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Sample records for bcr-abl mutant lacking

  1. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Energy Technology Data Exchange (ETDEWEB)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim; (OHSU- Cancer Instit.); (ARIAD)

    2010-09-07

    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  2. Comparative quantitative analysis of BCR-ABL transcripts with the T315I mutant clone by polymerase chain reaction (PCR)-Invader method.

    Science.gov (United States)

    Tadokoro, Kenichi; Ishikawa, Maho; Suzuki, Makoto; Saito, Tomoyoshi; Suzuki, Yoshie; Yamaguchi, Toshikazu; Yagasaki, Fumiharu

    2011-09-01

    Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts.

  3. Modeling the influence of stromal microenvironment in the selection of ENU-induced BCR-ABL1 mutants by tyrosine kinase inhibitors.

    Science.gov (United States)

    Aggoune, Djamel; Tosca, Lucie; Sorel, Nathalie; Bonnet, Marie-Laure; Dkhissi, Fatima; Tachdjian, Gérard; Bennaceur-Griscelli, Annelise; Chomel, Jean-Claude; Turhan, Ali G

    2014-01-01

    Tyrosine kinase inhibitors (TKIs) have profoundly changed the natural history of chronic myeloid leukemia (CML). However, acquired resistance to imatinib, dasatinib or nilotinib (1(st) and 2(nd) generation TKIs), due in part to BCR-ABL1 kinase mutations, has been largely described. These drugs are ineffective on the T315I gatekeeper substitution, which remains sensitive to 3(rd) generation TKI ponatinib. It has recently been suggested that the hematopoietic niche could protect leukemic cells from targeted therapy. In order to investigate the role of a stromal niche in mutation-related resistance, we developed a niche-based cell mutagenesis assay. For this purpose, ENU (N-ethyl-N-nitrosourea)-exposed UT-7 cells expressing non-mutated or T315I-mutated BCR-ABL1 were cultured with or without murine MS-5 stromal cells and in the presence of imatinib, dasatinib, nilotinib, or ponatinib. In the assays relative to 1(st) and 2(nd) generation TKIs, which were performed on non-mutated BCR-ABL1 cells, our data highlighted the increasing efficacy of the latter, but did not reveal any substantial effect of the niche. In ponatinib assays performed on both non-mutated and T315I-mutated BCR-ABL1 cells, an increased number of resistant clones were observed in the presence of MS-5. Present data suggested that T315I mutants need either compound mutations (e.g. E255K/T315I) or a stromal niche to escape from ponatinib. Using array-comparative genomic hybridization experiments, we found an increased number of variations (involving some recurrent chromosome regions) in clones cultured on MS-5 feeder. Overall, our study suggests that the hematopoietic niche could play a crucial role in conferring resistance to ponatinib, by providing survival signals and favoring genetic instability.

  4. shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance.

    Science.gov (United States)

    Khorashad, Jamshid S; Eiring, Anna M; Mason, Clinton C; Gantz, Kevin C; Bowler, Amber D; Redwine, Hannah M; Yu, Fan; Kraft, Ira L; Pomicter, Anthony D; Reynolds, Kimberly R; Iovino, Anthony J; Zabriskie, Matthew S; Heaton, William L; Tantravahi, Srinivas K; Kauffman, Michael; Shacham, Sharon; Chenchik, Alex; Bonneau, Kyle; Ullman, Katharine S; O'Hare, Thomas; Deininger, Michael W

    2015-03-12

    The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.

  5. A phase 2 study of MK-0457 in patients with BCR-ABL T315I mutant chronic myelogenous leukemia and philadelphia chromosome-positive acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Seymour, J F; Kim, D W; Rubin, E;

    2014-01-01

    Aurora kinase overexpression has been observed in patients with hematologic malignancies. MK-0457, a pan-aurora kinase inhibitor that also inhibits the ABL T315I mutant, was evaluated to treat patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome (Ph+) acute lymphoblastic...

  6. Expression of BCR/ABL p210 from a Knockin Allele Enhances Bone Marrow Engraftment without Inducing Neoplasia

    Directory of Open Access Journals (Sweden)

    Samantha B. Foley

    2013-10-01

    Full Text Available Chronic myeloid leukemia (CML and some acute lymphoblastic leukemias are characterized by the t(9;22 chromosome, which encodes the BCR/ABL oncogene. Multiple mouse models of CML express BCR/ABL at high levels from non-Bcr promoters, resulting in the development of leukemias. In contrast, a significant fraction of healthy humans have been found to have BCR/ABL-positive hematopoietic cells. To bridge the gap between the information derived from current mouse models and nonleukemic humans with the BCR/ABL oncogene, we generated a knockin model with BCR/ABL p210 expressed from the Bcr locus. Unlike previous models, expression of BCR/ABL from the knockin allele did not induce leukemia. BCR/ABL mutant cells did exhibit favorable bone marrow engraftment compared to control cells. These data suggest that BCR/ABL expression alone is insufficient to induce disease. This model allows for inducible spatial and temporal control of BCR/ABL expression for analysis of early steps in the pathogenesis of BCR/ABL-expressing leukemias.

  7. Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

    Science.gov (United States)

    Dixon, Andrew S; Miller, Geoffrey D; Bruno, Benjamin J; Constance, Jonathan E; Woessner, David W; Fidler, Trevor P; Robertson, James C; Cheatham, Thomas E; Lim, Carol S

    2012-01-01

    The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

  8. Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein.

    LENUS (Irish Health Repository)

    Elzinga, Baukje M

    2013-06-01

    Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1\\/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl\\/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.

  9. Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis

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    Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M.; Gish, Gerald D.; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio (AAS); (Mount Sinai Hospital); (Med U. Vienna); (UC); (IMP-CNRS)

    2012-10-25

    Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.

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

    Science.gov (United States)

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

    2014-09-08

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

  11. Oridonin Triggers Chaperon-mediated Proteasomal Degradation of BCR-ABL in Leukemia

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    Huang, Huilin; Weng, Hengyou; Dong, Bowen; Zhao, Panpan; Zhou, Hui; Qu, Lianghu

    2017-01-01

    Inducing degradation of oncoproteins by small molecule compounds has the potential to avoid drug resistance and therefore deserves to be exploited for new therapies. Oridonin is a natural compound with promising antitumor efficacy that can trigger the degradation of oncoproteins; however, the direct cellular targets and underlying mechanisms remain unclear. Here we report that oridonin depletes BCR-ABL through chaperon-mediated proteasomal degradation in leukemia. Mechanistically, oridonin poses oxidative stress in cancer cells and directly binds to cysteines of HSF1, leading to the activation of this master regulator of the chaperone system. The resulting induction of HSP70 and ubiquitin proteins and the enhanced binding to CHIP E3 ligase hence target BCR-ABL for ubiquitin-proteasome degradation. Both wild-type and mutant forms of BCR-ABL can be efficiently degraded by oridonin, supporting its efficacy observed in cultured cells as well as mouse tumor xenograft assays with either imatinib-sensitive or -resistant cells. Collectively, our results identify a novel mechanism by which oridonin induces rapid degradation of BCR-ABL as well as a novel pharmaceutical activator of HSF1 that represents a promising treatment for leukemia. PMID:28128329

  12. Activity of the Aurora kinase inhibitor VX-680 against Bcr/Abl-positive acute lymphoblastic leukemias.

    Science.gov (United States)

    Fei, Fei; Stoddart, Sonia; Groffen, John; Heisterkamp, Nora

    2010-05-01

    The emergence of resistance to tyrosine kinase inhibitors due to point mutations in Bcr/Abl is a challenging problem for Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukemia (ALL) patients, especially for those with the T315I mutation, against which neither nilotinib or dasatinib shows significant activity. VX-680 is a pan-Aurora kinase inhibitor active against all Bcr/Abl proteins but has not been extensively examined in preclinical models of Ph-positive ALL. Here, we have tested VX-680 for the treatment of Bcr/Abl-positive ALL when leukemic cells are protected by the presence of stroma. Under these conditions, VX-680 showed significant effects on primary human Ph-positive ALL cells both with and without the T315I mutation, including ablation of tyrosine phosphorylation downstream of Bcr/Abl, decreased viability, and induction of apoptosis. However, drug treatment of human Ph-positive ALL cells for 3 days followed by drug removal allowed the outgrowth of abnormal cells 21 days later, and on culture of mouse Bcr/Abl ALL cells on stroma with lower concentrations of VX-680, drug-resistant cells emerged. Combined treatment of human ALL cells lacking the T315I mutation with both VX-680 and dasatinib caused significantly more cytotoxicity than each drug alone. We suggest that use of VX-680 together with a second effective drug as first-line treatment for Ph-positive ALL is likely to be safer and more useful than second-line treatment with VX-680 as monotherapy for drug-resistant T315I Ph-positive ALL.

  13. BCR-ABL1 kinase inhibits uracil DNA glycosylase UNG2 to enhance oxidative DNA damage and stimulate genomic instability

    Science.gov (United States)

    Slupianek, Artur; Falinski, Rafal; Znojek, Pawel; Stoklosa, Tomasz; Flis, Sylwia; Doneddu, Valentina; Pytel, Dariusz; Synowiec, Ewelina; Blasiak, Janusz; Bellacosa, Alfonso; Skorski, Tomasz

    2013-01-01

    Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of CML-CP. Unfortunately, 25% of TKI-naive patients and 50–90% of TKI-responding patients carry CML clones expressing TKI resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species (ROS), which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil-DNA glycosylase UNG2 were inhibited in BCR-ABL1 –transformed cell lines and CD34+ CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na+/K+ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI-resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML. PMID:23047475

  14. INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-hong; SUN Bing-zhong; YUAN Yue-chuan

    1999-01-01

    Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of exvivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

  15. The impact of multiple low-level BCR-ABL1 mutations on response to ponatinib

    Science.gov (United States)

    Yeung, David T. O.; Yeoman, Alexandra L.; Altamura, Haley K.; Jamison, Bronte A.; Field, Chani R.; Hodgson, J. Graeme; Lustgarten, Stephanie; Rivera, Victor M.; Hughes, Timothy P.; Branford, Susan

    2016-01-01

    The third-generation tyrosine kinase inhibitor (TKI) ponatinib shows activity against all common BCR-ABL1 single mutants, including the highly resistant BCR-ABL1-T315I mutant, improving outcome for patients with refractory chronic myeloid leukemia (CML). However, responses are variable, and causal baseline factors have not been well-studied. The type and number of low-level BCR-ABL1 mutations present after imatinib resistance has prognostic significance for subsequent treatment with nilotinib or dasatinib as second-line therapy. We therefore investigated the impact of low-level mutations detected by sensitive mass-spectrometry before ponatinib initiation (baseline) on treatment response in 363 TKI-resistant patients enrolled in the PONATINIB for Chronic Myeloid Leukemia Evaluation and Ph+ Acute Lymphoblastic Leukemia trial, including 231 patients in chronic phase (CP-CML). Low-level mutations were detected in 53 patients (15%, including low-level T315I in 14 patients); most, however, did not undergo clonal expansion during ponatinib treatment and, moreover, no specific individual mutations were associated with inferior outcome. We demonstrate however, that the number of mutations detectable by mass spectrometry after TKI resistance is associated with response to ponatinib treatment and could be used to refine the therapeutic approach. Although CP-CML patients with T315I (63/231, 27%) had superior responses overall, those with multiple mutations detectable by mass spectrometry (20, 32%) had substantially inferior responses compared with those with T315I as the sole mutation detected (43, 68%). In contrast, for CP-CML patients without T315I, the inferior responses previously observed with nilotinib/dasatinib therapy for imatinib-resistant patients with multiple mutations were not seen with ponatinib treatment, suggesting that ponatinib may prove to be particularly advantageous for patients with multiple mutations detectable by mass spectrometry after TKI resistance

  16. BCR-ABL DERIVED PEPTIDE VACCINES FOR CHRONIC MYELOID LEUKAEMIA

    Directory of Open Access Journals (Sweden)

    M. Bocchia

    2012-01-01

    Full Text Available Chronic Myeloid Leukemia (CML is a myeloproliferative pluripotent stem cell disorder characterized by the presence of a cytogenetic hallmark, the Philadelphia (Ph chromosome, and accounts for 15% of adult leukemias. The disease progresses from a chronic phase through an accelerated phase to a blast phase and its natural course accounts for a median 4 years survival1. The Ph chromosome is derived by a reciprocal translocation termed t(9;22 in which the c-abl oncogene has moved from chromosome 9 into the breakpoint cluster region (bcr, within the bcr gene on chromosome 22, resulting in a chimeric bcr-abl fusion gene that encodes a 210 KD protein (p210 with constitutive tyrosine kinase activity. Two major alternative chimeric p210 can result from this fusion gene: p210-b2a2 where the junction occurs between bcr exon 2 (b2 and abl exon 2 (a2 and p210-b3a2 where the the junction occurs between bcr exon 3 (b3 and abl exon 2 (a2. About 40% of CML patients harbor the p210-b2a2 and about 60% of them show the p210-b3a2.

  17. BCR-ABL1 kinase inhibits uracil DNA glycosylase UNG2 to enhance oxidative DNA damage and stimulate genomic instability.

    Science.gov (United States)

    Slupianek, A; Falinski, R; Znojek, P; Stoklosa, T; Flis, S; Doneddu, V; Pytel, D; Synowiec, E; Blasiak, J; Bellacosa, A; Skorski, T

    2013-03-01

    Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50-90% of patients developing TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species, which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34(+) CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na(+)/K(+)ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.

  18. Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis

    OpenAIRE

    Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M.; Gish, Gerald D.; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio

    2011-01-01

    Summary Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for hig...

  19. Detection of p190BCR-ABL AND p210BCR-ABL fusion transcripts in patients with chronic myeloid leukemia (CML using qualitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Aya Bonilla, Carlos Alberto

    2014-10-01

    Full Text Available Introduction: Chronic myelogenous leukemia (CML is characterized by the presence of the Philadelphia chromosome (Ph, resulting from the balanced reciprocal translocation t(9;22(q34;q11. This marker chromosome is found less frequently in patients suffering from acute lymphoblastic leukemia. Objective: To determine the frequency of BCR-ABL gene fusions encoding the p210BCR-ABL y p190 BCR-ABL transcripts in Colombian patients diagnosed with CML in different stages of the disease and/or its treatment. Materials and methods: Cross sectional, descriptive study of thirty one CML patients (aged 15-78. Analysis was carried out through qualitative nested PCR for the isoforms P210 BCR-ABL (b3a2 e b2a2 and P190 BCR-ABL (e1a2, and based on peripheral blood samples. Results: In 29 of the 31 patients (93.6% transcript p210BCR-ABL was detected; b2a2 and b3a2 gene fusions and the coexpression b3a2 y b2a2 were identified in 55.2% (16/29, 34.5% (10/29 and 10.3% (3/29 of the cases, respectively. Conclusion: b2a2 gene fusion was the most frequent in this CML population.

  20. Combination of bortezomib and mitotic inhibitors down-modulate Bcr-Abl and efficiently eliminates tyrosine-kinase inhibitor sensitive and resistant Bcr-Abl-positive leukemic cells.

    Science.gov (United States)

    Bucur, Octavian; Stancu, Andreea Lucia; Goganau, Ioana; Petrescu, Stefana Maria; Pennarun, Bodvael; Bertomeu, Thierry; Dewar, Rajan; Khosravi-Far, Roya

    2013-01-01

    Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases.

  1. Structural Mechanism of the Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons for Overcoming Kinase Inhibitor Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Thomas, Mathew; Keats, Jeff; Xu, Qihong; Rivera, Victor M.; Shakespeare, William C.; Clackson, Tim; Dalgarno, David C.; Zhu, Xiaotian (ARIAD)

    2012-01-20

    The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

  2. BCR/ABL stimulates WRN to promote survival and genomic instability

    Science.gov (United States)

    Slupianek, Artur; Poplawski, Tomasz; Jozwiakowski, Stanislaw K.; Cramer, Kimberly; Pytel, Dariusz; Stoczynska, Ewelina; Nowicki, Michal O.; Blasiak, Janusz; Skorski, Tomasz

    2010-01-01

    BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA). Here we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC -mediated activation of transcription and Bcl-xL –dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability. PMID:21123451

  3. BCR/ABL stimulates WRN to promote survival and genomic instability.

    Science.gov (United States)

    Slupianek, Artur; Poplawski, Tomasz; Jozwiakowski, Stanislaw K; Cramer, Kimberly; Pytel, Dariusz; Stoczynska, Ewelina; Nowicki, Michal O; Blasiak, Janusz; Skorski, Tomasz

    2011-02-01

    BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSB) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), nonhomologous end-joining (NHEJ), and single-strand annealing (SSA). Here, we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTK), such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC-mediated activation of transcription and Bcl-xL-dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability.

  4. Outcomes of Children With BCR-ABL1–Like Acute Lymphoblastic Leukemia Treated With Risk-Directed Therapy Based on the Levels of Minimal Residual Disease

    Science.gov (United States)

    Roberts, Kathryn G.; Pei, Deqing; Campana, Dario; Payne-Turner, Debbie; Li, Yongjin; Cheng, Cheng; Sandlund, John T.; Jeha, Sima; Easton, John; Becksfort, Jared; Zhang, Jinghui; Coustan-Smith, Elaine; Raimondi, Susana C.; Leung, Wing H.; Relling, Mary V.; Evans, William E.; Downing, James R.; Mullighan, Charles G.; Pui, Ching-Hon

    2014-01-01

    Purpose BCR-ABL1–like acute lymphoblastic leukemia (ALL) is a recently identified B-cell ALL (B-ALL) subtype with poor outcome that exhibits a gene expression profile similar to BCR-ABL1-positive ALL but lacks the BCR-ABL1 fusion protein. We examined the outcome of children with BCR-ABL1–like ALL treated with risk-directed therapy based on minimal residual disease (MRD) levels during remission induction. Patients and Methods Among 422 patients with B-ALL enrolled onto the Total Therapy XV study between 2000 and 2007, 344 had adequate samples for gene expression profiling. Next-generation sequencing and/or analysis of genes known to be altered in B-ALL were performed in patients with BCR-ABL1–like ALL who had available material. Outcome was compared between patients with and those without BCR-ABL1–like ALL. Results Forty (11.6%) of the 344 patients had BCR-ABL1–like ALL. They were significantly more likely to be male, have Down syndrome, and have higher MRD levels on day 19 and at the end of induction than did other patients with B-ALL. Among 25 patients comprehensively studied for genetic abnormalities, 11 harbored a genomic rearrangement of CRLF2, six had fusion transcripts responsive to ABL tyrosine kinase inhibitors or JAK inhibitors, and seven had mutations involving the Ras signaling pathway. There were no significant differences in event-free survival (90.0% ± 4.7% [SE] v 88.4% ± 1.9% at 5 years; P = .41) or in overall survival (92.5% ± 4.2% v 95.1% ± 1.3% at 5 years; P = .41) between patients with and without BCR-ABL1–like ALL. Conclusion Patients who have BCR-ABL1–like ALL with poor initial treatment response can be salvaged with MRD-based risk-directed therapy and may benefit from identification of kinase-activating lesions for targeted therapies. PMID:25049327

  5. A Requirement for SOCS-1 and SOCS-3 Phosphorylation in Bcr-Abl-Induced Tumorigenesis

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    Xiaoxue Qiu

    2012-06-01

    Full Text Available Suppressors of cytokine signaling 1 and 3 (SOCS-1 and SOCS-3 are inhibitors of the Janus tyrosine kinase (JAK/signal transducers and activators of transcription (STAT pathway and function in a negative feedback loop during cytokine signaling. Abl transformation is associated with constitutive activation of JAK/STAT-dependent signaling. However, the mechanism by which Abl oncoproteins bypass SOCS inhibitory regulation remains poorly defined. Here, we demonstrate that coexpression of Bcr-Abl with SOCS-1 or SOCS-3 results in tyrosine phosphorylation of these SOCS proteins. Interestingly, SOCS-1 is highly tyrosine phosphorylated in one of five primary chronic myelogenous leukemia samples. Bcr-Abl-dependent tyrosine phosphorylation of SOCS-1 and SOCS-3 occurs mainly on Tyr 155 and Tyr 204 residues of SOCS-1 and on Tyr 221 residue of SOCS-3. We observed that phosphorylation of these SOCS proteins was associated with their binding to Bcr-Abl. Bcr-Abl-dependent phosphorylation of SOCS-1 and SOCS-3 diminished their inhibitory effects on the activation of JAK and STAT5 and thereby enhanced JAK/STAT5 signaling. Strikingly, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 impaired the expression of Bcl-XL protein and sensitized K562 leukemic cells to undergo apoptosis. Moreover, selective mutation of tyrosine phosphorylation sites of SOCS-1 or SOCS-3 significantly blocked Bcr-Abl-mediated tumorigenesis in nude mice and inhibited Bcr-Abl-mediated murine bone marrow transformation. Together, these results reveal a mechanism of how Bcr-Abl may overcome SOCS-1 and SOCS-3 inhibition to constitutively activate the JAK/STAT-dependent signaling, and suggest that Bcr-Abl may critically requires tyrosine phosphorylation of SOCS-1 and SOCS-3 to mediate tumorigenesis when these SOCS proteins are present in cells.

  6. A Non-ATP-Competitive Dual Inhibitor of JAK2 and BCR-ABL Kinases: Elucidation of a Novel Therapeutic Spectrum Based on Substrate Competitive Inhibition.

    Science.gov (United States)

    Jatiani, Shashidhar S; Cosenza, Stephen C; Reddy, M V Ramana; Ha, Ji Hee; Baker, Stacey J; Samanta, Ajoy K; Olnes, Matthew J; Pfannes, Loretta; Sloand, Elaine M; Arlinghaus, Ralph B; Reddy, E Premkumar

    2010-04-01

    Here we report the discovery of ON044580, an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases, JAK2 and BCR-ABL, and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non-ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2(V617F)-positive leukemic cells and blocks the IL-3-mediated phosphorylation of JAK2 and STAT5. Interestingly, this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally, ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate, STAT5, and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABL(T315I) and JAK2(V617F). Importantly, ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients, suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS, imatinib-resistant CML, and myeloproliferative neoplasms that develop resistance to ATP-competitive agents.

  7. The Study on BCR/ABL Fusion Gene in Adult Acute Lymphoblastic Leukemia

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to assess the significance of BCR/ABC fusion gene in adult acute lymphoblastic Leukemia (ALL), 28 patients who were diagnosed as ALL were enrolled to detect BCR/ABC gene using nested-RT PCR. The results showed that 9 cases (31.25%) were BCR/ABL positive ,and expressed P210 subtype. A mong them 7 cases were B-ALL, and one was T-ALL. The diagnosis was proved by monoclonal antibodies recognition by indirect immunofluorescence. Adult patients with BCR/ABL positive ALL were significantly older (p<0. 01) and had higher WBC count (p<0. 01) as compared with BCR/ABL-negative patients. There was no significant difference in sex, hemoglobin and splenomegaly between two group (p>0. 05). The induc tion failure rate was high in BCR/ABL positive patients and those who achieved complete remission usually relapsed earlier. In conclusion, adult ALL patients with BCR/ABL-positive have poorer prognosis.

  8. Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission

    Directory of Open Access Journals (Sweden)

    Costa Juliana

    2010-11-01

    Full Text Available Abstract Background The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR has become important to assess minimal residual disease (MRD and standard of care in the treatment of chronic myeloid leukemia (CML. In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP who achieved complete cytogenetic remission (CCyR and major molecular remission (MMR throughout imatinib treatment. Methods The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. Results Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A 57 (62.6% had no variation on sequential analysis; (B 30 (32.9% had a single positive variation result obtained in a single sample; and (C 4 (4.39% had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C, 19 (55.8% had a BCR-ABL/BCR ratio, 13 (38.2% patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5% patients, and none of whom lost CCyR. Conclusions Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of

  9. Frequency of BCR-ABL Transcript Types in Syrian CML Patients

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    Sulaf Farhat-Maghribi

    2016-01-01

    Full Text Available Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs and monitored until complete molecular response is achieved. BCR-ABL mRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of different BCR-ABL transcripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols. Methods. CML patients positive for BCR-ABL transcripts by quantitative RT-PCR were enrolled. BCR-ABL transcript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit. Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%, 3 (14.3%, 2 (9.5%, and 1 (4.8%, respectively. Three samples (14.3% contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P=0.047. Conclusion. It might be advisable to identify the BCR-ABL transcript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods.

  10. Evaluation of Morpholino Antisense Oligos’ Role on BCR-ABL Gene Silencing in the K562 Cell Line

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    Bahman Delalat

    2010-01-01

    Full Text Available Objective: Chronic myeloid leukemia (CML develops when a hematopoietic stem cellacquires the BCR/ABL fusion gene. This causes these transformed hematopoietic cellsto have a greater than normal proliferation rate. Scientists attempt to improve the CMLtreatment process by silencing the BCR/ABL oncogene. In this work, we used morpholinoantisense oligos to silence the BCR/ABL oncogene.Materials and Methods: In this study, the K562 was used as a BCR/ABL fusion-genepositive cell line and the Jurkat cell line as a control. We explored the inhibiting capacityof morpholino antisense oligos in the the expression of the BCR/ABL oncogene andstudied their p210 BCR/ABL suppression, inhibition of cell proliferation and stimulation ofapoptosis in the K562 cells after 24 and 48 hours. Endo-Porter was used for delivery ofmorpholino antisense oligos into cell cytosols. Meanwhile, flow cytometric analysis wasperformed in order to determine the appropriate concentration of morpholino antisenseoligos.Results: Prolonged exposure of the K562 cell line to the morpholino antisense oligostargeted against the BCR-ABL gene showed proliferation inhibition as its main feature.After western blotting, we found that complete silencing of BCR/ABL was achieved, butflow cytometric analysis showed no broad apoptosis.Conclusion: The results indicate that the Morpholino antisense oligo is able to inhibitp210 BCR/ABL; however, it cannot induce broad apoptosis due to co-silencing of BCR.

  11. Allogeneic stem cell transplantation for patients harboring T315I BCR-ABL mutated leukemias

    DEFF Research Database (Denmark)

    Nicolini, Franck Emmanuel; Basak, Grzegorz W; Soverini, Simona;

    2011-01-01

    T315I(+) Philadelphia chromosome-positive leukemias are inherently resistant to all licensed tyrosine kinase inhibitors, and therapeutic options remain limited. We report the outcome of allogeneic stem cell transplantation in 64 patients with documented BCR-ABL(T315I) mutations. Median follow...... myeloid leukemia. The occurrence of chronic GVHD had a positive impact on overall survival (P = .047). Transplant-related mortality rates were low. Multivariate analysis identified only blast phase at transplantation (hazard ratio 3.68, P = .0011) and unrelated stem cell donor (hazard ratio 2.98, P = .011......) as unfavorable factors. We conclude that allogeneic stem cell transplantation represents a valuable therapeutic tool for eligible patients with BCR-ABL(T315I) mutation, a tool that may or may not be replaced by third-generation tyrosine kinase inhibitors....

  12. Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Churchman, Michelle L; Low, Jonathan; Qu, Chunxu; Paietta, Elisabeth M; Kasper, Lawryn H; Chang, Yunchao; Payne-Turner, Debbie; Althoff, Mark J; Song, Guangchun; Chen, Shann-Ching; Ma, Jing; Rusch, Michael; McGoldrick, Dan; Edmonson, Michael; Gupta, Pankaj; Wang, Yong-Dong; Caufield, William; Freeman, Burgess; Li, Lie; Panetta, John C; Baker, Sharyn; Yang, Yung-Li; Roberts, Kathryn G; McCastlain, Kelly; Iacobucci, Ilaria; Peters, Jennifer L; Centonze, Victoria E; Notta, Faiyaz; Dobson, Stephanie M; Zandi, Sasan; Dick, John E; Janke, Laura; Peng, Junmin; Kodali, Kiran; Pagala, Vishwajeeth; Min, Jaeki; Mayasundari, Anand; Williams, Richard T; Willman, Cheryl L; Rowe, Jacob; Luger, Selina; Dickins, Ross A; Guy, R Kiplin; Chen, Taosheng; Mullighan, Charles G

    2015-09-14

    Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

  13. LMC y detección del gen de fusión BCR/ABL

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    C. G. Artigas Allaire

    2006-04-01

    Full Text Available La anormalidad citogenética más común en la leucemia mieloide crónica (LMC es el cromosoma Philadelphia producida por la translocación t(9:22, cuya expresión molecular es el gen de fusión BCR-ABL, que codifica una proteína con actividad tirosinquinasa.

