Overall, these results underline the strong impact that the W163C ZAP-70 mutation has on T cell-driven arthritis, and they support the use of the SKG model in black mice, which is useful for further investigations of this distinctive arthritis model to better understand autoimmunity. PMID:27040161

  8. RNA Polymerase II Mutations Conferring Defects in Poly(A) Site Cleavage and Termination in Saccharomyces cerevisiae

    PubMed Central

    Kubicek, Charles E.; Chisholm, Robert D.; Takayama, Sachiko; Hawley, Diane K.

    2013-01-01

    Transcription termination by RNA polymerase (Pol) II is an essential but poorly understood process. In eukaryotic nuclei, the 3′ ends of mRNAs are generated by cleavage and polyadenylation, and the same sequence elements that specify that process are required for downstream release of the polymerase from the DNA. Although Pol II is known to bind proteins required for both events, few studies have focused on Pol II mutations as a means to uncover the mechanisms that couple polyadenylation and termination. We performed a genetic screen in the yeast Saccharomyces cerevisiae to isolate mutations in the N-terminal half of Rpb2, the second largest Pol II subunit, that conferred either a decreased or increased response to a well-characterized poly(A) site. Most of the mutant alleles encoded substitutions affecting either surface residues or conserved active site amino acids at positions important for termination by other RNA polymerases. Reverse transcription polymerase chain reaction experiments revealed that transcript cleavage at the poly(A) site was impaired in both classes of increased readthrough mutants. Transcription into downstream sequences beyond where termination normally occurs was also probed. Although most of the tested readthrough mutants showed a reduction in termination concomitant with the reduced poly(A) usage, these processes were uncoupled in at least one mutant strain. Several rpb2 alleles were found to be similar or identical to published mutants associated with defective TFIIF function. Tests of these and additional mutations known to impair Rpb2−TFIIF interactions revealed similar decreased readthrough phenotypes, suggesting that TFIIF may have a role in 3′ end formation and termination. PMID:23390594

  9. Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process

    PubMed Central

    Iijima, Leo; Suzuki, Shingo; Hashimoto, Tomomi; Oyake, Ayana; Kobayashi, Hisaka; Someya, Yuki; Narisawa, Dai; Yomo, Tetsuya

    2015-01-01

    The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution. PMID:26177190

  10. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor

    PubMed Central

    Yao, Jia-Long; Dong, Yi-Hu; Morris, Bret A. M.

    2001-01-01

    Fruit development in higher plants normally requires pollination and fertilization to stimulate cell division of specific floral tissues. In some cases, parthenocarpic fruit development proceeds without either pollination or fertilization. Parthenocarpic fruit without seed has higher commercial value than seeded fruit. Several apple (Malus domestica) mutants (Rae Ime, Spencer Seedless and Wellington Bloomless) are known to produce only apetalous flowers that readily go on to develop into parthenocarpic fruit. Through genetics, a single recessive gene has been identified to control this trait in apple. Flower phenotypes of these apple mutants are strikingly similar to those of the Arabidopsis mutant pistillata (pi), which produces flowers where petals are transformed to sepals and stamens to carpels. In this study, we have cloned the apple PI homolog (MdPI) that shows 64% amino acid sequence identity and closely conserved intron positions and mRNA expression patterns to the Arabidopsis PI. We have identified that in the apetalous mutants MdPI has been mutated by a retrotransposon insertion in intron 4 in the case of Rae Ime and in intron 6 in the case of Spencer Seedless and Wellington Bloomless. The insertion apparently abolishes the normal expression of the MdPI gene. We conclude that the loss of function mutation in the MdPI MADS-box transcription factor confers parthenocarpic fruit development in these apple varieties and demonstrates another function for the MADS- box gene family. The knowledge generated here could be used to produce parthenocarpic fruit cultivars through genetic engineering. PMID:11158635

  11. 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

  12. Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance.

    PubMed

    Hattori, J; Rutledge, R; Labbé, H; Brown, D; Sunohara, G; Miki, B

    1992-03-01

    The acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3' end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides. PMID:1557022

  13. Adaptive evolution by recombination is not associated with increased mutation rates in Maize streak virus

    PubMed Central

    2012-01-01

    Background Single-stranded (ss) DNA viruses in the family Geminiviridae are proving to be very useful in real-time evolution studies. The high mutation rate of geminiviruses and other ssDNA viruses is somewhat mysterious in that their DNA genomes are replicated in host nuclei by high fidelity host polymerases. Although strand specific mutation biases observed in virus species from the geminivirus genus Mastrevirus indicate that the high mutation rates in viruses in this genus may be due to mutational processes that operate specifically on ssDNA, it is currently unknown whether viruses from other genera display similar strand specific mutation biases. Also, geminivirus genomes frequently recombine with one another and an alternative cause of their high mutation rates could be that the recombination process is either directly mutagenic or produces a selective environment in which the survival of mutants is favoured. To investigate whether there is an association between recombination and increased basal mutation rates or increased degrees of selection favoring the survival of mutations, we compared the mutation dynamics of the MSV-MatA and MSV-VW field isolates of Maize streak virus (MSV; Mastrevirus), with both a laboratory constructed MSV recombinant, and MSV recombinants closely resembling MSV-MatA. To determine whether strand specific mutation biases are a general characteristic of geminivirus evolution we compared mutation spectra arising during these MSV experiments with those arising during similar experiments involving the geminivirus Tomato yellow leaf curl virus (Begomovirus genus). Results Although both the genomic distribution of mutations and the occurrence of various convergent mutations at specific genomic sites indicated that either mutation hotspots or selection for adaptive mutations might elevate observed mutation rates in MSV, we found no association between recombination and mutation rates. Importantly, when comparing the mutation spectra of MSV

  14. Mutations in human cytomegalovirus UL97 gene confer clinical resistance to ganciclovir and can be detected directly in patient plasma.

    PubMed Central

    Wolf, D G; Smith, I L; Lee, D J; Freeman, W R; Flores-Aguilar, M; Spector, S A

    1995-01-01

    Specific mutations in the UL97 region of human cytomegalovirus (HCMV) have been found to confer resistance to laboratory-adapted strains subjected to ganciclovir selection. In this study, mutations in the UL97 region of HCMV isolates obtained from patients receiving ganciclovir therapy were examined to determine whether they would confer ganciclovir resistance, and if these mutations could be detected directly in the plasma of AIDS patients with progressive HCMV disease despite ganciclovir treatment. A single nucleotide change within a conserved region of UL97 was found in five resistant isolates, resulting in an amino acid substitution in residue 595: from leucine to phenylalanine in one, and from leucine to serine in four resistant isolates. A sixth resistant isolate demonstrated a single nucleotide change, leading to a threonine to isoleucine substitution in residue 659. The role of the 595 amino acid substitution in conferring ganciclovir resistance was confirmed by marker transfer experiments. In further studies, direct sequencing of HCMV DNA present in plasma obtained from persons with resistant viruses revealed the identical amino acid substitutions in plasma as those present in the cultured viruses. These findings indicate that clinical resistance to ganciclovir can result from specific point mutations in the UL97 gene, and that the emergence of the resistant genotype can be detected directly in patient plasma. Images PMID:7814623

  15. Pyrimidine base damage is increased in women with BRCA mutations.

    PubMed

    Budzinski, Edwin E; Patrzyc, Helen B; Dawidzik, Jean B; Freund, Harold G; Frederick, Peter; Godoy, Heidi E; Voian, Nicoleta C; Odunsi, Kunle; Box, Harold C

    2013-09-28

    Oxidatively-induced DNA damage was measured in the DNA of WBC from two groups of women: carriers of a BRCA mutation, but asymptomatic for disease, and healthy controls. Two oxidatively induced lesions were measured: a formamide remnant of pyrimidine base and the glycol modification of thymine. These lesions, employed previously in studies of the effects of smoking, antioxidant usage and ovarian cancer, are proving valuable indicators of oxidative stress. The BRCA carriers of mutations, with no overt sign of cancer, nevertheless had significantly higher levels of DNA damage than the controls. The level measured for the formamide lesion was 5.9 ± 1.0 (femtomoles/μg of DNA ± SEM) compared with 2.4 ± 0.3 in controls. The level of the glycol lesion was 2.9 ± 0.4 compared with 1.8 ± 0.2 in controls. The experimental design utilized DNA from WBC and employed LC-MS/MS to detect the lesions. PMID:23583677

  16. 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

  17. Sdt97: A Point Mutation in the 5' Untranslated Region Confers Semidwarfism in Rice.

    PubMed

    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

  18. Biochemical and Molecular Characterization of a Mutation That Confers a Decreased Raffinosaccharide and Phytic Acid Phenotype on Soybean Seeds

    PubMed Central

    Hitz, William D.; Carlson, Thomas J.; Kerr, Phil S.; Sebastian, Scott A.

    2002-01-01

    A single, recessive mutation in soybean (Glycine max L. Merr.), which confers a seed phenotype of increased inorganic phosphate, decreased phytic acid, and a decrease in total raffinosaccharides, has been previously disclosed (S.A. Sebastian, P.S. Kerr, R.W. Pearlstein, W.D. Hitz [2000] Soy in Animal Nutrition, pp 56–74). The genetic lesion causing the multiple changes in seed phenotype is a single base change in the third base of the codon for what is amino acid residue 396 of the mature peptide encoding a seed-expressed myo-inositol 1-phospate synthase gene. The base change causes residue 396 to change from lysine to asparagine. That amino acid change decreases the specific activity of the seed-expressed myo-inositol 1-phosphate synthase by about 90%. Radio tracer experiments indicate that the supply of myo-inositol to the reaction, which converts UDP-galactose and myo-inositol to galactinol is a controlling factor in the conversion of total carbohydrate into the raffinosaccharides in both wild-type and mutant lines. That same decrease in myo-inositol 1-phosphate synthetic capacity leads to a decreased capacity for the synthesis of myo-inositol hexaphosphate (phytic acid) and a concomitant increase in inorganic phosphate. PMID:11842168

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

    PubMed Central

    Hardouin, Emilie A.; Tautz, Diethard

    2013-01-01

    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. PMID:23389667

  20. 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.

  1. 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.

  2. BCL2 mutations are associated with increased risk of transformation and shortened survival in follicular lymphoma

    PubMed Central

    Correia, Cristina; Schneider, Paula A.; Dai, Haiming; Dogan, Ahmet; Maurer, Matthew J.; Church, Amy K.; Novak, Anne J.; Feldman, Andrew L.; Wu, Xiaosheng; Ding, Husheng; Meng, X. Wei; Cerhan, James R.; Slager, Susan L.; Macon, William R.; Habermann, Thomas M.; Karp, Judith E.; Gore, Steven D.; Kay, Neil E.; Jelinek, Diane F.; Witzig, Thomas E.; Nowakowski, Grzegorz S.

    2015-01-01

    Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P = .012). In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activation-induced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma. PMID:25452615

  3. Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype

    PubMed Central

    Cuyàs, Elisabet; Fernández-Arroyo, Salvador; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Bosch-Barrera, Joaquim; Martin-Castillo, Begoña; De Llorens, Rafael; Joven, Jorge; Menendez, Javier A.

    2015-01-01

    Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG

  4. Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic.

    PubMed

    Larimer, Curtis; Islam, Mohammad Shyful; Ojha, Anil; Nettleship, Ian

    2014-08-01

    Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc(2)155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO3 and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO3. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc(2)155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO4 and ZnSO4 than the mc(2)155 strain. PMID:24989695

  5. Maternal filaggrin mutations increase the risk of atopic dermatitis in children: an effect independent of mutation inheritance.

    PubMed

    Esparza-Gordillo, Jorge; Matanovic, Anja; Marenholz, Ingo; Bauerfeind, Anja; Rohde, Klaus; Nemat, Katja; Lee-Kirsch, Min-Ae; Nordenskjöld, Magnus; Winge, Marten C G; Keil, Thomas; Krüger, Renate; Lau, Susanne; Beyer, Kirsten; Kalb, Birgit; Niggemann, Bodo; Hübner, Norbert; Cordell, Heather J; Bradley, Maria; Lee, Young-Ae

    2015-03-01

    Epidemiological studies suggest that allergy risk is preferentially transmitted through mothers. This can be due to genomic imprinting, where the phenotype effect of an allele depends on its parental origin, or due to maternal effects reflecting the maternal genome's influence on the child during prenatal development. Loss-of-function mutations in the filaggrin gene (FLG) cause skin barrier deficiency and strongly predispose to atopic dermatitis (AD). We investigated the 4 most prevalent European FLG mutations (c.2282del4, p.R501X, p.R2447X, and p.S3247X) in two samples including 759 and 450 AD families. We used the multinomial and maximum-likelihood approach implemented in the PREMIM/EMIM tool to model parent-of-origin effects. Beyond the known role of FLG inheritance in AD (R1meta-analysis = 2.4, P = 1.0 x 10-36), we observed a strong maternal FLG genotype effect that was consistent in both independent family sets and for all 4 mutations analysed. Overall, children of FLG-carrier mothers had a 1.5-fold increased AD risk (S1 = 1.50, Pmeta-analysis = 8.4 x 10-8). Our data point to two independent and additive effects of FLG mutations: i) carrying a mutation and ii) having a mutation carrier mother. The maternal genotype effect was independent of mutation inheritance and can be seen as a non-genetic transmission of a genetic effect. The FLG maternal effect was observed only when mothers had allergic sensitization (elevated allergen-specific IgE antibody plasma levels), suggesting that FLG mutation-induced systemic immune responses in the mother may influence AD risk in the child. Notably, the maternal effect reported here was stronger than most common genetic risk factors for AD recently identified through genome-wide association studies (GWAS). Our study highlights the power of family-based studies in the identification of new etiological mechanisms and reveals, for the first time, a direct influence of the maternal genotype on the offspring's susceptibility to a

  6. Maternal Filaggrin Mutations Increase the Risk of Atopic Dermatitis in Children: An Effect Independent of Mutation Inheritance

    PubMed Central

    Esparza-Gordillo, Jorge; Matanovic, Anja; Marenholz, Ingo; Bauerfeind, Anja; Rohde, Klaus; Nemat, Katja; Lee-Kirsch, Min-Ae; Nordenskjöld, Magnus; Winge, Marten C. G.; Keil, Thomas; Krüger, Renate; Lau, Susanne; Beyer, Kirsten; Kalb, Birgit; Niggemann, Bodo; Hübner, Norbert; Cordell, Heather J.; Bradley, Maria; Lee, Young-Ae

    2015-01-01

    Epidemiological studies suggest that allergy risk is preferentially transmitted through mothers. This can be due to genomic imprinting, where the phenotype effect of an allele depends on its parental origin, or due to maternal effects reflecting the maternal genome's influence on the child during prenatal development. Loss-of-function mutations in the filaggrin gene (FLG) cause skin barrier deficiency and strongly predispose to atopic dermatitis (AD). We investigated the 4 most prevalent European FLG mutations (c.2282del4, p.R501X, p.R2447X, and p.S3247X) in two samples including 759 and 450 AD families. We used the multinomial and maximum-likelihood approach implemented in the PREMIM/EMIM tool to model parent-of-origin effects. Beyond the known role of FLG inheritance in AD (R1meta-analysis = 2.4, P = 1.0 x 10−36), we observed a strong maternal FLG genotype effect that was consistent in both independent family sets and for all 4 mutations analysed. Overall, children of FLG-carrier mothers had a 1.5-fold increased AD risk (S1 = 1.50, Pmeta-analysis = 8.4 x 10−8). Our data point to two independent and additive effects of FLG mutations: i) carrying a mutation and ii) having a mutation carrier mother. The maternal genotype effect was independent of mutation inheritance and can be seen as a non-genetic transmission of a genetic effect. The FLG maternal effect was observed only when mothers had allergic sensitization (elevated allergen-specific IgE antibody plasma levels), suggesting that FLG mutation-induced systemic immune responses in the mother may influence AD risk in the child. Notably, the maternal effect reported here was stronger than most common genetic risk factors for AD recently identified through genome-wide association studies (GWAS). Our study highlights the power of family-based studies in the identification of new etiological mechanisms and reveals, for the first time, a direct influence of the maternal genotype on the offspring’s susceptibility

  7. A mutation Ser213/Asn in the hexokinase 1 from Schizosaccharomyces pombe increases its affinity for glucose.

    PubMed

    Petit, T; Herrero, P; Gancedo, C

    1998-10-29

    Alignment of amino acids of the region implicated in glucose binding from a series of hexokinases showed that Schizosaccharomyces pombe hexokinase 1 had a Ser residue in a place where all other kinases had an Asn. We changed an AGT codon to AAT to place an Asn in the Ser213 position. This mutation decreased Km for glucose from 9.4 mM to 1.6 mM and the ratio Vmax (Fructose)/Vmax (Glucose) from 5 to 2.5. Also the Km for 2-deoxyglucose decreased from 2.7 mM to 0.8 mM. A mutation in the similar position of S. pombe hexokinase 2 (Asn196/Ser) increased the Km for glucose from 0.16 mM to 0.56 mM. Fermentation of glucose is not detectable in a S. pombe mutant with only hexokinase 1 activity but expression of the hxk1(S213/N) gene conferred ability to ferment the sugar. While the mutated hexokinase 1 partially mimicked S. cerevisiae hexokinase II in catabolite repression of invertase, the wild type one could not substitute for it. PMID:9790975

  8. 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). PMID:25890976

  9. Hair keratin mutations in tooth enamel increase dental decay risk

    PubMed Central

    Duverger, Olivier; Ohara, Takahiro; Shaffer, John R.; Donahue, Danielle; Zerfas, Patricia; Dullnig, Andrew; Crecelius, Christopher; Beniash, Elia; Marazita, Mary L.; Morasso, Maria I.

    2014-01-01

    Tooth enamel is the hardest substance in the human body and has a unique combination of hardness and fracture toughness that protects teeth from dental caries, the most common chronic disease worldwide. In addition to a high mineral content, tooth enamel comprises organic material that is important for mechanical performance and influences the initiation and progression of caries; however, the protein composition of tooth enamel has not been fully characterized. Here, we determined that epithelial hair keratins, which are crucial for maintaining the integrity of the sheaths that support the hair shaft, are expressed in the enamel organ and are essential organic components of mature enamel. Using genetic and intraoral examination data from 386 children and 706 adults, we found that individuals harboring known hair disorder–associated polymorphisms in the gene encoding keratin 75 (KRT75), KRT75A161T and KRT75E337K, are prone to increased dental caries. Analysis of teeth from individuals carrying the KRT75A161T variant revealed an altered enamel structure and a marked reduction of enamel hardness, suggesting that a functional keratin network is required for the mechanical stability of tooth enamel. Taken together, our results identify a genetic locus that influences enamel structure and establish a connection between hair disorders and susceptibility to dental caries. PMID:25347471

  10. Hair keratin mutations in tooth enamel increase dental decay risk.

    PubMed

    Duverger, Olivier; Ohara, Takahiro; Shaffer, John R; Donahue, Danielle; Zerfas, Patricia; Dullnig, Andrew; Crecelius, Christopher; Beniash, Elia; Marazita, Mary L; Morasso, Maria I

    2014-12-01

    Tooth enamel is the hardest substance in the human body and has a unique combination of hardness and fracture toughness that protects teeth from dental caries, the most common chronic disease worldwide. In addition to a high mineral content, tooth enamel comprises organic material that is important for mechanical performance and influences the initiation and progression of caries; however, the protein composition of tooth enamel has not been fully characterized. Here, we determined that epithelial hair keratins, which are crucial for maintaining the integrity of the sheaths that support the hair shaft, are expressed in the enamel organ and are essential organic components of mature enamel. Using genetic and intraoral examination data from 386 children and 706 adults, we found that individuals harboring known hair disorder-associated polymorphisms in the gene encoding keratin 75 (KRT75), KRT75(A161T) and KRT75(E337K), are prone to increased dental caries. Analysis of teeth from individuals carrying the KRT75(A161T) variant revealed an altered enamel structure and a marked reduction of enamel hardness, suggesting that a functional keratin network is required for the mechanical stability of tooth enamel. Taken together, our results identify a genetic locus that influences enamel structure and establish a connection between hair disorders and susceptibility to dental caries. PMID:25347471

  11. Autism Linked to Increased Oncogene Mutations but Decreased Cancer Rate

    PubMed Central

    Zimmerman, M. Bridget; Mahajan, Vinit B.; Bassuk, Alexander G.