  14. Antileukemia effects of xanthohumol in Bcr/Abl-transformed cells involve nuclear factor-kappaB and p53 modulation.

    Science.gov (United States)

    Monteghirfo, Stefano; Tosetti, Francesca; Ambrosini, Claudia; Stigliani, Sara; Pozzi, Sarah; Frassoni, Francesco; Fassina, Gianfranco; Soverini, Simona; Albini, Adriana; Ferrari, Nicoletta

    2008-09-01

    The oncogenic Bcr-Abl tyrosine kinase activates various signaling pathways including phosphoinositide 3-kinase/Akt and nuclear factor-kappaB that mediate proliferation, transformation, and apoptosis resistance in Bcr-Abl+ myeloid leukemia cells. The hop flavonoid xanthohumol inhibits tumor growth by targeting the nuclear factor-kappaB and Akt pathways and angiogenesis. Here, we show that xanthohumol has in vitro activity against Bcr-Abl+ cells and clinical samples and retained its cytotoxicity when imatinib mesylate-resistant K562 cells were examined. Xanthohumol inhibition of K562 cell viability was associated with induction of apoptosis, increased p21 and p53 expression, and decreased survivin levels. We show that xanthohumol strongly inhibited Bcr-Abl expression at both mRNA and protein levels and show that xanthohumol caused elevation of intracellular reactive oxygen species and that the antioxidant N-acetylcysteine blunted xanthohumol-induced events. Further, we observed that xanthohumol inhibits leukemia cell invasion, metalloprotease production, and adhesion to endothelial cells, potentially preventing in vivo life-threatening complications of leukostasis and tissue infiltration by leukemic cells. As structural mutations and/or gene amplification in Bcr-Abl can circumvent an otherwise potent anticancer drug such as imatinib, targeting Bcr-Abl expression as well as its kinase activity could be a novel additional therapeutic approach for the treatment of Bcr-Abl+ myeloid leukemia.

  15. MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia.

    Science.gov (United States)

    Xishan, Zhu; Ziying, Lin; Jing, Du; Gang, Liu

    2015-01-01

    Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including chronic myeloid leukemia (CML). We found that miR-320a expression was reduced in K562 and in CML cancer stem cells. Moreover, we found that miR-320a inhibited K562 cell migration, invasion, proliferation and promoted apoptosis by targeting BCR/ABL oncogene. As an upstream regulator of BCR/ABL, miR-320a directly targets BCR/ABL. The enhanced expression of miR-320a inhibited the phosphorylation of PI3K, AKT and NF-κB; however, the expression of phosphorylated PI3K, AKT and NF-κB were restored by the overexpression of BCR/ABL. In K562, infected with miR-320a or transfected with SiBCR/ABL, the protein levels of fibronectin, vimentin, and N-cadherin were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-320a-expressing cells was restored to normal levels by the restoration of BCR/ABL expression. Generally speaking, miR-320a acts as a novel tumor suppressor gene in CML and miR-320a can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as CML EMT, by attenuating the expression of BCR/ABL oncogene.

  16. [Standardization of quantitative detection of BCR-ABL gene expression by RQ-PCR in patients with chronic myeloid leukemia in cooperation with European Leukemia Net].

    Science.gov (United States)

    Sacha, Tomasz; Zawada, Magdalena; Czekalska, Sylwia; Florek, Izabela; Mueller, Martin; Gniot, Michał; Jaźwiec, Bozena; Kyrcz-Krzemień, Sławomira; Leszczyńska, Aleksandra; Lewandowski, Krzysztof; Matiakowska, Karolina; Solarska, Iwona; Stokłosa, Tomasz; Skotnicki, Aleksander B

    2010-01-01

    Monitoring of chronic myeloid leukemia treatment efficacy requires very sensitive methods of BCR-ABL gene detection based on polymerase chain reaction (PCR). The lack of comparability of BCR-ABL mRNA quantification results generated by various methodologies in different laboratories was the cause of an international multicenter trial initiation with the participation of 133 laboratories in 24 European countries cooperating within the "EUTOS for CML" project. Pracownia Diagnostyki Molekularnej Kliniki Hematologii is taking part in standardisation rounds organised since 2005. The compatibility of methodology used in Pracownia with European Leukemia Net (ELN) standards was confirmed, and correction factor for the expression of RQ-PCR results in an international scale was calculated. Pracownia was charge by ELN with a task of conducting the standardisation in polish molecular biology laboratories. Test probes were prepared and sent to eight cooperating laboratories. The results obtained in six laboratories were concordant with results from laboratory in Krakow after conversion to international scale, therefore it was possible to calculate individual correction factors. The participation of polish laboratories in international standardization process created the opportunity for unification of BCR-ABL quantification methodologies with recommendations of international experts, and showed that the quality of analyses performed in majority of them was satisfactory enough to calculate correction factor and to express the RQ-PCR results in widely accepted international scale.

  17. Molecular Imaging of Bcr-Abl Phosphokinase in a Xenograft Model*

    OpenAIRE

    Wu, Ji Yuan; David J. Yang; Angelo, Laura S.; Kohanim, Saady; Kurzrock, Razelle

    2009-01-01

    The purpose of this study was to determine whether the Bcr-Abl tyrosine kinase can be assessed by gamma imaging using an 111Indium-labeled anti-phosphotyrosine antibody, and if the response to treatment with imatinib could be detected using this imaging technique. Anti-phosphotyrosine antibody (APT) was labeled with indium (111In) using ethylenedicysteine (EC) as a chelator. To determine if 111In-EC-APT could assess a non-receptor tyrosine kinase, xenografts of the human chronic myelogenous l...

  18. Functionally deregulated AML1/RUNX1 cooperates with BCR-ABL to induce a blastic phase-like phenotype of chronic myelogenous leukemia in mice.

    Directory of Open Access Journals (Sweden)

    Kiyoko Yamamoto

    Full Text Available Patients in the chronic phase (CP of chronic myelogenous leukemia (CML have been treated successfully following the advent of ABL kinase inhibitors, but once they progress to the blast crisis (BC phase the prognosis becomes dismal. Although mechanisms underlying the progression are largely unknown, recent studies revealed the presence of alterations of key molecules for hematopoiesis, such as AML1/RUNX1. Our analysis of 13 BC cases revealed that three cases had AML1 mutations and the transcript levels of wild-type (wt. AML1 were elevated in BC compared with CP. Functional analysis of representative AML1 mutants using mouse hematopoietic cells revealed the possible contribution of some, but not all, mutants for the BC-phenotype. Specifically, K83Q and R139G, but neither R80C nor D171N mutants, conferred upon BCR-ABL-expressing cells a growth advantage over BCR-ABL-alone control cells in cytokine-free culture, and the cells thus grown killed mice upon intravenous transfer. Unexpectedly, wt.AML1 behaved similarly to K83Q and R139G mutants. In a bone marrow transplantation assay, K83Q and wt.AML1s induced the emergence of blast-like cells. The overall findings suggest the roles of altered functions of AML1 imposed by some, but not all, mutants, and the elevated expression of wt.AML1 for the disease progression of CML.

  19. Janus kinase 2 mutations in cases with BCR-ABL-negative chronic myeloproliferative disorders from Turkey

    Science.gov (United States)

    Yildiz, Ismail; Yokuş, Osman; Gedik, Habip

    2017-01-01

    Objective: We aimed to investigate the frequency of Janus kinase 2 (JAK2) mutations in cases with chronic myeloproliferative disorders (CMDs), and the relationship between the presence of JAK2 mutation and leukocytosis and splenomegaly, retrospectively. Materials and Methods: Patients, who were diagnosed with BCR-ABL-negative CMDs according to diagnosis criteria of the World Health Organization and followed up at the hematology clinic between 2013 and 2015, were investigated in terms of the frequency of JAK2 mutation in cases with CMDs, and the relationship between the presence of JAK2 mutation and leukocytosis and splenomegaly, retrospectively. Results: In total, 100 patients, who were diagnosed with BCR-ABL-negative CMDs, were evaluated retrospectively. The mean age of the patients with JAK2 positivity was significantly higher compared to patients with negative. JAK2-positivity rates in the age groups were significantly different. Gender, diagnosis, splenomegaly, and leukocytosis were not statistically different for JAK2 positivity between the groups. Conclusion: JAK2 V617F mutation is more commonly seen in older age as a risk for complications related to CDMS. Splenomegaly and leukocytosis are not associated with JAK2 V617F mutation. PMID:28182037

  20. Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia.

    Science.gov (United States)

    Trino, Stefania; De Luca, Luciana; Simeon, Vittorio; Laurenzana, Ilaria; Morano, Annalisa; Caivano, Antonella; La Rocca, Francesco; Pietrantuono, Giuseppe; Bianchino, Gabriella; Grieco, Vitina; Signorino, Elisabetta; Fragasso, Alberto; Bochicchio, Maria Teresa; Venturi, Claudia; Rosti, Gianantonio; Martinelli, Giovanni; Del Vecchio, Luigi; Cilloni, Daniela; Musto, Pellegrino

    2016-01-01

    Endocytosis is the major regulator process of tyrosine kinase receptor (RTK) functional activities. Bridging integrator 1 (BIN1) is a key protein involved in RTK intracellular trafficking. Here, we report, by studying 34 patients with chronic myeloid leukemia (CML) at diagnosis, that BIN1 gene is downregulated in CML as compared to healthy controls, suggesting an altered endocytosis of RTKs. Rab interactor 1 (RIN1), an activator of BIN1, displayed a similar behavior. Treatment of 57 patients by tyrosine kinase inhibitors caused, along with BCR-ABL1 inactivation, an increase of BIN1 and RIN1 expression, potentially restoring endocytosis. There was a significant inverse correlation between BIN1-RIN1 and BCR-ABL1 expression. In vitro experiments on both CML and nontumorigenic cell lines treated with Imatinib confirmed these results. In order to provide another proof in favor of BIN1 and RIN1 endocytosis function in CML, we demonstrated that Imatinib induced, in K562 cell line, BIN1-RIN1 upregulation accompanied by a parallel AXL receptor internalization into cytoplasmic compartment. This study shows a novel deregulated mechanism in CML patients, indicating BIN1 and RIN1 as players in the maintenance of the abnormal RTK signaling in this hematological disease.

  1. Susceptibility of Ph-positive all to TKI therapy associated with Bcr-Abl rearrangement patterns: a retrospective analysis.

    Directory of Open Access Journals (Sweden)

    Yu Jing

    Full Text Available BACKGROUND: Tyrosine kinase inhibitors (TKIs have demonstrated success in the treatment of acute lymphoblastic leukemia (ALL in patients that express BCR-ABL rearrangements (Philadelphia chromosome [Ph]. The current study aimed to assess the efficacy of TKIs and prognostic factors in the treatment of adults with Ph+-ALL. METHODS: In this multicenter retrospective study, the relationship between Ph+-ALL and treatment outcomes among Chinese patients receiving TKI-containing induction/consolidation chemotherapy was examined. A total of 86 Ph+-ALL patients were included and followed for 3.85 (0.43-9.30 years. Overall survival (OS and event-free survival (EFS were analyzed. RESULTS: A total of 86 Ph+-ALL patients (40 females and 46 males; median age: 34.0 years were enrolled, including those with BCR/ABL transcripts 190 (n = 52, 210 (n = 25, and 230 (n = 2; BCR/ABL isoform determination was not available for 7 patients. Mortality was influenced by variable BCR/ABL transcripts and TKI administration, and BCR/ABL transcripts, hematopoietic stem cell transplantation (HSCT, and TKI administration were associated with the occurrence of events. The OS rate in the TKI administration group during steady state was significantly higher compared with those patients who did not receive TKI administration (P = 0.008, the EFS rate in the TKI administration group during steady state was significantly higher compared with those patients who did not receive TKIs (P = 0.012, and also higher than those with TKI salvage administration (P = 0.004. BCR/ABL transcripts 210 showed preferable OS and EFS compared with BCR/ABL transcripts 190 and 230 (P<0.05 for each. CONCLUSIONS: The susceptibility of Ph+-ALL to TKI associated with the patterns of BCR-ABL rearrangement is demonstrated for the first time, thus adding another risk-stratifying molecular prognostic tool for the management of patients with Ph+-ALL.

  2. Autophagy induction by Bcr-Abl-expressing cells facilitates their recovery from a targeted or nontargeted treatment.

    LENUS (Irish Health Repository)

    Crowley, Lisa C

    2012-01-31

    Although Imatinib has transformed the treatment of chronic myeloid leukemia (CML), it is not curative due to the persistence of resistant cells that can regenerate the disease. We have examined how Bcr-Abl-expressing cells respond to two mechanistically different therapeutic agents, etoposide and Imatinib. We also examined Bcr-Abl expression at low and high levels as elevated expression has been associated with treatment failure. Cells expressing low levels of Bcr-Abl undergo apoptosis in response to the DNA-targeting agent (etoposide), whereas high-Bcr-Abl-expressing cells primarily induce autophagy. Autophagic populations engage a delayed nonapoptotic death; however, sufficient cells evade this and repopulate following the withdrawal of the drug. Non-Bcr-Abl-expressing 32D or Ba\\/F3 cells induce both apoptosis and autophagy in response to etoposide and can recover. Imatinib treatment induces both apoptosis and autophagy in all Bcr-Abl-expressing cells and populations rapidly recover. Inhibition of autophagy with ATG7 and Beclin1 siRNA significantly reduced the recovery of Imatinib-treated K562 cells, indicating the importance of autophagy for the recovery of treated cells. Combination regimes incorporating agents that disrupt Imatinib-induced autophagy would remain primarily targeted and may improve response to the treatment in CML.

  3. Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes.

    Science.gov (United States)

    Vinhas, Raquel; Correia, Cláudia; Ribeiro, Patricia; Lourenço, Alexandra; Botelho de Sousa, Aida; Fernandes, Alexandra R; Baptista, Pedro V

    2016-07-01

    Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

  4. Transferred BCR/ABL DNA from K562 extracellular vesicles causes chronic myeloid leukemia in immunodeficient mice.

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    Jin Cai

    Full Text Available Our previous study showed that besides mRNAs and microRNAs, there are DNA fragments within extracellular vesicles (EVs. The BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia (CML, could be transferred from K562 EVs to neutrophils and decrease their phagocytic activity in vitro. Our present study provides evidence that BCR/ABL DNAs transferred from EVs have pathophysiological significance in vivo. Two months after injection of K562 EVs into the tail vein of Sprague-Dawley (SD rats, they showed some characteristics of CML, e.g., feeble, febrile, and thin, with splenomegaly and neutrophilia but with reduced neutrophil phagocytic activity. These findings were also observed in immunodeficient NOD/SCID mice treated with K562 EVs; BCR/ABL mRNA and protein were found in their neutrophils. The administration of actinomycin D, an inhibitor of de novo mRNA synthesis, prevented the abnormalities caused by K562 EVs in NOD/SCID mice related to CML, including neutrophilia and bone marrow hyperplasia. As a specific inhibitor of tyrosine kinases, imatinib blocked the activity of tyrosine kinases and the expression of phospho-Crkl, induced by the de novo BCR/ABL protein caused by K562 EVs bearing BCR/ABL DNA. Our current study shows the pathophysiological significance of transferred tumor gene from EVs in vivo, which may represent an important mechanism for tumorigenesis, tumor progression, and metastasis.

  5. [Guidelines for molecular monitoring of BCR-ABL1 in chronic myeloid leukemia patients by RT-qPCR].

    Science.gov (United States)

    Larripa, Irene; Ruiz, María Sol; Gutiérrez, Marina; Bianchini, Michele

    2017-01-01

    Current clinical guidelines for managing chronic myeloid leukemia include molecular monitoring of BCR-ABL1 transcript quantitative reverse-transcription PCR. Despite the proven prognostic significance of molecular response, it is not widely appreciated that quantitative reverse-transcription PCR potentially produces highly variable data, which may affect the validity of results, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve comparable BCR-ABL1 values is the use of an international reporting scale. Conversion to the international scale is achieved by the application of laboratory specific conversion factor that is obtained by using validated secondary reference calibrators. Moreover, with the aim to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate local laboratory results interpretation and reporting, we decide to prepare laboratory guidelines that will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results, in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs.

  6. Guidelines for molecular monitoring of BCR-ABL1 in chronic myeloid leukemia patients by RT-qPCR

    Directory of Open Access Journals (Sweden)

    Irene Larripa

    2017-02-01

    Full Text Available Current clinical guidelines for managing chronic myeloid leukemia include molecular monitoring of BCR-ABL1 transcript quantitative reverse-transcription PCR. Despite the proven prognostic significance of molecular response, it is not widely appreciated that quantitative reverse-transcription PCR potentially produces highly variable data, which may affect the validity of results, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve comparable BCR-ABL1 values is the use of an international reporting scale. Conversion to the international scale is achieved by the application of laboratory specific conversion factor that is obtained by using validated secondary reference calibrators. Moreover, with the aim to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate local laboratory results interpretation and reporting, we decide to prepare laboratory guidelines that will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results, in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs

  7. Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia.

    Science.gov (United States)

    Zeng, Xian; Zhao, Hui; Li, Yubin; Fan, Jiajun; Sun, Yun; Wang, Shaofei; Wang, Ziyu; Song, Ping; Ju, Dianwen

    2015-01-01

    The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL(+) CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL(+) CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL(+) cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL(+) cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.

  8. A multiple reaction monitoring (MRM method to detect Bcr-Abl kinase activity in CML using a peptide biosensor.

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    Tzu-Yi Yang

    Full Text Available The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML, and is the target of the breakthrough drug imatinib (Gleevec™. While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1-5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient's Bcr-Abl kinase activity (relative to their level at diagnosis is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ~15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.

  9. ON012380: A Non-ATP Competitive Inhibitor of BCR-ABL for the Therapy of Imatinib-Resistant CMLs

    Science.gov (United States)

    2010-05-01

    diverse client proteins. Biopolymers 2009. 41. Tsuruma R, Ohbayashi N, Kamitani S, et al. Physical and functional interactions between STAT3 and...compounds to significantly inhibit the proliferation of K562 and Ba/F3:JAK2V617F cells suggests that this scaffold could be a promising lead for the... scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leuke- mic cells. 2010

  10. Quantification of BCR-ABL mRNA in Plasma/Serum of Patients with Chronic Myelogenous Leukemia

    Directory of Open Access Journals (Sweden)

    Miwako Narita, Anri Saito, Aya Kojima, Minami Iwabuchi, Naoya Satoh, Takayoshi Uchiyama, Akie Yamahira, Tatsuo Furukawa, Hirohito Sone, Masuhiro Takahashi

    2012-01-01

    Full Text Available Quantification of tumor-associated mRNA extracted from blood cells/tissues containing tumor cells is used for evaluation of treatment efficacy or residual tumor cell burden in tumors including leukemia. However, this method using tumor cell-containing blood/tissue is difficult to evaluate the whole tumor cell burden in the body. In order to establish an efficient method to evaluate the whole tumor cell burden in the body, we tried to quantify tumor-associated mRNA existing in plasma/serum instead of leukemia cell-containing blood cells in patients with chronic myelogenous leukemia (CML and compared the levels of BCR-ABL mRNA between plasma/serum and peripheral blood cells. mRNA of BCR-ABL, WT1 or GAPDH (control molecule was detected by real-time RT-PCR using RNA extracted from plasma/serum of almost all the patients with CML. Copy numbers of BCR-ABL mRNA were significantly correlated between plasma/serum and peripheral blood cells. However, levels of BCR-ABL mRNA extracted from serum were low compared with those extracted with peripheral blood cells. The present findings suggest that although real-time RT-PCR of mRNA existing in plasma/serum could be used for evaluating the whole tumor cell burden in the body, it's required to establish an efficient method to quantify plasma/serum mRNA by nature without degrading during the procedure.

  11. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  12. Patan hospital experience in treating philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukemia patients with gleevec (imatinib mesylate; the first generation specific tyrosine kinase inhibitor

    Directory of Open Access Journals (Sweden)

    Zimmerman Mark

    2010-12-01

    Full Text Available Abstract Background Chronic Myeloid Leukemia (CML is caused by the abnormal fusion protein BCR-ABL1, a constitutively active tyrosine kinase and product of the Philadelphia chromosome. Gleevec (Imatinib mesylate is a selective inhibitor of this kinase. Treatment with this agent is known to result in hematologic, cytogenetic, and molecular responses. Patan hospital (Patan, Nepal is one of the Gleevec International Patient Assistance Program (GIPAP centers for patients with CML. Methods A total of 106 Philadelphia positive CML patients were enrolled in our center between Feb 2003 and Jun 2008, and 103 of them were eligible for cytogenetic and/or hematologic response analyses. Results Out of 103 patients, 27% patients underwent cytogenetic analysis. Imatinib induced major cytogenetic responses in 89% and complete hematologic responses in almost 100% of the patients with confirmed CML. After a mean follow up of 27 months, an estimated 90% of the patients on imatinib remained in hematologic remission and more than 90% of the patients are still alive. About 30% of patients developed some form of manageable myelosuppression. A few patients developed non-hematologic toxic side effects such as edema and hepatotoxicity. Conclusions Our study demonstrates that imatinib is safe to use in a developing country. Furthermore, we demonstrate that imatinib is very effective and induced long lasting responses in a high proportion of patients with Ph chromosome/BCR-ABL1 positive CML. Imatinib is well tolerated by our patients. The lack of cytogenetic analysis in the majority of our patients hindered our ability to detect inadequate responses to imatinib and adjust therapy appropriately.

  13. Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance.

    Science.gov (United States)

    Pytel, Dariusz; Wysocki, Tomasz; Majsterek, Ireneusz

    2006-09-01

    The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.

  14. CCR7 is involved in BCR-ABL/STAP-2-mediated cell growth in hematopoietic Ba/F3 cells.

    Science.gov (United States)

    Kubo, Kaori; Iwakami, Masashi; Muromoto, Ryuta; Inagaki, Takuya; Kitai, Yuichi; Kon, Shigeyuki; Sekine, Yuichi; Oritani, Kenji; Matsuda, Tadashi

    2015-08-07

    Chronic myeloid leukemia is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We previously reported that in murine hematopoietic Ba/F3 cells, signal transducing adaptor protein-2 (STAP-2) binds to BCR-ABL and up-regulates BCR-ABL phosphorylation, leading to enhanced activation of its downstream signaling molecules. The binding of STAP-2 to BCR-ABL also influenced the expression levels of chemokine receptors, such as CXCR4 and CCR7. For the induction of CCR7 expression, signals mediated by the MAPK/ERK pathway were critical in Ba/F3 cells expressing BCR-ABL and STAP-2. In addition, STAP-2 cooperated with BCR-ABL to induce the production of CCR7 ligands, CCL19 and CCL21. Our results demonstrate a contribution of CCR7 to STAP-2-dependent enhancement of BCR-ABL-mediated cell growth in Ba/F3 cells.

  15. Fluorescent in-situ hybridization (FISH for BCR/ABL in chronic myeloid leukemia after bone marrow transplantation

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    Maria de Lourdes Lopes Ferrari Chauffaille

    2001-01-01

    Full Text Available CONTEXT: Identification of Philadelphia chromosome or BCR/ABL gene rearrangement in chronic myeloid leukemia is important at diagnosis as well as after treatment. OBJECTIVE: To compare the results of karyotyping using fluorescent in-situ hybridization (FISH upon diagnosis and 1 year after bone marrow transplantation in 12 patients. TYPE OF STUDY: Diagnostic test and residual disease detection. SETTING: Hematology and Hemotherapy Department, Federal University of São Paulo/Escola Paulista de Medicina, São Paulo, Brazil. SAMPLE: 12 patients with chronic myeloid leukemia at diagnosis and 1 year after bone marrow transplantation. DIAGNOSTIC TEST: Karyotyping was done in the usual way and the BCR/ABL gene-specific probe was used for FISH. MAIN MEASUREMENTS: Disease at diagnosis and residual. RESULTS: At diagnosis, 10 patients presented t(9;22(q34.1;q11 as well as positive FISH. Two cases did not have metaphases but FISH was positive. After bone marrow transplantation, 8 patients presented normal karyotype, 1 had persistence of identifiable Philadelphia chromosome and 3 had no metaphases. Two cases showed complete chimera and 2 had donor and host cells simultaneously. FISH was possible in all cases after bone marrow transplantation and confirmed the persistence of identifiable Philadelphia chromosome clone in one patient, and identified another that did not present metaphases for analysis. Cases that showed mixed chimera in karyotype were negative for BCR/ABL by FISH. CONCLUSION: The applicability of FISH is clear, particularly for residual disease detection. Classical and molecular cytogenetics are complementary methods.

  16. Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl

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    Cao Qi

    2010-05-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-κB transcriptional activity, and NF-κB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I. Results In this report, we discovered that pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFα-induced IκBα phosphorylation, translocation of p65, and expression of NF-κB-regulated genes. Pristimerin inhibited two steps in NF-κB signaling: TAK1→IKK and IKK→IκBα. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5 or imatinib-resistant (KBM5-T315I CML cells were reduced after pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFα-induced NF-κB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-κB inactivation and Bcr-Abl inhibition may be parallel independent pathways. Conclusion To our knowledge, this is the first report to show that pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-κB and Bcr-Abl. We concluded that pristimerin could be a lead compound for

  17. Molecular monitoring of BCR-ABL transcripts in patients with chronic myelogenous leukemia: is high sensitivity of clinical value?

    Science.gov (United States)

    Norkin, Maxim; Schiffer, Charles A

    2010-04-01

    Monitoring of disease response during treatment with tyrosine kinase inhibitors of patients with chronic myelogenous leukemia dramatically changed after the introduction of real-time PCR, which allows quantification of BCR-ABL transcript levels with high sensitivity and precision. However, its role in patients who have achieved complete cytogenetic response is not entirely clear; incorrect interpretation of results could lead to unnecessary changes from an effective treatment. This review discusses the current evidence regarding the benefits, uncertainties, and potential drawbacks of molecular monitoring in patients with chronic myelogenous leukemia in chronic phase.