    2016-01-01

    Autism spectrum disorder (ASD) is one phenotypic aspect of many monogenic, hereditary cancer syndromes. Pleiotropic effects of cancer genes on the autism phenotype could lead to repurposing of oncology medications to treat this increasingly prevalent neurodevelopmental condition for which there is currently no treatment. To explore this hypothesis we sought to discover whether autistic patients more often have rare coding, single-nucleotide variants within tumor suppressor and oncogenes and whether autistic patients are more often diagnosed with neoplasms. Exome-sequencing data from the ARRA Autism Sequencing Collaboration was compared to that of a control cohort from the Exome Variant Server database revealing that rare, coding variants within oncogenes were enriched for in the ARRA ASD cohort (p<1.0x10-8). In contrast, variants were not significantly enriched in tumor suppressor genes. Phenotypically, children and adults with ASD exhibited a protective effect against cancer, with a frequency of 1.3% vs. 3.9% (p<0.001), but the protective effect decreased with age. The odds ratio of neoplasm for those with ASD relative to controls was 0.06 (95% CI: 0.02, 0.19; p<0.0001) in the 0 to 14 age group; 0.35 (95% CI: 0.14, 0.87; p = 0.024) in the 15 to 29 age group; 0.41 (95% CI: 0.15, 1.17; p = 0.095) in the 30 to 54 age group; and 0.49 (95% CI: 0.14, 1.74; p = 0.267) in those 55 and older. Both males and females demonstrated the protective effect. These findings suggest that defects in cellular proliferation, and potentially senescence, might influence both autism and neoplasm, and already approved drugs targeting oncogenic pathways might also have therapeutic value for treating autism. PMID:26934580

  12. 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…

  13. A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum.

    PubMed

    Ohnishi, Junko; Katahira, Ritsuko; Mitsuhashi, Satoshi; Kakita, Shingo; Ikeda, Masato

    2005-01-15

    Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation. PMID:15621447

  14. 5-Azacytidine and RNA secondary structure increase the retrovirus mutation rate.

    PubMed Central

    Pathak, V K; Temin, H M

    1992-01-01

    A broad spectrum of mutations occurs at a high rate during a single round of retrovirus replication (V.K. Pathak and H. M. Temin, Proc. Natl. Acad. Sci. USA 87:6019-6023, 1990). We have now determined that this high rate of spontaneous mutation can be further increased by 5-azacytidine (AZC) treatment or by regions of potential RNA secondary structure. We found a 13-fold increase in the mutation rate after AZC treatment of retrovirus-producing cells and target cells. The AZC-induced substitutions were located at the same target sites as previously identified spontaneous substitutions. The concordance of the AZC-induced and spontaneous substitutions indicates the presence of reverse transcription "pause sites," where the growing point is error prone. An analysis of nucleotides that neighbored substitutions revealed that transversions occur primarily by transient template misalignment, whereas transitions occur primarily by misincorporation. We also introduced a 34-bp potential stem-loop structure as an in-frame insertion within a lacZ alpha gene that was inserted in the long terminal repeat (LTR) U3 region and determined whether this potential secondary structure increased the rate of retrovirus mutations. We found a threefold increase in the retrovirus mutation rate. Fifty-seven of 96 mutations were deletions associated with the potential stem-loop. We also determined that these deletion mutations occurred primarily during minus-strand DNA synthesis by comparing the frequencies of mutations in recovered provirus plasmids containing both LTRs and in provirus plasmids containing only one LTR. PMID:1373201

  15. 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

  16. 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

  17. Mutation in the bimD gene of Aspergillus nidulans confers a conditional mitotic block and sensitivity to DNA damaging agents

    SciTech Connect

    Denison, S.H.; May, G.S. ); Kaefer, E. )

    1993-08-01

    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, the authors 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[sub 1] or S phase of the cell cycle. The data suggest that bimD may play an essential regulatory role relating to DNA metabolism which is required for a successful mitosis. 7l refs., 7 figs., 1 tab.

  18. Mutations conferring resistance to quinol oxidation (Qz) inhibitors of the cyt bc1 complex of Rhodobacter capsulatus.

    PubMed Central

    Daldal, F; Tokito, M K; Davidson, E; Faham, M

    1989-01-01

    Several spontaneous mutants of the photosynthetic bacterium Rhodobacter capsulatus resistant to myxothiazol, stigmatellin and mucidin--inhibitors of the ubiquinol: cytochrome c oxidoreductase (cyt bc1 complex)--were isolated. They were grouped into eight different classes based on their genetic location, growth properties and inhibitor cross-resistance. The petABC (fbcFBC) cluster that encodes the structural genes for the Rieske FeS protein, cyt b and cyt c1 subunits of the cyt bc1 complex was cloned out of the representative isolates and the molecular basis of inhibitor-resistance was determined by DNA sequencing. These data indicated that while one group of mutations was located outside the petABC(fbcFBC) cluster, the remainder were single base pair changes in codons corresponding to phylogenetically conserved amino acid residues of cyt b. Of these substitutions, F144S conferred resistance to myxothiazol, T163A and V333A to stigmatellin, L106P and G152S to myxothiazol + mucidin and M140I and F144L to myxothiazol + stigmatellin. In addition, a mutation (aer126) which specifically impairs the quinol oxidase (Qz) activity of the cyt bc1 complex of a non-photosynthetic mutant (R126) was identified to be a glycine to an aspartic acid replacement at position 158 of cyt b. Six of these mutations were found between amino acid residues 140 and 163, in a region linking the putative third and fourth transmembrane helices of cyt b. The non-random clustering of several inhibitor-resistance mutations around the non-functional aer126 mutation suggests that this region may be involved in the formation of the Qz inhibitor binding/quinol oxidation domain(s) of the cyt bc1 complex. Of the two remaining mutations, the V333A replacement conferred resistance to stigmatellin exclusively and was located in another region toward the C terminus of cyt b. The L106P substitution, on the other hand, was situated in the transmembrane helix II that carries two conserved histidine residues

  19. Identification of alpha interferon-induced envelope mutations of hepatitis C virus in vitro associated with increased viral fitness and interferon resistance.

    PubMed

    Serre, Stéphanie B N; Krarup, Henrik B; Bukh, Jens; Gottwein, Judith M

    2013-12-01

    Alpha interferon (IFN-α) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of IFN-α resistance in vitro. Long-term culture with IFN-α2b in Huh7.5 cells resulted in viral spread with acquisition of putative escape mutations in HCV structural and nonstructural proteins. Reverse genetic studies showed that primarily amino acid changes I348T in 1a(H77) E1 and F345V/V414A in 3a(S52) E1/E2 increased viral fitness. Single-cycle assays revealed that I348T and F345V/V414A enhanced viral entry and release, respectively. In assays allowing viral spread, these mutations conferred a level of IFN-α resistance exceeding the observed fitness effect. The identified mutations acted in a subtype-specific manner but were not found in genotype 1a and 3a patients, who failed IFN-α therapy. Studies with HCV recombinants with different degrees of culture adaptation confirmed the correlation between viral fitness and IFN-α resistance. In conclusion, in vitro escape experiments led to identification of HCV envelope mutations resulting in increased viral fitness and conferring IFN-α resistance. While we established a close link between viral fitness and IFN-α resistance, identified mutations acted via different mechanisms and appeared to be relatively specific to the infecting virus, possibly explaining difficulties in identifying signature mutations for IFN resistance. PMID:24049176

  20. Increased MUTYH mutation frequency among Dutch families with breast cancer and colorectal cancer.

    PubMed

    Wasielewski, Marijke; Out, Astrid A; Vermeulen, Joyce; Nielsen, Maartje; van den Ouweland, Ans; Tops, Carli M J; Wijnen, Juul T; Vasen, Hans F A; Weiss, Marjan M; Klijn, Jan G M; Devilee, Peter; Hes, Frederik J; Schutte, Mieke

    2010-12-01

    Homozygous and compound heterozygous MUTYH mutations predispose for MUTYH-associated polyposis (MAP). The clinical phenotype of MAP is characterised by the multiple colorectal adenomas and colorectal carcinoma. We previously found that female MAP patients may also have an increased risk for breast cancer. Yet, the involvement of MUTYH mutations in families with both breast cancer and colorectal cancer is unclear. Here, we have genotyped the MUTYH p.Tyr179Cys, p.Gly396Asp and p.Pro405Leu founder mutations in 153 Dutch families with breast cancer patients and colorectal cancer patients. Families were classified as polyposis, revised Amsterdam criteria positive (FCRC-AMS positive), revised Amsterdam criteria negative (FCRC-AMS negative), hereditary breast and colorectal cancer (HBCC) and non-HBCC breast cancer families. As anticipated, biallelic MUTYH mutations were identified among 13% of 15 polyposis families, which was significantly increased compared to the absence of biallelic MUTYH mutations in the population (P = 0.0001). Importantly, six heterozygous MUTYH mutations were identified among non-polyposis families with breast and colorectal cancer. These mutations were identified specifically in FCRC-AMS negative and in HBCC breast cancer families (11% of 28 families and 4% of 74 families, respectively; P = 0.02 for both groups combined vs. controls). Importantly, the 11% MUTYH frequency among FCRC-AMS negative families was almost fivefold higher than the reported frequencies for FCRC-AMS negative families unselected for the presence of breast cancer patients (P = 0.03). Together, our results indicate that heterozygous MUTYH mutations are associated with families that include both breast cancer patients and colorectal cancer patients, independent of which tumour type is more prevalent in the family. PMID:20191381

  1. Germline mutations in RAD51D confer susceptibility to ovarian cancer.

    PubMed

    Loveday, Chey; Turnbull, Clare; Ramsay, Emma; Hughes, Deborah; Ruark, Elise; Frankum, Jessica R; Bowden, Georgina; Kalmyrzaev, Bolot; Warren-Perry, Margaret; Snape, Katie; Adlard, Julian W; Barwell, Julian; Berg, Jonathan; Brady, Angela F; Brewer, Carole; Brice, Glen; Chapman, Cyril; Cook, Jackie; Davidson, Rosemarie; Donaldson, Alan; Douglas, Fiona; Greenhalgh, Lynn; Henderson, Alex; Izatt, Louise; Kumar, Ajith; Lalloo, Fiona; Miedzybrodzka, Zosia; Morrison, Patrick J; Paterson, Joan; Porteous, Mary; Rogers, Mark T; Shanley, Susan; Walker, Lisa; Eccles, Diana; Evans, D Gareth; Renwick, Anthony; Seal, Sheila; Lord, Christopher J; Ashworth, Alan; Reis-Filho, Jorge S; Antoniou, Antonis C; Rahman, Nazneen

    2011-09-01

    Recently, RAD51C mutations were identified in families with breast and ovarian cancer. This observation prompted us to investigate the role of RAD51D in cancer susceptibility. We identified eight inactivating RAD51D mutations in unrelated individuals from 911 breast-ovarian cancer families compared with one inactivating mutation identified in 1,060 controls (P = 0.01). The association found here was principally with ovarian cancer, with three mutations identified in the 59 pedigrees with three or more individuals with ovarian cancer (P = 0.0005). The relative risk of ovarian cancer for RAD51D mutation carriers was estimated to be 6.30 (95% CI 2.86-13.85, P = 4.8 × 10(-6)). By contrast, we estimated the relative risk of breast cancer to be 1.32 (95% CI 0.59-2.96, P = 0.50). These data indicate that RAD51D mutation testing may have clinical utility in individuals with ovarian cancer and their families. Moreover, we show that cells deficient in RAD51D are sensitive to treatment with a PARP inhibitor, suggesting a possible therapeutic approach for cancers arising in RAD51D mutation carriers. PMID:21822267

  2. 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.

  3. Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

    PubMed Central

    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. PMID:23285292

  4. The E46K mutation in alpha-synuclein increases amyloid fibril formation.

    PubMed

    Greenbaum, Eric A; Graves, Charles L; Mishizen-Eberz, Amanda J; Lupoli, Michael A; Lynch, David R; Englander, S Walter; Axelsen, Paul H; Giasson, Benoit I

    2005-03-01

    The identification of a novel mutation (E46K) in one of the KTKEGV-type repeats in the amino-terminal region of alpha-synuclein suggests that this region and, more specifically, Glu residues in the repeats may be important in regulating the ability of alpha-synuclein to polymerize into amyloid fibrils. It was demonstrated that the E46K mutation increased the propensity of alpha-synuclein to fibrillize, but this effect was less than that of the A53T mutation. The substitution of Glu(46) for an Ala also increased the assembly of alpha-synuclein, but the polymers formed can have different ultrastructures, further indicating that this amino acid position has a significant effect on the assembly process. The effect of residue Glu(83) in the sixth repeat of alpha-synuclein, which lies closest to the amino acid stretch critical for filament assembly, was also studied. Mutation of Glu(83) to a Lys or Ala increased polymerization but perturbed some of the properties of mature amyloid. These results demonstrated that some of the Glu residues within the repeats can have significant effects on modulating the assembly of alpha-synuclein to form amyloid fibrils. The greater effect of the A53T mutation, even when compared with what may be predicted to be a more dramatic mutation such as E46K, underscores the importance of protein microenvironment in affecting protein structure. Moreover, the relative effects of the A53T and E46K mutations are consistent with the age of onset of disease. These findings support the notion that aberrant alpha-synuclein polymerization resulting in the formation of pathological inclusions can lead to disease. PMID:15632170

  5. Presence of multiple recurrent mutations confers poor trial outcome of relapsed/refractory CLL.

    PubMed

    Guièze, Romain; Robbe, Pauline; Clifford, Ruth; de Guibert, Sophie; Pereira, Bruno; Timbs, Adele; Dilhuydy, Marie-Sarah; Cabes, Maite; Ysebaert, Loïc; Burns, Adam; Nguyen-Khac, Florence; Davi, Frédéric; Véronèse, Lauren; Combes, Patricia; Le Garff-Tavernier, Magali; Leblond, Véronique; Merle-Béral, Hélène; Alsolami, Reem; Hamblin, Angela; Mason, Joanne; Pettitt, Andrew; Hillmen, Peter; Taylor, Jenny; Knight, Samantha J L; Tournilhac, Olivier; Schuh, Anna

    2015-10-29

    Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥ 2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥ 2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome. PMID:26316624

  6. Selection of mutations in the connection and RNase H domains of human immunodeficiency virus type 1 reverse transcriptase that increase resistance to 3'-azido-3'-dideoxythymidine.

    PubMed

    Brehm, Jessica H; Koontz, Dianna; Meteer, Jeffrey D; Pathak, Vinay; Sluis-Cremer, Nicolas; Mellors, John W

    2007-08-01

    Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3'-azido-3'-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1(LAI) or AZT-resistant HIV-1(LAI) (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1(LAI) were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance ( approximately 16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance approximately 10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain. PMID:17507476

  7. 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

    2014-09-01

    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. PMID:25132497

  8. 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

  9. Molecular and phenotypic characterization of Als1 and Als2 mutations conferring tolerance to acetolactate synthase herbicides in soybean

    PubMed Central

    Walter, Kay L; Strachan, Stephen D; Ferry, Nancy M; Albert, Henrik H; Castle, Linda A; Sebastian, Scott A

    2014-01-01

    BACKGROUND Sulfonylurea (SU) herbicides are effective because they inhibit acetolactate synthase (ALS), a key enzyme in branched-chain amino acid synthesis required for plant growth. A soybean line known as W4-4 was developed through rounds of seed mutagenesis and was demonstrated to have a high degree of ALS-based resistance to both post-emergence and pre-emergence applications of a variety of SU herbicides. This report describes the molecular and phenotypic characterization of the Als1 and Als2 mutations that confer herbicide resistance to SUs and other ALS inhibitors. RESULTS The mutations are shown to occur in two different ALS genes that reside on different chromosomes: Als1 (P178S) on chromosome 4 and Als2 (W560L) on chromosome 6 (P197S and W574L in Arabidopsis thaliana). CONCLUSION Although the Als1 and Als2 genes are unlinked, the combination of these two mutations is synergistic for improved tolerance of soybeans to ALS-inhibiting herbicides. © 2014 DuPont Pioneer. Pest Management Science published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:24425499

  10. 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

  11. 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

    2016-01-01

    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

  12. Mitochondrial mutations contribute to HIF1α accumulation via increased reactive oxygen species and upregulated PDK2 in head and neck squamous cell carcinoma

    PubMed Central

    Sun, Wenyue; Zhou, Shaoyu; Chang, Steven S.; McFate, Thomas; Verma, Ajay; Califano, Joseph A.

    2008-01-01

    Purpose Mitochondrial mutations have been identified in head and neck squamous cell carcinoma (HNSCC), but the pathways by which phenotypic effects of these mutations are exerted remain unclear. Previously, we found that mitochondrial ND2 mutations in primary HNSCC increased reactive oxygen species (ROS) and conferred an aerobic, glycolytic phenotype with HIF1α accumulation and increased cell growth. The purpose of present study was to examine the pathways relating these alterations. Experimental Design Mitochondrial mutant and wild-type ND2 constructs were transfected into oral keratinocyte immortal cell line OKF6 and head and neck cancer cell line JHU-O19 and established transfectants. The protein levels of HIF1α, pyruvate dehydrogenease (PDH), phospho-PDH, and pyruvate dehydrogenease kinase (PDK) 2, together with ROS generation, were compared between the mutant and wild type. Meanwhile, the effects of small molecule inhibitors targeting PDK2, and mitochondrial targeted catalase, were evaluated on the ND2 mutant transfectants. Results We determined that ND2 mutant downregulated PDH expression via upregulated PDK2, with an increase in phospho-PDH. Inhibition of PDK2 with dichloroacetate decreased HIF1α accumulation and reduced cell growth. Extracellular treatment with hydrogen peroxide, a ROS mimic, increased PDK2 expression and HIF1α expression, and introduction of mitochondrial targeted catalase decreased mitochondrial mutation mediated PDK2 and HIF1α expression and suppressed cell growth. Conclusions Our findings suggest that mitochondrial ND2 mutation contributes to HIF1α accumulation via increased ROS production, upregulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in HIF1α stabilization. This may provide insight into a potential mechanism by which mitochondrial mutations contribute to HNSCC development. PMID:19147752

  13. 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

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

    PubMed

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

    2014-02-15

    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 Ca(2+) 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 Ca(2+)-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 Ca(2+) channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression. PMID:24277868

  15. Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin.

    PubMed

    Wienert, Beeke; Funnell, Alister P W; Norton, Laura J; Pearson, Richard C M; Wilkinson-White, Lorna E; Lester, Krystal; Vadolas, Jim; Porteus, Matthew H; Matthews, Jacqueline M; Quinlan, Kate G R; Crossley, Merlin

    2015-01-01

    Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal γ-globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal γ-globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified γ-globin promoter. PMID:25971621

  16. Rare mutations in XRCC2 increase the risk of breast cancer.

    PubMed

    Park, D J; Lesueur, F; Nguyen-Dumont, T; Pertesi, M; Odefrey, F; Hammet, F; Neuhausen, S L; John, E M; Andrulis, I L; Terry, M B; Daly, M; Buys, S; Le Calvez-Kelm, F; Lonie, A; Pope, B J; Tsimiklis, H; Voegele, C; Hilbers, F M; Hoogerbrugge, N; Barroso, A; Osorio, A; Giles, G G; Devilee, P; Benitez, J; Hopper, J L; Tavtigian, S V; Goldgar, D E; Southey, M C

    2012-04-01

    An exome-sequencing study of families with multiple breast-cancer-affected individuals identified two families with XRCC2 mutations, one with a protein-truncating mutation and one with a probably deleterious missense mutation. We performed a population-based case-control mutation-screening study that identified six probably pathogenic coding variants in 1,308 cases with early-onset breast cancer and no variants in 1,120 controls (the severity grading was p < 0.02). We also performed additional mutation screening in 689 multiple-case families. We identified ten breast-cancer-affected families with protein-truncating or probably deleterious rare missense variants in XRCC2. Our identification of XRCC2 as a breast cancer susceptibility gene thus increases the proportion of breast cancers that are associated with homologous recombination-DNA-repair dysfunction and Fanconi anemia and could therefore benefit from specific targeted treatments such as PARP (poly ADP ribose polymerase) inhibitors. This study demonstrates the power of massively parallel sequencing for discovering susceptibility genes for common, complex diseases. PMID:22464251

  17. Rare Mutations in XRCC2 Increase the Risk of Breast Cancer

    PubMed Central

    Park, D.J.; Lesueur, F.; Nguyen-Dumont, T.; Pertesi, M.; Odefrey, F.; Hammet, F.; Neuhausen, S.L.; John, E.M.; Andrulis, I.L.; Terry, M.B.; Daly, M.; Buys, S.; Le Calvez-Kelm, F.; Lonie, A.; Pope, B.J.; Tsimiklis, H.; Voegele, C.; Hilbers, F.M.; Hoogerbrugge, N.; Barroso, A.; Osorio, A.; Giles, G.G.; Devilee, P.; Benitez, J.; Hopper, J.L.; Tavtigian, S.V.; Goldgar, D.E.; Southey, M.C.

    2012-01-01

    An exome-sequencing study of families with multiple breast-cancer-affected individuals identified two families with XRCC2 mutations, one with a protein-truncating mutation and one with a probably deleterious missense mutation. We performed a population-based case-control mutation-screening study that identified six probably pathogenic coding variants in 1,308 cases with early-onset breast cancer and no variants in 1,120 controls (the severity grading was p < 0.02). We also performed additional mutation screening in 689 multiple-case families. We identified ten breast-cancer-affected families with protein-truncating or probably deleterious rare missense variants in XRCC2. Our identification of XRCC2 as a breast cancer susceptibility gene thus increases the proportion of breast cancers that are associated with homologous recombination-DNA-repair dysfunction and Fanconi anemia and could therefore benefit from specific targeted treatments such as PARP (poly ADP ribose polymerase) inhibitors. This study demonstrates the power of massively parallel sequencing for discovering susceptibility genes for common, complex diseases. PMID:22464251

  18. Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation

    PubMed Central

    Chou, Hsin-Hung; Berthet, Julia; Marx, Christopher J.

    2009-01-01

    Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B12, a cobalt-containing cofactor, to sustain two vitamin B12–dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate–dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences. PMID:19763169

  19. C239S Mutation in the β-Tubulin of Phytophthora sojae Confers Resistance to Zoxamide.

    PubMed

    Cai, Meng; Miao, Jianqiang; Song, Xi; Lin, Dong; Bi, Yang; Chen, Lei; Liu, Xili; Tyler, Brett M

    2016-01-01

    Zoxamide is the sole β-tubulin inhibitor registered for the control of oomycete pathogens. The current study investigated the activity of zoxamide against Phytophthora sojae and baseline sensitivity was established with a mean EC50 of 0.048 μg/ml. The data is critical for monitoring changes in zoxamide-sensitivity in the field. Three stable resistant mutants with a high resistance level were obtained by selection on zoxamide amended media. Although the development of resistance occurred at a low frequency, there were no apparent fitness penalty in the acquired mutants in terms of growth rate, sporulation, germination and pathogenicity. Based on the biological profiles and low mutagenesis rate, the resistance risk of P. sojae to zoxamide can be estimated as low to medium. Further investigation revealed all the zoxamide-resistant mutants had a point mutation of C239S in their β-tubulin. Zoxamide also exhibited high activity against most species from the genus Pythium in which only Pythium aphanidermatum was found naturally resistant to zoxamide and harboring the natural point mutation S239 in the β-tubulin. Back-transformation in P. sojae with the mutated allele (S239) confirmed the C239S mutation can induce resistance to zoxamide, and the resistance level was positively related to the expression level of the mutated gene. In contrast, the overexpression of the wild type gene was unable to cause zoxamide resistance. It is the first report on the resistance molecular mechanism of zoxamide in oomycetes. Based on our study, C239 is supposed to be a key target site of zoxamide, which distinguishes zoxamide from benzimidazoles and accounts for its low resistance risk. The result can provide advice on the design of new β-tubulin inhibitors in future. PMID:27242773

  20. 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. PMID:27073078

  1. C239S Mutation in the β-Tubulin of Phytophthora sojae Confers Resistance to Zoxamide

    PubMed Central

    Cai, Meng; Miao, Jianqiang; Song, Xi; Lin, Dong; Bi, Yang; Chen, Lei; Liu, Xili; Tyler, Brett M.

    2016-01-01

    Zoxamide is the sole β-tubulin inhibitor registered for the control of oomycete pathogens. The current study investigated the activity of zoxamide against Phytophthora sojae and baseline sensitivity was established with a mean EC50 of 0.048 μg/ml. The data is critical for monitoring changes in zoxamide-sensitivity in the field. Three stable resistant mutants with a high resistance level were obtained by selection on zoxamide amended media. Although the development of resistance occurred at a low frequency, there were no apparent fitness penalty in the acquired mutants in terms of growth rate, sporulation, germination and pathogenicity. Based on the biological profiles and low mutagenesis rate, the resistance risk of P. sojae to zoxamide can be estimated as low to medium. Further investigation revealed all the zoxamide-resistant mutants had a point mutation of C239S in their β-tubulin. Zoxamide also exhibited high activity against most species from the genus Pythium in which only Pythium aphanidermatum was found naturally resistant to zoxamide and harboring the natural point mutation S239 in the β-tubulin. Back-transformation in P. sojae with the mutated allele (S239) confirmed the C239S mutation can induce resistance to zoxamide, and the resistance level was positively related to the expression level of the mutated gene. In contrast, the overexpression of the wild type gene was unable to cause zoxamide resistance. It is the first report on the resistance molecular mechanism of zoxamide in oomycetes. Based on our study, C239 is supposed to be a key target site of zoxamide, which distinguishes zoxamide from benzimidazoles and accounts for its low resistance risk. The result can provide advice on the design of new β-tubulin inhibitors in future. PMID:27242773

  2. A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B☆

    PubMed Central

    Robinson, Jerid W.; Dickey, Deborah M.; Miura, Kohji; Michigami, Toshimi; Ozono, Keiichi; Potter, Lincoln R.

    2015-01-01

    C-type natriuretic peptide (CNP) increases long bone growth by stimulating guanylyl cyclase (GC)-B/NPR-B/NPR2. Recently, a Val to Met missense mutation at position 883 in the catalytic domain of GC-B was identified in humans with increased blood cGMP levels that cause abnormally long bones. Here, we determined how this mutation activates GC-B. In the absence of CNP, cGMP levels in cells expressing V883M-GC-B were increased more than 20 fold compared to cells expressing wild-type (WT)-GC-B, and the addition of CNP only further increased cGMP levels 2-fold. In the absence of CNP, maximal enzymatic activity (Vmax) of V883M-GC-B was increased 15-fold compared to WT-GC-B but the affinity of the enzymes for substrate as revealed by the Michaelis constant (Km) was unaffected. Surprisingly, CNP decreased the Km of V883M-GC-B 10-fold in a concentration dependent manner without increasing Vmax. Unlike the WT enzyme the Km reduction of V883M-GC-B did not require ATP. Unexpectedly, V883M-GC-B, but not WT-GC-B, failed to inactivate with time. Phosphorylation elevated but was not required for the activity increase associated with the mutation because the Val to Met substitution also activated a GC-B mutant lacking all known phosphorylation sites. We conclude that the V883M mutation increases maximal velocity in the absence of CNP, eliminates the requirement for ATP in the CNP-dependent Km reduction, and disrupts the normal inactivation process. PMID:23827346

  3. Loss-of-Function Mutations in CsMLO1 Confer Durable Powdery Mildew Resistance in Cucumber (Cucumis sativus L.)

    PubMed Central

    Nie, Jingtao; Wang, Yunli; He, Huanle; Guo, Chunli; Zhu, Wenying; Pan, Jian; Li, Dandan; Lian, Hongli; Pan, Junsong; Cai, Run

    2015-01-01

    Powdery mildew (PM) is a serious fungal disease of cucumber worldwide. The identification of resistance genes is very important for resistance breeding to ensure cucumber production. Here, natural loss-of-function mutations at an MLO homologous locus, CsMLO1, were found to confer durable PM resistance in cucumber. CsMLO1 encoded a cell membrane protein, was mainly expressed in leaves and cotyledons, and was up-regulated by PM at the early stage of host–pathogen interaction. Ectopic expression of CsMLO1 rescued the phenotype of the PM resistant Atmlo2 Atmlo12 double mutant to PM susceptible in Arabidopsis. Domesticated and wild resistant cucumbers originating from various geographical regions of the world were found to harbor three independent natural mutations that resulted in CsMLO1 loss of function. In addition, between the near-isogenic lines (NILs) of PM resistant and susceptible, S1003 and NIL(Pm5.1), quantitative RT-PCR revealed that there is no difference at expression levels of several genes in the pathways of ethylene, jasmonic acid or salicylic acid. Moreover, the two NILs were used for transcriptome profiling to explore the mechanism underlying the resistance. Several genes correlated with plant cell wall thickening are possibly involved in the PM resistance. This study revealed that loss of function of CsMLO1 conferred durable PM resistance, and that this loss of function is necessary but alone may not be sufficient for PM resistance in cucumber. These findings will facilitate the molecular breeding of PM resistant varieties to control this destructive disease in cucumber. PMID:26734050

  4. 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

  5. Truncating mutation in the autophagy gene UVRAG confers oncogenic properties and chemosensitivity in colorectal cancers

    PubMed Central

    He, Shanshan; Zhao, Zhen; Yang, Yongfei; O'Connell, Douglas; Zhang, Xiaowei; Oh, Soohwan; Ma, Binyun; Lee, Joo-Hyung; Zhang, Tian; Varghese, Bino; Yip, Janae; Dolatshahi Pirooz, Sara; Li, Ming; Zhang, Yong; Li, Guo-Min; Ellen Martin, Sue; Machida, Keigo; Liang, Chengyu

    2015-01-01

    Autophagy-related factors are implicated in metabolic adaptation and cancer metastasis. However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive. Recent studies have shown that UVRAG, a key autophagic tumour suppressor, is mutated in common human cancers. Here we demonstrate that the cancer-related UVRAG frameshift (FS), which does not result in a null mutation, is expressed as a truncated UVRAGFS in colorectal cancer (CRC) with microsatellite instability (MSI), and promotes tumorigenesis. UVRAGFS abrogates the normal functions of UVRAG, including autophagy, in a dominant-negative manner. Furthermore, expression of UVRAGFS can trigger CRC metastatic spread through Rac1 activation and epithelial-to-mesenchymal transition, independently of autophagy. Interestingly, UVRAGFS expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect. These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response. PMID:26234763

  6. 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. PMID:25870226

  7. Mutation Conferring Apical-Targeting Motif on AE1 Exchanger Causes Autosomal Dominant Distal RTA

    PubMed Central

    Fry, Andrew C.; Su, Ya; Yiu, Vivian; Cuthbert, Alan W.; Trachtman, Howard

    2012-01-01

    Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the α-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1. PMID:22518001

  8. Mutation conferring apical-targeting motif on AE1 exchanger causes autosomal dominant distal RTA.

    PubMed

    Fry, Andrew C; Su, Ya; Yiu, Vivian; Cuthbert, Alan W; Trachtman, Howard; Karet Frankl, Fiona E

    2012-07-01

    Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the α-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1. PMID:22518001

  9. 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. PMID:25398256

  10. A chimeric vacuolar Na(+)/H(+) antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast.

    PubMed

    Wu, Guangxia; Wang, Gang; Ji, Jing; Li, Yong; Gao, Hailing; Wu, Jiang; Guan, Wenzhu

    2015-06-10

    The vacuolar Na(+)/H(+) antiporter plays an important role in maintaining ionic homeostasis and the osmotic balance of the cell with the environment by sequestering excessive cytoplasmic Na(+) into the vacuole. However, the relatively low Na(+)/H(+) exchange efficiency of the identified Na(+)/H(+) antiporter could limit its application in the molecular breeding of salt tolerant crops. In this study, DNA family shuffling was used to create chimeric Na(+)/H(+) antiporters with improved transport activity. Two homologous Na(+)/H(+) antiporters from halophytes Salicornia europaea (SeNHX1) and Suaeda salsa (SsNHX1) were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected. Expression of SseNHX1 in yeast mutant exhibited approximately 46% and 22% higher salt tolerance ability in yeast growth test than that of SsNHX1and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 protein in yeast cells accumulated more Na(+) and slightly more K(+) than the parental proteins did. Furthermore, this chimera also conferred increased tolerance to LiCl and a similar tolerance to hygromycin B compared with the parental proteins in yeast. PMID:25784157

  11. 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

  12. Mutations in ARS1 increase the rate of simple loss of plasmids in Saccharomyces cerevisiae.

    PubMed

    Strich, R; Woontner, M; Scott, J F

    1986-09-01

    Autonomously replicating sequence (ARS) elements are DNA sequences that promote extrachromosomal maintenance of plasmids in yeast. Mutations generated in vitro in the ARS1 region were examined for their effect on plasmid maintenance in a yeast centromeric plasmid. Our data show that mutations in the regions surrounding the ARS1 consensus sequence cause increases in the frequency of simple loss (1:0) events without affecting the rate of nondisjunction (2:0). Removal of the consensus sequence itself causes a drastic increase in the rate of simple loss. Sequences sensitive to mutagenesis were identified in each flanking region and differ with respect to their location and importance to ARS function. These results suggest that the role ARS1 plays in plasmid maintenance deals with the replication and/or localization of the plasmid in yeast. PMID:3333306

  13. Plasmodium falciparum Isolates in India Exhibit a Progressive Increase in Mutations Associated with Sulfadoxine-Pyrimethamine Resistance

    PubMed Central

    Ahmed, Anwar; Bararia, Deepak; Vinayak, Sumiti; Yameen, Mohammed; Biswas, Sukla; Dev, Vas; Kumar, Ashwani; Ansari, Musharraf A.; Sharma, Yagya D.

    2004-01-01

    The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant Plasmodium falciparum malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of P. falciparum collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double dhfr mutations and single dhps mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing. PMID:14982779

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

    PubMed

    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

    2014-10-21

    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

  15. 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

  16. 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

  17. Increased Susceptibility to DNA Virus Infection in Mice with a GCN2 Mutation

    PubMed Central

    Won, Sungyong; Eidenschenk, Celine; Arnold, Carrie N.; Siggs, Owen M.; Sun, Lei; Brandl, Katharina; Mullen, Tina-Marie; Nemerow, Glen R.; Moresco, Eva Marie Y.