  18. A Non-ATP Competitive Inhibitor of BCR-ABL for the Therapy of Imatinib-Resistant Cmls

    Science.gov (United States)

    2008-05-01

    10% SDS-PAGE and analyzed for indicated proteins by infrared labeled secondary antibodies and scanning with Odyssey scanner ( LiCor Technology). pBcr...proteins by infrared labeled secondary antibodies and scanning with Odyssey scanner ( LiCor Technology). 10 treated 32D/T315I-BCR-ABL cells with increasing...with Odyssey scanner ( LiCor Technology). V G 0.5 1.0 2.5 5.0 0.5 1.0 2.5 5.0 (µM

  19. Papel da P190 BCR-ABL como parâmetro de recaída na leucemia mielóide crônica P190 BCR-ABL role in myeloid chronic leukemia relapse

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    Gabriela V. Andrade

    2008-08-01

    Full Text Available A leucemia mielóide crônica (LMC é uma desordem hematológica mieloproloferativa clonal causada por uma mutação em uma célula-tronco pluripotente, resultando na proliferação e acúmulo de células mielóides e seus progenitores. O cromossomo Philadelphia é o resultado de uma translocação recíproca de material genético entre os genes abl (Abelson murine leukemia no cromossomo 9, e o bcr (breakpoint cluster region no cromossomo 22, resultando na formação do gene quimérico BCR-ABL. Neste trabalho, 45 pacientes foram acompanhados de forma seqüencial, de acordo com as avaliações periódicas individuais, e 360 amostras foram selecionadas e analisadas pela técnica da reação em cadeia da polimerase (PCR através da técnica qualitativa para as isoformas características da P210BCR-ABL (b3a2 e b2a2 e P190BCR-ABL (e1a2. No nosso estudo no pré-TMO, observou-se uma prevalência das isoformas características da LMC (b3a2 e/ou b2a2, fato imprescindível para que o paciente fosse acompanhado dentro do protocolo da LMC. A isoforma e1a2, característica da LLA, foi detectada em 11 pacientes em conjunto com essas isoformas. A detecção do transcrito e1a2 foi avaliada quanto ao seu provável papel na LMC, e foi um dos parâmetros de avaliação deste estudo.Chronic myeloid leukemia (CML is a myeloproliferative clonal disorder caused by a mutation in a stem cell, resulting in the proliferation and accumulation of myeloid cells and their progenitors. The Philadelphia chromosome (Ph1 is a result of a mutual translocation of genetic material between the abl gene (Abelson murine leukemia in chromosome 9 and bcr gene (breakpoint cluster region in chromosome 22, resulting in the formation of the chimerical gene, BCR-ABL. In this work 45 patients were sequentially followed up in individual periodic evaluations, and 360 samples were selected and analyzed by PCR using the qualitative technique for isoforms characteristic of P210CR-ABL (b3a2 and b2a2

  20. Biosensing of BCR/ABL fusion gene using an intensity-interrogation surface plasmon resonance imaging system

    Science.gov (United States)

    Wu, Jiangling; Huang, Yu; Bian, Xintong; Li, DanDan; Cheng, Quan; Ding, Shijia

    2016-10-01

    In this work, a custom-made intensity-interrogation surface plasmon resonance imaging (SPRi) system has been developed to directly detect a specific sequence of BCR/ABL fusion gene in chronic myelogenous leukemia (CML). The variation in the reflected light intensity detected from the sensor chip composed of gold islands array is proportional to the change of refractive index due to the selective hybridization of surface-bound DNA probes with target ssDNA. SPRi measurements were performed with different concentrations of synthetic target DNA sequence. The calibration curve of synthetic target sequence shows a good relationship between the concentration of synthetic target and the change of reflected light intensity. The detection limit of this SPRi measurement could approach 10.29 nM. By comparing SPRi images, the target ssDNA and non-complementary DNA sequence are able to be distinguished. This SPRi system has been applied for assay of BCR/ABL fusion gene extracted from real samples. This nucleic acid-based SPRi biosensor therefore offers an alternative high-effective, high-throughput label-free tool for DNA detection in biomedical research and molecular diagnosis.

  1. A target-disease network model of second-generation BCR-ABL inhibitor action in Ph+ ALL.

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    Uwe Rix

    Full Text Available Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL is in part driven by the tyrosine kinase bcr-abl, but imatinib does not produce long-term remission. Therefore, second-generation ABL inhibitors are currently in clinical investigation. Considering different target specificities and the pronounced genetic heterogeneity of Ph+ ALL, which contributes to the aggressiveness of the disease, drug candidates should be evaluated with regard to their effects on the entire Ph+ ALL-specific signaling network. Here, we applied an integrated experimental and computational approach that allowed us to estimate the differential impact of the bcr-abl inhibitors nilotinib, dasatinib, Bosutinib and Bafetinib. First, we determined drug-protein interactions in Ph+ ALL cell lines by chemical proteomics. We then mapped those interactions along with known genetic lesions onto public protein-protein interactions. Computation of global scores through correlation of target affinity, network topology, and distance to disease-relevant nodes assigned the highest impact to dasatinib, which was subsequently confirmed by proliferation assays. In future, combination of patient-specific genomic information with detailed drug target knowledge and network-based computational analysis should allow for an accurate and individualized prediction of therapy.

  2. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-04-15

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

  3. Prospective molecular monitoring of BCR/ABL transcript in children with Ph+ acute lymphoblastic leukaemia unravels differences in treatment response.

    Science.gov (United States)

    Cazzaniga, Giovanni; Lanciotti, Marina; Rossi, Vincenzo; Di Martino, Daniela; Aricò, Maurizio; Valsecchi, Maria Grazia; Basso, Giuseppe; Masera, Giuseppe; Micalizzi, Concetta; Biondi, Andrea

    2002-11-01

    Children with Philadelphia-chromosome-positive (Ph+) acute lymphoblastic leukaemia (ALL) represent a subgroup at very high risk for treatment failure, despite intensive chemotherapy. However, recent retrospective studies showed that Ph+ childhood ALL is a heterogeneous disease with regard to treatment response. We have prospectively monitored, by reverse transcription polymerase chain reaction (RT-PCR) during follow-up, the presence of the BCR/ABL fusion transcript in Ph+ ALL children diagnosed in the Italian multicentre Associazione Italiana Ematologia Oncologia Pediatrica ALL-AIEOP-95 therapy protocol. To our knowledge, this is the first report on the evaluation of minimal residual disease (MRD) in childhood Ph+ ALL prospectively enrolled in an intensive, Berlin-Frankfurt-Munster (BFM)-type treatment protocol. Twenty-seven of 36 (75.0%) Ph+ patients consecutively enrolled into the high-risk group of the AIEOP-ALL protocol between May 1995 and October 1999 were successfully analysed. Twenty were good responders to the pre-phase of prednisone/intrathecal methotrexate treatment (PGR) and seven were poor responders (PPR). Within the PPR group, the RT-PCR monitoring constantly showed positivity for the BCR/ABL fusion transcript and all the patients died of disease progression. In contrast, highly sensitive qualitative RT-PCR monitoring revealed heterogeneity within the PGR group of Ph+ childhood ALL patients. Three different subgroups could be defined, according to the clearance of Ph+ cells within the first 5 months of treatment. This provides useful information on the capability of chemotherapy to reduce the leukaemic clone, with prognostic implications.

  4. Frequency of p190 and p210 BCR-ABL rearrangements and survival in Brazilian adult patients with acute lymphoblastic leukemia

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    Ilana de França Azevedo

    2014-10-01

    Full Text Available Objective: This study investigated the occurrence of the p190 and p210 break point clusterregion-Abelson (BCR-ABL rearrangements in adults with acute lymphoblastic leukemia and possible associations with clinical and laboratory characteristics and survival. Methods: Forty-one over 18-year-old patients with acute lymphoblastic leukemia of both genders followed-up between January 2008 and May 2012 were included in this study. Clinical and laboratory data were obtained from the medical charts of the patients. Reverse transcription polymerase chain reaction (RT-PCR using specific primers was employed to identify molecular rearrangements. Results: At diagnosis, the median age was 33 years, and there was a predominance of males (61%. The most common immunophenotype was B lineage (76%. BCR-ABL rearrangements was detected in 14 (34% patients with the following distribution: p190 (28%, p210 (50% and double positive (22%. Overall survival of patients with a mean/median of 331/246 days of follow up was 39%, respectively, negative BCR-ABL (44% and positive BCR-ABL (28%. Conclusion: These results confirm the high frequency of BCR-ABL rearrangements and the low survival rate of adult Brazilian patients with acute lymphoblastic leukemia.

  5. Detection of residual bcr/abl translocation by polymerase chain reaction in chronic myeloid leukaemia patients after bone-marrow transplantation.

    Science.gov (United States)

    Gabert, J; Thuret, I; Lafage, M; Carcassonne, Y; Maraninchi, D; Mannoni, P

    1989-11-11

    The polymerase chain reaction was used to evaluate minimum residual disease in chronic myelogenous leukaemia (CML) patients after bone-marrow transplantation, by amplification of the transcript of the specific bcr/abl hybrid gene. Strict precautions were taken to avoid contamination. Peripheral blood cells from 22 patients transplanted for haematological malignant disorders were analysed. The results were clearcut for positive controls (patients with CML in relapse) and negative controls (patients with malignant disorders other than CML). In 11 of 12 CML patients in clinical and cytogenetic remission the bcr/abl transcript was detected 3 months to 6 years after transplantation. Thus, it appears that cells expressing the bcr/abl mRNA are not eradicated from the blood of CML patients in complete clinical remission even years after bone-marrow transplantation.

  6. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-02

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  7. Rapid Evolution to Blast Crisis Associated with a Q252H ABL1 Kinase Domain Mutation in e19a2 BCR-ABL1 Chronic Myeloid Leukaemia

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    Sarah L. McCarron

    2013-01-01

    Full Text Available A minority of chronic myeloid leukaemia (CML patients express variant transcripts of which the e19a2 BCR-ABL1 fusion is the most common. Instances of tyrosine kinase inhibitor (TKI resistance in e19a2 BCR-ABL1 CML patients have rarely been reported. A case of e19a2 BCR-ABL1 CML is described in whom imatinib resistance, associated with a Q252H ABL1 kinase domain mutation, became apparent soon after initiation of TKI therapy. The patient rapidly transformed to myeloid blast crisis (BC with considerable bone marrow fibrosis and no significant molecular response to a second generation TKI. The clinical course was complicated by comorbidities with the patient rapidly succumbing to advanced disease. This scenario of Q252H-associated TKI resistance with rapid BC transformation has not been previously documented in e19a2 BCR-ABL1 CML. This case highlights the considerable challenges remaining in the management of TKI-resistant BC CML, particularly in the elderly patient.

  8. miR-29b suppresses CML cell proliferation and induces apoptosis via regulation of BCR/ABL1 protein

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yajuan; Wang, Haixia; Tao, Kun [Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated of Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016 (China); Xiao, Qing [Department of Hematology, The First Affiliated Hospital, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016 (China); Huang, Zhenglan; Zhong, Liang; Cao, Weixi [Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated of Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016 (China); Wen, Jianping [Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 (Canada); Feng, Wenli, E-mail: fengwlcqmu@sina.com [Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated of Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016 (China)

    2013-05-01

    MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally and are critical for many cellular pathways. Recent evidence has shown that aberrant miRNA expression profiles and unique miRNA signaling pathways are present in many cancers. Here, we demonstrate that miR-29b is markedly lower expressed in CML patient samples. Bioinformatics analysis reveals a conserved target site for miR-29b in the 3′-untranslated region (UTR) of ABL1. miR-29b significantly suppresses the activity of a luciferase reporter containing ABL1-3′UTR and this activity is not observed in cells transfected with mutated ABL1-3′UTR. Enforced expression of miR-29b in K562 cells inhibits cell growth and colony formation ability thereby inducing apoptosis through cleavage of procaspase 3 and PARP. Furthermore, K562 cells transfected with a siRNA targeting ABL1 show similar growth and apoptosis phenotypes as cells overexpression of miR-29b. Collectively, our results suggest that miR-29b may function as a tumor suppressor by targeting ABL1 and BCR/ABL1. - Highlights: ► miR-29b expression was downregulated in CML patients. ► ABL1 was identified as a direct target gene of miR-29b. ► Enforced expression of miR-29b inhibits cell proliferation and induces apoptosis. ► miR-29b might be a therapeutic target to CML.

  9. Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells.

    Science.gov (United States)

    Li, Qianyin; Huang, Zhenglan; Gao, Miao; Cao, Weixi; Xiao, Qin; Luo, Hongwei; Feng, Wenli

    2017-03-02

    The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML). The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein) and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR) mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag), HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag) and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells) apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI)-resistance.

  10. Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Qianyin Li

    2017-03-01

    Full Text Available The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML. The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag, HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK and signal transducer and activator of transcription 5 (STAT5 pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI-resistance.

  11. Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

    Science.gov (United States)

    Shi, Yan; Li, Li-Zhen; Sun, Jian-Zhi; Zhang, Ti; Peng, Jun; Xu, Cong-Gao

    2008-01-01

    Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

  12. Targeting of heme oxygenase-1 attenuates the negative impact of Ikaros isoform 6 in adult BCR-ABL1-positive B-ALL.

    Science.gov (United States)

    Lin, Xiaojing; Zou, Xingli; Wang, Ziming; Fang, Qin; Chen, Shuya; Huang, Jun; Zhe, Nana; Yu, Meisheng; Zhang, Yaming; Wang, Jishi

    2016-08-16

    The correlation between Heme oxygenase-1 (HO-1) and dominant-negative Ikaros isoform 6 (IK6) is unclear. Firstly, we detected that IK6 existed in 20 of 42 (47.6%) adult BCR-ABL1-positive B-lineage acute lymphoblastic leukemia (BCR-ABL1-positive B-ALL) by using reverse transcribed polymerase chain reaction (PCR) and nucleotide sequencing. IK6-positive patients had an unfavorable outcome compared with IK6-negative ones. Further study showed that the level of HO-1 expression was higher in IK6-positive patients' samples than that in IK6-negative ones. And there was a strong correlation between the expression of IK6 and HO-1. The growth of primary CD34+ leukemic cells derived from our IK6-positive patients' pool was prohibited by silencing HO-1, further promoting their apoptosis. Furthermore, primary CD34+ leukemic cells derived from IK6-positive patients shown poor responses to imatinib in comparison with wild-type (IK1) patients, suggesting that the expression of IK6 resisted to imatinib in adult BCR-ABL1-positive B-ALL. Importantly, inhibition of HO-1 also increased their sensitivity to tyrosine kinase inhibitors (TKIs). Finally, we found that IK6 activated downstream STAT5, and HO-1 was one of the downstream target genes of STAT5. In conclusion, HO-1 is an essential survival factor in BCR-ABL1-positive B-ALL with IK6, and targeting HO-1 can attenuate the negative impact of IK6.

  13. UV Differentially Induces Oxidative Stress, DNA Damage and Apoptosis in BCR-ABL1-Positive Cells Sensitive and Resistant to Imatinib

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    Ewelina Synowiec

    2015-08-01

    Full Text Available Chronic myeloid leukemia (CML cells express the active BCR-ABL1 protein, which has been targeted by imatinib in CML therapy, but resistance to this drug is an emerging problem. BCR-ABL1 induces endogenous oxidative stress promoting genomic instability and imatinib resistance. In the present work, we investigated the extent of oxidative stress, DNA damage, apoptosis and expression of apoptosis-related genes in BCR-ABL1 cells sensitive and resistant to imatinib. The resistance resulted either from the Y253H mutation in the BCR-ABL1 gene or incubation in increasing concentrations of imatinib (AR. UV irradiation at a dose rate of 0.12 J/(m2·s induced more DNA damage detected by the T4 pyrimidine dimers glycosylase and hOGG1, recognizing oxidative modifications to DNA bases in imatinib-resistant than -sensitive cells. The resistant cells displayed also higher susceptibility to UV-induced apoptosis. These cells had lower native mitochondrial membrane potential than imatinib-sensitive cells, but UV-irradiation reversed that relationship. We observed a significant lowering of the expression of the succinate dehydrogenase (SDHB gene, encoding a component of the complex II of the mitochondrial respiratory chain, which is involved in apoptosis sensing. Although detailed mechanism of imatinib resistance in AR cells in unknown, we detected the presence of the Y253H mutation in a fraction of these cells. In conclusion, imatinib-resistant cells may display a different extent of genome instability than their imatinib-sensitive counterparts, which may follow their different reactions to both endogenous and exogenous DNA-damaging factors, including DNA repair and apoptosis.

  14. Coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites for the detection of BCR/ABL fusion gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xueping [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Wang, Li [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Department of Medical Laboratory, Chongqing Emergency Medical Center (Chongqing The Fourth Hospital), Chongqing, 400016 (China); Sheng, Shangchun [The No.2 Peoples' Hospital of Yibin, Sichuan, 644000 (China); Wang, Teng; Yang, Juan [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Xie, Guoming, E-mail: guomingxie@cqmu.edu.cn [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Feng, Wenli, E-mail: fengwlcqmu@sina.com [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China)

    2015-08-19

    This article described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites (GS/PANI/AuNPs) for highly sensitive and specific detection of BCR/ABL fusion gene (bcr/abl) in chronic myeloid leukemia (CML). DNA circuit known as catalyzed hairpin assembly (CHA) is enzyme-free and can be simply operated to achieve exponential amplification, which has been widely employed in biosensing. However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input. Herein, a transducer hairpin (HP) was designed to obtain a universal DNA circuit with favorable signal-to-background ratio. To further improve signal amplification, GS/PANI/AuNPs with excellent conductivity and enlarged effective area were introduced into this DNA circuit. Consequently, by combining the advantages of CHA and GS/PANI/AuNPs, bcr/abl could be detected in a linear range from 10 pM to 20 nM with a detection limit of 1.05 pM. Moreover, this protocol showed excellent specificity, good stability and was successfully applied for the detection of real sample, which demonstrated its great potential in clinical application. - Highlights: • A transducer hairpin was designed to improve the versatility of DNA circuit. • GS/PANI/AuNPs were introduced to the DNA circuit for further signal amplification. • The established biosensor displayed high sensitivity and good specificity.

  15. Low expression of miR-196b enhances the expression of BCR-ABL1 and HOXA9 oncogenes in chronic myeloid leukemogenesis.

    Directory of Open Access Journals (Sweden)

    Yue Liu

    Full Text Available MicroRNAs (miRNAs can function as tumor suppressors or oncogene promoters during tumor development. In this study, low levels of expression of miR-196b were detected in patients with chronic myeloid leukemia. Bisulfite genomic sequencing PCR and methylation-specific PCR were used to examine the methylation status of the CpG islands in the miR-196b promoter in K562 cells, patients with leukemia and healthy individuals. The CpG islands showed more methylation in patients with chronic myeloid leukemia compared with healthy individuals (P<0.05, which indicated that low expression of miR-196b may be associated with an increase in the methylation of CpG islands. The dual-luciferase reporter assay system demonstrated that BCR-ABL1 and HOXA9 are the target genes of miR-196b, which was consistent with predictions from bioinformatics software analyses. Further examination of cell function indicated that miR-196b acts to reduce BCR-ABL1 and HOXA9 protein levels, decrease cell proliferation rate and retard the cell cycle. A low level of expression of miR-196b can cause up-regulation of BCR-ABL1 and HOXA9 expression, which leads to the development of chronic myeloid leukemia. MiR-196b may represent an effective target for chronic myeloid leukemia therapy.

  16. Sensitive detection of pre-existing BCR-ABL kinase domain mutations in CD34+ cells of newly diagnosed chronic-phase chronic myeloid leukemia patients is associated with imatinib resistance: implications in the post-imatinib era.

    Directory of Open Access Journals (Sweden)

    Zafar Iqbal

    Full Text Available BACKGROUND: BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. METHODS: We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. RESULTS: Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48, all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. CONCLUSION: Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid

  17. Multiple defects in Escherichia coli mutants lacking HU protein.

    OpenAIRE

    Huisman, O; Faelen, M; Girard, D; Jaffé, A; Toussaint, A; Rouvière-Yaniv, J

    1989-01-01

    The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, alpha and beta, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid. We report here the construction of double mutants totally lacking both subunits of HU protein. These mutants exhibited poor growth and a perturbation of cell division, resulting in the formation of anucleate cells. In the absence of HU, phage Mu was unable to grow, to lysogenize,...

  18. Attomolar electrochemical detection of the BCR/ABL fusion gene based on an amplifying self-signal metal nanoparticle-conducting polymer hybrid composite.

    Science.gov (United States)

    Avelino, Karen Y P S; Frias, Isaac A M; Lucena-Silva, Norma; Gomes, Renan G; de Melo, Celso P; Oliveira, Maria D L; Andrade, César A S

    2016-12-01

    In the last ten years, conjugated polymers started to be used in the immobilization of nucleic acids via non-covalent interactions. In the present study, we describe the construction and use of an electrochemical DNA biosensor based on a nanostructured polyaniline-gold composite, specifically developed for the detection of the BCR/ABL chimeric oncogene. This chromosome translocation is used as a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The working principle of the biosensor rests on measuring the conductivity resulting from the non-covalent interactions between the hybrid nanocomposite and the DNA probe. The nanostructured platform exhibits a large surface area that enhances the conductivity. Positive cases, which result from the hybridization between DNA probe and targeted gene, induce changes in the amperometric current and in the charge transfer resistance (RCT) responses. Atomic force microscopy (AFM) images showed changes in the genosensor surface after exposure to cDNA sample of patient with leukemia, evidencing the hybridization process. This new hybrid sensing-platform displayed high specificity and selectivity, and its detection limit is estimated to be as low as 69.4 aM. The biosensor showed excellent analytical performance for the detection of the BCR/ABL oncogene in clinical samples of patients with leukemia. Hence, this electrochemical sensor appears as a simple and attractive tool for the molecular diagnosis of the BCR/ABL oncogene even in early-stage cases of leukemia and for the monitoring of minimum levels of residual disease.

  19. The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia.

    Science.gov (United States)

    García-Tuñón, Ignacio; Hernández-Sánchez, María; Ordoñez, José Luis; Alonso-Pérez, Veronica; Álamo-Quijada, Miguel; Benito, Rocio; Guerrero, Carmen; Hernández-Rivas, Jesús María; Sánchez-Martín, Manuel

    2017-02-09

    CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line.CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients.

  20. Epidemiologic study on survival of chronic myeloid leukemia and Ph(+) acute lymphoblastic leukemia patients with BCR-ABL T315I mutation

    DEFF Research Database (Denmark)

    Nicolini, Franck E; Mauro, Michael J; Martinelli, Giovanni;

    2009-01-01

    The BCR-ABL T315I mutation represents a major mechanism of resistance to tyrosine kinase inhibitors (TKIs). The objectives of this retrospective observational study were to estimate overall and progression-free survival for chronic myeloid leukemia in chronic-phase (CP), accelerated-phase (AP......), or blastic-phase (BP) and Philadelphia chromosome-positive (Ph)(+) acute lymphoblastic leukemia (ALL) patients with T315I mutation. Medical records of 222 patients from 9 countries were reviewed; data were analyzed using log-rank tests and Cox proportional hazard models. Median age at T315I mutation...

  1. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F+ HEL Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Axel Weber

    2015-03-01

    Full Text Available Signal transducers and activators of transcription (Stats play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML and Jak2(V617F in other myeloproliferative diseases (MPD. We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL

  2. Immunologic evaluation of peptides derived from BCR/ABL-out-of-frame fusion protein in HLA A2.1 transgenic mice.

    Science.gov (United States)

    Casnici, Claudia; Volpe, Gisella; Crotta, Katia; Lattuada, Donatella; Saglio, Giuseppe; Marelli, Ornella

    2012-05-01

    Philadelphia chromosome-positive chronic myelogenous leukemia and acute lymphocytic leukemia express, besides the main BCR/ABL transcripts, novel BCR/ABL transcripts derived from alternative splicing between BCR exons 1, 13, or 14 with ABL exons 4 and 5. Their translational products present at C-terminus an amino acid portion derived from out-of-frame (OOF) reading of the ABL gene. The presence of OOF-peptide-specific T cells in chronic myelogenous leukemia patients was demonstrated and a first study in in vivo model demonstrated that OOF ABL portion was immunogenic in human leukcocyte antigen (HLA)-A2.1 transgenic mice. Here we immunized HLA A2.1 mice with novel peptides designed on the ABL OOF sequence, containing epitopes with high affinity for HLA A2.1 molecule. The specific immune response, cellular and humoral, obtained ex vivo against HLA A2.1-positive human chronic myelogenous leukemia cells using peptide 22-53 and the cytotoxic activity induced by peptide 32mer confirm the possibility to use the ABL OOF portion as target to evoke a specific and multiple immune response in Philadelphia positive leukemic patients in cytogenetic remission.

  3. Sensitivity of imatinib-resistant T315I BCR-ABL CML to a synergistic combination of ponatinib and forskolin treatment.

    Science.gov (United States)

    Oaxaca, Derrick M; Yang-Reid, Sun Ah; Ross, Jeremy A; Rodriguez, Georgialina; Staniswalis, Joan G; Kirken, Robert A

    2016-09-01

    Tyrosine kinase inhibitors (TKIs) have dramatically improved the life expectancy of patients suffering from chronic myeloid leukemia (CML); however, patients will eventually develop resistance to TKI therapy or adverse side effects due to secondary off-target mechanisms associated with TKIs. CML patients exhibiting TKI resistance are at greater risk of developing an aggressive and drug-insensitive disease. Drug-resistant CML typically arises in response to spontaneous mutations within the drug binding sites of the targeted oncoproteins. To better understand the mechanism of drug resistance in TKI-resistant CML patients, the BCR-ABL transformed cell line KCL22 was grown with increasing concentrations of imatinib for a period of 6 weeks. Subsequently, a drug-resistant derivative of the parental KCL22 cell line harboring the T315I gatekeeper mutation was isolated and investigated for TKI drug sensitivity via multi-agent drug screens. A synergistic combination of ponatinib- and forskolin-reduced cell viability was identified in this clinically relevant imatinib-resistant CML cell line, which also proved efficacious in other CML cell lines. In summary, this study provides new insight into the biological underpinnings of BCR-ABL-driven CML and potential rationale for investigating novel treatment strategies for patients with T315I CML.

  4. Isolation and characterization of Escherichia coli mutants lacking inducible cyanase.

    Science.gov (United States)

    Guilloton, M; Karst, F

    1987-03-01

    To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

  5. Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission.

    Science.gov (United States)

    van Kooten Niekerk, Peter B; Petersen, Charlotte C; Nyvold, Charlotte G; Ommen, Hans B; Roug, Anne S; Nederby, Line; Hokland, Peter; Kjeldsen, Eigil

    2014-01-01

    The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD34(+) stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph(+) ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR(4) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR(4) , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.

  6. A BCR-ABL1 cutoff of 1.5% at 3 months, determined by the GeneXpert system, predicts an optimal response in patients with chronic myeloid leukemia

    Science.gov (United States)

    García-Gutiérrez, Valentín; Gómez-Casares, María T.; Puerta, José M.; Alonso-Domínguez, Juan M.; Osorio, Santiago; Hernández-Boluda, Juan C.; Collado, Rosa; Ramírez, María J.; Ibáñez, Fátima; Martín, María L.; Rodríguez-Gambarte, Juan D.; Martínez-Laperche, Carolina; Gómez, Montse; Fiallo, Dolly V.; Redondo, Sara; Rodríguez, Alicia; Ruiz-Nuño, Concepción; Steegmann, Juan L.

    2017-01-01

    In chronic myeloid leukemia (CML) patients, 3-month BCR-ABL1 levels have consistently been correlated with further outcomes. Monitoring molecular responses in CML using the GeneXpert (Cepheid) platform has shown an optimal correlation with standardized RQ-PCR (IS) when measuring BCR-ABL1 levels lower than 10%, as it is not accurate for values over 10%. The aim of the present study was to determine the predictive molecular value at three months on different outcome variables using the Xpert BCR-ABL1 MonitorTM assay (Xpert BCR-ABL1). We monitored 125 newly diagnosed consecutive CML patients in the chronic phase (CML-CP) using an automated method: Xpert BCR-ABL1. Only 5% of patients did not achieve an optimal response at 3 months, and the 10% BCR-ABL1 cutoff defined by RQ-PCR (IS) methods was unable to identify significant differences in the probabilities of achieving a complete cytogenetic response (CCyR) (50% vs. 87%, p = 0.1) or a major molecular response (MMR) (60% vs. 80%, p = 0.29) by 12 months. In contrast, a cutoff of 1.5% more accurately identified differences in the probabilities of achieving CCyR (98% vs. 54%, p<0.001) and MMR (88% vs. 56%, p<0.001) by 12 months, as well as probabilities of treatment changes (p = 0.005). Therefore, when using the Xpert BCR-ABL1 assay, a cutoff of 1.5% at 3 months could with high probability identify patients able to achieve an optimal response at 12 months. PMID:28278193

  7. Pharmacophore modeling of nilotinib as an inhibitor of ATP-binding cassette drug transporters and BCR-ABL kinase using a three-dimensional quantitative structure-activity relationship approach.

    Science.gov (United States)

    Shukla, Suneet; Kouanda, Abdul; Silverton, Latoya; Talele, Tanaji T; Ambudkar, Suresh V

    2014-07-07

    Nilotinib (Tasigna) is a tyrosine kinase inhibitor approved by the FDA to treat chronic phase chronic myeloid leukemia patients. It is also a transport substrate of the ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-glycoprotein, P-gp) and ABCG2 (BCRP), which may have an effect on the pharmacokinetics and toxicity of this drug. The goal of this study was to identify pharmacophoric features of nilotinib in order to potentially develop specific inhibitors of BCR-ABL kinase with minimal interactions with ABC drug transporters. Three-dimensional pharmacophore modeling and quantitative structure-activity relationship (QSAR) studies were carried out on a series of nilotinib analogues to identify chemical features that contribute to inhibitory activity of nilotinib against BCR-ABL kinase activity, P-gp, and ABCG2. Twenty-five derivatives of nilotinib were synthesized and were then tested to measure their activity to inhibit BCR-ABL kinase and to inhibit the function of ABC drug transporters. A set of in vitro experiments including kinase activity and cell-based transport assays and photolabeling of P-gp and ABCG2 with a transport substrate, [(125)I]-iodoarylazido-prazosin (IAAP), were carried out in isolated membranes to evaluate the potency of the derivatives to inhibit the function of ABC drug transporters and BCR-ABL kinase. Sixteen, fourteen, and ten compounds were selected as QSAR data sets, respectively, to generate PHASE v3.1 pharmacophore models for BCR-ABL kinase, ABCG2, and P-gp inhibitors. The IC50 values of these derivatives against P-gp, ABCG2, or BCR-ABL kinase were used to generate pharmacophore features required for optimal interactions with these targets. A seven-point pharmacophore (AADDRRR) for BCR-ABL kinase inhibitory activity, a six-point pharmacophore (ADHRRR) for ABCG2 inhibitory activity, and a seven-point pharmacophore (AADDRRR) for P-gp inhibitory activity were generated. The derived models clearly demonstrate high predictive power

  8. Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia = Determinação da lactate desidrogenase (LDH e do transcrito Bcr-Abl em pacientes com leucemia mielóide crônica

    Directory of Open Access Journals (Sweden)

    Roberto Iemitsu Tatakihara

    2010-07-01

    Full Text Available Chronic myeloid leukemia (CML is a malignant myeloproliferative disorder that originates from a pluripotent stem cell characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow into the bloodstream. The vast majority of patients with CML present Bcr-Abl transcripts. Lactate dehydrogenase (LDH is considered a biochemical marker common for tumor growth, anaerobic glycolysis and has been considered a poor prognostic factor for acute myeloid leukemia. Therefore, this study aimed to evaluate the concentration of LDH in plasma and the detection of the Bcr-Abl transcripts in patients with CML and healthy donors. We analyzed 22 patients demonstrably diagnosed with CML and 56 healthy donors. LDH concentration in plasma was higher in patients with CML. All patients with CML in this study were under treatment, but even so four patients had the Bcr-Abl (b3a2 transcript in peripheral blood. Two out of the four patients with b3a2 showed higher LDH (486 U L-1 and 589 U L-1. Thus, although the study was conducted with small numbers of samples, it is possible to suggest therapy alteration for two patients who presented transcript b3a2 in the peripheral blood samples and whose LDH concentration was high, in order to improve the disease. Leucemia mieloide crônica (LMC é uma desordem mieloproliferativa maligna que é originada de célula-tronco pluripotente caracterizada por expansão anormal, maligna de clones de células tronco da medula óssea na circulação. A grande maioria dos pacientes com LMC apresentam transcritos Bcr-Abl. Lactato desidrogenase (LDH,considerado um marcador bioquímico para crescimento tumoral, glicólise anaeróbica, e tem sido considerado um fator de pior prognóstico da LMC. Portanto, este estudo visa avaliar a concentraçãode LDH no plasma e a detecção do transcrito Bcr-Abl em 22 pacientes com LMC e 56 indivíduos saudáveis. Foram avaliados 22 pacientes com LMC e 56 doadores saudáveis. A

  9. BCR-ABL1: Test

    Science.gov (United States)

    ... molecular genetic test, and/or fluorescence in situ hybridization (FISH) . These help establish the initial diagnosis of ... Health Professionals ©2001 - by American Association for Clinical Chemistry • Contact Us | Terms of Use | Privacy We comply ...

  10. Overexpression or knock-down of runt-related transcription factor 1 affects BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo in mice

    Institute of Scientific and Technical Information of China (English)

    YANG Li-jun; YU Wei-dong; DU Jun-bao; CHAO Shuang; CHEN Min-xia; ZHAO He-hua; GUO Jing-zhu

    2009-01-01

    Background Runt-related transcription factor 1 (Runx1) plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runx1 will induce leukemia and how the change of Runx1 expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runx1 in BaF3 cells alone would induce leukemogenesis. And we also wanted to know if abnormal expression of Runx1 in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runx1 in BaF3 cells would induce leukemogenesis.Methods Plasmids containing full-length Runx1 cDNA were transduced into BaF3 cells and BaF3-P185wt cells (BCR-ABL transformed BaF3 cells) by electroporation. Plasmids containing a short hairpin RNA of Runx1 were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runx1 expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runx1 on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-FGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes.Results In vitro analysis revealed that overexpression of Runx1 in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runx1 could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe

  11. 用COLD-PCR方法提高BCR-ABL1激酶区耐药突变检测灵敏度%Application of COLD-PCR for improved detection of BCR-ABL1 kinase domain drug-resistant mutation

    Institute of Scientific and Technical Information of China (English)

    李庆; 刘红星; 孙慧; 王倩; 张之芬; 武焕玲; 李元堂; 陈永金; 田文君

    2013-01-01

    Objective To evaluate the feasibility of the co-amplification using a low denaturation temperature PCR (COLD-PCR) method in improving the detection of BCR-ABL1 fusion gene kinase domain drug-resistant mutation.Methods Conventional nested PCR (CN-PCR) and COLD-PCR protocol were designed to amplify full-length of BCR-ABL1 kinase domain.PCR products were purified and then Sanger sequenced to detect BCR-ABL1 kinase domain mutation (KDM).20 newly diagnosed CML patients and 32 Imatinib (IM) resistant patients were chosen and peripheral blood or bone marrow samples were collected.PCR products of the clinical specimens were purified and then cloned to evaluate the proportion of the KDM allele.Standard samples with different proportion of KDMs were prepared using T-A vectors with KDM and wild type BCR-ABL1 kinase domain sequence,and then CN-PCR,COLD-PCR and Sanger sequencing were performed to compare the detection sensitivity.Results In the 32 IM-resistant patients,23 were identified KDM-positive using CN-PCR and 28 KDM-positive using COLD-PCR.In the 20 newly diagnosed CML patients,none was identified KDM-positive using CN-PCR and 1 was identified carrying a lower proportion of KDM using COLD-PCR.For 16 different types of KDMs,COLD-PCR method could improve the detection sensitivity 4-12 times.Conclusion Detection sensitivity of BCR-ABL1 drug-resistant kinase domain mutation can be effectively improved by using COLD-PCR method and may contribute to early detection of resistance KDMs.%目的 探讨用低变性温度共扩增PCR(COLD-PCR)方法提高BCR-ABL1融合基因激酶区耐药突变检测灵敏度的可行性.方法 本研究为实验诊断研究.设计常规巢式PCR(CN-PCR)和COLD-PCR方案扩增BCR-ABL1激酶区全长,扩增产物纯化后用Sanger测序法检测激酶区突变(KDM).采集32例经伊马替尼(IM)治疗并出现耐药的慢性粒细胞性白血病(CML)患者和20例初诊CML患者外周血或骨髓标本,用上述2种方案检测标本中的KDM.

  12. Doxorubicin Differentially Induces Apoptosis, Expression of Mitochondrial Apoptosis-Related Genes, and Mitochondrial Potential in BCR-ABL1-Expressing Cells Sensitive and Resistant to Imatinib

    Directory of Open Access Journals (Sweden)

    Ewelina Synowiec

    2015-01-01

    Full Text Available Imatinib resistance is an emerging problem in the therapy of chronic myeloid leukemia (CML. Because imatinib induces apoptosis, which may be coupled with mitochondria and DNA damage is a prototype apoptosis-inducing factor, we hypothesized that imatinib-sensitive and -resistant CML cells might differentially express apoptosis-related mitochondrially encoded genes in response to genotoxic stress. We investigated the effect of doxorubicin (DOX, a DNA-damaging anticancer drug, on apoptosis and the expression of the mitochondrial NADH dehydrogenase 3 (MT-ND3 and cytochrome b (MT-CYB in model CML cells showing imatinib resistance caused by Y253H mutation in the BCR-ABL1 gene (253 or culturing imatinib-sensitive (S cells in increasing concentrations of imatinib (AR. The imatinib-resistant 253 cells displayed higher sensitivity to apoptosis induced by 1 μM DOX and this was confirmed by an increased activity of executioner caspases 3 and 7 in those cells. Native mitochondrial potential was lower in imatinib-resistant cells than in their sensitive counterparts and DOX lowered it. MT-CYB mRNA expression in 253 cells was lower than that in S cells and 0.1 μM DOX kept this relationship. In conclusion, imatinib resistance may be associated with altered mitochondrial response to genotoxic stress, which may be further exploited in CML therapy in patients with imatinib resistance.

  13. Application of BCR-ABL in chronic myelogenous leukemia%BCR-ABL探针在慢性粒细胞白血病中的应用

    Institute of Scientific and Technical Information of China (English)

    李娟; 陆莉; 蒲育栋; 杨柳; 赵丽

    2009-01-01

    目的 用常规细胞遗传学(conventional cytogenetics,CC)和荧光原位杂交(fluorescence chromosomal in situ hybridization,FISH)技术检测Ph染色体.方法 常规细胞遗传学分析(CC),荧光原位杂交(FISH)技术.结果 7例患者4例Ph染色体阴性,其中2例分别伴有t(18;22)和t(17;22)异常,其余2例为异基因造血干细胞移植后正常核型.一例培养后无中期分裂相.2例Ph染色体阳性,FISH结果bcr/abl(+)细胞检出率分别为63.87%,84.51%,7.56%,4.0%,74.45%,67%,47%.结论 常规细胞遗传学与荧光原位杂交技术相结合对CML患者诊断治疗过程中肿瘤负荷动态检测有显著意义.

  14. Induction of apoptosis by shikonin through a ROS/JNKmediated process in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia (CML) cells (e.g., K562, LAMA84). Treatment of K562 cells with shikonin (e.g., 0.5 μM) resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species (ROS), striking activation of c-Jun-N-terminai kinase (JNK) and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events (i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis) following shikonin treatment. Inhibition of JNK and knock-down of JNKI significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.

  15. Expression patterns of WT-1 and Bcr-Abl measured by TaqMan quantitative real-time RT-PCR during follow-up of leukemia patients with the Ph chromosome

    Institute of Scientific and Technical Information of China (English)

    CHEN Zi-xing陈子兴; Jaspal Kaeda; Sue Saunders; John M Goldman

    2004-01-01

    Background This study was designed to quantitatively measure WT-1 expression levels in patients with chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) during follow-up and to clarify the value of WT-1 as a molecular marker in minimal residual disease monitoring.Methods The TaqMan quantitative real-time RT-PCR method was established by using cloned WT-1 cDNA or synthesized oligonucleotides resembling WT-1 cDNA fragments in limit dilution as template until a stable and reliable standard curve was obtained. In a 25-month follow-up, the transcriptional levels of WT-1, Bcr-Abl, and Abl gene, were quantitatively measured in bone marrow cells from 25 CML or acute lymphoblastic leukemia (ALL) patients with the Ph chromosome. In addition, the expression of these genes in 40 samples of normal peripheral blood was also examined using the same method. The ratios of WT-1/Abl and Bcr-Abl/Abl were both plotted, and the two expression patterns were compared as well as their clinical significance.Results The levels of WT-1 expression in normal peripheral blood were detectable. In CML and Ph positive ALL patients, WT-1 expression levels changed in parallel with the Bcr-Abl expression pattern as the disease progressed or responded to effective treatment.Conclusion WT-1 expression provides a novel molecular marker in addition to Bcr-Abl for monitoring minimal residual disease (MRD) and targeting therapy in Ph chromosome-positive leukemia patients.

  16. Increased acetylation of lysine 317/320 of p53 caused by BCR-ABL protects from cytoplasmic translocation of p53 and mitochondria-dependent apoptosis in response to DNA damage

    NARCIS (Netherlands)

    Kusio-Kobialka, Monika; Wolanin, Kamila; Podszywalow-Bartnicka, Paulina; Sikora, Ewa; Skowronek, Krzysztof; McKenna, Sharon L.; Ghizzoni, Massimo; Dekker, Frank J.; Piwocka, Katarzyna

    2012-01-01

    Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by the expression of BCR-ABL. Loss of p53 has not been implicated as important for the development of CML. Mutations in p53 protein are infrequent, however they correlate with the disease progression. The absence of p53

  17. Characterization of the CDR3 structure of the Vβ21 T cell clone in patients with P210(BCR-ABL)-positive chronic myeloid leukemia and B-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Zha, Xianfeng; Chen, Shaohua; Yang, Lijian; Li, Bo; Chen, Yu; Yan, Xiaojuan; Li, Yangqiu

    2011-10-01

    The clonally expanded T cells identified in most cancer patients that respond to tumor-associated antigen such as P210(BCR-ABL) protein have definite, specific antitumor cytotoxicity. T cell receptor (TCR) Vβ CDR3 repertoire diversity was analyzed in patients with chronic myeloid leukemia (CML) and BCR-ABL(+) B-cell acute lymphoblastic leukemia (B-ALL) by GeneScan. A high frequency of oligoclonal expansion of the TCR Vβ21 subfamily was observed in the peripheral blood of CML and B-ALL patients. These clonally expanded Vβ21 T cells were correlated with the pathophysiologic process of CML. A conserved amino acid motif (SLxxV) was observed within the CDR3 region in only 3 patients with CML. Preferential usage of the Jβ segments was also observed in a minority of patients. The 3-dimensional structures of the CDR3 region containing the same motif or using the same Jβ segment displayed low similarity; on the contrary, the conformation of the CDR3 region containing no conserved motif in some T cell clones was highly similar. In conclusion, our findings indicate a high frequency of TCR Vβ21 subfamily expansion in p210(BCR-ABL)-positive CML and B-ALL patients. The characterization of the CDR3 structure was complex. Regrettably, at this time it was not possible to confirm that the Vβ21 T cell clones were derived from the stimulation of p210(BCR-ABL) protein.

  18. Which method better evaluates the molecular response in newly diagnosed chronic phase chronic myeloid leukemia patients with imatinib treatment, BCR-ABL(IS) or log reduction from the baseline level?

    Science.gov (United States)

    Qin, Ya-Zhen; Jiang, Qian; Jiang, Hao; Li, Jin-Lan; Li, Ling-Di; Zhu, Hong-Hu; Lai, Yue-Yun; Lu, Xi-Jing; Liu, Yan-Rong; Jiang, Bin; Huang, Xiao-Jun

    2013-09-01

    The molecular response of chronic myeloid leukemia (CML) patients to tyrosine kinase inhibitor treatment can be evaluated either by BCR-ABL mRNA levels on international scale (IS) or by log reduction from the baseline level of the laboratory. Both methods were compared in 248 newly diagnosed chronic phase CML patients treated with imatinib. The major molecular responses (MMR) obtained by both methods predict progression-free survival (PFS, all Plog reduction method, had the same PFS as MMR patients identified by both methods. The molecular responses of patients at 3 and 6 months, as evaluated by the two methods, have similar predictive values on their cytogenetic responses at 12 months and on their molecular responses at 18 months. Both ≤ 10%(IS) and ≥ 1 log reduction at 3 months and ≤ 1%(IS) at 6 months were significantly associated with PFS (P=0.0011, 0.0090, and 0.0064). The percentages of patients with BCR-ABL(IS) of ≤ 1%, >1-10%, and of >10% at 3 months and 6 months in the German CML Study IV were similar with those with corresponding BCR-ABL(IS) in our center, but was significantly different with those evaluated by the log reduction method. Therefore, the molecular response evaluated by BCR-ABL(IS) has similar trends in PFS and in response prediction, but can better differentiate patients than that by the log reduction method. Furthermore, the IS method allows comparison among molecular response results from different laboratories.

  19. 自身淬灭荧光定量PCR检测慢性粒细胞白血病bcr/abl mRNA%Quantification of bcr/abl mRNA in patients with chronic myeloid leukemia by using real-time quantitative fluorescence PCR with self-quenched primer

    Institute of Scientific and Technical Information of China (English)

    彭辉; 冯文莉; 王小中; 曾建明; 肖青; 潘健; 曹唯希; 罗云萍; 黄宗干

    2007-01-01

    目的 利用自身淬灭探针技术建立一种能检测慢性粒细胞白血病(CML)bcr/abl融合基因mRNA的实时荧光定量PCR方法,为CML的诊断、疗效观察以及微量残留白血病(MRD)的监测提供有效手段.方法 用逆转录PCR方法(RT-PCR)扩增K562细胞的bcr/abl融合基因,A-T克隆法构建定量标准模板;设计自身淬灭荧光引物(探针),建立自身淬灭荧光定量逆转录PCR方法(FQRT-PCR),并对该方法的线性检测范围、灵敏度、重复性、稳定性进行检测;然后检测临床白血病患者骨髓标本bcr/abl mRNA.结果 建立的FQ-RT-PCR方法可检测10 copies/μl的bcr/abl重组质粒,并能从105个正常细胞中检出1个白血病细胞,该方法的批内、批间变异系数(CV)分别为2.1%、6.1%,线性检测范围为102~109 copies/μl.25例CML患者bcr/abl融合基因mRNA表达量中位数为4.50×104[(0.45~89.00)×104]copies/μg RNA,其中11例慢性期初诊患者bcr/abl mRNA表达量中位数为5.45×104[(2.95~19.30)×104]copies/μg RNA,6例急变期患者bcr/abl mRNA表达量中位数为13.00×104[(4.10~89.00)×104]copies/μg RNA,8例慢性期治疗后复查患者bcr/ablmRNA表达量中位数为2.35×104[(0.45~5.12)×104]copies/μg RNA,CML急变期患者bcr/abl融合基因表达水平与慢性期患者之间差异有统计学意义(q=3.41,P<0.05).PCR产物经电泳分析,其中21例CML患者为b3a2型,4例CML患者为b2a2型;3例急性淋巴细胞白血病(ALL)患者中,1例有bcr/abl mRNA表达,为b2a2型.结论 所建立的基于自身淬灭探针技术的实时荧光定量PCR检测方法灵敏、特异、重复性好,结果用拷贝数表示,准确可靠,利于标准统一.可广泛用于CML的诊断、疗效观察以及MRD的监测.

  20. SPR Detection and Discrimination of the Oligonucleotides Related to the Normal and the Hybrid bcr-abl Genes by Two Stringency Control Strategies

    Science.gov (United States)

    Matsishin, M. J.; Ushenin, Iu. V.; Rachkov, A. E.; Solatkin, A. P.

    2016-01-01

    In this study, we applied two stringency control strategies for surface plasmon resonance (SPR) detection of DNA hybridization and discrimination of completely and partially complementary 24-mer sequences. These sequences are specific to the human normal bcr and the hybrid bcr-abl genes, protein products of which are responsible for some leukemia. SPR sensors based on resonance phenomena in nanoscale gold films are well suited for label-free, real-time investigations of the macromolecule interactions. Thermodynamic parameters obtained using the web server DINAMelt allowed supposing the possibility for realization (a) stringency control based on the ionic strength of the hybridization buffer and (b) stringency control based on the temperature elevation. The first one resulted in that the discrimination index of completely complementary and partially complementary oligonucleotides depending on the target concentration varied from 1.3 to 1.8 in 2 × SSC and from 2.0 to 2.9 in 0.5 × SSC. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~50 °C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 °C. Thus, the absolutely effective thermodiscrimination of this pair of oligonucleotides was achieved in this temperature range (40-50 °C).

  1. BCR/ABL DCDF-FISH信号特征与染色体核型的关系及其在慢性粒细胞白血病中的意义%Significance of BCR/ABL DCDF-FISH signal pattern and karyotype in chronic myeloid leukemia

    Institute of Scientific and Technical Information of China (English)

    杜庆华; 应逸; 陈晓燕

    2010-01-01

    目的 探讨慢性粒细胞白血病(CML)BCR/ABL DCCF-FISH的信号特点及其与染色体核型的关系.结果 使用BCR/ABL的DCDF探针对65例慢性粒细胞白血病骨髓标本进行荧光原位杂交及染色体核型检查.结果 65例CML核型43例Ph阳性,1例阴性,其余21例未行核性检查或无可分析分裂相;FISH结果65例均为BCR/ABL阳性,其中9例伴ASS基因缺失,5例复杂易位,1例+Ph伴ASS基因缺失.结论 传统核型与DCDF-FISH,应互相结合,方可对遗传学特征作出更准确分析.%Objective To investigate the relation between the BCR/ABL DCDF-FISH signal pattern and karyotype in chronic myeloid leukemia(CML). Methods 65 cases of CML were performed FISH and karyotyping. Results In 65 cases CML. 43 cases were Ph positive, 1 case was Ph negative, 21 cases had no performed karyotyping analysis. All of 65 cases were FISH positive,9 cases with ASS gene deletion, 5 cases with complex variant translocation, 1 case with (+) Ph and ASS gene deletion. Conclusion More precise analysis can be got by combining the result of FISH and karyotyping.

  2. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

    Science.gov (United States)

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-01

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  3. Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia - doi: 10.4025/actascihealthsci.v32i2.6408 Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia - doi: 10.4025/actascihealthsci.v32i2.6408

    Directory of Open Access Journals (Sweden)

    Thiago Cezar Fujita

    2010-09-01

    Full Text Available Chronic myeloid leukemia (CML is a malignant myeloproliferative disorder that originates from a pluripotent stem cell characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow into the bloodstream. The vast majority of patients with CML present Bcr-Abl transcripts. Lactate dehydrogenase (LDH is considered a biochemical marker common for tumor growth, anaerobic glycolysis and has been considered a poor prognostic factor for acute myeloid leukemia. Therefore, this study aimed to evaluate the concentration of LDH in plasma and the detection of the Bcr-Abl transcripts in patients with CML and healthy donors. We analyzed 22 patients demonstrably diagnosed with CML and 56 healthy donors. LDH concentration in plasma was higher in patients with CML. All patients with CML in this study were under treatment, but even so four patients had the Bcr-Abl (b3a2 transcript in peripheral blood. Two out of the four patients with b3a2 showed higher LDH (486 U L-1 and 589 U L-1. Thus, although the study was conducted with small numbers of samples, it is possible to suggest therapy alteration for two patients who presented transcript b3a2 in the peripheral blood samples and whose LDH concentration was high, in order to improve the disease.Chronic myeloid leukemia (CML is a malignant myeloproliferative disorder that originates from a pluripotent stem cell characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow into the bloodstream. The vast majority of patients with CML present Bcr-Abl transcripts. Lactate dehydrogenase (LDH is considered a biochemical marker common for tumor growth, anaerobic glycolysis and has been considered a poor prognostic factor for acute myeloid leukemia. Therefore, this study aimed to evaluate the concentration of LDH in plasma and the detection of the Bcr-Abl transcripts in patients with CML and healthy donors. We analyzed 22 patients demonstrably diagnosed

  4. Establishment and characterization of A novel Philadelphia-chromosome positive chronic myeloid leukemia cell line, TCC-S, expressing P210 and P190 BCR/ABL transcripts but missing normal ABL gene.

    Science.gov (United States)

    Van, Phan Nguyen Thanh; Xinh, Phan Thi; Kano, Yasuhiko; Tokunaga, Katsushi; Sato, Yuko

    2005-03-01

    A novel Philadelphia-chromosome positive (Ph+) cell line, TCC-S, has been established from a patient with Ph+ chronic myeloid leukemia (CML) in the blastic crisis. TCC-S cells were shown to express both P210 and P190 BCR/ABL transcripts by reverse transcriptase-polymerase chain reaction (PCR), although quantitative-PCR revealed that TCC-S cells mainly expressed P210 BCR/ABL transcript. Karyotype analysis revealed several triploid clones which constantly harbored two der(9)del(9) (p12)t(9;22) (q34;qll)s and two del(9) (q21)s. The der(9)del(9) (p12)t(9;22) (q34;q11) is rarely found in other CML cell lines. Moreover, to the best of our knowledge, del(9) (q21) resulting in missing of a restrict region including normal ABL gene has not been found among CML cell lines previously described. Thus, TCC-S cells with only BCR/ABL gene and no normal ABL gene may be a useful tool for functional study of ABL in Ph+ CML.

  5. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

    2015-03-19

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl{sup +} K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells

  6. Frequency of the ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and MLL-AFF1 fusion genes in Guatemalan pediatric acute lymphoblastic leukemia patients and their ethnic associations.

    Science.gov (United States)

    Carranza, Claudia; Granados, Lilian; Morales, Oneida; Jo, Wendy; Villagran, Swuanny; Tinti, Damaris; Villegas, Mauricio; Antillón, Federico; Torselli, Silvana; Silva, Gabriel

    2013-06-01

    Fusion genes involved in acute lymphoblastic leukemia (ALL) occur mostly due to genetic and environmental factors, and only a limited number of studies have reported any ethnic influence. This study assesses whether an ethnic influence has an effect on the frequency of any of the four fusion genes: BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, and MLL-AFF1 found in ALL. To study this ethnic influence, mononuclear cells were obtained from bone marrow samples from 143 patients with ALL. We performed RNA extraction and reverse transcription, then assessed the quality of the cDNA by amplifying the ABL1 control gene, and finally evaluated the presence of the four transcripts by multiplex polymerase chain reaction. We found 10 patients who had the BCR-ABL1 fusion gene (7%); 3 patients (2%) were TCF3-PBX1 positive; and 6 patients (4.5%) were ETV6-RUNX1 positive. The incidence of this last fusion gene is quite low when compared to the values reported in most countries. The low incidence of the ETV6-RUNX1 fusion gene found in Guatemala matches the incidence rates that have been reported in Spain and Indian Romani. Since it is known that an ethnic resemblance exists among these three populations, as shown by ancestral marker studies, the ALL data suggests an ethnic influence on the occurrence and frequency of this particular fusion gene.

  7. Persistence of BCR/ABL mRNA-expressing bone-marrow cells in patients with chronic myelogenous leukemia in complete cytogenetic remission induced by interferon-alpha therapy.

    Science.gov (United States)

    Eberlé, F; Toiron, Y; Camerlo, J; Lafage, M; Sainty, D; Arnoulet, C; Raineri, V; Gabert, J; Maraninchi, D; Mannoni, P

    1995-06-01

    Complete hematologic and cytogenetic responses can be obtained with interferon-alpha (IFN-alpha) in 15-25% of the patients with chronic myelogenous leukemia (CML). In these patients, reverse-transcription polymerase chain reaction (RT-PCR) can be used to evaluate minimal residual disease. We studied 12 patients who remained Philadelphia-negative for a median period of 21 months on IFN-alpha therapy. Using RT-PCR, the specific transcript was found in all bone marrow (BM) samples. Ten patients still exhibiting a persistent residual clone remained in cytogenetic remission for a median period of 14 months. As we observed a dissociation between bcr-abl expression in BM and peripheral blood (PB) cells, and given the known fluctuations of the bcr-abl PCR results, we suggest that PB negative results should be confirmed on BM specimens. Alternatively, it remains to be demonstrated whether longitudinal monitoring of residual disease would benefit from quantitative PCR or double fluorescence in situ hybridization.

  8. RT-PCR ANALYSIS OF E2A-PBX1, TEL-AML1, BCR-ABL AND MLL-AF4 FUSION GENE TRANSCRIPTS IN B-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA

    Directory of Open Access Journals (Sweden)

    Iuliu-Cristian Ivanov

    2013-11-01

    Full Text Available Acute lymphoblastic leukemia represents a heterogeneous group of hematological malignancies, defined by clonal proliferation of lymphoid cells. Immunophenotyping by flow cytometry and molecular analysis for the detection of genetic anomalies are clinical standard procedures for diagnosis, sub-classification and post-therapeutic evaluation. Samples from 105 patients diagnosed with acute lymphoblastic leukemia were immunophenotyped at diagnosis and were investigated by molecular analysis in order to identify the occurrence of four fusion genes: MLL-AF4, TEL-AML-1, BCR-ABL-p190, E2A-PBX-1. There were no associations found between the immunophenotype and the presence of any fusion genes evaluated. Both methods in combination remain a prerequisite for an improved subclassification of hematological malignancies, therapeutic decision, and evaluation of treatment response.