    2012-01-01

    The downregulation of translation through eIF2α phosphorylation is a cellular response to diverse stresses, including viral infection, and is mediated by the GCN2 kinase, protein kinase R (PKR), protein kinase-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase (HRI). Although PKR plays a major role in defense against viruses, other eIF2α kinases also may respond to viral infection and contribute to the shutdown of protein synthesis. Here we describe the recessive, loss-of-function mutation atchoum (atc) in Eif2ak4, encoding GCN2, which increased susceptibility to infection by the double-stranded DNA viruses mouse cytomegalovirus (MCMV) and human adenovirus. This mutation was identified by screening macrophages isolated from mice carrying N-ethyl-N-nitrosourea (ENU)-induced mutations. Cells from Eif2ak4atc/atc mice failed to phosphorylate eIF2α in response to MCMV. Importantly, homozygous Eif2ak4atc mice showed a modest increase in susceptibility to MCMV infection, demonstrating that translational arrest dependent on GCN2 contributes to the antiviral response in vivo. PMID:22114338

  18. Heightened BTK-dependent cell proliferation in unmutated chronic lymphocytic leukemia confers increased sensitivity to ibrutinib

    PubMed Central

    Galanina, Natalie; Nabhan, Chadi; Smith, Sonali M.; Coleman, Morton; Wang, Y. Lynn

    2016-01-01

    In chronic lymphocytic leukemia (CLL), patients with unmutated immunoglobulin heavy chain variable region gene (UM-CLL) have worse outcomes than mutated CLL (M-CLL) following chemotherapy or chemoimmunotherapy. However, in the era of BCR-targeted therapies, the adverse prognostic impact of unmutated IGHV seems to be diminishing, and there are clinical datasets showing unexpected improved responses in UM-CLL. We investigated the biological differences of BTK activity between these subgroups and further compared the impact of ibrutinib on molecular and cellular behaviors. Immunoblotting analysis revealed that phosphorylated active BTK is significantly higher in UM-CLL. Moreover, UM-CLL, compared to M-CLL, displayed a much higher proliferative capacity that was correlated with higher phospho-BTK and greater sensitivity to ibrutinib. In addition, BTK depletion with siRNA led to a more prominent reduction in the proliferation of UM-CLL, suggesting that elevated BTK activity is responsible for increased cell proliferation. Further, cell signaling activity by multiple measurements was consistently higher in UM-CLL accompanied by a higher sensitivity to ibrutinib. These studies link UM-CLL to elevated BCR signaling, heightened BTK-dependent cell proliferation and increased sensitivity to ibrutinib. The prognostic significance of IGHV mutation should be reevaluated in the era of new therapies targeting BCR signaling. PMID:26717038

  19. 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

  20. 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

  1. 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. PMID:26872031

  2. Isoniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase, peroxidase, and INH-NADH adduct formation activities

    PubMed Central

    Cade, Christine E; Dlouhy, Adrienne C; Medzihradszky, Katalin F; Salas-Castillo, Saida Patricia; Ghiladi, Reza A

    2010-01-01

    Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD+/NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-Tyr-Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t-BuOOH and H2O2), superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct. PMID:20054829

  3. 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

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

    PubMed

    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

    2015-11-01

    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

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

    PubMed

    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

  6. 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

  7. Mutations in the herpes simplex virus DNA polymerase gene can confer resistance to 9-beta-D-arabinofuranosyladenine.

    PubMed Central

    Coen, D M; Furman, P A; Gelep, P T; Schaffer, P A

    1982-01-01

    Mutants of herpes simplex virus type 1 resistant to the antiviral drug 9-beta-D-arabinofuranosyladenine (araA) have been isolated and characterized. AraA-resistant mutants can be isolated readily and appear at an appreciable frequency in low-passage stocks of wild-type virus. Of 13 newly isolated mutants, at least 11 were also resistant to phosphonoacetic acid (PAA). Of four previously described PAA-resistant mutants, two exhibited substantial araA resistance. The araA resistance phenotype of one of these mutants, PAAr5, has been mapped to the HpaI-B fragment of herpes simplex virus DNA by marker transfer, and araA resistance behaved in marker transfer experiments as if it were closely linked to PAA resistance, a recognized marker for the viral DNA polymerase locus. PAAr5 induced viral DNA polymerase activity which was much less susceptible to inhibition by the triphosphate derivative of araA than was wild-type DNA polymerase. These genetic and biochemical data indicate that the herpes simplex virus DNA polymerase gene is a locus which, when mutated, can confer resistance to araA and thus that the herpes simplex virus DNA polymerase is a target for this antiviral drug. PMID:6284981

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

    PubMed

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

    2016-05-01

    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 elongates 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 ofS. 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

  9. M233I Mutation in the β-Tubulin of Botrytis cinerea Confers Resistance to Zoxamide

    PubMed Central

    Cai, Meng; Lin, Dong; Chen, Lei; Bi, Yang; Xiao, Lu; Liu, Xi-li

    2015-01-01

    Three phenotypes were detected in 161 Botrytis cinerea field isolates, including ZoxSCarS (sensitive to zoxamide and carbendazim), ZoxSCarR (sensitive to zoxamide and resistant to carbendazim), and ZoxRCarR (resistant to zoxamide and carbendazim), but not ZoxRCarS (resistant to zoxamide and sensitive to carbendazim). The baseline sensitivity to zoxamide was determined with a mean EC50 of 0.76 μg/ml. Two stable ZoxRCarS isolates were obtained with a resistance factor of 13.28 and 20.43; there was a fitness penalty in mycelial growth rate, sporulation, virulence and sclerotium production. The results suggest that the resistance risk of B. cinerea to zoxamide is low where benzimidazoles have not been used. E198V, E198K and M233I, were detected in the β-tubulin of ZoxSCarR, ZoxRCarR, ZoxRCarS, respectively. Molecular docking indicated that position 198 in β-tubulin were targets for both zoxamide and carbendazim. The mutations at 198 prevented formation of hydrogen bonds between β-tubulin and carbendazim (E198V/K), and changed the conformation of the binding pocket of zoxamide (E198K). M233I had no effect on the binding of carbendazim but resulted in loss of a hydrogen bond between zoxamide and F200. M233 is suggested to be a unique target site for zoxamide and be very important in the function of β tubulin. PMID:26596626

  10. GBA mutations increase risk for Lewy body disease with and without Alzheimer disease pathology

    PubMed Central

    Leverenz, James B.; Lopez, Oscar L.; Hamilton, Ronald L.; Bennett, David A.; Schneider, Julie A.; Buchman, Aron S.; Larson, Eric B.; Crane, Paul K.; Kaye, Jeffrey A.; Kramer, Patricia; Woltjer, Randy; Kukull, Walter; Nelson, Peter T.; Jicha, Gregory A.; Neltner, Janna H.; Galasko, Doug; Masliah, Eliezer; Trojanowski, John Q.; Schellenberg, Gerard D.; Yearout, Dora; Huston, Haley; Fritts-Penniman, Allison; Mata, Ignacio F.; Wan, Jia Y.; Edwards, Karen L.; Montine, Thomas J.

    2012-01-01

    Objectives: Mutations in the GBA gene occur in 7% of patients with Parkinson disease (PD) and are a well-established susceptibility factor for PD, which is characterized by Lewy body disease (LBD) neuropathologic changes (LBDNCs). We sought to determine whether GBA influences risk of dementia with LBDNCs, Alzheimer disease (AD) neuropathologic changes (ADNCs), or both. Methods: We screened the entire GBA coding region for mutations in controls and in subjects with dementia and LBDNCs and no or low levels of ADNCs (pure dementia with Lewy bodies [pDLB]), LBDNCs and high-level ADNCs (LBD-AD), and high-level ADNCs but without LBDNCs (AD). Results: Among white subjects, pathogenic GBA mutations were identified in 6 of 79 pDLB cases (7.6%), 8 of 222 LBD-AD cases (3.6%), 2 of 243 AD cases (0.8%), and 3 of 381 controls (0.8%). Subjects with pDLB and LBD-AD were more likely to carry mutations than controls (pDLB: odds ratio [OR] = 7.6; 95% confidence interval [CI] = 1.8–31.9; p = 0.006; LBD-AD: OR = 4.6; CI = 1.2–17.6; p = 0.025), but there was no significant difference in frequencies between the AD and control groups (OR = 1.1; CI = 0.2–6.6; p = 0.92). There was a highly significant trend test across groups (χ2(1) = 19.3; p = 1.1 × 10−5), with the likelihood of carrying a GBA mutation increasing in the following direction: control/AD < LBD-AD < pDLB. Conclusions: GBA is a susceptibility gene across the LBD spectrum, but not in AD, and appears to convey a higher risk for PD and pDLB than for LBD-AD. PD and pDLB might be more similar to one another in genetic determinants and pathophysiology than either disease is to LBD-AD. PMID:23035075

  11. Increased Incidence of Mitochondrial Cytochrome C Oxidase 1 Gene Mutations in Patients with Primary Ovarian Insufficiency

    PubMed Central

    Zhen, Xiumei; Wu, Bailin; Wang, Jian; Lu, Cuiling; Gao, Huafang; Qiao, Jie

    2015-01-01

    Primary ovarian insufficiency (POI), also known as premature ovarian failure (POF), is defined as more than six months of cessation of menses before the age of 40 years, with two serum follicle stimulating hormone (FSH) levels (at least 1 month apart) falling in the menopause range. The cause of POI remains undetermined in the majority of cases, although some studies have reported increased levels of reactive oxygen species (ROS) in idiopathic POF. The role of mitochondrial DNA in the pathogenesis of POI has not been studied extensively. This aim of this study was to uncover underlying mitochondrial genetic defects in patients with POI. The entire region of the mitochondrial genome was amplified in subjects with idiopathic POI (n=63) and age-matched healthy female controls (n=63) using nine pair sets of primers, followed by screening of the mitochondrial genome using an Illumina MiSeq. We identified a total of 96 non-synonymous mitochondrial variations in POI patients and 93 non-synonymous variations in control subjects. Of these, 21 (9 in POI and 12 in control) non-synonymous variations had not been reported previously. Eight mitochondrial cytochrome coxidase 1 (MT-CO1) missense variants were identified in POI patients, whereas only four missense mutations were observed in controls. A high incidence of MT-CO1 missense variants were identified in POI patients compared with controls, and the difference between the groups was statistically significant (13/63 vs. 5/63, p=0.042). Our results show that patients with primary ovarian insufficiency exhibit an increased incidence of mitochondrial cytochrome c oxidase 1 gene mutations, suggesting that MT-CO1 gene mutation may be causal in POI. PMID:26225554

  12. Organic Cation Transporter 2 Overexpression May Confer an Increased Risk of Gentamicin-Induced Nephrotoxicity.

    PubMed

    Gai, Zhibo; Visentin, Michele; Hiller, Christian; Krajnc, Evelin; Li, Tongzhou; Zhen, Junhui; Kullak-Ublick, Gerd A

    2016-09-01

    Nephrotoxicity is a relevant limitation of gentamicin, and obese patients have an increased risk for gentamicin-induced kidney injury. This damage is thought to depend on the accumulation of the drug in the renal cortex. Obese rats showed substantially higher levels of gentamicin in the kidney than did lean animals. This study characterized the role of organic cation transporters (OCTs) in gentamicin transport and elucidated their possible contribution in the increased renal accumulation of gentamicin in obesity. The mRNA and protein expression levels of the organic cation transporters Oct2 (Slc22a2) and Oct3 (Slc22a3) were increased in kidney samples from obese mice fed a high-fat diet. Similarly, OCT2 (∼2-fold) and OCT3 (∼3-fold) showed increased protein expression in the kidneys of obese patients compared with those of nonobese individuals. Using HEK293 cells overexpressing the different OCTs, human OCT2 was found to transport [(3)H]gentamicin with unique sigmoidal kinetics typical of homotropic positive cooperativity (autoactivation). In mouse primary proximal tubular cells, [(3)H]gentamicin uptake was reduced by approximately 40% when the cells were coincubated with the OCT2 substrate metformin. The basolateral localization of OCT2 suggests that gentamicin can enter proximal tubular cells from the blood side, probably as part of a slow tubular secretion process that may influence intracellular drug concentrations and exposure time. Increased expression of OCT2 may explain the higher accumulation of gentamicin, thereby conferring an increased risk of renal toxicity in obese patients. PMID:27401566

  13. ALDH2(E487K) mutation increases protein turnover and promotes murine hepatocarcinogenesis.

    PubMed

    Jin, Shengfang; Chen, Jiang; Chen, Lizao; Histen, Gavin; Lin, Zhizhong; Gross, Stefan; Hixon, Jeffrey; Chen, Yue; Kung, Charles; Chen, Yiwei; Fu, Yufei; Lu, Yuxuan; Lin, Hui; Cai, Xiujun; Yang, Hua; Cairns, Rob A; Dorsch, Marion; Su, Shinsan M; Biller, Scott; Mak, Tak W; Cang, Yong

    2015-07-21

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated. PMID:26150517

  14. 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

  15. Improved survival of very high light and oxidative stress is conferred by spontaneous gain-of-function mutations in Chlamydomonas.

    PubMed

    Förster, Britta; Osmond, C Barry; Pogson, Barry J

    2005-08-15

    Investigations into high light and oxidative stress in photosynthetic organisms have focussed primarily on genetic impairment of different photoprotective functions. There are few reports of "gain-of-function" mutations that provide enhanced resistance to high light and/or oxidative stress without reduced productivity. We have isolated at least four such very high light resistant (VHL(R)) mutations in the green alga, Chlamydomonas reinhardtii, that permit near maximal growth rates at light intensities lethal to wild type. This resistance is not due to an alteration in electron transport rate or quantity and functionality of the two photosystems that could have enhanced photochemical quenching. Nor is it due to reduced excitation pressure by downregulation of the light harvesting antennae or increased nonphotochemical quenching. In fact, photosynthetic activity is unaffected in more than 30 VHL(R) isolates. Instead, the basis of the VHL(R) phenotype is a combination of traits, which appears to be dominated by enhanced capacity to tolerate reactive oxygen species generated by excess light, methylviologen, rose bengal or hydrogen peroxide. This is further evidenced in lower levels of ROS after exposure to very high light in the VHL(R)-S9 mutant. Additionally, the VHL(R) phenotype is associated with increased zeaxanthin accumulation, maintenance of fast synthesis and degradation rates of the D1 protein, and sustained balanced electron flow into and out of PSI under very high light. We conclude that the VHL(R) mutations arose from a selection pressure that favors changes to the regulatory system(s) that coordinates several photoprotective processes amongst which repair of PSII and enhanced detoxification of reactive oxygen species play seminal roles. PMID:16002040

  16. 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. PMID:24699865

  17. 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

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

    PubMed

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

    2015-08-01

    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

  19. 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

  20. 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

  1. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate.

    PubMed

    Sharkhuu, Altanbadralt; Narasimhan, Meena L; Merzaban, Jasmeen S; Bressan, Ray A; Weller, Steve; Gehring, Chris

    2014-06-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide. PMID:24654847

  2. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate

    PubMed Central

    Sharkhuu, Altanbadralt; Narasimhan, Meena L; Merzaban, Jasmeen S; Bressan, Ray A; Weller, Steve; Gehring, Chris

    2014-01-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide. PMID:24654847

  3. The fidelity of transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in S. cerevisiae.

    PubMed

    Strathern, Jeffrey; Malagon, Francisco; Irvin, Jordan; Gotte, Deanna; Shafer, Brenda; Kireeva, Maria; Lubkowska, Lucyna; Jin, Ding Jun; Kashlev, Mikhail

    2013-01-25

    The fidelity of RNA synthesis depends on both accurate template-mediated nucleotide selection and proper maintenance of register between template and RNA. Loss of register, or transcriptional slippage, is particularly likely on homopolymeric runs in the template. Transcriptional slippage can alter the coding capacity of mRNAs and is used as a regulatory mechanism. Here we describe mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II that substantially increase the level of transcriptional slippage. Alleles of RPB1 (RPO21) with elevated slippage rates were identified among 6-azauracil-sensitive mutants and were also isolated using a slippage-dependent reporter gene. Biochemical characterization of polymerase II isolated from these mutants confirms elevated levels of transcriptional slippage. PMID:23223234

  4. 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

  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. Two Mutations associated with Macrolide Resistance in Treponema pallidum: Increasing Prevalence and Correlation with Molecular Strain Type in Seattle, Washington

    PubMed Central

    Grimes, Matthew; Sahi, Sharon K.; Godornes, B. Charmie; Tantalo, Lauren C.; Roberts, Neal; Bostick, David; Marra, Christina M.; Lukehart, Sheila A.

    2013-01-01

    Background Although azithromycin promised to be a safe and effective single dose oral treatment for early syphilis, azithromycin treatment failure has been documented and is associated with mutations in the 23S rDNA of corresponding Treponema pallidum strains. The prevalence of strains harboring these mutations varies throughout the US and the world. We examined T. pallidum strains circulating in Seattle, Washington, from 2001–2010 to determine the prevalence of two mutations associated with macrolide resistance, and to determine whether these mutations were associated with certain T. pallidum strain types. Methods Subjects were enrolled in a separate ongoing study of cerebrospinal fluid (CSF) abnormalities in patients with syphilis. T. pallidum DNA purified from blood and T. pallidum strains isolated from blood or CSF were analyzed for two 23S rDNA mutations and for the molecular targets used in an enhanced molecular stain typing system. Results Nine molecular strain types of T. pallidum were identified in Seattle from 2001–2010. Both macrolide resistance mutations were identified in Seattle strains, and the prevalence of resistant T. pallidum exceeded 80% in 2005 and increased through 2010. Resistance mutations were associated with discrete molecular strain types of T. pallidum. Conclusions Macrolide resistant T. pallidum strains are highly prevalent in Seattle, and each mutation is associated with discrete strain types. Macrolides should not be considered for treatment of syphilis in regions where prevalence of the mutations is high. Combining the resistance mutations with molecular strain typing permits a finer analysis of the epidemiology of syphilis in a community. PMID:23191949

  7. A Single Missense Mutation in a Coiled-Coil Domain of Escherichia coli Ribosomal Protein S2 Confers a Thermosensitive Phenotype That Can Be Suppressed by Ribosomal Protein S1

    PubMed Central

    Aseev, Leonid V.; Chugunov, Anton O.; Efremov, Roman G.

    2013-01-01

    Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation. PMID:23104805

  8. Increased neuronal firing in computer simulations of sodium channel mutations that cause generalized epilepsy with febrile seizures plus.

    PubMed

    Spampanato, Jay; Aradi, Ildiko; Soltesz, Ivan; Goldin, Alan L

    2004-05-01

    Generalized epilepsy with febrile seizures plus (GEFS+) is an autosomal dominant familial syndrome with a complex seizure phenotype. It is caused by mutations in one of 3 voltage-gated sodium channel subunit genes (SCN1B, SCN1A, and SCN2A) and the GABA(A) receptor gamma2 subunit gene (GBRG2). The biophysical characterization of 3 mutations (T875M, W1204R, and R1648H) in SCN1A, the gene encoding the CNS voltage-gated sodium channel alpha subunit Na(v)1.1, demonstrated a variety of functional effects. The T875M mutation enhanced slow inactivation, the W1204R mutation shifted the voltage dependency of activation and inactivation in the negative direction, and the R1648H mutation accelerated recovery from inactivation. To determine how these changes affect neuronal firing, we used the NEURON simulation software to design a computational model based on the experimentally determined properties of each GEFS+ mutant sodium channel and a delayed rectifier potassium channel. The model predicted that W1204R decreased the threshold, T875M increased the threshold, and R1648H did not affect the threshold for firing a single action potential. Despite the different effects on the threshold for firing a single action potential, all of the mutations resulted in an increased propensity to fire repetitive action potentials. In addition, each mutation was capable of driving repetitive firing in a mixed population of mutant and wild-type channels, consistent with the dominant nature of these mutations. These results suggest a common physiological mechanism for epileptogenesis resulting from sodium channel mutations that cause GEFS+. PMID:14702334

  9. 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

  10. Somatic mutations in MAP3K5 attenuate its pro-apoptotic function in melanoma through increased binding to Thioredoxin

    PubMed Central

    Prickett, Todd D.; Zerlanko, Brad; Gartner, Jared J.; Parker, Stephen C. J.; Dutton-Regester, Ken; Lin, Jimmy C.; Teer, Jamie K.; Wei, Xiaomu; Jiang, Jiji; Chen, Guo; Davies, Michael A.; Gershenwald, Jeffrey E.; Robinson, William; Robinson, Steven; Hayward, Nicholas K.; Rosenberg, Steven, A.; Margulies, Elliott H.; Samuels, Yardena

    2013-01-01

    Patients with advanced metastatic melanoma have poor prognosis and the genetics underlying its pathogenesis are poorly understood. High throughput sequencing has allowed comprehensive discovery of somatic mutations in cancer samples. Here, upon analysis of our whole-genome and whole-exome sequencing data of 29 melanoma samples we identified several genes that harbor recurrent non-synonymous mutations. These included MAP3K5, which in a prevalence screen of 288 melanomas was found to harbor a R256C substitution in 5 cases. All MAP3K5 mutated samples were wild-type for BRAF, suggesting a mutual exclusivity for these mutations. Functional analysis of the MAP3K5 R256C mutation revealed attenuation of MKK4 activation through increased binding of the inhibitory protein thioredoxin (TXN/TRX-1/Trx); resulting in increased proliferation and anchorage-independent growth of melanoma cells. This mutation represents a potential target for the design of new therapies to treat melanoma. PMID:24008424

  11. 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.

  12. 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

  13. Target site insensitivity mutations in the AChE enzyme confer resistance to organophosphorous insecticides in Leptinotarsa decemlineata (Say).