  9. Detection of BCR/ABL Fusion Gene by Fluorescence In Situ Hybridization and Its Clinical Application%荧光原位杂交技术检测BCR/ABL融合基因的临床应用

    Institute of Scientific and Technical Information of China (English)

    周瑞莲; 莫耀禧; 蓝梅; 林金盈

    2011-01-01

    This study was aimed to investigate the clinical value of detecting BCR/ABL fusion gene by fluorescence in situ hybridization (FISH). The conventional cytogenetic test and detection of BCR/ABL fusion gene by FISH for bone marrow of patients with newly diagnosed chronic myeloproliferative disease or myelodysplastic and myeloproliferative disorders, acute lymphocytic leukemia and chronic myelogeneous leukemia (CML) after allogeneic hematopoietic stem cell transplantation were carried out. The results showed that ( 1 ) out of 46 newly diagnosed as chronic myeloproliferative dsease or myelodysplastic and myeloproliferative disorders, 22 cases were diagnosed as CML, the FISH detection showed all positive (100% ), while cytogenetic test showed 86.4% (19/22) positive, in the other 24 patients who were diagnosed as other chronic myeloproliferative disease or myelodysplastic and myeloproliferative disorders, BCR/ABL fusion gene all were be detected as regative 100% by FISH, while the cytogenetic test of bone marrow in 3 cases supported the diagnosis of CML, and the diagnosis of myelodysplastic disorder in 1 case; (2) in 3 out of 7 acute lymphocytic leukemia cases the BCR/ABL fusion gene could not be detected by FISH; (3) the BCR/ABL fusion gene could be detected by FISH in 2 cases of CML received allogeneic hematopoietic stem cell transplantation, with abnormal threshold 6.5% and 1. 2% respectively. It is concluded that the detection of BCR/ABL fusion gene by FISH is sensitive and reliable, which is very important for the diagnosis and differential dignosis of chronic myeloproliferative disorders, myelodysplastic and myeloproliferative disease, as well as definite diagnosis of Ph* acute lymphoblastic leukemia. This method also has an important significance for monitor of minimal residual disease in CML patients received allogeneic hematopoietic stem cell transplantation.%本研究探讨应用荧光原位杂交技术( FISH)检测BCR/ABL融合基因在临床中的应用价

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

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses...... to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA...... mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning...

  11. NADP+-dependent glutamate dehydrogenase activity is impaired in mutants of Saccharomyces cerevisiae that lack aconitase.

    Science.gov (United States)

    González, A; Rodríguez, L; Olivera, H; Soberón, M

    1985-10-01

    A mutant of Saccharomyces cerevisiae lacking aconitase did not grow on minimal medium (MM) and had five- to tenfold less NADP+-dependent glutamate dehydrogenase (GDH) activity than the wild-type, although its glutamine synthetase (GS) activity was still inducible. When this mutant was incubated with glutamate as the sole nitrogen source, the 2-oxoglutarate content rose, and the NADP+-dependent GDH activity increased. Furthermore, carbon-limited cultures showed a direct relation between NADP+-dependent GDH activity and the intracellular 2-oxoglutarate content. We propose that the low NADP+-dependent GDH activity found in the mutant was due to the lack of 2-oxoglutarate or some other intermediate of the tricarboxylic acid cycle.

  12. Regulation of Amidase Formation in Mutants from Pseudomonas aeruginosa PAO Lacking Glutamine Synthetase Activity

    NARCIS (Netherlands)

    Janssen, Dick B.; Herst, Patricia M.; Joosten, Han M.L.J.; Drift, Chris van der

    1982-01-01

    The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation

  13. Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

    NARCIS (Netherlands)

    Simeonova, I.; Jaber, S.; Draskovic, I.; Bardot, B.; Fang, M.; Bouarich-Bourimi, R.; Lejour, V.; Charbonnier, L.; Soudais, C.; Bourdon, J.C.; Huerre, M.; Londono-Vallejo, A.; Toledo, F.

    2013-01-01

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53(Delta31), a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis,

  14. Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system

    OpenAIRE

    Narita, Yuka; Sato, Keiko; Yukitake, Hideharu; Shoji, Mikio; Nakane, Daisuke; Nagano, Keiji; Yoshimura, Fuminobu; Naito, Mariko; Nakayama, Koji

    2014-01-01

    Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, t...

  15. Imipenem- and meropenem-resistant mutants of Enterobacter cloacae and Proteus rettgeri lack porins.

    Science.gov (United States)

    Raimondi, A; Traverso, A; Nikaido, H

    1991-01-01

    Carbapenems such as imipenem and meropenem are not rapidly hydrolyzed by commonly occurring beta-lactamases. Nevertheless, it was possible, by mutagenesis and selection, to isolate mutant strains of Enterobacter cloacae and Proteus rettgeri that are highly resistant to meropenem and imipenem. Two alterations were noted in the E. cloacae mutants. First, the mutant strains appeared to be strongly derepressed in the production of beta-lactamases, which reached a very high level when the strains were grown in the presence of imipenem. Second, these mutants were deficient in the production of nonspecific porins, as judged by the pattern of outer membrane proteins as well as by reconstitution assays of permeability. As with most porin-deficient mutants, their cultures were unstable, and their cultivation in the absence of carbapenems rapidly led to an overgrowth of porin-producing revertants. Analysis of the data suggests that the synergism between the lowered outer membrane permeability and the slow but significant hydrolysis of carbapenems by the overproduced enzymes can explain the resistance phenotypes quantitatively, although the possibility of alteration of the target cannot be excluded at present. With P. rettgeri mutants, there was no indication of further derepression of beta-lactamase, but the enzyme hydrolyzed imipenem much more efficiently than the E. cloacae enzyme did. In addition, the major porin was absent in one mutant strain. These results suggest that a major factor for the carbapenem resistance of these enteric bacteria is the porin deficiency, and this conclusion forms a contrast to the situation in Pseudomonas aeruginosa, in which the most prevalent class of imipenem-resistant mutants appears to lack the specific channel protein D2 yet retains the major nonspecific porin F. Images PMID:1656855

  16. Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex.

    Science.gov (United States)

    Fromm, Steffanie; Senkler, Jennifer; Eubel, Holger; Peterhänsel, Christoph; Braun, Hans-Peter

    2016-05-01

    The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific 'carbonic anhydrase domain' of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe 'life without complex I'.

  17. First Case of Biphenotypic/bilineal (B/myeloid, B/monocytic) Mixed Phenotype Acute Leukemia with t(9;22)(q34;q11.2);BCR-ABL1.

    Science.gov (United States)

    Kim, Hyeong Nyeon; Hur, Mina; Kim, Hanah; Ji, Misuk; Moon, Hee-Won; Yun, Yeo-Min; Lee, Mark Hong

    2016-07-01

    Mixed phenotype acute leukemia (MPAL) includes biphenotypic leukemia, bilineal leukemia, or its combination by the 2008 WHO classification. A few cases of combined biphenotypic/bilineal MPAL have been reported so far; they all had biphenotypic expressions in only one of the two distinct leukemic populations. A 43-year-old female presented with leukocytosis and bicytopenia. Her complete blood counts were: hemoglobin, 6.9 g/dL; white blood cells, 62.8×10(9)/L; and platelets, 83×10(9)/L. Neither lymphadenopathy nor organomegaly was observed. Blasts and promonocytes/monoblasts were increased in her peripheral blood (42%) and bone marrow (60.1%). Flow cytometric analysis revealed two distinct populations of leukemic cells, which expressed CD11c, CD19, and cytoplasmic CD79a in common. Additionally, the first population expressed CD10 and CD117 (B/myeloid), and the second one expressed CD14 and CD20 (B/monocytic). She had a karyotype of 46,XX,inv(9)(p12q13),t(9;22)(q34;q11.2)[20] and BCR/ABL1 rearrangement. To the best of our knowledge, this is the first reported case of biphenotypic/bilineal MPAL with B/myeloid and B/monocytic expressions.

  18. Switching to second-generation tyrosine kinase inhibitor improves the response and outcome of frontline imatinib-treated patients with chronic myeloid leukemia with more than 10% of BCR-ABL/ABL ratio at 3 months

    Science.gov (United States)

    Casado, Luis-Felipe; García-Gutiérrez, José-Valentín; Massagué, Isabel; Giraldo, Pilar; Pérez-Encinas, Manuel; de Paz, Raquel; Martínez-López, Joaquín; Bautista, Guiomar; Osorio, Santiago; Requena, María-José; Palomera, Luis; Peñarrubia, María-Jesús; Calle, Carmen; Hernández-Rivas, José-Ángel; Burgaleta, Carmen; Maestro, Begoña; García-Ormeña, Nuria; Steegmann, Juan-Luis

    2015-01-01

    Chronic myeloid leukemia patients display heterogeneous responses to imatinib. Survival depends on baseline clinical characteristics (including prognostic scoring systems) and on early response (such as >10% BCR-ABL/ABL ratio at 3 months of therapy). The results of switching to second-generation tyrosine kinase inhibitors (2GTKIs) may contain a bias since, in the majority of these studies, patients who switch treatment due to intolerance or failure are censored or excluded. We analyzed the Spanish Registry data on switching in an intention-to-treat analysis of patients in standard clinical practice. Switching to 2GTKIs improves responses from 45% to 75% of complete cytogenetic response (CCyR) and from 15% to 45% of major molecular response (MMR) in the group without molecular response 1 (MR1) at 3 months and from 70% to 87% in CCyR and from 52% to 87% in MMR in the group with MR1. The final response rate is poorer in the group with no MR1 at 3 months. Nevertheless, the differences in the rates of response were not translated into differences in major events (transformations or deaths), and the final progression-free survival and overall survival were similar. PMID:25756742

  19. Estandarización de la TR-PCR para la detección de las fusiones génicas BCR-ABL en pacientes con leucemia Mieloide Crónica (LMC y Linfoide Aguda (LLA provenientes de HUSVP y Clíncia León XIII

    Directory of Open Access Journals (Sweden)

    Gonzálo Vásquez Palacio

    2006-04-01

    Full Text Available La translocación recíproca t(9:22(q34;q11 involucra el proto-oncogen ABL y el gen BCR, originando un gen de fusión BCR-ABL, que codifica una proteína con elevada actividad tirosina quinasa, implicada en la leucemogénesis.

  20. The synthetic heat shock protein 90 (Hsp90) inhibitor EC141 induces degradation of Bcr-Abl p190 protein and apoptosis of Ph-positive acute lymphoblastic leukemia cells.

    Science.gov (United States)

    Tong, Wei-Gang; Estrov, Zeev; Wang, Yongtao; O'Brien, Susan; Faderl, Stefan; Harris, David M; Van Pham, Quin; Hazan-Halevy, Inbal; Liu, Zhiming; Koch, Patricia; Kantarjian, Hagop; Keating, Michael J; Ferrajoli, Alessandra

    2011-12-01

    The prognosis of patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is poor. Chemotherapy is rarely curative and tyrosine kinase inhibitors (TKIs) induce only transient responses. Heat shock protein 90 (Hsp90) is a chaperone protein that is important in signal transduction, cell cycle control, and transcription regulation in both normal and leukemia cells. In the current study, we tested the growth inhibitory and apoptotic effects of a novel Hsp90 inhibitor, EC141 on the Ph+ ALL lines Z-119, Z-181, and Z-33, as well as primary bone marrow-derived blasts from patients with newly diagnosed Ph+ ALL. We found that EC141 inhibited the growth of Ph+ ALL cells in a concentration-dependent manner with IC(50) ranged from 1 to 10 nM. EC141 also inhibited the proliferation of primary bone marrow-derived blasts using the ALL blast colony assay. EC141 down-regulated Hsp90 and up-regulated Hsp70 protein levels, inhibited CrkL phosphorylation, and induced degradation of Bcr-Abl p190 protein through ubiquitin-dependent proteasomal pathway. Furthermore, exposure of Ph+ ALL cells to EC141 resulted in activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and induction of apoptosis. In conclusion, our data suggest that EC141 is a potent Hsp90 inhibitor with activity against Ph+ ALL. Further studies to investigate the anticancer effect of EC141 either as a single agent, or in combination in Ph+ ALL and other hematological malignancies are warranted.

  1. Potent, transient inhibition of BCR-ABL with dasatinib 100 mg daily achieves rapid and durable cytogenetic responses and high transformation-free survival rates in chronic phase chronic myeloid leukemia patients with resistance, suboptimal response or intolerance to imatinib

    Science.gov (United States)

    Shah, Neil P.; Kim, Dong-Wook; Kantarjian, Hagop; Rousselot, Philippe; Llacer, Pedro Enrique Dorlhiac; Enrico, Alicia; Vela-Ojeda, Jorge; Silver, Richard T.; Khoury, Hanna Jean; Müller, Martin C.; Lambert, Alexandre; Matloub, Yousif; Hochhaus, Andreas

    2010-01-01

    Background Dasatinib 100 mg once daily achieves intermittent BCR-ABL kinase inhibition and is approved for chronic-phase chronic myeloid leukemia patients resistant or intolerant to imatinib. To better assess durability of response to and tolerability of dasatinib, data from a 2-year minimum follow-up for a dose-optimization study in chronic-phase chronic myeloid leukemia are reported here. Design and Methods In a phase 3 study, 670 chronic-phase chronic myeloid leukemia patients with resistance, intolerance, or suboptimal response to imatinib were randomized to dasatinib 100 mg once-daily, 50 mg twice-daily, 140 mg once-daily, or 70 mg twice-daily. Results Data from a 2-year minimum follow-up demonstrate that dasatinib 100 mg once daily achieves major cytogenetic response and complete cytogenetic response rates comparable to those in the other treatment arms, and reduces the frequency of key side effects. Comparable 2-year progression-free survival and overall survival rates were observed (80% and 91%, respectively, for 100 mg once daily, and 75%–76% and 88%–94%, respectively, in other arms). Complete cytogenetic responses were achieved rapidly, typically by 6 months. In patients treated with dasatinib 100 mg once daily for 6 months without complete cytogenetic response, the likelihood of achieving such a response by 2 years was 50% for patients who had achieved a partial cytogenetic response, and only 8% or less for patients with minor, minimal, or no cytogenetic response. Less than 3% of patients suffered disease transformation to accelerated or blast phase. Conclusions Intermittent kinase inhibition can achieve rapid and durable responses, indistinguishable from those achieved with more continuous inhibition. PMID:20139391

  2. Cross-protection provided by live Shigella mutants lacking major antigens.

    Science.gov (United States)

    Szijártó, Valéria; Hunyadi-Gulyás, Eva; Emődy, Levente; Pál, Tibor; Nagy, Gábor

    2013-05-01

    The immune response elicited by Shigella infections is dominated by serotype-specific antibodies recognizing the LPS O-antigens. Although a marked antibody response to invasion plasmid antigens (Ipa-s) shared by all virulent strains is also induced, the varying level of immunity elicited by natural infections is serotype-restricted. Previous vaccines have tried to mimic and achieve this serotype-specific, infection-induced immunity. As, however, the four Shigella species can express 50 different types of O-antigens, current approaches with the aim to induce a broad coverage use a mixture of the most common O-antigens combined in single vaccines. In the current study we present data on an alternative approach to generate immunity protective against multiple serotypes. Mutants lacking both major immune-determinant structures (i.e. the Ipa and O-antigens) were not only highly attenuated, but, unlike their avirulent counterparts still expressing these antigens, elicited a protective immune response to heterologous serotypes in a murine model. Evidence is provided that protection was mediated by the enhanced immunogenic potential of minor conserved antigens. Furthermore, the rough, non-invasive double mutants triggered an immune response different from that induced by the smooth, invasive strains regarding the isotype of antibodies generated. These non-invasive, rough mutants may represent promising candidates for further development into live vaccines for the prophylaxis of bacillary dysentery in areas with multiple endemic serotypes.

  3. Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system.

    Science.gov (United States)

    Narita, Yuka; Sato, Keiko; Yukitake, Hideharu; Shoji, Mikio; Nakane, Daisuke; Nagano, Keiji; Yoshimura, Fuminobu; Naito, Mariko; Nakayama, Koji

    2014-10-01

    Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, these mutants exhibited decreased haemagglutination and increased biofilm formation. Comparison of the proteins secreted by the porK and WT strains revealed that the secretion of several proteins containing C-terminal domain (CTD)-like sequences is dependent on the porK gene. These results indicate that the T9SS is functional in T. forsythia and contributes to the translocation of CTD proteins to the cell surface or into the extracellular milieu.

  4. Efficacy of Dasatinib in Treatment of Imatinib-Resistant BCR/ABL Positive Leukemia%达沙替尼治疗伊马替尼耐药的BCR/ABL阳性白血病的临床研究

    Institute of Scientific and Technical Information of China (English)

    朱雨; 潘良琴; 钱思轩; 宋萍; 于慧; 张苏江; 葛峥; 洪鸣; 田甜

    2013-01-01

    本研究评价达沙替尼治疗原发或继发伊马替尼耐药的BCR/ABL阳性白血病的疗效和安全性.对27例原发或继发伊马替尼耐药的慢性髓系白血病(CML)或Ph阳性急性淋巴细胞白血病(Ph+ ALL)患者,给予达沙替尼100-140 mg/d口服治疗,评估疗效、总体生存和耐受情况.结果表明:27例伊马替尼耐药的BCR/ABL阳性白血病中位达沙替尼治疗时间8(1-66)个月,中位随访时间54(3-75)个月.27例接受达沙替尼治疗的患者中,88.8%获得完全血液学反应(CHR),44.4%获得主要细胞遗传学反应(mCyR),37%获得完全遗传学反应(CCyR),18.5%获得主要分子学反应(MMR).伊马替尼耐药的进展期(CML-AP、CML-BC、骨髓复发Ph+ ALL)患者接受达沙替尼治疗获CCyR率低于疾病稳定期(CML-CP、骨髓缓解Ph+ ALL)患者(P=0.0377),且3-4级不良反应发生率明显增高.达沙替尼治疗后获得CCyR的患者生存期(OS)较未达CCyR者明显延长(63个月vs9个月,P=0.0126).达沙替尼治疗后最常见的3-4级不良反应包括血液学反应如血小板减少(51.8%)、中性粒细胞减少(48.1%)、贫血(33.3%)和非血液学反应如胸腔积液(18.5%)、肺部感染(18.5%)、心包积液(11.1%).3-4级不良反应主要发生在疾病进展期时改服达沙替尼者,均发生于服药12个月内.结论:达沙替尼治疗伊马替尼耐药的BCR/ABL阳性白血病有效,且在疾病稳定期改服达沙替尼疗效和耐受性更好.%This study was aimed to evaluate the efficacy and safety of dasatinib in BCR/ABL positive leukemia patients with primary or secondary resistance to imatinib.27 patients with primary or secondary imatinib-resistant chronic myelogenous leukemia (CML) or Philadelphia chromosome positive acute lymphocytic leukemia (Ph + ALL) received 100-140 mg/d dasatinib orally.Their overall survival and tolerance were evaluated.The results showed that the median duration of dasatinib therapy was 8 (1-66) months in the 27

  5. Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase.

    Science.gov (United States)

    García-Calderón, Margarita; Pons-Ferrer, Teresa; Mrázova, Anna; Pal'ove-Balang, Peter; Vilková, Mária; Pérez-Delgado, Carmen M; Vega, José M; Eliášová, Adriana; Repčák, Miroslav; Márquez, Antonio J; Betti, Marco

    2015-01-01

    This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2) in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L. japonicus plants in response to stress.

  6. Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase

    Directory of Open Access Journals (Sweden)

    Margarita eGarcía-Calderón

    2015-09-01

    Full Text Available This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2 in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L .japonicus plants in response to stress.

  7. Evidence for dynamic network regulation of Drosophila photoreceptor function from mutants lacking the neurotransmitter histamine

    Directory of Open Access Journals (Sweden)

    An eDau

    2016-03-01

    Full Text Available Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. Here, we investigate this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the hdcJK910 mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that hdcJK910 photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected hdcJK910 photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to hdcJK910 R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in hdcJK910 mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons.

  8. Evidence for Dynamic Network Regulation of Drosophila Photoreceptor Function from Mutants Lacking the Neurotransmitter Histamine.

    Science.gov (United States)

    Dau, An; Friederich, Uwe; Dongre, Sidhartha; Li, Xiaofeng; Bollepalli, Murali K; Hardie, Roger C; Juusola, Mikko

    2016-01-01

    Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. Here, we investigate this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the hdc (JK910) mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that hdc (JK910) photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected hdc (JK910) photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to hdc (JK910) R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in hdc (JK910) mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons.

  9. 达沙替尼治疗伊马替尼耐药的 BCR/ABL阳性白血病%Dasatinib in treatment of imatinib-resistant BCR/ABL positive leukemia

    Institute of Scientific and Technical Information of China (English)

    严红; 赵海军

    2014-01-01

    This study was aimed to evaluate the efficacy and safety of dasatinib in BCR /ABL positive leukemia patients with resistance to imatinib.Method 9 patients with imatinib -resistant chronic myelogenous leukemia ( CML) or Philadelphia chromosome positive acute lymphocytic leukemia (Ph+ALL) received 100~140 mg· d-1 dasatinib orally.Their overall survival and tolerance were evalua-ted.Results After the dasatinib treatment ,2 patients with CML in chronic phase achieved complete hematologic response ( CHR) ,one of them achieved complete cytogenetic response (CCyR);4 of 5 patients with CML in blast crisis achieved CHR and partly cytogenetic response (PCyR);one patient had no response .Treated with dasatinib,one patient with Ph +ALL,presented with E255V mutation,a-chieved CHR and PCyR .The other died of infection .Conlusion Receiving therapy of dasatinib , patients with resistance to imatinib could achieve CHR ,even PCyR .%目的:评价达沙替尼治疗伊马替尼耐药的BCR/ABL阳性白血病的疗效和安全性。方法对9例伊马替尼耐药的慢性髓系白血病( CML)或Ph阳性急性淋巴细胞白血病( Ph+ALL)患者,给予达沙替尼100~140 mg· d-1口服治疗,评估疗效和耐受情况。结果9例伊马替尼耐药的BCR/ABL阳性白血病,2例CML-CP患者均获得CHR,1例达CCyR;5例CML-BC患者中4例获得CHR和PCyR,1例NR;2例Ph+ALL患者中1例检测到E255V突变,采用达沙替尼治疗达CHR和PCyR,1例诱导缓解时,同时行VDP方案化疗,继发严重感染死亡。结论达沙替尼治疗伊马替尼耐药的BCR/ABL阳性白血病患者可获得血液学甚至细胞遗传学缓解,且耐受性好。

  10. Behavioral characterization of a mutant mouse strain lacking D-amino acid oxidase activity.

    Science.gov (United States)

    Zhang, Min; Ballard, Michael E; Basso, Ana M; Bratcher, Natalie; Browman, Kaitlin E; Curzon, Pete; Konno, Ryuichi; Meyer, Axel H; Rueter, Lynne E

    2011-02-02

    D-amino acid oxidase (DAO), an enzyme that degrades d-serine, has been suggested as a susceptibility factor for schizophrenia. Here we sought to understand more about the behavioral consequence of lacking DAO and the potential therapeutic implication of DAO inhibition by characterizing a mouse strain (ddY/DAO(-)) lacking DAO activity. We found that the mutant mice showed enhanced prepulse inhibition responses (PPI). Intriguingly, DAO-/- mice had increased sensitivity to the PPI-disruptive effect induced by the competitive NMDA antagonist, SDZ 220-581. In the 24-h inhibitory avoidance test, DAO-/- mice were not different from DAO+/+ mice during the recall. In Barnes Maze, we found that DAO-/- mice had a shortened latency to enter the escape tunnel. Interestingly, although these mice were hypoactive when tested in a protected open field, they showed a profound increase of activity on the edge of the unprotected open field of the Barnes Maze even with the escape tunnel removed. This increased edge activity does not appear to be related to a reduced level of anxiety given that there were no significant genotype effects on the measures of anxiety-like behaviors in two standard animal models of anxiety, elevated plus maze and novelty suppressed feeding. Our data suggest that DAO-/- mice might have altered functioning of NMDARs. However, these results provide only modest support for manipulations of DAO activity as a potential therapeutic approach to treat schizophrenia.

  11. PESV对K562细胞BCR/ABL融合基因及凋亡调控因子Bcl-2、Bad表达的影响%The Effects of PESV on the Expression of BCR/ABL Fusion Gene and Bcl-2, Bad of Apoptosis Regulators on the K562 Cells

    Institute of Scientific and Technical Information of China (English)

    于文俊; 杨文华; 杨向东; 史哲新; 王兴丽; 郝征; 张佳

    2012-01-01

    Objective: To investigate the PESV of K562 cells BCR / ABL fusion gene and apoptosis regulators bcl-2 and bad expression. Methods: K562 cells were cultured in vitro, by PESV for different times, the apoptosis rate by flow cytometry, fluorescence quantitative RT-PCR detection of BCR / ABL, Bcl-2, Bad mRNA level changes. Results: Compared with the control group, PESV treated K562 cells, apoptosis increased, BCR / ABL fusion gene reduced expression, anti-apoptotic gene Bcl-2 mRNA expression decreased, pro-apoptotic gene Bad mRNA expression. Conclusion: PESV reduced in K562 cells can reduce the BCR / ABL fusion gene, may regulate the expression of Bcl-2 and Bad, inhibit proliferation of K562 cells and promote their apoptosis.%目的:探讨PESV对K562细胞BCR/ABL融合基因及凋亡调控因子bcl-2和bad表达的影响.方法:将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,荧光定量RT-PCR检测BCR/ABL、Bcl-2、Bad mRNA水平变化.结果:与对照组相比,PESV处理后K562细胞,凋亡率增加,BCR/ABL融合基因表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加.结论:PESV能降低降低K562细胞BCR/ABL融合基因的表达,可能通过调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.

  12. Mutant huntingtin regulates EGF receptor fate in non-neuronal cells lacking wild-type protein.

    Science.gov (United States)

    Melone, Mariarosa A B; Calarco, Anna; Petillo, Orsolina; Margarucci, Sabrina; Colucci-D'Amato, Luca; Galderisi, Umberto; Koverech, Guido; Peluso, Gianfranco

    2013-01-01

    Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild-type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

  13. Mutant mice lacking the p53 C-terminal domain model telomere syndromes.

    Science.gov (United States)

    Simeonova, Iva; Jaber, Sara; Draskovic, Irena; Bardot, Boris; Fang, Ming; Bouarich-Bourimi, Rachida; Lejour, Vincent; Charbonnier, Laure; Soudais, Claire; Bourdon, Jean-Christophe; Huerre, Michel; Londono-Vallejo, Arturo; Toledo, Franck

    2013-06-27

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.

  14. Cadmium toxicity to Microcystis aeruginosa PCC 7806 and its microcystin-lacking mutant.

    Directory of Open Access Journals (Sweden)

    Bin Huang

    Full Text Available The adverse effects of microcystin (MC produced by cyanobacteria have drawn considerable attention from the public. Yet it remains unclear whether MC confers any benefits to the cyanobacteria themselves. One suggested function of MC is complexation, which may influence the bioaccumulation and toxicity of trace metals. To test this hypothesis, we examined Cd toxicity to wild-type Microcystis aeruginosa PCC 7806 (WT and its MC-lacking mutant (MT under nutrient-enriched (+NP, phosphorus-limited (-P, and nitrogen-limited (-N conditions. The accumulation of Cd and the biochemical parameters associated with its detoxification [total phosphorus (TP, inorganic polyphosphate (Poly-P, and glutathione (GSH in the cells as well as intra- and extra-cellular carbohydrates] were quantified. Although the -P cyanobacteria accumulated less Cd than their +NP and -N counterparts, the different nutrient-conditioned cyanobacteria were similarly inhibited by similar free ion concentration of Cd in the medium ([Cd2+]F. Such good toxicity predictability of [Cd2+]F was ascribed to the synchronous decrease in the intracellular concentrations of Cd and TP. Nevertheless, Cd toxicity was still determined by the intracellular Cd to phosphorus ratio (Cd/P, in accordance with what has been reported in the literature. On the other hand, the concentrations of TP, Poly-P, and carbohydrates went up, but GSH concentration dropped down with the enhancement of [Cd2+]F, indicating their association with Cd detoxification. Although the inactivation of MC peptide synthetase gene had some nutrient and Cd concentration dependent effects on the parameters above, both cyanobacterial strains showed the same Cd accumulation ability and displayed similar Cd sensitivity. These results suggest that MC cannot affect metal toxicity either by regulating metal accumulation or by altering the detoxification ability of the cyanobacteria. Other possible functions of MC need to be further investigated.

  15. A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase.

    Science.gov (United States)

    Ray, A; Williams, H D

    1996-01-01

    Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c-type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway's terminal oxidase is a quinol oxidase.

  16. A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase. Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain.

    Science.gov (United States)

    Middelhoven, W J; van Eijk, J; van Renesse, R; Blijham, J M

    1978-01-01

    NAD-specific glutamate dehydrogenase (GDH-B) was induced in a wild-type strain derived of alpha-sigma 1278b by alpha-amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing alpha-amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate. Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases area lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.

  17. Reduced activity of glutamine synthetase in Rhodospirillum rubrum mutants lacking the adenylyltransferase GlnE.

    Science.gov (United States)

    Jonsson, Anders; Nordlund, Stefan; Teixeira, Pedro Filipe

    2009-10-01

    In the nitrogen-fixing bacterium Rhodospirillum rubrum, the GlnE adenylyltransferase (encoded by glnE) catalyzes reversible adenylylation of glutamine synthetase, thereby regulating nitrogen assimilation. We have generated glnE mutant strains that are unable to adenylylate glutamine synthetase (GS). Surprisingly, the activity of GS was lower in the mutants than in the wild type, even when grown in nitrogen-fixing conditions. Our results support the proposal that R. rubrum can only cope with the absence of an adenylylation system in the presence of lowered GS expression or activity. In general terms, this report also provides further support for the central role of GS in bacterial metabolism.

  18. Characterization of yeast mutants lacking alkaline ceramidases YPC1 and YDC1

    DEFF Research Database (Denmark)

    Voynova, Natalia S; Mallela, Shamroop K; Vazquez, Hector M;

    2014-01-01

    /conditions that would alter the growth of ypc1∆ydc1∆ double mutants. These screens were essentially negative, demonstrating that ceramidase activity is not required for cell growth even under genetic stresses. A previously reported protein targeting defect of ypc1∆ could not be reproduced and reported abnormalities...

  19. Classical ethylene insensitive mutants of the Arabidopsis EIN2 orthologue lack the expected 'hypernodulation' response in Lotus japonicus.

    Science.gov (United States)

    Chan, Pick Kuen; Biswas, Bandana; Gresshoff, Peter M

    2013-04-01

    Three independent ethylene insensitive mutants were selected from an EMS- mutagenized population of Lotus japonicus MG-20 (Miyakojima). The mutants, called 'Enigma', were mutated in the LjEIN2a gene from Lotus chromosome 1, sharing significant homology with Arabidopsis EIN2 (ethylene-insensitive2). All three alleles showed classical ethylene insensitivity phenotypes (e.g., Triple Response), but lacked the increased nodulation phenotype commonly associated with ethylene insensitivity. Indeed, all showed a marginal reduction in nodule number per plant, a phenotype that is enigmatic to sickle, an ethylene-insensitive EIN2 mutant in Medicago truncatula. In contrast to wild type, but similar to an ETR1-1 ethylene ethylene-insensitive transgenic of L. japonicus, enigma mutants formed nodules in between the protoxylem poles, demonstrating the influence of ethylene on radial positioning. Suppression of nodule numbers by nitrate and colonisation by mycorrhizal fungi in the enigma-1 mutant were indistinguishable from the wild-type MG-20. However, reflecting endogenous ethylene feedback, the enigma-1 mutant released more than twice the wild-type amount of ethylene. enigma-1 had a moderate reduction in growth, greater root mass (and lateral root formation), delayed flowering and ripening, smaller pods and seeds. Expression analysis of ethylene-regulated genes, such as ETR1, NRL1 (neverripe-like 1), and EIL3 in shoots and roots of enigma-1 and MG-20 illustrated that the ethylene-insensitive mutation strongly affected transcriptional responses in the root. These mutants open the possibility that EIN2 in L. japonicus, a determinate nodulating legume, acts in a more complex fashion possibly through the presence of a duplicated copy of LjEIN2.

  20. Classical Ethylene Insensitive Mutants of the Arabidopsis EIN2Orthologue Lack the Expected 'hypernodulation' Response in Lotus japonicus

    Institute of Scientific and Technical Information of China (English)

    Pick Kuen Chan; Bandana Biswas; Peter M.Gresshoff

    2013-01-01

    Three independent ethylene insensitive mutants were selected from an EMS-mutagenized population of Lotus japonicus MG-20 (Miyakojima).The mutants,called 'Enigma',were mutated in the LjEIN2a gene from Lotus chromosome 1,sharing significant homology with Arabidopsis EIN2 (ethylene-insensitive2).All three alleles showed classical ethylene insensitivity phenotypes (e.g.,Triple Response),but lacked the increased nodulation phenotype commonly associated with ethylene insensitivity.Indeed,all showed a marginal reduction in nodule number per plant,a phenotype that is enigmatic to sickle,an ethyleneinsensitive EIN2 mutant in Medicago truncatula.In contrast to wild type,but similar to an ETR1-1 ethylene ethylene-insensitive transgenic of L.japonicus,enigma mutants formed nodules in between the protoxylem poles,demonstrating the influence of ethylene on radial positioning.Suppression of nodule numbers by nitrate and colonisation by mycorrhizal fungi in the enigma-1 mutant were indistinguishable from the wild-type MG-20.However,reflecting endogenous ethylene feedback,the enigma-1 mutant released more than twice the wild-type amount of ethylene.enigma-1 had a moderate reduction in growth,greater root mass (and lateral root formation),delayed flowering and ripening,smaller pods and seeds.Expression analysis of ethylene-regulated genes,such as ETR1,NRL1 (neverripe-like 1),and ElL3 in shoots and roots of enigma-1 and MG-20 illustrated that the ethylene-insensitive mutation strongly affected transcriptional responses in the root.These mutants open the possibility that EIN2 in L.japonicus,a determinate nodulating legume,acts in a more complex fashion possibly through the presence of a duplicated copy of LjEIN2.

  1. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

    Directory of Open Access Journals (Sweden)

    Carlos Bueno

    Full Text Available Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the

  2. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands

    Science.gov (United States)

    Bueno, Carlos; Tabares-Seisdedos, Rafael; Moraleda, Jose M.; Martinez, Salvador

    2016-01-01

    Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may

  3. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

    Science.gov (United States)

    Bueno, Carlos; Tabares-Seisdedos, Rafael; Moraleda, Jose M; Martinez, Salvador

    2016-01-01

    Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may

  4. 荧光原位杂交在慢性粒细胞白血病细胞BCR/ABL融合基因检测中的应用及其意义%The detection of BCR/ABL fusion gene by interphase fluorescence in situ hybridization in patients with chronic myeloid leukemia and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    张济; 李君君; 颜家运; 邹礼衡; 刘静

    2012-01-01

    目的:探讨荧光原位杂交(FISH)技术检测慢性粒细胞白血病(CML)骨髓和外周血细胞的BCR /ABL融合基因的临床价值.方法:应用FISH 对正常对照组及CML患者骨髓和外周血细胞BCR/ ABL融合基因进行检测和分析.结果:慢性期CML 患者骨髓和外周血细胞该融合基因阳性细胞率分别为(61.9 ± 22.3)%和(68.4 ± 19.8)%,加速期为(77.2 ± 16.7)% 和(86.8 ± 12.1)%,急变期为(80.6 ± 17.5)%和(81.4 ± 18.0)%,两者细胞中BCR/ABL 基因的阳性率未见统计学差异(P>0.05),且骨髓和外周血细胞之间融合基因阳性细胞比率呈直线正相关.同时发现在完全临床缓解患者中,经伊马替尼治疗的CML 患者(71.4%)较经干扰素和羟基脲联合治疗者(10.0%)有更高的分子生物学缓解(P 0.05). A significant correlation was found between the BCR/ABL positive cells in peripheral blood and in bone marrow samples. Moreover, the higher rate of complete molecular remission was achieved by treating with imatinib than with interferon and hydroxyurea during complete remission phase (P < 0.05). Conclusions Detecting BCR/ABL fusion gene in peripheral blood and bone marrow samples by FISH are beneficial to diagnosis, treatment and minimal residual disease monitor in CML.

  5. Mutant mice lacking acetyl-CoA carboxylase 1 are embryonically lethal

    Science.gov (United States)

    Abu-Elheiga, Lutfi; Matzuk, Martin M.; Kordari, Parichher; Oh, WonKeun; Shaikenov, Tattym; Gu, Ziwei; Wakil, Salih J.

    2005-01-01

    Acetyl-CoA carboxylases (ACC1 and ACC2) catalyze the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate metabolite that plays a pivotal role in the regulation of fatty acid metabolism. We previously reported that ACC2 null mice are viable, and that ACC2 plays an important role in the regulation of fatty acid oxidation through the inhibition of carnitine palmitoyltransferase I, a mitochondrial component of the fatty-acyl shuttle system. Herein, we used gene targeting to knock out the ACC1 gene. The heterozygous mutant mice (Acc1+/–) had normal fertility and lifespans and maintained a similar body weight to that of their wild-type cohorts. The mRNA level of ACC1 in the tissues of Acc1+/– mice was half that of the wild type; however, the protein level of ACC1 and the total malonyl-CoA level were similar. In addition, there was no difference in the acetate incorporation into fatty acids nor in the fatty acid oxidation between the hepatocytes of Acc1+/– mice and those of the wild type. In contrast to Acc2–/– mice, Acc1–/– mice were not detected after mating. Timed pregnancies of heterozygotes revealed that Acc–/– embryos are already undeveloped at embryonic day (E)7.5, they die by E8.5, and are completely resorbed at E11.5. Our previous results of the ACC2 knockout mice and current studies of ACC1 knockout mice further confirm our hypotheses that malonyl-CoA exists in two independent pools, and that ACC1 and ACC2 have distinct roles in fatty acid metabolism. PMID:16103361

  6. D-Amino acids in the brain and mutant rodents lacking D-amino-acid oxidase activity.

    Science.gov (United States)

    Yamanaka, Masahiro; Miyoshi, Yurika; Ohide, Hiroko; Hamase, Kenji; Konno, Ryuichi

    2012-11-01

    D-Amino acids are stereoisomers of L-amino acids. They are often called unnatural amino acids, but several D-amino acids have been found in mammalian brains. Among them, D-serine is abundant in the forebrain and functions as a co-agonist of NMDA receptors to enhance neurotransmission. D-Amino-acid oxidase (DAO), which degrades neutral and basic D-amino acids, is mainly present in the hindbrain. DAO catabolizes D-serine and, therefore, modulates neurotransmission. In the brains of mutant mice and rats lacking DAO activity, the amounts of D-serine and other D-amino acids are markedly increased. Mutant mice manifested behavioral changes characteristic of altered NMDA receptor activity, likely due to increased levels of D-serine. D-Serine and DAO have been demonstrated to play important roles in cerebellar development and synaptic plasticity. They have also implicated in amyotrophic lateral sclerosis and pain response. There have also been several lines of evidence correlating DAO with schizophrenia. Taken together, the experiments indicate that D-amino acids and DAO have pivotal functions in the central nervous system.

  7. 伴不典型BCR-ABL融合基因的慢性髓性白血病的临床和实验研究%A clinical and laboratory study of chronic myeloid leukemia with atypical BCR-ABL fusion gene subtypes

    Institute of Scientific and Technical Information of China (English)

    桂晓敏; 潘金兰; 仇惠英; 岑建农; 薛永权; 陈苏宁; 沈宏杰; 姚利; 张俊

    2014-01-01

    目的 探讨伴不典型BCR-ABL融合基因亚型el4a3和e19a2型慢性髓性白血病(CML)的临床和实验室特点.方法 对2004至2012年染色体核型分析有t(9;22) (q34;q11),荧光原位杂交(FISH)证实为BCR-ABL融合基因阳性,而常规实时定量PCR(RQ-PCR)检测常见BCR-ABL融合基因(b3a2、b2a2和e1a2)阴性的6例CML患者,重新设计引物进行PCR扩增,并将扩增产物测序,以明确不典型BCR-ABL融合基因类型.对BCR-ABL融合基因扩增产物进行突变检测.对患者的临床资料进行回顾性分析.结果 6例患者PCR扩增产物经测序分析,其中5例为e14a3型,1例为e19a2型.5例e14a3型CML患者中,男4例,女1例,中位年龄48岁,慢性期4例,加速期1例;1例e19a2型患者为女性,40岁,CML慢性期,PLT>1 000× 109/L.5例e14a3型CML患者中4例在羟基脲或IFN治疗无效后予以伊马替尼(IM)治疗,1例行造血干细胞移植(HSCT).前4例患者中1例因有E255K突变而对IM耐药,改用达沙替尼后获完全细胞遗传学反应(CCyR);1例在IM治疗获CCyR后3个月复发并急变,最终死亡;2例IM治疗后获CCyR,目前状态稳定,仍处于CCyR,尽管其中1例伴有I293T突变.行HSCT治疗患者目前处于CCyR.1例e19a2型CML患者羟基脲治疗后获得完全血液学反应,后改用IM治疗,很快获得CCyR.结论 伴不典型BCR-ABL融合基因的CML发病率极低,酪氨酸激酶抑制剂或HSCT都可以取得疗效,常规RQ-PCR可能漏检少见的不典型BCR-ABL融合基因亚型.%Objective To explore the clinical and laboratory features of chronic myeloid leukemia (CML) with atypical e14a3 and e19a2 BCR-ABL fusion gene subtypes.Methods We retrospectively analyzed a cohort of CML patients with Ph chromosome positive confirmed by cytogenetic and FISH but classical el3a3 (b2a2),e14a2 (b3a2)and ela2 fusion transcripts negative identified by conventional realtime quantification RT-PCR (RQ-PCR).Further RQ-PCR was done with the forward primer and reverse primer designed to

  8. A Temporarily Red Light-Insensitive Mutant of Tomato Lacks a Light-Stable, B-Like Phytochrome.

    Science.gov (United States)

    Van Tuinen, A.; Kerckhoffs, LHJ.; Nagatani, A.; Kendrick, R. E.; Koornneef, M.

    1995-07-01

    We have selected four recessive mutants in tomato (Lycopersicon esculentum Mill.) that, under continuous red light (R), have long hypocotyls and small cotyledons compared to wild type (WT), a phenotype typical of phytochrome B (phyB) mutants of other species. These mutants, which are allelic, are only insensitive to R during the first 2 days upon transition from darkness to R, and therefore we propose the gene symbol tri (temporarily red light insensitive). White light-grown mutant plants have a more elongated growth habit than that of the WT. An immunochemically and spectrophotometrically detectable phyB-like polypeptide detectable in the WT is absent or below detection limits in the tri1 mutant. In contrast to the absence of an elongation growth response to far-red light (FR) given at the end of the daily photoperiod (EODFR) in all phyB-deficient mutants so far characterized, the tri1 mutant responds to EODFR treatment. The tri1 mutant also shows a strong response to supplementary daytime far-red light. We propose that the phyB-like phytochrome deficient in the tri mutants plays a major role during de-etiolation and that other light-stable phytochromes can regulate the EODFR and shade-avoidance responses in tomato.

  9. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  10. BCR/ABL阴性骨髓增生性疾病患者JAK2-V617F点突变与外周血三系变化的相关性%Correlation between JAK2-V617F mutations and variation of peripheral blood cells among BCR/ABL-negative MPD patients

    Institute of Scientific and Technical Information of China (English)

    叶远馨; 宋兴勃; 周易; 应斌武; 陆小军

    2012-01-01

    目的 探讨BCR/ABL阴性骨髓增生性疾病患者JAK2-V617F突变的阳性率以及突变量与外周血三系变化的相关性.方法 对191例确诊为BCR/ABL阴性的骨髓增生性疾病患者进行JAK2-V617F突变检测,分别以外周血中红细胞、白细胞、血小板计数进行分类统计,分析不同类型的标本JAK2-V617F基因点突变的发生率及相对定量变化.结果 外周血红细胞、白细胞、血小板计数不同范围的标本JAK2-V617F突变阳性率有统计学差异.随着外周血红细胞、血小板计数增高,JAK2-V617F突变阳性率也随之增高,且以三系均增多者JAK2-V617F突变率为最高(92.86%).结论 BCR/ABL阴性的骨髓增生性疾病JAK2-V617F点突变发生率与外周血三系变化存在明显的相关性.外周血三系变化、尤其是红细胞计数与BCR/ABL阴性的骨髓增生性疾病JAK2-V617F突变的阳性率以及突变相对定量存在一定的相关性.%Objective To assess the correlation between JAK2-V617F mutation and complete blood counts among patients with BCR/ABL-negative myeloproliferative diseases (MPD).Methods One hundred and ninety one patients were recruited.Retrospectively,their laboratory data were analyzed for the counts of red blood cells (RBC),white blood cells (WBC) and platelets (PLT).And the incidence of JAK2-V617F mutation was determined.Results There was significant difference in the incidence of JAK2-V617F mutation between patients with different cell counts (P<0.01).The incidence of JAK2-V617F mutation has increased with the counts of RBC and PLT,which was the highest (92.86%) among those featuring simultaneous increase in all three series.Conclusion The incidence of JAK2-V617F mutation seems to be strongly associated with variation of peripheral blood cell counts among patients with BCR/ABL-negative MPD.Variation of peripheral blood cells,particularly RBC,may be correlated with the rate of JAK2-V617F mutation.

  11. Dasatinib treatment based on BCR-ABL mutation detection in imatinib-resistant patients with chronic myeloid leukemia%BCR-ABL突变检测指导下的达沙替尼治疗伊马替尼耐药的慢性髓性白血病疗效分析

    Institute of Scientific and Technical Information of China (English)

    江倩; 秦亚溱; 赖悦云; 江浩; 石红霞

    2016-01-01

    Objective To evaluate the efficiency of dasatinib as the second-or third-line tyrosine kinase inhibitor(TKI)in imatinib-resistant patients with chronic myeloid leukemia(CML)based on BCR-ABL mutation detection. Methods 122 CML patients received dasatinib treatment, including 83 with imatinib-resistance and 39 with both imatinib-and nilotinib-resistance, 55 in the chronic-phase(CP), 21 in the accelerated-phase (AP) and 46 in the blast-phase (BP). Those harboring dasatinib highly-resistant mutations(T315I/A, F317L/V/C and V299L)were excluded based on BCR-ABL kinase domain mutation screening by Sanger sequencing at baseline. Hematologic, cytogenetic and molecular responses were evaluated regularly, and rates of progression-free-survival(PFS)and overall survival(OS)were analyzed. BCR-ABL mutation detection was performed once the patients failed on dasatinib. Results In the CP patients, the rates of complete hematological response (CHR), complete cytogenetic response (CCyR), major molecular response(MMR)and molecular response 4.5(MR4.5)were 92.7%, 53.7%, 29.6%and 14.8%, respectively. 4-year PFS and OS rates were 84.4%and 89.5%, respectively. In the AP patients, HR and CCyR rates were 81.0%and 35.0%;and 3-year PFS and OS rates were 56.1%and 59.3%, respectively. In the BP patients, HR and CCyR rates were 63.0%and 21.4%;and 1-year PFS and OS rates were 43.6%and 61.8%, respectively. Outcomes were similar when dasatinib was used as the second-line TKI or thethird-line TKI. Of the 75 patients who were resistant to dasatinib, 37(48.7%)developed new mutation(s), and T315I(59.5%)was the most common mutation type. The patients who already harbored mutation(s) before dasatinib therapy achieved similar responses and outcomes to those with no mutation at baseline. However, they had higher likelihood of developing additional mutations associated with resistance to dasatinib(65.7%vs 34.1%, P=0.006). Conclusions Dasatinib was proved to be effective in the treatment of imatinib

  12. A new osteopetrosis mutant mouse strain (ntl) with odontoma-like proliferations and lack of tooth roots.

    Science.gov (United States)

    Lu, Xincheng; Rios, Hector F; Jiang, Baichun; Xing, Lianping; Kadlcek, Renata; Greenfield, Edward M; Luo, Guangbin; Feng, Jian Q

    2009-12-01

    A new spontaneous mouse mutant (ntl) with autosomal-recessive osteopetrosis was characterized. These mice formed tartrate-resistant acid phosphate (TRAP)-positive osteoclasts but their osteoclasts had no ruffled border and did not resorb bone. These mice displayed no tooth eruption or tooth root formation. Adult mutant mice developed odontoma-like proliferations near the proximal ends of the incisors. Intraperitoneal injection of progenitor cells from the liver of 16.5 days postcoitum wild-type embryos into newborn mutants rescued the osteopetrosis phenotype, indicating that the defects were intrinsic to the osteoclasts. Our findings not only provide further support for a critical role of osteoclasts in tooth eruption and tooth root development, but also suggest that the perturbation of the homeostasis of the odontogenic precursors of the incisors is primarily responsible for the development of the odontoma-like proliferations in this osteopetrosis mutant. Genetic mapping has narrowed down the location of the mutant allele to a genetic interval of 3.2 cM on mouse chromosome 17.

  13. Carbon dioxide fixation and photoevolution of hydrogen and oxygen in a mutant of Chlamydomonas lacking Photosystem I

    Energy Technology Data Exchange (ETDEWEB)

    Greenbaum, E.; Lee, J.W.; Tevault, C.V. [Oak Ridge National Lab., TN (United States)] [and others

    1995-09-01

    Sustained photoassimilation of atmospheric CO{sub 2} and simultaneous photoevolution of molecular hydrogen and oxygen has been observed in a Photosystem I deficient mutant B4 of Chlamydomonas reinhardtii that contains only Photosystem II. The data indicate that Photosystem II alone is capable of spanning the potential difference between water oxidation/oxygen evolution and ferredoxin reduction. The rates of both CO{sub 2} fixation and hydrogen and oxygen evolution are similar in the mutant to that of the wild-type C. reinhardtii 137c containing both photosystems. The wild-type had stable photosynthetic activity, measured as CO{sub 2} fixation, under both air and anaerobic conditions, while the mutant was stable only under anaerobic conditions. The results are discussed in terms of the fundamental mechanisms and energetics of photosynthesis and possible implications for the evolution of oxygenic photosynthesis.

  14. Redox crisis underlies conditional light-dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA.

    Science.gov (United States)

    Diamond, Spencer; Rubin, Benjamin E; Shultzaberger, Ryan K; Chen, You; Barber, Chase D; Golden, Susan S

    2017-01-24

    Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily light-dark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day.

  15. The lack of mitochondrial complex I in a CMSII mutant of Nicotiana sylvestris increases photorespiration through an increased internal resistance to CO2 diffusion.

    Science.gov (United States)

    Priault, P; Tcherkez, G; Cornic, G; De Paepe, R; Naik, R; Ghashghaie, J; Streb, P

    2006-01-01

    The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO(2), molar fraction (c(i)) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO(2) diffusion (g(m)) in mutant leaves was half that of WT leaves, thus decreasing the c(c) and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (J(C)/J(O)) and as a function of the calculated chloroplastic CO(2) molar fraction (c(c)) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower c(c) in ambient air, since initial Rubisco activity on the basis of equal c(c) values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO(2) concentrations, showing that limiting CO(2) supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.

  16. Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein

    Directory of Open Access Journals (Sweden)

    Roncaglia Enrica

    2011-04-01

    Full Text Available Abstract Background Reactive oxygen species (ROS are unavoidable by-products of oxygenic photosynthesis, causing progressive oxidative damage and ultimately cell death. Despite their destructive activity they are also signalling molecules, priming the acclimatory response to stress stimuli. Results To investigate this role further, we exposed wild type Arabidopsis thaliana plants and the double mutant npq1lut2 to excess light. The mutant does not produce the xanthophylls lutein and zeaxanthin, whose key roles include ROS scavenging and prevention of ROS synthesis. Biochemical analysis revealed that singlet oxygen (1O2 accumulated to higher levels in the mutant while other ROS were unaffected, allowing to define the transcriptomic signature of the acclimatory response mediated by 1O2 which is enhanced by the lack of these xanthophylls species. The group of genes differentially regulated in npq1lut2 is enriched in sequences encoding chloroplast proteins involved in cell protection against the damaging effect of ROS. Among the early fine-tuned components, are proteins involved in tetrapyrrole biosynthesis, chlorophyll catabolism, protein import, folding and turnover, synthesis and membrane insertion of photosynthetic subunits. Up to now, the flu mutant was the only biological system adopted to define the regulation of gene expression by 1O2. In this work, we propose the use of mutants accumulating 1O2 by mechanisms different from those activated in flu to better identify ROS signalling. Conclusions We propose that the lack of zeaxanthin and lutein leads to 1O2 accumulation and this represents a signalling pathway in the early stages of stress acclimation, beside the response to ADP/ATP ratio and to the redox state of both plastoquinone pool. Chloroplasts respond to 1O2 accumulation by undergoing a significant change in composition and function towards a fast acclimatory response. The physiological implications of this signalling specificity are

  17. Restoration of growth by manganese in a mutant strain of Escherichia coli lacking most known iron and manganese uptake systems

    DEFF Research Database (Denmark)

    Taudte, Nadine; German, Nadezhda; Zhu, Yong-Guan;

    2016-01-01

    The interplay of manganese and iron homeostasis and oxidative stress in Escherichia coli can give important insights into survival of bacteria in the phagosome and under differing iron or manganese bioavailabilities. Here, we characterized a mutant strain devoid of all know iron....../manganese-uptake systems relevant for growth in defined medium. Based on these results an exit strategy enabling the cell to cope with iron depletion and use of manganese as an alternative for iron could be shown. Such a strategy would also explain why E. coli harbors some iron- or manganese-dependent iso......-enzymes such as superoxide dismutases or ribonucleotide reductases. The benefits for gaining a means for survival would be bought with the cost of less efficient metabolism as indicated in our experiments by lower cell densities with manganese than with iron. In addition, this strain was extremely sensitive to the metalloid...

  18. Excitation energy transfer and charge separation are affected in Arabidopsis thaliana mutants lacking light-harvesting chlorophyll a/b binding protein Lhcb3.

    Science.gov (United States)

    Adamiec, Małgorzata; Gibasiewicz, Krzysztof; Luciński, Robert; Giera, Wojciech; Chełminiak, Przemysław; Szewczyk, Sebastian; Sipińska, Weronika; van Grondelle, Rienk; Jackowski, Grzegorz

    2015-12-01

    The composition of LHCII trimers as well as excitation energy transfer and charge separation in grana cores of Arabidopsis thaliana mutant lacking chlorophyll a/b binding protein Lhcb3 have been investigated and compared to those in wild-type plants. In grana cores of lhcb3 plants we observed increased amounts of Lhcb1 and Lhcb2 apoproteins per PSII core. The additional copies of Lhcb1 and Lhcb2 are expected to substitute for Lhcb3 in LHCII trimers M as well as in the LHCII "extra" pool, which was found to be modestly enlarged as a result of the absence of Lhcb3. Time-resolved fluorescence measurements reveal a deceleration of the fast phase of excitation dynamics in grana cores of the mutant by ~15 ps, whereas the average fluorescence lifetime is not significantly altered. Monte Carlo modeling predicts a slowing down of the mean hopping time and an increased stabilization of the primary charge separation in the mutant. Thus our data imply that absence of apoprotein Lhcb3 results in detectable differences in excitation energy transfer and charge separation.

  19. Action potential generation in the small intestine of W mutant mice that lack interstitial cells of Cajal

    DEFF Research Database (Denmark)

    Malysz, J; Thuneberg, L; Mikkelsen, Hanne Birte

    1996-01-01

    The small intestine of W/Wv mice lacks both the network of interstitial cells of Cajal (ICC), associated with Auerbach's plexus, and pacemaker activity, i.e., it does not generate slow-wave-type action potentials. The W/Wv muscle preparations showed a wide variety of electrical activities, ranging...... from total quiescence to generation of action potentials at regular or irregular frequency with or without periods of quiescence. The action potentials consisted of a slow component with superimposed spikes, preceded by a slowly developing depolarization and followed by a transient hyperpolarization....... The action potentials were completely abolished by L-type Ca2+ channel blockers. W/Wv mice responded to K+ channel blockade (0.5 mM Ba2+ or 10 mM tetraethylammonium chloride) with effects on amplitude, frequency, rate of rise, and duration of the action potentials. In quiescent tissues from W/Wv mice, K...