    PubMed

    Malekmohammadi, M; Galehdari, H

    2016-01-01

    In the present study, we demonstrated the use and optimization of the tetra-primer ARMS-PCR procedure to detect and analyze the frequency of the R30K and I392T mutations in resistant field populations of CPB. The R30K mutation was detected in 72%, 84%, 52% and 64% of Bahar, Dehpiaz, Aliabad and Yengijeh populations, respectively. Overall frequencies of the I392T mutation were 12%, 8% and 16% of Bahar, Aliabad and Yengijeh populations, respectively. No I392T point mutation was found among samples from Dehpiaz field population. Moreover, only 31% and 2% of samples from the resistant field populations were homozygous for R30K and I392T mutations, respectively. No individual simultaneously had both I392T and S291G/R30K point mutations. The incidence of individuals with both S291G and R30K point mutations in the samples from Bahar, Dehpiaz, Aliabad, and Yengijeh populations were 31.5%, 44.7%, 41.6%, and 27.3% respectively. Genotypes determined by the tetra-primer ARMS-PCR method were consistent with those determined by PCR sequencing. There was no significant correlation between the mutation frequencies and resistance levels in the resistant populations, indicating that other mutations may contribute to this variation. Polymorphism in the partial L. decemlineata cDNA AChE gene Ldace2 of four field populations was identified by direct sequencing of PCR-amplified fragments. Among 45 novel mutations detected in this study, T29P mutation was found across all four field populations that likely contribute to the AChE insensitivity. Site-directed mutagenesis and protein expression experiments are needed for a more complete evaluation. PMID:26778439

  14. Phospholipid mass is increased in fibroblasts bearing the Swedish amyloid precursor mutation.

    PubMed

    Murphy, Eric J; Huang, Hsueh-Meei; Cowburn, Richard F; Lannfelt, Lars; Gibson, Gary E

    2006-03-15

    Phospholipid changes occur in brain regions affected by Alzheimer disease (AD), including a marked reduction in plasmalogens, which could diminish brain function either by directly altering signaling events or by bulk membrane effects. However, model systems for studying the dynamics of lipid biosynthesis in AD are lacking. To determine if fibroblasts bearing the Swedish amyloid precursor protein (swAPP) mutation are a useful model to study the mechanism(s) associated with altered phospholipid biosynthesis in AD, we examined the steady-state phospholipid mass and composition of fibroblasts, including plasmalogens. We found a 15% increase in total phospholipid mass, accounted for by a 24% increase in the combined total of phosphatidylethanolamine and plasmanylethanolamine mass and a 19% increase in the combined total of phosphatidylcholine (PtdCho) and plasmanycholine (PakCho) mass in the swAPP mutant bearing fibroblasts. Cholesterol mass was unchanged in these cells. The changes in phospholipid mass did not alter the cellular molar composition of the phospholipids nor the cholesterol to phospholipid ratio. While plasmalogen mass was not altered, the ratio of choline plasmalogen (PlsCho) mass to PtdCho+PakCho mass was decreased 16% and there was a 14% reduction in the proportion of PlsCho as a percent of total phospholipids in the swAPP mutant bearing fibroblasts. This change in choline plasmalogen is consistent with the reported decreases in plasmalogen proportions in affected regions of AD brain, suggesting that these cells may serve as a useful model to determine the mechanism underlying changes in plasmalogen biosynthesis in AD brain. PMID:16464688

  15. 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

  16. 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

  17. The Dual MEK/FLT3 Inhibitor E6201 Exerts Cytotoxic Activity against Acute Myeloid Leukemia Cells Harboring Resistance-Conferring FLT3 Mutations.

    PubMed

    Zhang, Weiguo; Borthakur, Gautam; Gao, Chen; Chen, Ye; Mu, Hong; Ruvolo, Vivian R; Nomoto, Kenichi; Zhao, Nanding; Konopleva, Marina; Andreeff, Michael

    2016-03-15

    Fms-like tyrosine kinase 3 (FLT3) inhibition has elicited encouraging responses in acute myeloid leukemia (AML) therapy. Unfortunately, unless combined with a bone marrow transplant, disease relapse is frequent. In addition to the acquired point mutations in the FLT3 kinase domain that contribute to FLT3 inhibitor resistance, MEK/ERK signaling is persistently activated in AML cells even when FLT3 phosphorylation is continually suppressed. Thus, concomitant targeting of FLT3 and MAPK may potentially exert synergistic activity to counteract the resistance of AML cells to FLT3-targeted therapy. In this study, we investigated the antileukemia activity of a MEK1 and FLT3 dual inhibitor, E6201, in AML cells resistant to FLT3 inhibition. We found that E6201 exerted profound apoptogenic effects on AML cells harboring resistance-conferring FLT3 mutations. This activity appeared to be p53 dependent, and E6201-induced cytotoxicity was retained under hypoxic culture conditions and during coculture with mesenchymal stem cells that mimic the AML microenvironment. Furthermore, E6201 markedly reduced leukemia burden and improved the survival of mice in a human FLT3-mutated AML model. Collectively, our data provide a preclinical basis for the clinical evaluation of E6201 in AML patients harboring FLT3 mutations, including those who relapse following FLT3-targeted monotherapy. PMID:26822154

  18. Tipping the mutation-selection balance: Limited migration increases the frequency of deleterious mutants.

    PubMed

    Cooper, Jacob D; Neuhauser, Claudia; Dean, Antony M; Kerr, Benjamin

    2015-09-01

    Typical mutation-selection models assume well-mixed populations, but dispersal and migration within many natural populations is spatially limited. Such limitations can lead to enhanced variation among locations as different types become clustered in different places. Such clustering weakens competition between unlike types relative to competition between like types; thus, the rate by which a fitter type displaces an inferior competitor can be affected by the spatial scale of movement. In this paper, we use a birth-death model to show that limited migration can affect asexual populations by creating competitive refugia. We use a moment closure approach to show that as population structure is introduced by limiting migration, the equilibrial frequency of deleterious mutants increases. We support and extend the model through stochastic simulation, and we use a spatially explicit cellular automaton approach to corroborate the results. We discuss the implications of these results for standing variation in structured populations and adaptive valley crossing in Wright's "shifting balance" process. PMID:25983046

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

    PubMed

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

    2015-11-01

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

  20. 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. PMID:17825069

  1. G2385R and I2020T Mutations Increase LRRK2 GTPase Activity

    PubMed Central

    Jang, Jihoon; Joe, Eun-hye; Son, Ilhong; Seol, Wongi

    2016-01-01

    The LRRK2 mutation is a major causal mutation in familial Parkinson's disease. Although LRRK2 contains functional GTPase and kinase domains and their activities are altered by pathogenic mutations, most studies focused on LRRK2 kinase activity because the most prevalent mutant, G2019S, enhances kinase activity. However, the G2019S mutation is extremely rare in the Asian population. Instead, the G2385R mutation was reported as a major risk factor in the Asian population. Similar to other LRRK2 studies, G2385R studies have also focused on kinase activity. Here, we investigated GTPase activities of G2385R with other LRRK2 mutants, such as G2019S, R1441C, and I2020T, as well as wild type (WT). Our results suggest that both I2020T and G2385R contain GTPase activities stronger than that of WT. A kinase assay using the commercial recombinant proteins showed that I2020T harbored stronger activity, whereas G2385R had weaker activity than that of WT, as reported previously. This is the first report of LRRK2 I2020T and G2385R GTPase activities and shows that most of the LRRK2 mutations that are pathogenic or a risk factor altered either kinase or GTPase activity, suggesting that their physiological consequences are caused by altered enzyme activities. PMID:27314038

  2. No evidence of increased mutation rates at microsatellite loci in offspring of A-bomb survivors.

    PubMed

    Kodaira, M; Ryo, H; Kamada, N; Furukawa, K; Takahashi, N; Nakajima, H; Nomura, T; Nakamura, N

    2010-02-01

    To evaluate the genetic effects of A-bomb radiation, we examined mutations at 40 microsatellite loci in exposed families (father-mother-offspring, mostly uni-parental exposures), which consisted of 66 offspring having a mean paternal dose of 1.87 Gy and a mean maternal dose of 1.27 Gy. The control families consisted of 63 offspring whose parents either were exposed to low doses of radiation (< 0.01 Gy) or were not in the cities of Hiroshima or Nagasaki at the time of the bombs. We found seven mutations in the exposed alleles (7/2,789; mutation rate 0.25 x 10(-2)/locus/generation) and 26 in the unexposed alleles (26/7,465; 0.35 x 10(-2)/locus/generation), which does not indicate an effect from parental exposure to radiation. Although we could not assign the parental origins of four mutations, the conclusion may hold since even if we assume that these four mutations had occurred in the exposed alleles, the estimated mean mutation rate would be 0.39 x 10(-2) in the exposed group [(7 + 4)/2,789)], which is slightly higher than 0.35 x 10(-2) in the control group, but the difference is not statistically significant. PMID:20095853

  3. Increased Missense Mutation Burden of Fatty Acid Metabolism Related Genes in Nunavik Inuit Population

    PubMed Central

    Zhou, Sirui; Xiong, Lan; Xie, Pingxing; Ambalavanan, Amirthagowri; Bourassa, Cynthia V.; Dionne-Laporte, Alexandre; Spiegelman, Dan; Turcotte Gauthier, Maude; Henrion, Edouard; Diallo, Ousmane; Dion, Patrick A.; Rouleau, Guy A.

    2015-01-01

    Background Nunavik Inuit (northern Quebec, Canada) reside along the arctic coastline where for generations their daily energy intake has mainly been derived from animal fat. Given this particular diet it has been hypothesized that natural selection would lead to population specific allele frequency differences and unique variants in genes related to fatty acid metabolism. A group of genes, namely CPT1A, CPT1B, CPT1C, CPT2, CRAT and CROT, encode for three carnitine acyltransferases that are important for the oxidation of fatty acids, a critical step in their metabolism. Methods Exome sequencing and SNP array genotyping were used to examine the genetic variations in the six genes encoding for the carnitine acyltransferases in 113 Nunavik Inuit individuals. Results Altogether ten missense variants were found in genes CPT1A, CPT1B, CPT1C, CPT2 and CRAT, including three novel variants and one Inuit specific variant CPT1A p.P479L (rs80356779). The latter has the highest frequency (0.955) compared to other Inuit populations. We found that by comparison to Asians or Europeans, the Nunavik Inuit have an increased mutation burden in CPT1A, CPT2 and CRAT; there is also a high level of population differentiation based on carnitine acyltransferase gene variations between Nunavik Inuit and Asians. Conclusion The increased number and frequency of deleterious variants in these fatty acid metabolism genes in Nunavik Inuit may be the result of genetic adaptation to their diet and/or the extremely cold climate. In addition, the identification of these variants may help to understand some of the specific health risks of Nunavik Inuit. PMID:26010953

  4. 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. PMID:17469202

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

    PubMed

    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

  6. 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

  7. 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

  8. 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.

  9. Genetic basis for increased intestinal permeability in families with Crohn's disease: role of CARD15 3020insC mutation?

    PubMed Central

    Buhner, S; Buning, C; Genschel, J; Kling, K; Herrmann, D; Dignass, A; Kuechler, I; Krueger, S; Schmidt, H H‐J; Lochs, H

    2006-01-01

    Background and aim A genetically impaired intestinal barrier function has long been suspected to be a predisposing factor for Crohn's disease (CD). Recently, mutations of the capsase recruitment domain family, member 15 (CARD15) gene have been identified and associated with CD. We hypothesise that a CARD15 mutation may be associated with an impaired intestinal barrier. Methods We studied 128 patients with quiescent CD, 129 first degree relatives (CD‐R), 66 non‐related household members (CD‐NR), and 96 healthy controls. The three most common CARD15 polymorphisms (R702W, G908R, and 3020insC) were analysed and intestinal permeability was determined by the lactulose/mannitol ratio. Results Intestinal permeability was significantly increased in CD and CD‐R groups compared with CD‐NR and controls. Values above the normal range were seen in 44% of CD and 26% of CD‐R but only in 6% of CD‐NR, and in none of the controls. A household community with CD patients, representing a common environment, was not associated with increased intestinal permeability in family members. However, 40% of CD first degree relatives carrying a CARD15 3020insC mutation and 75% (3/4) of those CD‐R with combined 3020insC and R702W mutations had increased intestinal permeability compared with only 15% of wild‐types, indicating a genetic influence on barrier function. R702W and G908R mutations were not associated with high permeability. Conclusions In healthy first degree relatives, high mucosal permeability is associated with the presence of a CARD15 3020insC mutation. This indicates that genetic factors may be involved in impairment of intestinal barrier function in families with IBD. PMID:16000642

  10. 3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase inhibitors.

    PubMed Central

    Larder, B A

    1992-01-01

    Nonnucleoside reverse transcriptase (NNRT) inhibitors (R82913; (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione; Cl-TIBO; and BI-RG-587, nevirapine) were used to select resistant human immunodeficiency virus type 1 (HIV-1) variants by passage in cell cultures of wild-type or 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant strains. Similar to other NNRT inhibitors, Cl-TIBO induced a single mutation (Y181 to C) in reverse transcriptase (RT) that accounted for the resistance. BI-RG-587 induced a different mutation (V106-->A) in AZT resistance backgrounds. A series of viable HIV-1 variants was constructed by site-directed mutagenesis of the RT, which harbored multiple drug resistance mutations, including Y181 to C. HIV-1 that was co-resistant to NNRT inhibitors and 2',3'-dideoxyinosine resulted when a 2',3'-dideoxyinosine resistance mutation (L74 to V) was also present in RT. By contrast, however, the Y181 to C mutation in an AZT resistance background significantly suppressed resistance to AZT, while it conferred resistance to NNRT inhibitors. However, the V106-->A substitution did not cause suppression of preexisting AZT resistance. Since certain combinations of nucleoside analogs and NNRT inhibitors might result in the development of co-resistance, careful analysis of clinical isolates obtained during combination therapy will be needed to determine the potential significance of these observations. PMID:1282792

  11. The A2'N mutation of the RDL gamma-aminobutyric acid receptor conferring fipronil resistance in Laodelphax striatellus (Hemiptera: Delphacidae).

    PubMed

    Nakao, Toshifumi; Kawase, Ayumi; Kinoshita, Ayako; Abe, Reiko; Hama, Masako; Kawahara, Nobuyuki; Hirase, Kangetsu

    2011-04-01

    The planthopper Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae) is a serious insect pest of rice, Oryza sativa L., and has developed resistance to fipronil in Japan. Sequence analysis of L. striatellus RDL gamma-aminobutyric acid (GABA) receptor subunit (LS-RDL) genes from a fipronil-resistant population and a fipronil-susceptible strain identified the A2'N mutation (index number for M2 membrane-spanning region), that was previously implicated in fipronil resistance in the planthopper Sogatella furcifera (Horváth) (Hemiptera: Delphacidae). Nineteen of 21 fipronil-resistant L. striatellus individuals were genotyped as heterozygous for the A2'N mutation, suggesting that this mutation is associated with fipronil resistance and that most fipronil-resistant L. striatellus express wild-type and A2'N mutant LS-RDL simultaneously. To confirm the role of the A2'N mutation of LS-RDL, Drosophila Mel-2 cells were transfected with wild-type and A2'N mutant LS-RDL genes, either individually or together. A membrane potential assay showed that fipronil had no inhibitory effect at 10 microM on cells transfected with the A2'N mutant LS-RDL gene with or without the wild-type LS-RDL gene. By contrast, the IC50 value of fipronil for wild-type LS-RDL homomers was 14 nM. These results suggest that the A2'N mutation of the RDL GABA receptor subunit confers fipronil resistance in L. striatellus as well as S. furcifera. PMID:21510217

  12. Does a SCN1A gene mutation confer earlier age of onset of febrile seizures in GEFS+?

    PubMed

    Sijben, Angelique E J; Sithinamsuwan, Pasiri; Radhakrishnan, Ashalata; Badawy, Radwa A B; Dibbens, Leanne; Mazarib, Aziz; Lev, Dorit; Lerman-Sagie, Tally; Straussberg, Rachel; Berkovic, Samuel F; Scheffer, Ingrid E

    2009-04-01

    SCN1A is the most clinically relevant epilepsy gene and is associated with generalized epilepsy and febrile seizure plus (GEFS+) and Dravet syndrome. We postulated that earlier onset of febrile seizures in the febrile seizure (FS) and febrile seizure plus (FS+) phenotypes may occur in the presence of a SCN1A mutation. This was because of the age-related onset of Dravet syndrome, which typically begins in the first year of life. We found that patients with FS and FS+ with SCN1A mutations had earlier median onset of febrile seizures compared to the population median. Patients with GABRG2 mutations had a similar early onset in contrast to patients with SCN1B mutations where onset was later. This study is the first to demonstrate that a specific genetic abnormality directly influences the FS and FS+ phenotype in terms of age of onset. PMID:19292758

  13. Increased prevalence of Mediterranean and Muslim populations in mutation-related research literature.

    PubMed

    Birenbaum-Carmeli, Daphna

    2005-01-01

    This paper assesses the prevalence of 569 population groups in mutation-related research literature by means of prevalence scores, calculated on the basis of a systematic search of the PubMed database. The main finding is that Mediterranean and Muslim populations are mentioned more often than other groups. The observed overrepresentation is attributed to the pervasiveness of hemoglobin disorders in these populations, the early decoding of hemoglobin and the relative commonness of consanguineous marriage in some of these populations. The paper points at potential benefits as well as predicaments that the intensive targeting of these populations for mutation research may entail. PMID:15925887

  14. Double heterozygous mutations of MITF and PAX3 result in Waardenburg syndrome with increased penetrance in pigmentary defects.