  20. Shank3-mutant mice lacking exon 9 show altered excitation/inhibition balance, enhanced rearing, and spatial memory deficit

    Directory of Open Access Journals (Sweden)

    Jiseok eLee

    2015-03-01

    Full Text Available Shank3 is a postsynaptic scaffolding protein implicated in synapse development and autism spectrum disorders. The Shank3 gene is known to produce diverse splice variants whose functions have not been fully explored. In the present study, we generated mice lacking Shank3 exon 9 (Shank3∆9 mice, and thus missing 5 out of 10 known Shank3 splice variants containing the N-terminal ankyrin repeat region, including the longest splice variant, Shank3a. Our X-gal staining results revealed that Shank3 proteins encoded by exon 9-containing splice variants are abundant in upper cortical layers, striatum, hippocampus, and thalamus, but not in the olfactory bulb or cerebellum, despite the significant Shank3 mRNA levels in these regions. The hippocampal CA1 region of Shank3∆9 mice exhibited reduced excitatory transmission at Schaffer collateral synapses and increased frequency of spontaneous inhibitory synaptic events in pyramidal neurons. In contrast, prelimbic layer 2/3 pyramidal neurons in the medial prefrontal cortex displayed decreased frequency of spontaneous inhibitory synaptic events, indicating alterations in the ratio of excitation/inhibition (E/I ratio in the Shank3∆9 brain. These mice displayed a mild increase in rearing in a novel environment and mildly impaired spatial memory, but showed normal social interaction and repetitive behavior. These results suggest that ankyrin repeat-containing Shank3 splice variants are important for E/I balance, rearing behavior, and spatial memory.

  1. BCR-ABL融合基因T315I突变的白血病行异基因HSCT的疗效及复发后的治疗分析%Curative effectiveness of allogeneic stem cell transplantation and treatment of recurrence in patients harboring T315I BCR-ABL mutated leukemias

    Institute of Scientific and Technical Information of China (English)

    桂晓敏; 仇惠英; 潘金兰; 周敏; 鲍协炳; 陈苏宁; 孙爱宁; 吴德沛; 岑建农

    2014-01-01

    Objective To retrospectively analyze the curative effectiveness of allogeneic stem cell transplantation (allo-SCT) for the leukemia patients with the T315I mutation,and to discuss the treatments for the relapsing patients after allo-SCT.Method Through observing 10 leukemia patients harboring a T315I BCR-ABL mutation who underwent allo-SCT,we analyzed the remission rate,recurrence rate and long-term survival situation of these patients and explored the treatments for relapsing patients after allo-SCT.Result Of 10 patients,4 patients were female and 6 were male.The median age was 29 years (range,18-49).Before HSCT,5 were given imatinib,3 nilotinib and 2 dasatinib,respectively.Ten patients (100%) obtained complete remission (CR) and the median remission time was 23 days (range,14-56).During the follow-up period,1 case of CML and 3 cases of Ph+-ALL (40%) obtained CR.The survival time was 75 to 741 days.In the 6 dead patients,1 died of upper gastrointestinal hemorrhage on the day 137 after allo-SCT; 2 died of graft versus host disease on the day 53 and 617 respectively after allo-SCT; 1 case of CML and 2 cases of Ph+ ALL relapsed on the day 27,50 and 130 respectively after allo-SCT,and 2 of them died on the day 5 and 40 respectively after relapse,and 1 recurrent case did not obtain remission after chemotherapy and died on the day 97 after relapse.Conclusion The allo-SCT can be used to treat the leukemia patients with T315I mutation,and high remission rate can be obtained,tut there is the high risk of recurrence.The patients with recurrent leukemia can take further relevant treatment to prolong survival time.%目的 分析BCR-ABL融合基因T315I突变的白血病患者接受异基因造血干细胞移植(HSCT)的资料,探讨移植疗效及复发后的治疗.方法 观察10例伴T315I突变的费城染色体阳性(Ph+)的急性淋巴细胞白血病(ALL)和慢性粒细胞白血病(CML)接受异基因HSCT后缓解率、复发率以及长期存活情况,分析这

  2. Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.

    Science.gov (United States)

    Preuner, Sandra; Barna, Agnes; Frommlet, Florian; Czurda, Stefan; Konstantin, Byrgazov; Alikian, Mary; Machova Polakova, Katerina; Sacha, Tomasz; Richter, Johan; Lion, Thomas; Gabriel, Christian

    2016-04-29

    Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.

  3. Transport and arrangement of the outer-dynein-arm docking complex in the flagella of Chlamydomonas mutants that lack outer dynein arms.

    Science.gov (United States)

    Wakabayashi, K; Takada, S; Witman, G B; Kamiya, R

    2001-04-01

    The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC.

  4. Combinations of Novel Histone Deacetylase and Bcr-Abl Inhibitors in the Therapy of Imatinib Mesylate-Sensitive and -Refractory Bcr-Abl Expressing Leukemia

    Science.gov (United States)

    2008-12-01

    changes to the principles of treatment for both basal cell and squamous cell skin cancers and changes in the consideration of radiotherapy . 550...including those that contain FLT-3 mutations, while relatively sparing NBMCs.26,28,29 On the basis of the strong rationale generated by these observations...neutropenia (14 percent) were mainly grade 3 or 4 and appeared to increase with increasing doses of the drug. Pruritus, rash, and dry skin were almost

  5. A model of sensitivity: 1,3-butadiene increases mutant frequencies and genomic damage in mice lacking a functional microsomal epoxide hydrolase gene.

    Science.gov (United States)

    Wickliffe, Jeffrey K; Ammenheuser, Marinel M; Salazar, James J; Abdel-Rahman, Sherif Z; Hastings-Smith, Darlene A; Postlethwait, Edward M; Lloyd, R Stephen; Ward, Jonathan B

    2003-01-01

    The specific role that polymorphisms in xenobiotic metabolizing enzymes play in modulating sensitivity to 1,3-butadiene (BD) genotoxicity has been relatively unexplored. The enzyme microsomal epoxide hydrolase (mEH) is important in detoxifying the mutagenic epoxides of BD (butadiene monoepoxide [BDO], butadiene diepoxide [BDO(2)]). Polymorphisms in the human mEH gene appear to affect the function of the enzyme. We exposed mice with normal mEH activity (WT) and knockout mice without mEH activity (KO) to 20 ppm BD (inhalation) or 30 mg/kg BDO(2) (intraperitoneal [IP] injection). We then compared Hprt mutant frequencies (MFs) among these groups. KO mice exposed to BD exhibited a significant (P damage in WT and KO mice (comet tail moment) following IP exposure to 3 mg/kg and 30 mg/kg BDO(2). KO mice exposed to 3 mg/kg exhibited significantly more DNA damage than controls (7.5-12.1-fold increase) and exposed WT mice (3 mg/kg; 4.8-fold increase). KO mice exposed to 30 mg/kg BDO(2) exhibited significantly more DNA damage than all other groups (2.3-27.9-fold increase). Correlation analysis indicated that a significant, positive relationship (r(2) = 0.92) exists between comet-measured damage and Hprt MFs. The lack of mEH activity increases the genetic sensitivity of mice exposed to BD and BDO(2). This model should facilitate a mechanistic understanding of the observed variation in human genetic sensitivity following exposure to BD.

  6. A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin.

    Science.gov (United States)

    Ochi, K

    1990-12-01

    A relaxed (rel) mutant was found among 70 thiopeptin-resistant isolates of Streptomyces coelicolor A3(2) which arose spontaneously. The ability of the rel mutant to accumulate ppGpp during Casamino acid deprivation was reduced 10-fold compared to the wild-type. Analysis of the ribosomal proteins by two-dimensional PAGE revealed that the mutant lacked a ribosomal protein, tentatively designated ST-L11. It was therefore classified as a relC mutant. The mutant was defective in producing A-factor and the pigmented antibiotic prodigiosin, in both liquid and agar cultures, but produced agarase normally. Production of actinorhodin, another pigmented antibiotic, was also abnormal; it appeared suddenly in agar cultures after 10 d incubation. Although aerial mycelium still formed, its appearance was markedly delayed. Whereas liquid cultures of the parent strain accumulated ppGpp, agar cultures accumulated only trace amounts. Instead, a substance characterized only as an unidentified HPLC peak accumulated intracellularly in the late growth phase, just before aerial mycelium formation and antibiotic production. This substance did not accumulate in mutant cells. It was found in S. lividans 66 and S. parvulus, but not in seven other Streptomyces species tested. The significance of these observations, and the relationship of the mutant to earlier rel isolates of Streptomyces is discussed.

  7. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase[W

    Science.gov (United States)

    Catalanotti, Claudia; Dubini, Alexandra; Subramanian, Venkataramanan; Yang, Wenqiang; Magneschi, Leonardo; Mus, Florence; Seibert, Michael; Posewitz, Matthew C.; Grossman, Arthur R.

    2012-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism. PMID:22353371

  8. Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL– and mutant FLT3-expressing cells

    Science.gov (United States)

    Weisberg, Ellen; Banerji, Lolita; Wright, Renee D.; Barrett, Rosemary; Ray, Arghya; Moreno, Daisy; Catley, Laurence; Jiang, Jingrui; Hall-Meyers, Elizabeth; Sauveur-Michel, Maira; Stone, Richard; Galinsky, Ilene; Fox, Edward; Kung, Andrew L.

    2008-01-01

    Mediators of PI3K/AKT signaling have been implicated in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Studies have shown that inhibitors of PI3K/AKT signaling, such as wortmannin and LY294002, are able to inhibit CML and AML cell proliferation and synergize with targeted tyrosine kinase inhi-bitors. We investigated the ability of BAG956, a dual PI3K/PDK-1 inhibitor, to be used in combination with inhibitors of BCR-ABL and mutant FLT3, as well as with the mTOR inhibitor, rapamycin, and the rapamycin derivative, RAD001. BAG956 was shown to block AKT phosphorylation induced by BCR-ABL–, and induce apoptosis of BCR-ABL–expressing cell lines and patient bone marrow cells at concentrations that also inhibit PI3K signaling. Enhancement of the inhibitory effects of the tyrosine kinase inhibitors, imatinib and nilotinib, by BAG956 was demonstrated against BCR-ABL expressing cells both in vitro and in vivo. We have also shown that BAG956 is effective against mutant FLT3-expressing cell lines and AML patient bone marrow cells. Enhancement of the inhibitory effects of the tyrosine kinase inhibitor, PKC412, by BAG956 was demonstrated against mutant FLT3-expressing cells. Finally, BAG956 and rapamycin/RAD001 were shown to combine in a nonantagonistic fashion against BCR-ABL– and mutant FLT3-expressing cells both in vitro and in vivo. PMID:18184863

  9. The transcriptional response of Lactobacillus sanfranciscensis DSM 20451T and its tcyB mutant lacking a functional cystine transporter to diamide stress.

    Science.gov (United States)

    Stetina, Mandy; Behr, Jürgen; Vogel, Rudi F

    2014-07-01

    As a result of its strong adaptation to wheat and rye sourdoughs, Lactobacillus sanfranciscensis has the smallest genome within the genus Lactobacillus. The concomitant absence of some important antioxidative enzymes and the inability to synthesize glutathione suggest a role of cystine transport in maintenance of an intracellular thiol balance. Diamide [synonym 1,1'-azobis(N,N-dimethylformamide)] disturbs intracellular and membrane thiol levels in oxidizing protein thiols depending on its initial concentration. In this study, RNA sequencing was used to reveal the transcriptional response of L. sanfranciscensis DSM 20451(T) (wild type [WT]) and its ΔtcyB mutant with a nonfunctional cystine transporter after thiol stress caused by diamide. Along with the different expression of genes involved in amino acid starvation, pyrimidine synthesis, and energy production, our results show that thiol stress in the wild type can be compensated through activation of diverse chaperones and proteases whereas the ΔtcyB mutant shifts its metabolism in the direction of survival. Only a small set of genes are significantly differentially expressed between the wild type and the mutant. In the WT, mainly genes which are associated with a heat shock response are upregulated whereas glutamine import and synthesis genes are downregulated. In the ΔtcyB mutant, the whole opp operon was more highly expressed, as well as a protein which probably includes enzymes for methionine transport. The two proteins encoded by spxA and nrdH, which are involved in direct or indirect oxidative stress responses, are also upregulated in the mutant. This work emphasizes that even in the absence of definitive antioxidative enzymes, bacteria with a small genome and a high frequency of gene inactivation and elimination use small molecules such as the cysteine/cystine couple to overcome potential cell damage resulting from oxidative stress.

  10. Loss-of-function mutations of retromer large subunit genes suppress the phenotype of an Arabidopsis zig mutant that lacks Qb-SNARE VTI11.

    Science.gov (United States)

    Hashiguchi, Yasuko; Niihama, Mitsuru; Takahashi, Tetsuya; Saito, Chieko; Nakano, Akihiko; Tasaka, Masao; Morita, Miyo Terao

    2010-01-01

    Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions.

  11. Loss-of-Function Mutations of Retromer Large Subunit Genes Suppress the Phenotype of an Arabidopsis zig Mutant That Lacks Qb-SNARE VTI11[C][W

    Science.gov (United States)

    Hashiguchi, Yasuko; Niihama, Mitsuru; Takahashi, Tetsuya; Saito, Chieko; Nakano, Akihiko; Tasaka, Masao; Morita, Miyo Terao

    2010-01-01

    Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions. PMID:20086190

  12. Optic nerve compression and retinal degeneration in Tcirg1 mutant mice lacking the vacuolar-type H-ATPase a3 subunit.

    Directory of Open Access Journals (Sweden)

    Nobuyuki Kawamura

    Full Text Available BACKGROUND: Vacuolar-type proton transporting ATPase (V-ATPase is involved in the proper development of visual function. Mutations in the Tcirg1 (also known as Atp6V0a3 locus, which encodes the a3 subunit of V-ATPase, cause severe autosomal recessive osteopetrosis (ARO in humans. ARO is often associated with impaired vision most likely because of nerve compression at the optic canal. We examined the ocular phenotype of mice deficient in Tcirg1 function. METHODOLOGY/PRINCIPAL FINDINGS: X-ray microtomography showed narrowed foramina in the skull, suggesting that optic nerve compression occurred in the a3-deficient (Tcirg1-/- mice. The retina of the mutant mice had normal architecture, but the number of apoptotic cells was increased at 2-3 wks after birth. In the ocular system, the a3 subunit accumulated in the choriocapillary meshwork in uveal tissues. Two other subunit isoforms a1 and a2 accumulated in the retinal photoreceptor layer. We found that the a4 subunit, whose expression has previously been shown to be restricted to several transporting epithelia, was enriched in pigmented epithelial cells of the retina and ciliary bodies. The expression of a4 in the uveal tissue was below the level of detection in wild-type mice, but it was increased in the mutant choriocapillary meshwork, suggesting that compensation may have occurred among the a subunit isoforms in the mutant tissues. CONCLUSIONS: Our findings suggest that a similar etiology of visual impairment is involved in both humans and mice; thus, a3-deficient mice may provide a suitable model for clinical and diagnostic purposes in cases of ARO.

  13. Sinorhizobium meliloti mutants lacking phosphotransferase system enzyme HPr or EIIA are altered in diverse processes, including carbon metabolism, cobalt requirements, and succinoglycan production.

    Science.gov (United States)

    Pinedo, Catalina Arango; Bringhurst, Ryan M; Gage, Daniel J

    2008-04-01

    Sinorhizobium meliloti is a member of the Alphaproteobacteria that fixes nitrogen when it is in a symbiotic relationship. Genes for an incomplete phosphotransferase system (PTS) have been found in the genome of S. meliloti. The genes present code for Hpr and ManX (an EIIA(Man)-type enzyme). HPr and EIIA regulate carbon utilization in other bacteria. hpr and manX in-frame deletion mutants exhibited altered carbon metabolism and other phenotypes. Loss of HPr resulted in partial relief of succinate-mediated catabolite repression, extreme sensitivity to cobalt limitation, rapid die-off during stationary phase, and altered succinoglycan production. Loss of ManX decreased expression of melA-agp and lac, the operons needed for utilization of alpha- and beta-galactosides, slowed growth on diverse carbon sources, and enhanced accumulation of high-molecular-weight succinoglycan. A strain with both hpr and manX deletions exhibited phenotypes similar to those of the strain with a single hpr deletion. Despite these strong phenotypes, deletion mutants exhibited wild-type nodulation and nitrogen fixation when they were inoculated onto Medicago sativa. The results show that HPr and ManX (EIIA(Man)) are involved in more than carbon regulation in S. meliloti and suggest that the phenotypes observed occur due to activity of HPr or one of its phosphorylated forms.

  14. Coexistance of JAK2V617F mutation and BCR/ABL translocation in one patient

    Directory of Open Access Journals (Sweden)

    Murat Albayrak

    2010-09-01

    Full Text Available Dear Editor,The myeloproliferative disorders (MPDs constitute a subcategory of chronic myeloid disorders and include chronic myeloid leukemia (CML, essential thrombocytemia (ET, polycythemia vera (PV and myelofibrosis (MF. In 1960, the discovery of the Philadelphia chromosome (Ph became a cornerstone in CML treatment and led to the development of moleculary targeted therapy. Recently, an acquired mutation in the Janus kinase 2 (JAK2 gene has been discovered in nearly all patents with PV and approximately half of the patients with primary MF and ET. Subsequently, the mutation has been demonstrated in atypical MPDs (chronic neutrophilic leukemia, unclassified, de novo myelodysplastic syndrome or acute myeloid leukemia.1 It has been hoped that targeted inhibition of JAK2V617F should achieve similar disease control as thyrosine kinases has produced in CML.

  15. Management of side effects of BCR/ABL-negative chronic myeloproliferative neoplasm therapies. Focus on anagrelide.

    Science.gov (United States)

    Antelo, María Luisa; de Las Heras, Natalia; Gonzalez Porras, Jose Ramón; Kerguelen, Ana; Raya, Jose María

    2015-12-01

    Although hydroxyurea is considered the first-line cytoreductive therapy in high-risk patients with polycythemia vera or essential thrombocythemia, approximately 20-25% of patients develop resistance or intolerance and they need an alternative therapy. Anagrelide is the treatment of choice in patients with essential thrombocythemia intolerant or with resistance to hydroxyurea. Anagrelide is usually well tolerated. Although there is concern about the increased risk of cardiac side effects, in most cases these are mild, and easily manageable. In this paper, the available evidence about the management of patients with myeloproliferative neoplasms, with a special focus on the side effects of drug therapies is reviewed.

  16. Cleft palate defect of Dlx1/2-/- mutant mice is caused by lack of vertical outgrowth in the posterior palate

    NARCIS (Netherlands)

    Jeong, J.; Cesario, J.; Zhao, Y.; Burns, L.; Westphal, H.; Rubenstein, J.L.

    2012-01-01

    BACKGROUND: Mice lacking the activities of Dlx1 and Dlx2 (Dlx1/2-/-) exhibit cleft palate, one of the most common human congenital defects, but the etiology behind this phenotype has been unknown. Therefore, we analyzed the morphological, cellular, and molecular changes caused by inactivation of Dlx

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

    Science.gov (United States)

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

    2014-05-01

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

  18. Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

    Science.gov (United States)

    Kameyama, K; Haga, K; Haga, T; Moro, O; Sadée, W

    1994-12-01

    A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

  19. Mice lacking Ras-GRF1 show contextual fear conditioning but not spatial memory impairments: convergent evidence from two independently generated mouse mutant lines

    Directory of Open Access Journals (Sweden)

    Raffaele ed'Isa

    2011-12-01

    Full Text Available Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning the Brambilla’s mice with a third mouse line (GENA53 in which a nonsense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in the Brambilla’s mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdalar functions but possibly to some distinct hippocampal connections specific to

  20. Staphylococcus aureus mutants lacking cell wall-bound protein A found in isolates from bacteraemia, MRSA infection and a healthy nasal carrier.

    Science.gov (United States)

    Sørum, Marit; Sangvik, Maria; Stegger, Marc; Olsen, Renate S; Johannessen, Mona; Skov, Robert; Sollid, Johanna U E

    2013-02-01

    Staphylococcus aureus is a major human pathogen and a multitude of virulence factors enables it to cause infections, from superficial lesions to life-threatening systemic conditions. Staphylococcal protein A (SpA) is a surface protein contributing to S. aureus pathogenesis by interfering with immune responses and activating inflammation. Seven isolates with frameshift mutations in the spa repeat region were investigated to determine whether these mutations lead to truncation and secretion of SpA into the extracellular environment. Five isolates originated from blood cultures, one from an MRSA infection and one from a persistent nasal carrier. Full-length spa genes from the seven isolates were sequenced, and Western blot experiments were performed to localize SpA. Three isolates had identical deviating 25-bp spa repeats, but all isolates displayed different repeat successions. The DNA sequence revealed that the frameshift mutations created premature stop codons in all seven isolates, resulting in truncated SpA of different lengths, however, all lacking the XC region with the C-terminal sorting signal. SpA was detected by Western blot in six of the seven isolates, mainly extracellularly. Our findings demonstrate that S. aureus isolates with truncated SpA, not anchored to the cell wall, can still be found in bacteraemia, infection and among carriers.

  1. Depletion of SAM50 Specifically Targets BCR-ABL-Expressing Leukemic Stem and Progenitor Cells by Interfering with Mitochondrial Functions

    NARCIS (Netherlands)

    Capala, Marta E; Pruis, Maurien; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    A high proliferation rate of malignant cells requires an increased energy production, both by anaerobic glucose metabolism and mitochondrial respiration. Moreover, increased levels of mitochondria-produced reactive oxygen species (ROS) promote survival of transformed cells and contribute to the dise

  2. A natural-like synthetic small molecule impairs bcr-abl signaling cascades and induces megakaryocyte differentiation in erythroleukemia cells.

    Directory of Open Access Journals (Sweden)

    Silvia Turroni

    Full Text Available Over the past years, we synthesized a series of new molecules that are hybrids of spirocyclic ketones as complexity-bearing cores with bi- and ter-phenyls as privileged fragments. Some of these newly-shaped small molecules showed antiproliferative, pro-apoptotic and differentiating activity in leukemia cell lines. In the present study, to investigate more in depth the mechanisms of action of these molecules, the protein expression profiles of K562 cells treated with or without the compounds IND_S1, MEL_T1, IND_S7 and MEL_S3 were analyzed using two-dimensional gel electrophoresis coupled with mass spectrometry. Proteome comparisons revealed several differentially expressed proteins, mainly related to cellular metabolism, chaperone activity, cytoskeletal organization and RNA biogenesis. The major results were validated by Western blot and qPCR. To attempt integrating findings into a cellular signaling context, proteomic data were explored using MetaCore. Network analysis highlighted relevant relationships between the identified proteins and additional potential effectors. Notably, qPCR validation of central hubs showed that the compound MEL_S3 induced high mRNA levels of the transcriptional factors EGR1 and HNF4-alpha; the latter to our knowledge is reported here for the first time to be present in K562 cells. Consistently with the known EGR1 involvement in the regulation of differentiation along megakaryocyte lineage, MEL_S3-treated leukemia cells showed a marked expression of glycoprotein IIb/IIIa (CD41 and glycoprotein Ib (CD42, two important cell markers in megakaryocytic differentiation, together with morphological aspects of megakaryoblasts and megakaryocytes.

  3. Unsupervised explorative data analysis of normal human leukocytes and BCR/ABL positive leukemic cells mid-infrared spectra

    NARCIS (Netherlands)

    Bellisola, G.; Bolomini-Vittori, M.; Cinque, G.; Dumas, P.; Fiorini, Z.; Laudanna, C.; Mirenda, M.; Sandt, C.; Silvestri, G.; Tomasello, L.; Vezzalini, M.; Wehbe, K.; Sorio, C.

    2015-01-01

    We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived p

  4. Ph+, BCR-ABL+ chronic myeloid leukemia (CML) in early chronic phase | EU Clinical Trials Register [EU Clinical Trials Register

    Lifescience Database Archive (English)

    Full Text Available ALT ≤ 2.5 x valore massimo della normalità o ≤ 5.0 x valore massimo della normalità se considerato secondario alla leucemia...o venga considerato secondario alla leucemia - Bilirubina diretta ≤ 1.5 x valore massimo della normalità, sa

  5. Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation

    NARCIS (Netherlands)

    Sontakke, Pallavi; Koczula, Katarzyna M; Jaques, Jennifer; Wierenga, Albertus T J; Brouwers-Vos, Annet Z; Pruis, Maurien; Günther, Ulrich L; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduce

  6. “Preleukemic or smoldering” chronic myelogenous leukemia (CML:BCR-ABL1 positive: A brief case report

    Directory of Open Access Journals (Sweden)

    John M. Bennett

    2015-01-01

    The most common feature of CML is an elevated WBC count, usually above 25×103/µL, and frequently above 100×103/µL. We report a case of confirmed Ph+CML with a normal CBC detected because of the presence of rare myelocytes and 2% basophils [Fig. 1]. Previous leukocyte counts for the preceding eight years were normal with the exception of one done four months prior to his presentation that showed an abnormal differential with 1% basophils, 2% metamyelocytes and 2% myelocytes.

  7. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus.

  8. Production of the Bioactive Compounds Violacein and Indolmycin Is Conditional in a maeA Mutant of Pseudoalteromonas luteoviolacea S4054 Lacking the Malic Enzyme

    DEFF Research Database (Denmark)

    Schmidt Thøgersen, Mariane; Delpin, Marina; Melchiorsen, Jette;

    2016-01-01

    It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relati...... of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea....... role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene...... produced by the mutant strain was about 300% that of the wild type. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production...

  9. Chlorobium tepidum mutant lacking bacteriochlorophyll c made by inactivation of the bchK gene, encoding bacteriochlorophyll c synthase

    DEFF Research Database (Denmark)

    Frigaard, Niels-Ulrik; Voigt, Ginny D; Bryant, Donald A

    2002-01-01

    , a fraction containing vestigial chlorosomes, denoted "carotenosomes," was partly purified by density centrifugation; these structures contained carotenoids, isoprenoid quinones, and a 798-nm-absorbing BChl a species that is probably protein associated. Because of the absence of the strong BChl c absorption...... found in the wild type, the bchK mutant should prove valuable for future analyses of the photosynthetic reaction center and of the roles of BChl a in photosynthesis in green bacteria. An evolutionary implication of our findings is that the photosynthetic ancestor of green sulfur bacteria could have...

  10. Structural Analysis of Free N-Glycans in α-Glucosidase Mutants of Saccharomyces cerevisiae: Lack of the Evidence for the Occurrence of Catabolic α-Glucosidase Acting on the N-Glycans.

    Science.gov (United States)

    Hossain, Tanim Jabid; Harada, Yoichiro; Hirayama, Hiroto; Tomotake, Haruna; Seko, Akira; Suzuki, Tadashi

    2016-01-01

    Saccharomyces cerevisiae produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from N-glycans (Glc3Man9GlcNAc2) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated N-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in S. cerevisiae. To this end, the precise structures of cytosolic free N-glycans (FNGs), mainly derived from the peptide:N-glycanase (Png1) mediated deglycosylation of N-glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on N-glycans are produced by this yeast.

  11. Lack of TNF-alpha receptor type 2 protects motor neurons in a cellular model of amyotrophic lateral sclerosis and in mutant SOD1 mice but does not affect disease progression.

    Science.gov (United States)

    Tortarolo, Massimo; Vallarola, Antonio; Lidonnici, Dario; Battaglia, Elisa; Gensano, Francesco; Spaltro, Gabriella; Fiordaliso, Fabio; Corbelli, Alessandro; Garetto, Stefano; Martini, Elisa; Pasetto, Laura; Kallikourdis, Marinos; Bonetto, Valentina; Bendotti, Caterina

    2015-10-01

    Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1-G93A co-cultures. Deleting TNFR2 from SOD1-G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1-G93A/TNFR2-/- mice showed high phospho-TAR DNA-binding protein 43 (TDP-43) accumulation and low levels of acetyl-tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane-bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology. We show evidence of the involvement of neuronal and astroglial TNFR2 in the motor neuron degeneration in ALS. Both concur to cause motor neuron death in primary astrocyte/spinal neuron co-cultures. TNFR2 deletion partially protects motor neurons and sciatic nerves in SOD1-G93A mice but does not improve their symptoms and survival. However, TNFR2 could be a new target for multi-intervention therapies.