    PubMed

    Yang, T; Li, X; Huang, Q; Li, L; Chai, Y; Sun, L; Wang, X; Zhu, Y; Wang, Z; Huang, Z; Li, Y; Wu, H

    2013-01-01

    Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentary defects of the hair, skin, and iris. Heterozygous mutations of MITF and its transactivator gene PAX3 are associated with Waardenburg syndrome type II (WS2) and type I (WS1), respectively. Most patients with MITF or PAX3 mutations, however, show variable penetrance of WS-associated phenotypes even within families segregating the same mutation, possibly mediated by genetic background or specific modifiers. In this study, we reported a rare Waardenburg syndrome simplex family in which a pair of WS parents gave birth to a child with double heterozygous mutations of MITF and PAX3. Compared to his parents who carried a single mutation in either MITF or PAX3, this child showed increased penetrance of pigmentary defects including white forelock, white eyebrows and eyelashes, and patchy facial depigmentation. This observation suggested that the expression level of MITF is closely correlated to the penetrance of WS, and variants in transcription regulator genes of MITF may modify the relevant clinical phenotypes. PMID:22320238

  15. 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

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

    PubMed Central

    Huang, Haixiu; Wu, Kan; Wang, Bing; Chen, Xufeng; Ma, Shenglin

    2015-01-01

    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. PMID:25714021

  17. Mutation of the Dominant Endocytosis Motif in Human Immunodeficiency Virus Type 1 gp41 Can Complement Matrix Mutations without Increasing Env Incorporation

    PubMed Central

    West, John T.; Weldon, Sally K.; Wyss, Stephanie; Lin, Xiaoxu; Yu, Qin; Thali, Markus; Hunter, Eric

    2002-01-01

    The human immunodeficiency virus type 1 transmembrane glycoprotein (TM) is efficiently endocytosed in a clathrin-dependent manner. Internalization is mediated by a tyrosine-containing motif within the cytoplasmic domain, and replacement of the cytoplasmic tyrosine by cysteine or phenylalanine increased expression of mutant glycoprotein on the surface of transfected cells by as much as 2.5-fold. Because interactions between the cytoplasmic domain of Env and the matrix protein (MA) have been suggested to mediate incorporation of Env in virus particles, we examined whether perturbation of endocytosis would alter incorporation. Proviruses were constructed to contain the wild-type or mutant Env in conjunction with point mutations in MA that had previously been shown to block Env incorporation. These constructs were used to evaluate the effect of glycoprotein endocytosis on incorporation into virus particles and to test the necessity for a specific interaction between Env and MA to mediate incorporation. Viruses produced from transfected 293T cells were used to infect various cell lines, including MAGI, H9, and CEMx174. Viruses encoding both a disrupted endocytosis motif signal and mutations within MA were significantly more infectious in MAGI cells than their counterparts encoding a mutant MA and wild-type Env. This complementation of infectivity for the MA incorporation mutant viruses was not due to increased glycoprotein incorporation into particles but instead reflected an enhanced fusogenicity of the mutated Env proteins. Our findings further support the concept that a specific interaction between the long cytoplasmic domain of TM and MA is required for efficient incorporation of Env into assembling virions. Alteration of the endocytosis signal of Env, and the resulting increase in cell surface glycoprotein, has no effect on incorporation despite demonstrable effects on fusion, virus entry, and infectivity. PMID:11884559

  18. Evolutionary Action score of TP53 (EAp53) identifies high risk mutations associated with decreased survival and increased distant metastases in head and neck cancer

    PubMed Central

    Neskey, David M.; Osman, Abdullah A.; Ow, Thomas J.; Katsonis, Panagiotis; McDonald, Thomas; Hicks, Stephanie C.; Hsu, Teng-Kuei; Pickering, Curtis R.; Ward, Alexandra; Patel, Ameeta; Yordy, John S.; Skinner, Heath D.; Giri, Uma; Sano, Daisuke; Story, Michael D.; Beadle, Beth M.; El-Naggar, Adel K.; Kies, Merrill S.; William, William N.; Caulin, Carlos; Frederick, Mitchell; Kimmel, Marek; Myers, Jeffrey N.; Lichtarge, Olivier

    2015-01-01

    TP53 is the most frequently altered gene in head and neck squamous cell carcinoma (HNSCC) with mutations occurring in over two third of cases, but the prognostic significance of these mutations remains elusive. In the current study, we evaluated a novel computational approach termed Evolutionary Action (EAp53) to stratify patients with tumors harboring TP53 mutations as high or low risk, and validated this system in both in vivo and in vitro models. Patients with high risk TP53 mutations had the poorest survival outcomes and the shortest time to the development of distant metastases. Tumor cells expressing high risk TP53 mutations were more invasive and tumorigenic and they exhibited a higher incidence of lung metastases. We also documented an association between the presence of high risk mutations and decreased expression of TP53 target genes, highlighting key cellular pathways that are likely to be dysregulated by this subset of p53 mutations which confer particularly aggressive tumor behavior. Overall, our work validated EAp53 as a novel computational tool that may be useful in clinical prognosis of tumors harboring p53 mutations. PMID:25634208

  19. Systematic review of allelic exchange experiments aimed at identifying mutations that confer drug resistance in Mycobacterium tuberculosis

    PubMed Central

    Nebenzahl-Guimaraes, Hanna; Jacobson, Karen R.; Farhat, Maha R.; Murray, Megan B.

    2014-01-01

    Background Improving our understanding of the relationship between the genotype and the drug resistance phenotype of Mycobacterium tuberculosis will aid the development of more accurate molecular diagnostics for drug-resistant tuberculosis. Studies that use direct genetic manipulation to identify the mutations that cause M. tuberculosis drug resistance are superior to associational studies in elucidating an individual mutation's contribution to the drug resistance phenotype. Methods We systematically reviewed the literature for publications reporting allelic exchange experiments in any of the resistance-associated M. tuberculosis genes. We included studies that introduced single point mutations using specialized linkage transduction or site-directed/in vitro mutagenesis and documented a change in the resistance phenotype. Results We summarize evidence supporting the causal relationship of 54 different mutations in eight genes (katG, inhA, kasA, embB, embC, rpoB, gyrA and gyrB) and one intergenic region (furA-katG) with resistance to isoniazid, the rifamycins, ethambutol and fluoroquinolones. We observed a significant role for the strain genomic background in modulating the resistance phenotype of 21 of these mutations and found examples of where the same drug resistance mutations caused varying levels of resistance to different members of the same drug class. Conclusions This systematic review highlights those mutations that have been shown to causally change phenotypic resistance in M. tuberculosis and brings attention to a notable lack of allelic exchange data for several of the genes known to be associated with drug resistance. PMID:24055765

  20. 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. PMID:27126074

  1. Increasing Productivity in Higher Education. Proceedings of the Conference to Mark the Dedication of the Henry Chauncey Conference Center, May 1974.

    ERIC Educational Resources Information Center

    Educational Testing Service, Princeton, NJ.

    Presented in this document are the proceedings of the conference to mark the dedication of the Henry Chauncey Conference Center at the Educational Testing Service in Princeton, New Jersey. Included in the document are the opening remarks by William W. Turnbull; Higher Education is a Chauncey Business by Roger W. Heyns; Higher Education in a Steady…

  2. 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

  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. Phosphorylation of Mutationally Introduced Tyrosine in the Activation Loop of HER2 Confers Gain-of-Function Activity

    PubMed Central

    Hu, Zexi; Wan, Xiaobo; Hao, Rui; Zhang, Heng; Li, Li; Li, Lin; Xie, Qiang; Wang, Peng; Gao, Yibo; Chen, She; Wei, Min; Luan, Zhidong; Zhang, Aiqun; Huang, Niu; Chen, Liang

    2015-01-01

    Amplification, overexpression, and somatic mutation of the HER2 gene have been reported to play a critical role in tumorigenesis of various cancers. The HER2 H878Y mutation was recently reported in 11% of hepatocellular carcinoma (HCC) patients. However, its functional impact on the HER2 protein and its role in tumorigenesis has not been determined. Here, we show that HER2 H878Y is a gain-of-function mutation. Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity. H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors. Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity. PMID:25853726

  5. 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

  6. Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates.

    PubMed

    Bayliss, Christopher D; Sweetman, Wendy A; Moxon, E Richard

    2004-05-01

    High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5' TA repeats located between the -10 and -35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5' AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5' AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5' TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5' TA-mediated pilin PV. PMID:15126452

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

    PubMed

    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

    2015-08-15

    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) CAPN5(R243L) only in the retina. The resulting hCAPN5(R243L) transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5(R243L) 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 hCAPN5(R243L) 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

  8. 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

  9. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa.

    PubMed

    Wondji, Charles S; Dabire, Roch K; Tukur, Zainab; Irving, Helen; Djouaka, Rousseau; Morgan, John C

    2011-07-01

    Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction. Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Partial sequencing of the dieldrin target site, the γ-aminobutyric acid (GABA) receptor, identified two amino acid substitutions, A296S and V327I. The A296S mutation has been associated with dieldrin resistance in other species. The V327I mutations was detected in the resistant sample from Burkina Faso and Cameroon and consistently associated with the A296S substitution. The full-length of the An. funestus GABA-receptor gene, amplified by RT-PCR, generated a sequence of 1674 bp encoding 557 amino acid of the protein in An. funestus with 98% similarity to that of Anopheles gambiae. Two diagnostic assays were developed to genotype the A296S mutation (pyrosequencing and PCR-RFLP), and use of these assays revealed high frequency of the resistant allele in Burkina Faso (60%) and Cameroon (82%), moderate level in Benin (16%) while low frequency or absence of the mutation was observed respectively in Uganda (7.5%) or 0% in Malawi and Mozambique. The distribution of the Rdl(R) mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions. PMID:21501685

  10. 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

    2009-01-01

    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. PMID:19551630

  11. Activating Mutations in PIK3CB Confer Resistance to PI3K Inhibition and Define a Novel Oncogenic Role for p110β.

    PubMed

    Nakanishi, Yoshito; Walter, Kimberly; Spoerke, Jill M; O'Brien, Carol; Huw, Ling Y; Hampton, Garret M; Lackner, Mark R

    2016-03-01

    Activation of the PI3K pathway occurs commonly in a wide variety of cancers. Experience with other successful targeted agents suggests that clinical resistance is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN-deficient cancers. We generated cell lines resistant to the pan-PI3K inhibitor GDC-0941 from parental PTEN-null breast cancer cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines that was recurrent in cancer patients. Stable expression of mutant PIK3CB variants conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. Furthermore, we show that the p110β D1067Y mutant was highly activated and induced PIP3 levels at the cell membrane, subsequently promoting the localization and activation of AKT and PDK1 at the membrane and driving PI3K signaling to a level that could withstand treatment with proximal inhibitors. Finally, we demonstrate that the PIK3CB D1067Y mutant behaved as an oncogene and transformed normal cells, an activity that was enhanced by PTEN depletion. Collectively, these novel preclinical and clinical findings implicate the acquisition of activating PIK3CB D1067 mutations as an important event underlying the resistance of cancer cells to selective PI3K inhibitors. PMID:26759240

  12. 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

  13. 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

  14. Increased ethanol consumption despite taste aversion in mice with a human tryptophan hydroxylase 2 loss of function mutation.

    PubMed

    Lemay, Francis; Doré, François Y; Beaulieu, Jean-Martin

    2015-11-16

    Polymorphisms in the gene encoding the brain serotonin synthesis enzyme Tph2 have been identified in mental illnesses, with co-morbidity of substance use disorder. However, little is known about the impact of Tph2 gene variants on addiction. Mice expressing a human Tph2 loss of function variant were used to investigate consequences of aversive conditions on ethanol intake. Mice were familiarized either with ethanol or a solution containing both ethanol and the bittering agent quinine. Effect of familiarization to ethanol or an ethanol-quinine solution was then evaluated using a two-bottles preference test in Tph2-KI and control littermates. Mice from both genotypes displayed similar levels of ethanol consumption and quinine avoidance when habituated to ethanol alone. In contrast, addition of quinine to ethanol during the familiarization period resulted in a reduction of avoidance for the quinine-ethanol solution only in mutant mice. These results indicate that loss of function mutation in Tph2 results in greater motivation for ethanol consumption under aversive conditions and may confer enhanced sensitivity to alcohol use disorder. PMID:26497913

  15. Assessment of the origins and spread of putative resistance-conferring mutations in Plasmodium vivax dihydropteroate synthase.

    PubMed

    Hawkins, Vivian N; Suzuki, Stephanie M; Rungsihirunrat, Kanchana; Hapuarachchi, Hapuarachchige C; Maestre, Amanda; Na-Bangchang, Kesara; Sibley, Carol Hopkins

    2009-08-01

    Infection with Plasmodium vivax is usually treated with chloroquine, but parasites are often exposed inadvertently to sulfadoxine-pyrimethamine. To infer patterns of selection and spread of resistant parasites in natural populations, we determined haplotypes of P. vivax dihydropteroate synthase ( dhps ) alleles that could confer resistance to sulfadoxine. We amplified the P. vivax pyrophosphokinase ( pppk )- dhps region and its flanking intergenic regions from 92 contemporary global isolates. Introns and exons of pppk-dhps were highly polymorphic, as were the flanking intergenic regions. Eighteen haplotypes were associated with wild-type alleles, but several different putatively sulfadoxine-resistant alleles have arisen in areas of intensive sulfadoxine-pyrimethamine use. Even when they encoded changes to the same amino acid, these mutant alleles were associated with multiple different haplotypes. Two main conclusions can be drawn from these data. First, dhps alleles resistant to sulfadoxine have arisen multiple times under drug pressure. Second, there has been convergent evolution of a variety of alleles that could confer resistance to sulfa drugs. PMID:19635897

  16. Single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

    SciTech Connect

    Ulaszewski, S.; Van Herck, J.C.; Dufour, J.P.; Kulpa, J.; Nieuwenhuis, B.; Goffeau, A.

    1987-01-05

    A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by (gamma-/sup 32/P) ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors.

  17. A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper).

    PubMed

    Liu, Zewen; Williamson, Martin S; Lansdell, Stuart J; Denholm, Ian; Han, Zhaojun; Millar, Neil S

    2005-06-14

    Neonicotinoids, such as imidacloprid, are nicotinic acetylcholine receptor (nAChR) agonists with potent insecticidal activity. Since its introduction in the early 1990s, imidacloprid has become one of the most extensively used insecticides for both crop protection and animal health applications. As with other classes of insecticides, resistance to neonicotinoids is a significant threat and has been identified in several pest species, including the brown planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. In this study, radioligand binding experiments have been conducted with whole-body membranes prepared from imidacloprid-susceptible and imidacloprid-resistant strains of N. lugens. The results reveal a much higher level of [3H]imidacloprid-specific binding to the susceptible strain than to the resistant strain (16.7 +/- 1.0 and 0.34 +/- 0.21 fmol/mg of protein, respectively). With the aim of understanding the molecular basis of imidacloprid resistance, five nAChR subunits (Nlalpha1-Nlalpha4 and Nlbeta1) have been cloned from N. lugens.A comparison of nAChR subunit genes from imidacloprid-sensitive and imidacloprid-resistant populations has identified a single point mutation at a conserved position (Y151S) in two nAChR subunits, Nlalpha1 and Nlalpha3. A strong correlation between the frequency of the Y151S point mutation and the level of resistance to imidacloprid has been demonstrated by allele-specific PCR. By expression of hybrid nAChRs containing N. lugens alpha and rat beta2 subunits, evidence was obtained that demonstrates that mutation Y151S is responsible for a substantial reduction in specific [3H]imidacloprid binding. This study provides direct evidence for the occurrence of target-site resistance to a neonicotinoid insecticide. PMID:15937112

  18. 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

  19. Does selection on increased cold tolerance in the adult stage confer resistance throughout development?

    PubMed

    Dierks, A; Kölzow, N; Franke, K; Fischer, K

    2012-08-01

    Artificial selection is a powerful approach to unravel constraints on genetic adaptation. Although it has been frequently used to reveal genetic trade-offs among different fitness-related traits, only a few studies have targeted genetic correlations across developmental stages. Here, we test whether selection on increased cold tolerance in the adult stage increases cold resistance throughout ontogeny in the butterfly Bicyclus anynana. We used lines selected for decreased chill-coma recovery time and corresponding controls, which had originally been set up from three levels of inbreeding (outbred control, one or two full-sib matings). Four generations after having terminated selection, a response to selection was found in 1-day-old butterflies (the age at which selection took place). Older adults showed a very similar although weaker response. Nevertheless, cold resistance did not increase in either egg, larval or pupal stage in the selection lines but was even lower compared to control lines for eggs and young larvae. These findings suggest a cost of increased adult cold tolerance, presumably reducing resource availability for offspring provisioning and thereby stress tolerance during development, which may substantially affect evolutionary trajectories. PMID:22686583

  20. Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations

    PubMed Central

    Godena, Vinay K.; Brookes-Hocking, Nicholas; Moller, Annekathrin; Shaw, Gary; Oswald, Matthew; Sancho, Rosa M.; Miller, Christopher C. J.; Whitworth, Alexander J.; De Vos, Kurt J.

    2014-01-01

    Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson’s disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson’s disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson’s disease. PMID:25316291

  1. Distinct Mutations in IRAK-4 Confer Hyporesponsiveness to Lipopolysaccharide and Interleukin-1 in a Patient with Recurrent Bacterial Infections

    PubMed Central

    Medvedev, Andrei E.; Lentschat, Arnd; Kuhns, Douglas B.; Blanco, Jorge C.G.; Salkowski, Cindy; Zhang, Shuling; Arditi, Moshe; Gallin, John I.; Vogel, Stefanie N.

    2003-01-01

    We identified previously a patient with recurrent bacterial infections who failed to respond to gram-negative LPS in vivo, and whose leukocytes were profoundly hyporesponsive to LPS and IL-1 in vitro. We now demonstrate that this patient also exhibits deficient responses in a skin blister model of aseptic inflammation. A lack of IL-18 responsiveness, coupled with diminished LPS and/or IL-1–induced nuclear factor–κB and activator protein-1 translocation, p38 phosphorylation, gene expression, and dysregulated IL-1R–associated kinase (IRAK)–1 activity in vitro support the hypothesis that the defect lies within the signaling pathway common to toll-like receptor 4, IL-1R, and IL-18R. This patient expresses a “compound heterozygous” genotype, with a point mutation (C877T in cDNA) and a two-nucleotide, AC deletion (620–621del in cDNA) encoded by distinct alleles of the IRAK-4 gene (GenBank/EMBL/DDBJ accession nos. AF445802 and AY186092). Both mutations encode proteins with an intact death domain, but a truncated kinase domain, thereby precluding expression of full-length IRAK-4 (i.e., a recessive phenotype). When overexpressed in HEK293T cells, neither truncated form augmented endogenous IRAK-1 kinase activity, and both inhibited endogenous IRAK-1 activity modestly. Thus, IRAK-4 is pivotal in the development of a normal inflammatory response initiated by bacterial or nonbacterial insults. PMID:12925671

  2. Prevalence of mutations conferring resistance among multi- and extensively drug-resistant Mycobacterium tuberculosis isolates in China.

    PubMed

    Chen, Yan; Zhao, Bing; Liu, Hai-can; Sun, Qing; Zhao, Xiu-qin; Liu, Zhi-guang; Wan, Kang-lin; Zhao, Li-li

    2016-03-01

    To identify the mutations in multi- and extensively drug-resistant tuberculosis isolates and to evaluate the use of molecular markers of resistance, we analyzed 257 multi- and extensively drug-resistant isolates and 64 pan-sensitive isolates from 23 provinces in China. Seven loci associated with drug resistance, including rpoB for rifampin (RIF), katG, inhA and oxyR-ahpC for isoniazid (INH), gyrA and gyrB for ofloxacin (OFX), and rrs for kanmycin (KAN), were examined by DNA sequencing. Compared with the phenotypic data, the sensitivity and specificity for DNA sequencing were 91.1% and 98.4% for RIF, 80.2% and 98.4% for INH, 72.2% and 98.3% for OFX and 40% and 98.2% for KAN, respectively. The most common mutations found in RIF, INH, OFX and KAN resistance were Ser531Leu (48.2%) in rpoB, Ser315Thr (49.8%) in katG, C(-15)T (10.5%) in inhA, Asp94Gly (20.3%), Asp94Ala (12.7%) and Ala90Val (21.5%) in gyrA, and A1401G (40%) in rrs. This molecular information will be helpful to establish new molecular biology-based methods for diagnosing multi- and extensively drug-resistant tuberculosis in China. PMID:26486879

  3. Increased frequency of in vivo hprt gene-mutated T cells in the peripheral blood of patients with systemic sclerosis.

    PubMed Central

    Sfikakis, P P; Tesar, J; Theocharis, S; Klipple, G L; Tsokos, G C

    1994-01-01

    OBJECTIVE--Activated T lymphocytes are involved in the pathogenesis of scleroderma (systemic sclerosis, SSc); such cells rapidly divide in vivo and are thus theoretically subject to random mutation more frequently than resting cells. To study whether SSc is associated with rapidly expanding T cell clones the frequency was determined of in vivo mutated T cells (MF) at the hypoxanthine guanine phosphoribosyl transferase (hprt) gene in the peripheral blood from patients with SSc. Specific clinical or serological associations were also investigated. METHODS--Peripheral blood lymphocytes from 16 healthy individuals and 20 patients with SSc were cultured using an hprt clonal assay; mutated and wild T cell clones were established to assess individual values of T cell MF. T cell clones were further expanded in vitro and their phenotype was determined by standard immunofluorescence technique. Enzyme-linked immunosorbent assays were used for simultaneous measurements of plasma levels of soluble Interleukin-2 receptors (s-IL-2R) and Intercellular adhesion molecule-1 (s-ICAM-1). RESULT--Mean (SD) value of T cell MF in patients with SSc was 2.5-fold higher than the normal mean (SD) value [10.6 (6.6) x 10(-6) v [4.4 (2.8) x 10(-6), p = 0.0007]. Eleven of 20 patients with SSc (55%) had T cell MF values greater than two SD above the normal mean value. The majority (84%) of mutated T cells had a helper/inducer, memory phenotype while 12% were cytotoxic/suppressor T cells. There was no association between T cell MF and the extent of skin involvement or the duration of Raynaud's phenomenon. High individual T cell MF values were not related to a possible concurrent immune overactivity as assessed by plasma levels of s-IL-2R and s-ICAM-1. Patients with long standing skin disease, however, had almost double T cell MF values than patients with early skin disease [(13.6 (7.4)) x 10(-6) v (7.5 (4.3)) x 10(-6), p = 0.03], suggesting that increased T cell MF in SSc may reflect an ongoing

  4. Specific inhibition of barley alpha-amylase 2 by barley alpha-amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation.

    PubMed

    Rodenburg, K W; Vallée, F; Juge, N; Aghajari, N; Guo, X; Haser, R; Svensson, B

    2000-02-01

    alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nM. AMY2 is a (beta/alpha)8-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-strand 3 of the (beta/alpha)8-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2-BASI crystal structure [F. Vallée, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, B. Svensson & R. Haser (1998) Structure 6, 649-659] Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt-bridge with Lys140BASI. These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor. These mutations were performed in the well-expressed, inhibitor-sensitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with Ki = 0.33 nM, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403). The binding energy was reduced by 2.7-3.0 kcal.mol-1 as determined from Ki after the mutations R128Q and D142N. This corresponds to loss of a charged interaction between the protein molecules. In contrast, sensitivity to the inhibitor was gained (Ki = 7 microM) by the mutation T129R/K130P in the insensitive isozyme hybrid. Charge screening raised Ki 14-20-fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme-inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2-BASI complex. PMID:10672010

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

    PubMed Central

    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. PMID:26973671

  6. 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

  7. Recent Mitochondrial DNA Mutations Increase the Risk of Developing Common Late-Onset Human Diseases

    PubMed Central

    Hudson, Gavin; Gomez-Duran, Aurora; Wilson, Ian J.; Chinnery, Patrick F.

    2014-01-01

    Mitochondrial DNA (mtDNA) is highly polymorphic at the population level, and specific mtDNA variants affect mitochondrial function. With emerging evidence that mitochondrial mechanisms are central to common human diseases, it is plausible that mtDNA variants contribute to the “missing heritability” of several complex traits. Given the central role of mtDNA genes in oxidative phosphorylation, the same genetic variants would be expected to alter the risk of developing several different disorders, but this has not been shown to date. Here we studied 38,638 individuals with 11 major diseases, and 17,483 healthy controls. Imputing missing variants from 7,729 complete mitochondrial genomes, we captured 40.41% of European mtDNA variation. We show that mtDNA variants modifying the risk of developing one disease also modify the risk of developing other diseases, thus providing independent replication of a disease association in different case and control cohorts. High-risk alleles were more common than protective alleles, indicating that mtDNA is not at equilibrium in the human population, and that recent mutations interact with nuclear loci to modify the risk of developing multiple common diseases. PMID:24852434

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

    PubMed

    Li, Ting; Wong, Vincent Kam Wai; 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

    2015-10-13

    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-β C46A transgenic mouse model. We show that a novel IKK-β inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-β C46A 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

  9. A missense variant in FGD6 confers increased risk of polypoidal choroidal vasculopathy.

    PubMed

    Huang, Lulin; Zhang, Houbin; Cheng, Ching-Yu; Wen, Feng; Tam, Pancy O S; Zhao, Peiquan; Chen, Haoyu; Li, Zheng; Chen, Lijia; Tai, Zhengfu; Yamashiro, Kenji; Deng, Shaoping; Zhu, Xianjun; Chen, Weiqi; Cai, Li; Lu, Fang; Li, Yuanfeng; Cheung, Chui-Ming G; Shi, Yi; Miyake, Masahiro; Lin, Yin; Gong, Bo; Liu, Xiaoqi; Sim, Kar-Seng; Yang, Jiyun; Mori, Keisuke; Zhang, Xiongzhe; Cackett, Peter D; Tsujikawa, Motokazu; Nishida, Kohji; Hao, Fang; Ma, Shi; Lin, He; Cheng, Jing; Fei, Ping; Lai, Timothy Y Y; Tang, Sibo; Laude, Augustinus; Inoue, Satoshi; Yeo, Ian Y; Sakurada, Yoichi; Zhou, Yu; Iijima, Hiroyuki; Honda, Shigeru; Lei, Chuntao; Zhang, Lin; Zheng, Hong; Jiang, Dan; Zhu, Xiong; Wong, Tien-Ying; Khor, Chiea-Chuen; Pang, Chi-Pui; Yoshimura, Nagahisa; Yang, Zhenglin

    2016-06-01

    Polypoidal choroidal vasculopathy (PCV), a subtype of 'wet' age-related macular degeneration (AMD), constitutes up to 55% of cases of wet AMD in Asian patients. In contrast to the choroidal neovascularization (CNV) subtype, the genetic risk factors for PCV are relatively unknown. Exome sequencing analysis of a Han Chinese cohort followed by replication in four independent cohorts identified a rare c.986A>G (p.Lys329Arg) variant in the FGD6 gene as significantly associated with PCV (P = 2.19 × 10(-16), odds ratio (OR) = 2.12) but not with CNV (P = 0.26, OR = 1.13). The intracellular localization of FGD6-Arg329 is distinct from that of FGD6-Lys329. In vitro, FGD6 could regulate proangiogenic activity, and oxidized phospholipids increased expression of FGD6. FGD6-Arg329 promoted more abnormal vessel development in the mouse retina than FGD6-Lys329. Collectively, our data suggest that oxidized phospholipids and FGD6-Arg329 might act synergistically to increase susceptibility to PCV. PMID:27089177

  10. Increased Susceptibility to Skin Carcinogenesis Associated with a Spontaneous Mouse Mutation in the Palmitoyl Transferase Zdhhc13 Gene.

    PubMed

    Perez, Carlos J; Mecklenburg, Lars; Jaubert, Jean; Martinez-Santamaria, Lucia; Iritani, Brian M; Espejo, Alexsandra; Napoli, Eleonora; Song, Gyu; del Río, Marcela; DiGiovanni, John; Giulivi, Cecilia; Bedford, Mark T; Dent, Sharon Y R; Wood, Richard D; Kusewitt, Donna F; Guénet, Jean-Louis; Conti, Claudio J; Benavides, Fernando

    2015-12-01

    Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis, and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared with wild-type (WT) epidermis in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than WT littermates. To our knowledge, this is the first report of a protective role for PAT in skin carcinogenesis. PMID:26288350

  11. Increased Susceptibility to Skin Carcinogenesis Associated with a Spontaneous Mouse Mutation in the Palmitoyl Transferase Zdhhc13 gene

    PubMed Central

    Perez, Carlos J.; Mecklenburg, Lars; Jaubert, Jean; Santamaria, Lucia Martinez; Iritani, Brian M.; Espejo, Alexsandra; Napoli, Eleonora; Song, Gyu; del Río, Marcela; DiGiovanni, John; Giulivi, Cecilia; Bedford, Mark T.; Dent, Sharon Y.R.; Wood, Richard D.; Kusewitt, Donna F.; Guénet, Jean Louis; Conti, Claudio J.; Benavides, Fernando

    2016-01-01

    Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared to wild-type epidermis, in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than wild-type littermates. To our knowledge, this is the first report of a protective role for a PAT in skin carcinogenesis. PMID:26288350

  12. 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. PMID:27335141

  13. Caveolin-1 (P132L), a Common Breast Cancer Mutation, Confers Mammary Cell Invasiveness and Defines a Novel Stem Cell/Metastasis-Associated Gene Signature

    PubMed Central

    Bonuccelli, Gloria; Casimiro, Mathew C.; Sotgia, Federica; Wang, Chenguang; Liu, Manran; Katiyar, Sanjay; Zhou, Jie; Dew, Elliott; Capozza, Franco; Daumer, Kristin M.; Minetti, Carlo; Milliman, Janet N.; Alpy, Fabien; Rio, Marie-Christine; Tomasetto, Catherine; Mercier, Isabelle; Flomenberg, Neal; Frank, Philippe G.; Pestell, Richard G.; Lisanti, Michael P.

    2009-01-01

    Here we used the Met-1 cell line in an orthotopic transplantation model in FVB/N mice to dissect the role of the Cav-1(P132L) mutation in human breast cancer. Identical experiments were performed in parallel with wild-type Cav-1. Cav-1(P132L) up-regulated the expression of estrogen receptor-α as predicted, because only estrogen receptor-α-positive patients have been shown to harbor Cav-1(P132L) mutations. In the context of primary tumor formation, Cav-1(P132L) behaved as a loss-of-function mutation, lacking any tumor suppressor activity. In contrast, Cav-1(P132L) caused significant increases in cell migration, invasion, and experimental metastasis, consistent with a gain-of-function mutation. To identify possible molecular mechanism(s) underlying this invasive gain-of-function activity, we performed unbiased gene expression profiling. From this analysis, we show that the Cav-1(P132L) expression signature contains numerous genes that have been previously associated with cell migration, invasion, and metastasis. These include i) secreted growth factors and extracellular matrix proteins (Cyr61, Plf, Pthlh, Serpinb5, Tnc, and Wnt10a), ii) proteases that generate EGF and HGF (Adamts1 and St14), and iii) tyrosine kinase substrates and integrin signaling/adapter proteins (Akap13, Cdcp1, Ddef1, Eps15, Foxf1a, Gab2, Hs2st1, and Itgb4). Several of the P132L-specific genes are also highly expressed in stem/progenitor cells or are associated with myoepithelial cells, suggestive of an epithelial-mesenchymal transition. These results directly support clinical data showing that patients harboring Cav-1 mutations are more likely to undergo recurrence and metastasis. PMID:19395651

  14. 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

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

    PubMed

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

    2015-07-01

    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

  16. Resistance to the Novel Fungicide Pyrimorph in Phytophthora capsici: Risk Assessment and Detection of Point Mutations in CesA3 That Confer Resistance

    PubMed Central

    Pang, Zhili; Shao, Jingpeng; Chen, Lei; Lu, Xiaohong; Hu, Jian; Qin, Zhaohai; Liu, Xili

    2013-01-01

    Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC50 value of 1.4261 (±0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1×10−4. The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC50 values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations. PMID:23431382

  17. 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

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

    PubMed Central

    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.

    2016-01-01

    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

  19. Role of Different Pfcrt and Pfmdr-1 Mutations in Conferring Resistance to Antimalaria Drugs in Plasmodium falciparum.

    PubMed

    Ibraheem, Zaid O; Abd Majid, R; Noor, S Mohd; Sedik, H Mohd; Basir, R

    2014-01-01

    Emergence of drugs resistant strains of Plasmodium falciparum has augmented the scourge of malaria in endemic areas. Antimalaria drugs act on different intracellular targets. The majority of them interfere with digestive vacuoles (DVs) while others affect other organelles, namely, apicoplast and mitochondria. Prevention of drug accumulation or access into the target site is one of the mechanisms that plasmodium adopts to develop resistance. Plasmodia are endowed with series of transporters that shuffle drugs away from the target site, namely, pfmdr (Plasmodium falciparum multidrug resistance transporter) and pfcrt (Plasmodium falciparum chloroquine resistance transporter) which exist in DV membrane and are considered as putative markers of CQ resistance. They are homologues to human P-glycoproteins (P-gh or multidrug resistance system) and members of drug metabolite transporter (DMT) family, respectively. The former mediates drifting of xenobiotics towards the DV while the latter chucks them outside. Resistance to drugs whose target site of action is intravacuolar develops when the transporters expel them outside the DVs and vice versa for those whose target is extravacuolar. In this review, we are going to summarize the possible pfcrt and pfmdr mutation and their role in changing plasmodium sensitivity to different anti-Plasmodium drugs. PMID:25506039

  20. The growth advantage in stationary-phase phenotype conferred by rpoS mutations is dependent on the pH and nutrient environment.

    PubMed

    Farrell, Michael J; Finkel, Steven E

    2003-12-01

    Escherichia coli cells that are aged in batch culture display an increased fitness referred to as the growth advantage in stationary phase, or GASP, phenotype. A common early adaptation to this culture environment is a mutant rpoS allele, such as rpoS819, that results in attenuated RpoS activity. However, it is important to note that during long-term batch culture, environmental conditions are in flux. To date, most studies of the GASP phenotype have focused on identifying alleles that render an advantage in a specific environment, Luria-Bertani broth (LB) batch culture. To determine what role environmental conditions play in rendering relative fitness advantages to E. coli cells carrying either the wild-type or rpoS819 alleles, we performed competitions under a variety of culture conditions in which either the available nutrients, the pH, or both were manipulated. In LB medium, we found that while the rpoS819 allele confers a strong competitive fitness advantage at basic pH, it confers a reduced advantage under neutral conditions, and it is disadvantageous under acidic conditions. Similar results were found using other media. rpoS819 conferred its greatest advantage in basic minimal medium in which either glucose or Casamino Acids were the sole source of carbon and energy. In acidic medium supplemented with either Casamino Acids or glucose, the wild-type allele conferred a slight advantage. In addition, populations were dynamic under all pH conditions tested, with neither the wild-type nor mutant rpoS alleles sweeping a culture. We also found that the strength of the fitness advantage gained during a 10-day incubation is pH dependent. PMID:14645263

  1. The Growth Advantage in Stationary-Phase Phenotype Conferred by rpoS Mutations Is Dependent on the pH and Nutrient Environment

    PubMed Central

    Farrell, Michael J.; Finkel, Steven E.

    2003-01-01

    Escherichia coli cells that are aged in batch culture display an increased fitness referred to as the growth advantage in stationary phase, or GASP, phenotype. A common early adaptation to this culture environment is a mutant rpoS allele, such as rpoS819, that results in attenuated RpoS activity. However, it is important to note that during long-term batch culture, environmental conditions are in flux. To date, most studies of the GASP phenotype have focused on identifying alleles that render an advantage in a specific environment, Luria-Bertani broth (LB) batch culture. To determine what role environmental conditions play in rendering relative fitness advantages to E. coli cells carrying either the wild-type or rpoS819 alleles, we performed competitions under a variety of culture conditions in which either the available nutrients, the pH, or both were manipulated. In LB medium, we found that while the rpoS819 allele confers a strong competitive fitness advantage at basic pH, it confers a reduced advantage under neutral conditions, and it is disadvantageous under acidic conditions. Similar results were found using other media. rpoS819 conferred its greatest advantage in basic minimal medium in which either glucose or Casamino Acids were the sole source of carbon and energy. In acidic medium supplemented with either Casamino Acids or glucose, the wild-type allele conferred a slight advantage. In addition, populations were dynamic under all pH conditions tested, with neither the wild-type nor mutant rpoS alleles sweeping a culture. We also found that the strength of the fitness advantage gained during a 10-day incubation is pH dependent. PMID:14645263

  2. 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

  3. Selected cysteine point mutations confer mercurial sensitivity to the mercurial-insensitive water channel MIWC/AQP-4.

    PubMed

    Shi, L B; Verkman, A S

    1996-01-16

    The mercurial-insensitive water channel (MIWC or AQP-4) is a 30-32 kDA integral membrane protein expressed widely in fluid-transporting epithelia [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500]. To investigate the mercurial insensitivity and key residues involved in MIWC-mediated water transport, amino acids just proximal to the conserved NPA motifs (residues 69-74 and 187-190) were mutated individually to cysteine. Complementary RNAs were expressed in Xenopus oocytes for assay of osmotic water permeability (Pf) and HgCl2 inhibition dose-response. Oocytes expressing the cysteine mutants were highly water permeable, with Pf values (24-33 x 10(-3) cm/s) not different from that of wild-type (WT) MIWC. Pf was reversibly inhibited by HgCl2 in mutants S70C, G71C, G72C, H73C, and S189C but insensitive to HgCl2 in the other mutants. K1/2 values for 50% inhibition of Pf by HgCl2 were as follows (in millimolar): 0.40 (S70C), 0.36 (G71C), 0.14 (G72C), 0.45 (H73C), 0.24 (S189C), and > 1 for WT MIWC and the other mutants. To test the hypothesis that these residues are near the MIWC aqueous pore, residues 72 and 188 were mutated individually to the larger amino acid tryptophan. Pf in oocytes expressing mutants G72W or A188W (1.3-1.4 x 10(-3) cm/s) was not greater than that in water-injected oocytes even though these proteins were expressed at the oocyte plasma membrane as shown by quantitative immunofluorescence. Coinjection of cRNAs encoding WT MIWC and G72W or A188W indicated a dominant negative effect; Pf (x 10(-3) cm/s) was 22 (0.25 ng of WT), 10 (0.25 ng of WT + 0.25 ng of G72W), and 12 (0.25 ng of WT + 0.25 ng of A188W). Taken together, these results suggest the MIWC is mercurial-insensitive because of absence of a cysteine residue near the NPA motifs and that residues 70-73 and 189 are located at or near the MIWC aqueous pore. In contrast to previous data for the channel-forming integral protein of 28kDa (CHIP28), the finding of a dominant negative phenotype for

  4. Rapid, high-throughput, multiplex, real-time PCR for identification of mutations in the cyp51A gene of Aspergillus fumigatus that confer resistance to itraconazole.

    PubMed

    Balashov, Sergey V; Gardiner, Rebecca; Park, Steven; Perlin, David S

    2005-01-01

    Aspergillus fumigatus is an important cause of life-threatening invasive fungal disease in patients with compromised immune systems. Resistance to itraconazole in A. fumigatus is closely linked to amino acid substitutions in Cyp51A that replace Gly54. In an effort to develop a new class of molecular diagnostic assay that can rapidly assess drug resistance, a multiplexed assay was established. This assay uses molecular beacons corresponding to the wild-type cyp51A gene and seven mutant alleles encoding either Arg54, Lys54, Val54, Trp54, or Glu54. Molecular beacon structure design and real-time PCR conditions were optimized to increase the assay specificity. The multiplex assay was applied to the analysis of chromosomal DNA samples from a collection of 48 A. fumigatus clinical and laboratory-derived isolates, most with reduced susceptibility to itraconazole. The cyp51A allelic identities for codon 54 were established for all of the strains tested, and mutations altering Gly54 in 23 strains were revealed. These mutations included G(54)W (n = 1), G(54)E (n = 12), G(54)K (n = 3), G(54)R (n = 3), and G(54)V (n = 4). Molecular beacon assay results were confirmed by DNA sequencing. Multiplex real-time PCR with molecular beacons is a powerful technique for allele differentiation and analysis of resistance mutations that is dynamic and suitable for rapid high-throughput assessment of drug resistance. PMID:15634974

  5. 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; Ruas-Madiedo, Patricia

    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

  6. Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

    PubMed Central

    Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

    1998-01-01

    The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding

  7. Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent

    PubMed Central

    Liu, Ying; Pike, Malcolm C.; Wu, Nancy; Lin, Yvonne G.; Mucowski, Sara; Punj, Vasu; Tang, Yuan; Yen, Hai-Yun; Stanczyk, Frank Z.; Enbom, Elena; Austria, Theresa; Widschwendter, Martin; Maxson, Robert; Dubeau, Louis

    2015-01-01

    We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type Brca1 to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional Brca1 gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that Brca1-/- female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by Brca1-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers. PMID:26488398

  8. Mutational anatomy of an HIV-1 protease variant conferring cross-resistance to protease inhibitors in clinical trials. Compensatory modulations of binding and activity.

    PubMed

    Schock, H B; Garsky, V M; Kuo, L C

    1996-12-13

    Site-specific substitutions of as few as four amino acids (M46I/L63P/V82T/I84V) of the human immunodeficiency virus type 1 (HIV-1) protease engenders cross-resistance to a panel of protease inhibitors that are either in clinical trials or have recently been approved for HIV therapy (Condra, J. H., Schleif, W. A., Blahy, O. M. , Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). These four substitutions are among the prominent mutations found in primary HIV isolates obtained from patients undergoing therapy with several protease inhibitors. Two of these mutations (V82T/I84V) are located in, while the other two (M46I/L63P) are away from, the binding cleft of the enzyme. The functional role of these mutations has now been delineated in terms of their influence on the binding affinity and catalytic efficiency of the protease. We have found that the double substitutions of M46I and L63P do not affect binding but instead endow the enzyme with a catalytic efficiency significantly exceeding (110-360%) that of the wild-type enzyme. In contrast, the double substitutions of V82T and I84V are detrimental to the ability of the protease to bind and, thereby, to catalyze. When combined, the four amino acid replacements institute in the protease resistance against inhibitors and a significantly higher catalytic activity than one containing only mutations in its active site. The results suggest that in raising drug resistance, these four site-specific mutations of the protease are compensatory in function; those in the active site diminish equilibrium binding (by increasing Ki), and those away from the active site enhance catalysis (by increasing kcat/KM). This conclusion is further supported by energy estimates in that the Gibbs free energies of binding and catalysis for the quadruple mutant are quantitatively

  9. 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. PMID:26234742

  10. Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis.

    PubMed

    Li, Zhao; Zhou, Miaoping; Zhang, Zengyan; Ren, Lijuan; Du, Lipu; Zhang, Boqiao; Xu, Huijun; Xin, Zhiyong

    2011-03-01

    Fusarium head blight (scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat (Triticum aestivum L.) worldwide. Wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, is one of the major diseases of wheat in China. The defensin RsAFP2, a small cyteine-rich antifungal protein from radish (Raphanus sativus), was shown to inhibit growth in vitro of agronomically important fungal pathogens, such as F. graminearum and R. cerealis. The RsAFP2 gene was transformed into Chinese wheat variety Yangmai 12 via biolistic bombardment to assess the effectiveness of the defensin in protecting wheat from the fungal pathogens in multiple locations and years. The genomic PCR and Southern blot analyses indicated that RsAFP2 was integrated into the genomes of the transgenic wheat lines and heritable. RT-PCR and Western blot proved that the RsAFP2 was expressed in these transgenic wheat lines. Disease tests showed that four RsAFP2 transgenic lines (RA1-RA4) displayed enhanced resistance to F. graminearum compared to the untransformed Yangmai 12 and the null-segregated plants. Assays on Q-RT-PCR and disease severity showed that the express level of RsAFP2 was associated with the enhanced resistance degree. Two of these transgenic lines (RA1 and RA2) also exhibited enhanced resistance to R. cerealis. These results indicated that the expression of RsAFP2 conferred increased resistance to F. graminearum and R. cerealis in transgenic wheat. PMID:21279533

  11. A novel recurrent CHEK2 Y390C mutation identified in high-risk Chinese breast cancer patients impairs its activity and is associated with increased breast cancer risk.

    PubMed

    Wang, N; Ding, H; Liu, C; Li, X; Wei, L; Yu, J; Liu, M; Ying, M; Gao, W; Jiang, H; Wang, Y

    2015-10-01

    Certain predisposition factors such as BRCA1/2 and CHEK2 mutations cause familial breast cancers that occur early. In China, breast cancers are diagnosed at relatively younger age, and higher percentage of patients are diagnosed before 40 years, than that in Caucasians. However, the prevalence for BRCA1/2 mutations and reported CHEK2 germline mutations is much lower or absent in Chinese population, arguing for the need to study other novel risk alleles among Chinese breast cancer patients. In this study, we searched for CHEK2 mutations in young, high-risk breast cancer patients in China and detected a missense variant Y390C (1169A > G) in 12 of 150 patients (8.0%) and 2 in 250 healthy controls (0.8%, P = 0.0002). Four of the Y390C carriers have family history of breast and/or ovarian cancer. In patients without family history, Y390C carriers tend to develop breast cancer early, before 35 years of age. The codon change at Y390, a highly conserved residue located in CHEK2's kinase domain, appeared to significantly impair CHEK2 activity. Functional analysis suggested that the CHEK2 Y390C mutation is deleterious as judged by the mutant protein's inability to inactivate CDC25A or to activate p53 after DNA damage. Cells expressing the CHEK2 Y390C variant showed impaired p21 and Puma expression after DNA damage, and the deregulated cell cycle checkpoint and apoptotic response may help conserve mutations and therefore contribute to tumorigeneisis. Taken together, our results not only identified a novel CHEK2 allele that is associated with cancer families and confers increased breast cancer risk, but also showed that this allele significantly impairs CHEK2 function during DNA damage response. Our results provide further insight on how the function of such an important cancer gene may be impaired by existing mutations to facilitate tumorigenesis. It also offers a new subject for breast cancer monitoring, prevention and management. PMID:25619829

  12. Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations.

    PubMed

    Wanngren, Johanna; Lara, Patricia; Ojemalm, Karin; Maioli, Silvia; Moradi, Nasim; Chen, Lu; Tjernberg, Lars O; Lundkvist, Johan; Nilsson, IngMarie; Karlström, Helena

    2014-01-01

    The enzyme complex γ-secretase generates amyloid β-peptide (Aβ), a 37-43-residue peptide associated with Alzheimer disease (AD). Mutations in presenilin 1 (PS1), the catalytical subunit of γ-secretase, result in familial AD (FAD). A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio), but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP) processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD) formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity. PMID:24918054

  13. Characterization of pbt genes conferring increased Pb2+ and Cd2+ tolerance upon Achromobacter xylosoxidans A8.

    PubMed

    Hložková, Kateřina; Suman, Jáchym; Strnad, Hynek; Ruml, Tomas; Paces, Vaclav; Kotrba, Pavel

    2013-12-01

    The cluster of pbtTFYRABC genes is carried by plasmid pA81. Its elimination from Achromobacter xylosoxidans A8 resulted in increased sensitivity towards Pb(2+) and Cd(2+). Predicted pbtTRABC products share strong similarities with Pb(2+) uptake transporter PbrT, transcriptional regulator PbrR, metal efflux P1-ATPases PbrA and CadA, undecaprenyl pyrophosphatase PbrB and its signal peptidase PbrC from Cupriavidus metallidurans CH34. Expression of pbtABC or pbtA in a metal-sensitive Escherichia coli GG48 rendered the strain Pb(2+)-, Cd(2+)- and Zn(2+)-tolerant and caused decreased accumulation of the metal ions. Accumulation of Pb(2+), but not of Cd(2+) or Zn(2+), was promoted in E. coli expressing pbtT. Additional genes of the pbt cluster are pbtF and pbtY, which encode the cation diffusion facilitator (CDF)-like transporter and a putative fatty acid hydroxylase of unknown function, respectively. Expression of pbtF did not confer increased metal tolerance upon E. coli GG48, although the protein showed measurable Pb(2+)-efflux activity. Unlike the pbtT promoter, promoters of pbtABC, pbtF and pbtY contain features characteristic of promoters controlled by metal-responsive transcriptional regulators of the MerR family. Upregulation of pbtABC, pbtF and pbtY upon Pb(2+), Cd(2+) and Zn(2+) exposure was confirmed in wild-type Achromobacter xylosoxidans A8. Gel shift assays proved binding of purified PbtR to the respective promoters. PMID:24125695

  14. Increased Potassium Absorption Confers Resistance to Group IA Cations in Rubidium-Selected Suspension Cells of Brassica napus1

    PubMed Central

    Lefebvre, Daniel D.

    1989-01-01

    Cell lines of suspension cultures of Brassica napus cv. Jet Neuf were identified for their ability to tolerate 100 millimolar Rb+, a level which was double the normally lethal concentration. Ten spontaneous isolates were obtained from approximately 5 × 107 cells, one of which was reestablished as a cell suspension. This cell line, JL5, was also resistant to the other group IA cations— Li+, Na+, K+, and Cs+—and this trait was stable for at least 30 cell generations in the absence of Rb+ selection pressure. The growth characteristics were similar to those of sensitive cells under nonselective conditions. The selected JL5 cells were shown by analysis to have effected more net accumulation of K+ and Rb+ and less of Na+ than did the unselected cells. JL5 and unselected cells after 14 days of culture in basal medium contained 597.2 and 258.2 micromoles of K per gram dry weight, respectively. Michaelis-Menten kinetic analysis of K+ influx showed that JL5 possessed an elevated phase 1 Vmax, but there was no alteration in its Km. This is the first time that a plant mutation has been shown to have both increased influx and net absorption of a major essential cation. Images Figure 1 PMID:16667201

  15. 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.

  16. A Single Mutation at PB1 Residue 319 Dramatically Increases the Safety of PR8 Live Attenuated Influenza Vaccine in a Murine Model without Compromising Vaccine Efficacy

    PubMed Central

    Cox, Andrew

    2015-01-01

    The live attenuated influenza vaccine (LAIV) is preferentially recommended for use in most children yet remains unsafe for the groups most at risk. Here we have improved the safety of a mouse-adapted live attenuated influenza vaccine containing the same attenuating amino acid mutations as in human LAIV by adding an additional mutation at PB1 residue 319. This results in a vaccine with a 20-fold decrease in protective efficacy and a 10,000-fold increase in safety. PMID:26676793

  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. PMID:25854174

  18. 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

  19. Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

    PubMed

    Messer, Andrew E; Bayliss, Christopher R; El-Mezgueldi, Mohammed; Redwood, Charles S; Ward, Douglas G; Leung, Man-Ching; Papadaki, Maria; Dos Remedios, Cristobal; Marston, Steven B

    2016-07-01

    We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca(2+)-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca(2+)-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca(2+)-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca(2+)-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca(2+)-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential. PMID:27036851

  20. Mutations in the Cytoplasmic Domain of the Newcastle Disease Virus Fusion Protein Confer Hyperfusogenic Phenotypes Modulating Viral Replication and Pathogenicity

    PubMed Central

    Samal, Sweety; Khattar, Sunil K.; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L.

    2013-01-01

    The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis. PMID:23843643

  1. Early Virological Failure in Naive Human Immunodeficiency Virus Patients Receiving Saquinavir (Soft Gel Capsule)-Stavudine-Zalcitabine (MIKADO Trial) Is Not Associated with Mutations Conferring Viral Resistance

    PubMed Central

    Mouroux, M.; Yvon-Groussin, A.; Peytavin, G.; Delaugerre, C.; Legrand, M.; Bossi, P.; Do, B.; Trylesinski, A.; Diquet, B.; Dohin, E.; Delfraissy, J. F.; Katlama, C.; Calvez, V.

    2000-01-01

    The MIKADO trial was designed to evaluate the efficacy of stavudine-zalcitabine-saquinavir (soft gel capsule) [d4T-ddC-SQV(SGC)] in 36 naive patients (−3.3 log10 units at week 24 [W24]). Among the 29 patients remaining on d4T-ddC-SQV(SGC) until W24, 10 harbored a virological failure (viral load of >200 copies/ml at W24) (group 1). To determine the reasons for therapeutic failure, genotypic and phenotypic resistance test results and SQV concentrations in plasma were analyzed and compared to those in successfully treated patients (viral load of <200 copies/ml at W24) (group 2). Reverse transcriptase and protease genotypic analyses in group 1 revealed the acquisition of only one SQV-associated mutation (L90M) in only two patients. There was no significant increase in the 50 or 90% inhibitory concentration of SQV in patients with or without the L90M mutation. However, the fact that two patients developed an L90M mutation only 4 weeks after relapse points to the need for genotypic resistance testing in the context of an initial failure of the antiretroviral regimen. At W24, the median SQV concentration in group 1 (71 ng/ml) was significantly lower than in group 2 (475 ng/ml), and the plasma SQV concentration was correlated with the viral load at W24 (r = −0.5; P < 0.05) and with the drop in viral load between day 0 and W24 (r = −0.5; P < 0.01). These results and the fact that the plasma SQV concentrations in the two groups prior to relapse (W12) were not significantly different strongly suggest that the early failure of this combination is not due to viral resistance but to a lack of compliance, pharmacological variability, and drug interactions or a combination of these factors. PMID:10878071

  2. 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

  3. Amyloid precursor protein mutation E682K at the alternative β-secretase cleavage β′-site increases Aβ generation†

    PubMed Central

    Zhou, Lujia; Brouwers, Nathalie; Benilova, Iryna; Vandersteen, Annelies; Mercken, Marc; Van Laere, Koen; Van Damme, Philip; Demedts, David; Van Leuven, Fred; Sleegers, Kristel; Broersen, Kerensa; Van Broeckhoven, Christine; Vandenberghe, Rik; De Strooper, Bart

    2011-01-01

    BACE1 cleaves the amyloid precursor protein (APP) at the β-cleavage site (Met671–Asp672) to initiate the generation of amyloid peptide Aβ. BACE1 is also known to cleave APP at a much less well-characterized β′-cleavage site (Tyr681–Glu682). We describe here the identification of a novel APP mutation E682K located at this β′-site in an early onset Alzheimer's disease (AD) case. Functional analysis revealed that this E682K mutation blocked the β′-site and shifted cleavage of APP to the β-site, causing increased Aβ production. This work demonstrates the functional importance of APP processing at the β′-site and shows how disruption of the balance between β- and β′-site cleavage may enhance the amyloidogenic processing and consequentially risk for AD. Increasing exon- and exome-based sequencing efforts will identify many more putative pathogenic mutations without conclusive segregation-based evidence in a single family. Our study shows how functional analysis of such mutations allows to determine the potential pathogenic nature of these mutations. We propose to classify the E682K mutation as probable pathogenic awaiting further independent confirmation of its association with AD in other patients. PMID:21500352

  4. Congenital microcornea-cataract syndrome-causing mutation X253R increases βB1-crystallin hydrophobicity to promote aggregate formation.

    PubMed

    Leng, Xiao-Yao; Li, Hai-Yun; Wang, Jing; Qi, Liang-Bo; Xi, Yi-Bo; Yan, Yong-Bin

    2016-07-15

    The high solubility and lifelong stability of crystallins are crucial to the maintenance of lens transparency and optical properties. Numerous crystallin mutations have been linked to congenital cataract, which is one of the leading causes of newborn blindness. Besides cataract, several crystallin mutations have also been linked to syndromes such as congenital microcornea-cataract syndrome (CMCC). However, the molecular mechanism of CMCC caused by crystallin mutations remains elusive. In the present study, we investigated the mechanism of CMCC caused by the X253R mutation in βB1-crystallin. The exogenously expressed X253R proteins were prone to form p62-negative aggregates in HeLa cells, strongly inhibited cell proliferation and induced cell apoptosis. The intracellular X253R aggregates could be successfully redissolved by lanosterol but not cholesterol. The extra 26 residues at the C-terminus of βB1-crystallin introduced by the X253R mutation had little impact on βB1-crystallin structure and stability, but increased βB1-crystallin hydrophobicity and decreased its solubility. Interestingly, the X253R mutant fully abolished the aggregatory propensity of βB1- and βA3/βB1-crystallins at high temperatures, suggesting that X253R was an aggregation-inhibition mutation of β-crystallin homomers and heteromers in dilute solutions. Our results suggest that an increase in hydrophobicity and a decrease in solubility might be responsible for cataractogenesis induced by the X253R mutation, while the cytotoxic effect of X253R aggregates might contribute to the defects in ocular development. Our results also highlight that, at least in some cases, the aggregatory propensity in dilute solutions could not fully mimic the behaviours of mutated proteins in the crowded cytoplasm of the cells. PMID:27208166

  5. 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

  6. 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

  7. A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1

    PubMed Central

    2011-01-01

    Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects. Results SOD1(G93A) mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice). Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A) mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective. Conclusions These findings suggest that the protection against SOD1(G93A) offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons. PMID:21521523

  8. A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens

    PubMed Central

    Veits, Jutta; Tauscher, Kerstin; Ziller, Mario; Teifke, Jens P.; Stech, Jürgen; Mettenleiter, Thomas C.

    2015-01-01

    ABSTRACT In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding

  9. Increased susceptibility to beta-lactam antibiotics and decreased porin content caused by envB mutations of Salmonella typhimurium.

    PubMed Central

    Oppezzo, O J; Avanzati, B; Antón, D N

    1991-01-01

    Isogenic derivatives carrying envB6, envB9, or envB+ alleles were obtained from a strain of Salmonella typhimurium that was partially resistant to mecillinam, a beta-lactam antibiotic specific for penicillin-binding protein 2 (PBP 2). Testing of the isogenic strains with several antibacterial agents demonstrated that envB mutations either increased resistance (mecillinam) or did not affect the response (imipemen) to beta-lactams that act primarily on PBP 2, while susceptibilities to beta-lactams that act on PBP 1B, PBP 3, or both were increased. Furthermore, the susceptibilities of envB strains to hydrophobic compounds such as rifampin, novobiocin, or chloramphenicol were not modified, even though their susceptibilities to deoxycholate and crystal violet were enhanced. Outer cell membranes of envB mutants presented a 50% reduction in protein content compared with that of the isogenic envB+ strains, and OmpF and OmpD porins were particularly affected by the reduction. No alteration in the amount or pattern of periplasmic proteins was noticed, and lipopolysaccharides from envB mutants appeared to be normal by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. By using derivatives that produced a plasmid-encoded beta-lactamase, it was demonstrated that envB cells are slightly less permeable to cephalothin than envB+ bacteria are. It is concluded that the high susceptibility of envB mutants to beta-lactams is due to the increased effectiveness of the antibiotics on PBP 1B, PBP 3, or both. Images PMID:1656857

  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