  12. Reduced alcohol consumption in mice lacking preprodynorphin.

    OpenAIRE

    Blednov, Yuri A.; Walker, Danielle; Martinez, Marni; Harris, R. Adron

    2006-01-01

    Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the κ-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower a...

  13. The HDAC inhibitor SB939 overcomes resistance to BCR-ABL kinase Inhibitors conferred by the BIM deletion polymorphism in chronic myeloid leukemia.

    Science.gov (United States)

    Rauzan, Muhammad; Chuah, Charles T H; Ko, Tun Kiat; Ong, S Tiong

    2017-01-01

    Chronic myeloid leukemia (CML) treatment has been improved by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate (IM) but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM) gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat) is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance.

  14. BCR-ABL isoforms associated with intrinsic or acquired resistance to imatinib : more heterogeneous than just ABL kinase domain point mutations?

    NARCIS (Netherlands)

    Gruber, Franz X.; Lundan, Tuija; Goll, Rasmus; Silye, Aleksandra; Mikkola, Ingvild; Rekvig, Ole Petter; Knuutila, Sakari; Remes, Kari; Gedde-Dahl, Tobias; Porkka, Kimmo; Hjorth-Hansen, Henrik

    2012-01-01

    Imatinib, a small molecule inhibitor of ABL, PDGFR and C-KIT, has revolutionized treatment of chronic myeloid leukaemia (CML). However, resistance to treatment is of increasing importance and often is due to point mutations in the Abl kinase domain (Abl KD). Here, we analysed clinical outcome and mu

  15. The HDAC inhibitor SB939 overcomes resistance to BCR-ABL kinase Inhibitors conferred by the BIM deletion polymorphism in chronic myeloid leukemia

    Science.gov (United States)

    Rauzan, Muhammad; Chuah, Charles T. H.; Ko, Tun Kiat; Ong, S. Tiong

    2017-01-01

    Chronic myeloid leukemia (CML) treatment has been improved by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate (IM) but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM) gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat) is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance. PMID:28301600

  16. Dasatinib targets B-lineage cells but does not provide an effective therapy for myeloproliferative disease in c-Cbl RING finger mutant mice.

    Directory of Open Access Journals (Sweden)

    Johanna M Duyvestyn

    Full Text Available This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL.

  17. Dasatinib Targets B-Lineage Cells but Does Not Provide an Effective Therapy for Myeloproliferative Disease in c-Cbl RING Finger Mutant Mice

    Science.gov (United States)

    Duyvestyn, Johanna M.; Taylor, Samuel J.; Dagger, Samantha A.; Orandle, Marlene; Morse, Herbert C.; Thien, Christine B. F.; Langdon, Wallace Y.

    2014-01-01

    This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL. PMID:24718698

  18. Co-expression of the CBFβ-MYH11 and BCR-ABL fusion genes in chronic myeloid leukaemia / Coexistenţa genelor de fuziune CBFβ-MYH11 şi BCR-ABL în leucemia mieloidă cronică

    Directory of Open Access Journals (Sweden)

    Popescu Roxana

    2015-06-01

    Full Text Available Coexistenţa t(9;22 şi a inv(16 a fost descrisă într-un număr limitat de cazuri de LMC, LAM de novo sau LAM post chimioterapie. Raportăm un pacient cu LMC care a prezentat atât inversie de 16 cât şi cromozom Philadelphia şi care a evoluat spre criză blastică sub tratament cu Imatinib. Diagnosicul de laborator şi monitorizarea s-a realizat prin citometrie în flux, citogenetică convenţională şi tehnici de genetică moleculară. Inv(16, detectată prin cariotipare în clona Philadelphia pozitivă la momentul transformării blastice, a fost evaluată retrospective prin metoda real-time PCR, şi s-a dovedit a fi fost prezentă încă de la diagnostic. Biopsia de măduvă osoasă, efectuată în faza blastică a LMC, a confirmat prezenţa blaştilor aparţinând liniei mieloide, cu indicii de diferenţiere monocitoidă, frecvent asociată cu inv (16. De asemenea, cazul a asociat şi mutaţia F359V în domeniul kinazic al ABL, care determină rezistenţă intermediară la Imatinib şi Nilotinib, ceea ce a impus schimbarea terapiei cu Dasatinib. În cazul prezentat evoluţia a fost progresivă, urmată de deces ca urmare a lipsei de răspuns la inhibitorii de tirozin kinază, la 18 luni de la diagnosticare. Coexistenţa t(9; 22 şi inv(16 în LMC pare a fi asociată cu o evoluţie clinică agresivă şi rezistenţă la terapia cu inhibitori de tirozin kinază. Având în vedere numărul foarte mic de cazuri descrise în literatura de specialitate, deciziile terapeutice în cazul pacienţilor care prezintă aceste anomalii sunt încă dificile

  19. Mutant p53: multiple mechanisms define biologic activity in cancer

    Directory of Open Access Journals (Sweden)

    Michael Paul Kim

    2015-11-01

    Full Text Available The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of p53 alterations involve missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may acquire novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in multiple model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 are reviewed and their limitations discussed.

  20. Mice lacking neurofibromin develop gastric hyperplasia

    Science.gov (United States)

    Lin, Lu; Chen, Jian; Richardson, James A.

    2009-01-01

    Gastrointestinal (GI) neoplasms are among many manifestations of the genetic disease neurofibromatosis type 1 (NF1). However, the physiological and pathological functions of the Nf1 gene in the GI system have not been fully studied, possibly because of a lack of mouse models. In this study, we generated conditional knockout mice with Nf1 deficiency in the GI tract. These mice develop gastric epithelial hyperplasia and inflammation together with increased cell proliferation and apoptosis. The gastric phenotypes observed in these mutant mice seem to be the consequence of loss of Nf1 in gastric fibroblasts, resulting in paracrine hyperactivation of the ERK pathway in the gastric epithelium. These mice provide a useful model to study the pathogenesis of GI lesions in a subset of patients with NF1 and to investigate the role of the Nf1 gene in the development of GI neoplasms. PMID:19661150

  1. Forward genetic screen for auxin-deficient mutants by cytokinin.

    Science.gov (United States)

    Wu, Lei; Luo, Pan; Di, Dong-Wei; Wang, Li; Wang, Ming; Lu, Cheng-Kai; Wei, Shao-Dong; Zhang, Li; Zhang, Tian-Zi; Amakorová, Petra; Strnad, Miroslav; Novák, Ondřej; Guo, Guang-Qin

    2015-07-06

    Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

  2. Gibberellins and Carotenoids in the Wild Type and Mutants of Gibberella fujikuroi

    OpenAIRE

    1991-01-01

    A new screening procedure was used to isolate 14 gib mut ts of Gi!'berella_ fujikurof wit modi cations the production of gibberellins. The production of carotenmds and g bberellms was mvestlgated m the gib mutants and in representative car mutants with various modifications of carotenoid biosynthesis. The determinations of gibberellins were carried out with a simplified ftuorescence method. One of the mutants lacked both gibberellins and carotenoids. In many mutants the two path...

  3. Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma.

    Science.gov (United States)

    Fedorenko, Inna V; Fang, Bin; Koomen, John M; Gibney, Geoffrey T; Smalley, Keiran S M

    2014-10-01

    Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

  4. Lacking power impairs executive functions

    NARCIS (Netherlands)

    Smith, P.K.; Jostmann, N.B.; Galinsky, A.D.; Dijk, W.W. van

    2008-01-01

    Four experiments explored whether lacking power impairs executive functioning, testing the hypothesis that the cognitive presses of powerlessness increase vulnerability to performance decrements during complex executive tasks. In the first three experiments, low power impaired performance on executi

  5. Connexin mutants and cataracts

    Directory of Open Access Journals (Sweden)

    Eric C Beyer

    2013-04-01

    Full Text Available The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8 have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death and formation of cytoplasmic accumulations (that may act as light scattering particles. These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.

  6. AFM images of complexes between amylose and Aspergillus niger glucoamylase mutants, native and mutant starch binding domains: a model for the action of glucoamylase

    DEFF Research Database (Denmark)

    Morris, V. M.; Gunning, A. P.; Faults, C. B.

    2005-01-01

    Atomic force microscopy has been used to investigate the complexes formed between high molecular weight amylose chains and Aspergillus niger glucoamylase mutants (E400Q and W52F), wild-type A. niger starch binding domains (SBDS), and mutant SBDs (W563K and W590K) lacking either of the two starch ...

  7. Lack of an antibacterial response defect in Drosophila Toll-9 mutant.

    Directory of Open Access Journals (Sweden)

    Karine Narbonne-Reveau

    Full Text Available Toll and Toll-like receptors represent families of receptors involved in mediating innate immunity response in insects and mammals. Although Drosophila proteome contains multiple Toll paralogs, Toll-1 is, so far, the only receptor to which an immune role has been attributed. In contrast, every single mammalian TLR is a key membrane receptor upstream of the vertebrate immune signaling cascades. The prevailing view is that TLR-mediated immunity is ancient. Structural analysis reveals that Drosophila Toll-9 is the most closely related to vertebrate TLRs and utilizes similar signaling components as Toll-1. This suggests that Toll-9 could be an ancestor of TLR-like receptors and could have immune function. Consistently, it has been reported that over-expression of Toll-9 in immune tissues is sufficient to induce the expression of some antimicrobial peptides in flies. These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms. To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9. Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection.

  8. Analysis of the aspartic acid metabolic pathway using mutant genes.

    Science.gov (United States)

    Azevedo, R A

    2002-01-01

    Amino acid metabolism is a fundamental process for plant growth and development. Although a considerable amount of information is available, little is known about the genetic control of enzymatic steps or regulation of several pathways. Much of the information about biochemical pathways has arisen from the use of mutants lacking key enzymes. Although mutants were largely used already in the 60's, by bacterial and fungal geneticists, it took plant research a long time to catch up. The advance in this area was rapid in the 80's, which was followed in the 90's by the development of techniques of plant transformation. In this review we present an overview of the aspartic acid metabolic pathway, the key regulatory enzymes and the mutants and transgenic plants produced for lysine and threonine metabolism. We also discuss and propose a new study of high-lysine mutants.

  9. Detection of BCR/ABL Fusion Gene in Interphase Cens with Bi-labelling Hybridization in Situ%双标记原位杂交法检测间期细胞BCR/ABL融合基因

    Institute of Scientific and Technical Information of China (English)

    袁长吉; 王建伟; 李舜华; 易永林

    2003-01-01

    目的:应用双标记原位杂交方法检测BCR/ABL融合基因.方法:BCR基因探针用地高辛标记,碱性磷酸酶显色;ABL基因用3H-dATP标记,核子乳胶放射自显影.结果:检测9例初治的慢性粒细胞白血病(CML)病人均为阳性,阳性细胞比例为93%;检测CML来源的K562细胞株阳性细胞占98.8%;正常人假阳性率为0.75%.结论:该方法简便、快速,适用于CML微小残留病的检测.

  10. Isolation and characterization of Rhizobium meliloti mutants affected in exopolysaccharide production.

    Science.gov (United States)

    Rodríguez-Navarro, D N; Palomares, A J; Casadesús, J

    1991-06-01

    Rhizobium meliloti mutants affected in the production of exopolysaccharide (EPS) were isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutants were classified into three phenotypic classes: (I) Exo-, rough mutants lacking exopolysaccharide; (II) Exos (for "small") which form tiny, compact colonies and synthesize reduced amounts of EPS; and (III) Exoc (for "constitutive"), hypermucoid mutants which overproduce EPS. Hypermucoid strains showed increased resistance to desiccation. All the mutants were able to nodulate, although a significant decrease in infectivity degree and/or competitiveness was found in rough and compact strains. Two mutants proved to be deficient in nitrogen fixation. Complementation analysis with cloned R. meliloti exo genes could not be applied to the study of these Fix- mutants because introduction of plasmids derived from cosmid vector pLAFR1 caused loss of nodulating ability. However, complementation of calcofluor staining and EPS production was observed. Complementation with certain exo genes also caused a marked increase in motility.

  11. Auxin physiology of the tomato mutant diageotropica

    Science.gov (United States)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  12. atp mutants of Escherichia coli fail to grow on succinate due to a transport deficiency

    DEFF Research Database (Denmark)

    Boogerd, Fred; Boe, Lars; Michelsen, Ole;

    1998-01-01

    Escherichia coli atp mutants, which lack a functional Hf-ATPase complex, are capable of growth on glucose but not on succinate or other C-4-dicarboxylates (Suc(-) phenotype). Suc(+) revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack ...

  13. Defective glycinergic synaptic transmission in zebrafish motility mutants

    Directory of Open Access Journals (Sweden)

    Hiromi Hirata

    2010-01-01

    Full Text Available Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs.

  14. Lack of Neuronal IFN-β-IFNAR Causes Lewy Body- and Parkinson's Disease-like Dementia

    DEFF Research Database (Denmark)

    Ejlerskov, Patrick; Hultberg, Jeanette Göransdotter; Wang, JunYang;

    2015-01-01

    -causing mutant proteins. Mice lacking Ifnb function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-β signaling caused...... neuron loss in a familial Parkinson's disease model. These results indicate a protective role for IFN-β in neuronal homeostasis and validate Ifnb mutant mice as a model for sporadic Lewy body and Parkinson's disease dementia.......Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-β (IFN-β) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease...

  15. Impaired exercise tolerance and skeletal muscle myopathy in sulfonylurea receptor-2 mutant mice

    Science.gov (United States)

    Stoller, Douglas; Pytel, Peter; Katz, Sophie; Earley, Judy U.; Collins, Keith; Metcalfe, Jamie; Lang, Roberto M.

    2009-01-01

    By sensing intracellular energy levels, ATP-sensitive potassium (KATP) channels help regulate vascular tone, glucose metabolism, and cardioprotection. SUR2 mutant mice lack full-length KATP channels in striated and smooth muscle and display a complex phenotype of hypertension and coronary vasospasm. SUR2 mutant mice also display baseline cardioprotection and can withstand acute sympathetic stress better than normal mice. We now studied response to a form of chronic stress, namely that induced by 4 wk of daily exercise on SUR2 mutant mice. Control mice increased exercise capacity by 400% over the training period, while SUR2 mutant mice showed little increase in exercise capacity. Unexercised SUR2 mutant showed necrotic and regenerating fibers in multiple muscle skeletal muscles, including quadriceps, tibialis anterior, and diaphragm muscles. Unlike exercised control animals, SUR2 mutant mice did not lose weight, presumably due to less overall exertion. Unexercised SUR2 mutant mice showed a trend of mildly reduced cardiac function, measured by fractional shortening, (46 ± 4% vs. 57 ± 7% for SUR2 mutant and control, respectively), and this decrease was not exacerbated by chronic exercise exposure. Despite an improved response to acute sympathetic stress and baseline cardioprotection, exercise intolerance results from lack of SUR2 KATP channels in mice. PMID:19675276

  16. Wild Accessions and Mutant Resources

    DEFF Research Database (Denmark)

    Kawaguchi, Masayoshi; Sandal, Niels Nørgaard

    2014-01-01

    Lotus japonicus, Lotus burttii, and Lotus filicaulis are species of Lotus genus that are utilized for molecular genetic analysis such as the construction of a linkage map and QTL analysis. Among them, a number of mutants have been isolated from two wild accessions: L. japonicus Gifu B-129...

  17. Isolation of mutants deficient in acetyl-CoA synthetase and a possible regulator of acetate induction in Aspergillus niger.

    Science.gov (United States)

    Sealy-Lewis, H M; Fairhurst, V

    1998-07-01

    Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.

  18. Lack of Set Theory Relevant Prerequisite Knowledge

    Science.gov (United States)

    Dogan-Dunlap, Hamide

    2006-01-01

    Many students struggle with college mathematics topics due to a lack of mastery of prerequisite knowledge. Set theory language is one such prerequisite for linear algebra courses. Many students' mistakes on linear algebra questions reveal a lack of mastery of set theory knowledge. This paper reports the findings of a qualitative analysis of a…

  19. Isolation of a Defective Prion Mutant from Natural Scrapie

    Science.gov (United States)

    Migliore, Sergio; Cosseddu, Gian Mario; Pirisinu, Laura; Riccardi, Geraldina; Nonno, Romolo

    2016-01-01

    It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of

  20. Purine transport by malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster.

    Science.gov (United States)

    Sullivan, D T; Bell, L A; Paton, D R; Sullivan, M C

    1979-06-01

    Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (pp) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.

  1. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    Science.gov (United States)

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A

    1989-12-01

    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

  2. Construction of a doramectin producer mutant from an avermectin-overproducing industrial strain of Streptomyces avermitilis.

    Science.gov (United States)

    Zhao, Xuejin; Wang, Yuanxin; Wang, Shiwei; Chen, Zhi; Wen, Ying; Song, Yuan

    2009-12-01

    The avermectin analogue doramectin (CHC-B1), which is produced in mutants that have an altered biosynthesis pathway of avermectin, is one of the most effective agricultural pesticides and antiparasitics. We report here the construction of a bkdF olmA double-deletion mutant lacking one of the branched-chain alpha-keto acid dehydrogenase encoding genes (bkdF) and the oligomycin PKS encoding gene cluster (olmA) in Streptomyces avermitilis 76-05. We then characterized the production of various antibiotics in cultures of the deletion mutant. In a fermentation medium supplemented with cyclohexanecarboxylic acid, this double mutant produced doramectin and its analogues but no oligomycin. The mutant proved to be genetically stable, without any antibiotic resistance markers inserted into its chromosome, and could potentially become an industrial doramectin-producing strain after further improvement.

  3. Molecular and biochemical characterization of xrs mutants defective in Ku80.

    Science.gov (United States)

    Singleton, B K; Priestley, A; Steingrimsdottir, H; Gell, D; Blunt, T; Jackson, S P; Lehmann, A R; Jeggo, P A

    1997-01-01

    The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities. PMID:9032253

  4. Properties of mutants of haemophilus influenzae deficient in ATP-dependent deoxyribonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.

    1976-01-01

    Eight isogenic Haemophilus influenzae strains whose extracts lack ATP-dependent deoxyribonuclease activity (Add/sup -/ mutants) form three complementation and genetic linkage groups. Since there are known to be three subunits of the enzyme, these data suggest that each of the three genes specifies a different subunit. Gel electrophoresis of partially purified mutant extracts indicates that the smallest subunit is missing in one of the groups but is present in all the other mutants. The mutants are more sensitive to a variety of chemical agents than the wild type. The most sensitive mutants lack the ATPase activity associated with the enzyme. These strains exhibit aberrant incorporation of tritiated thymidine, which starts up more rapidly and shuts off sooner than in the wild type. An extracellular compound is responsible for most of this effect, in that wild type cells put into medium in which Add/sup -/ cells have been growing show a similar aberrant incorporation. The effect of these media can be mimicked by cyclic AMP and cyclic GMP, although millimolar concentrations are required. It is postulated that the Add/sup -/ mutants are more permeable to many substances than the wild type, partly because of the extracellular compound usually surrounding them, and the increased permeability might be responsible for the mutants' nonviability.

  5. Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Strazielle, C; Lefevre, A; Jacquelin, C; Lalonde, R

    2012-07-15

    Dab1(scm) mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1(scm) mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1(scm) mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar cortex affects grooming activity and is consequently involved in executing various components, but not in its sequential organization, which requires other brain regions such as cerebral cortices or basal ganglia.

  6. Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma

    Science.gov (United States)

    Konen, Jessica; Koo, Junghui; Robinson, Brian S.; Wiles, Walter Guy; Huang, Chunzi; Martin, W. David; Behera, Madhusmita; Smith, Geoffrey H.; Hill, Charles E.; Rossi, Michael R.; Sica, Gabriel L.; Rupji, Manali; Chen, Zhengjia; Kowalski, Jeanne; Kasinski, Andrea L.; Ramalingam, Suresh S.; Khuri, Fadlo R.; Marcus, Adam I.

    2017-01-01

    Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre–induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

  7. Bacterial mutants for enhanced succinate production

    NARCIS (Netherlands)

    Baart, G.J.E.; Beauprez, J.J.R.; Foulquie, M.M.R.; Heijnen, J.J.; Maertens, J.

    2010-01-01

    The present invention relates to a method for obtaining enhanced metabolite production in micro-organisms, and to mutants and/or transformants obtained with said method. More particularly, it relates to bacterial mutants and/or transformants for enhanced succinate production, especially mutants and/

  8. Problem-Solving Test: Tryptophan Operon Mutants

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  9. Lack of Exercise Might Invite Dementia

    Science.gov (United States)

    ... fullstory_163264.html Lack of Exercise Might Invite Dementia Study found being sedentary may make you as ... TV may make you as likely to develop dementia as people genetically predisposed to the condition, a ...

  10. LACK - a VoIP Steganographic Method

    CERN Document Server

    Mazurczyk, Wojciech

    2008-01-01

    The paper presents a new steganographic method called LACK (Lost Audio PaCKets Steganography) which is intended mainly for VoIP. The method is presented in a broader context of network steganography and of VoIP steganography in particular. The analytical results presented in the paper concern the influence of LACK's hidden data insertion procedure on the method's impact on quality of voice transmission and its resistance to steganalysis.

  11. Pharmacological correctors of mutant CFTR mistrafficking

    Directory of Open Access Journals (Sweden)

    Nicoletta ePedemonte

    2012-10-01

    Full Text Available The lack of phenylalanine 508 (∆F508 mutation in the CFTR Cl- channel represents the most frequent cause of cystic fibrosis (CF, a genetic disease affecting multiple organs such lung, pancreas, and liver. ∆F508 causes instability and misfolding of CFTR protein leading to early degradation in the endoplasmic reticulum and accelerated removal from the plasma membrane. Pharmacological correctors of mutant CFTR protein have been identified by high-throughput screening of large chemical libraries, by in silico docking of virtual compounds on CFTR structure models, or by using compounds that affect the whole proteome (e.g. histone deacetylase inhibitors or a single CFTR-interacting protein. The presence of multiple defects caused at the CFTR protein level by ∆F508 mutation and the redundancy of quality control mechanisms detecting ∆F508-CFTR as a defective protein impose a ceiling to the maximal effect that a single compound (corrector may obtain. Therefore, treatment of patients with the most frequent CF mutation may require the optimized combination of two drugs having additive or synergic effects.

  12. Identification of a Long Rice Spikelet Mutant

    Institute of Scientific and Technical Information of China (English)

    WU Xian-jun; WANG Bin; HAN Zan-ping; XIE Zhao-hui; MOU Chun-hong; WANG Xu-dong

    2004-01-01

    A spontaneously occurring rice (Oryza sativa L. ) mutant, characterized by homeotic conversion in glumes and stamens, was found in the progeny of a cross. The mutant showed long glumes and glumaceous lodicules and morphological transformation of stamens into pistils. Mutant florets consisted of 1 to 3 completely developed pistils, some pistilloid stamens with filaments, but tipped by bulged tissue and 0 to 3 stigmas. It seens that the mutant phenotype of the homeotic conversions in glumes and stamens is similar to that of the B loss-of-function mutants in Arabidopsis and Antirrhinum. The mutant is controlled by a single recessive gene as a segregation ratio of 3:1 (wild type to mutant plants) was observed in the F2 generation.

  13. Mutants of GABA transaminase (POP2 suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh mutants in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Frank Ludewig

    Full Text Available BACKGROUND: The gamma-aminubutyrate (GABA shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD, the mitochondrial enzymes GABA transaminase (GABA-T; POP2 and succinic semialdehyde dehydrogenase (SSADH. We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported. PRINCIPAL FINDINGS: To elucidate the role of succinic semialdehyde (SSA, gamma-hydroxybutyrate (GHB and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants. SIGNIFICANCE: We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.

  14. Biochemical and histological characterization of tomato mutants

    Directory of Open Access Journals (Sweden)

    Carolina C. Monteiro

    2012-06-01

    Full Text Available Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT, we observed that the malondialdehyde (MDA content was enhanced in the diageotropica (dgt and lutescent (l mutants, whilst the highest levels of hydrogen peroxide (H2O2 were observed in high pigment 1 (hp1 and aurea (au mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT activity when compared to MT. Guaiacol peroxidase (GPOX was enhanced in both sitiens (sit and notabilis (not mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX. Based on PAGE analysis, the activities of glutathione reductase (GR isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD isoform III was reduced in leaves of sit, epi, Never ripe (Nr and green flesh (gf mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.Neste trabalho, analisamos as respostas bioquímicas inerentes ao sistema antioxidante, assim como propriedades morfológicas e anatômicas de mutantes fotomorfogenéticos e hormonais de tomateiro. Comparados ao não mutante Micro-Tom (MT, observamos que o conteúdo de malondialdeído (MDA aumentou nos mutantes diageotropica (dgt e lutescent (l, enquanto os maiores níveis de H2O2 foram encontrados nos mutantes high pigment 1 (hp1 e aurea (au. Análises de enzimas antioxidantes mostraram que todos os mutantes reduziram a atividade de catalase (CAT quando comparado a MT. A

  15. Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: II. Polypeptide Composition.

    Science.gov (United States)

    Cunningham, F X; Schiff, J A

    1986-01-01

    Chlorophyll-protein complexes (CPs) obtained from thylakoids of Euglena gracilis Klebs var bacillaris Cori contain the following polypeptides (listed in parentheses in order of prominence after Coomassie R-250 staining of polyacrylamide gels): CP Ia (66, 18, 22, 22.5, 27.5, 21, 28, 24, 25.5, and 26 kilodaltons [kD]); CP I (66 kD); CPx (41 kD); LHCP(2) (an oligomer of LHCP) (26.5, 28, and 26 kD); CPy (27 and 19 kD); CPa (54 kD); and LHCP (26.5, 28, and 26 kD). Mutants of bacillaris low in chlorophyll b (Gr(1)BSL, G(1)BU, and O(4)BSL; Chl a/b [mol/mol] = 50-100) which lack CP Ia, LHCP(2), and LHCP also lack or are deficient in polypeptides associated with these complexes in wild-type cells. Mutants G(1) and O(4), which also lack CPy, lack the CPy-associated polypeptides found in wild-type and Gr(1). Using an antiserum which was elicited by and reacts strongly and selectively with the SDS-treated major polypeptide (26.5 kD) of the LHCP complexes of wild-type, this polypeptide is undetectable in the mutants (saturation, consistent with the selective loss of major antenna components but not CP I or CPa from the mutants.

  16. Characterization of nonconventional hepatitis B viruses lacking the core promoter.

    Science.gov (United States)

    Chang, Shau-Feng; Chang, Shih-Hsuan; Li, Bi-Chen; Will, Hans; Netter, Hans Jürgen

    2004-12-20

    The core gene (C-gene) promoter and regulatory sequences play a central role in the hepatitis B virus (HBV) life cycle. They are essential for the synthesis of the pregenomic and precore mRNA. The pregenomic RNA is the template required for replication and also the template for the synthesis of the core protein and polymerase. Here, we report the in vivo existence and functional characterization of HBV variants that lack the C-gene promoter region and the regulatory sequences located therein. HBV promoter fragments were isolated by PCR from sera of chronic carriers and characterized. Truncated promoter elements were identified, and then tested in the context of wild-type genomes in the HuH-7 cell line. The expression of the recombinant HBV genome resulted in the synthesis of surface proteins, and low level of core protein as well as a transcript pattern similar to, but smaller in size to wild-type virus. The recombinant HBV genome with the truncated promoter region produced pregenomic RNA-like transcripts. These transcripts were encapsidated and reverse transcribed when complemented by sufficient core and polymerase protein. These date provide an explanation as to why such deletion mutants of HBV can be produced at all, they highlight the functional potentials of viral sequences activated by mutations and may be of relevance for viral evolution and persistence.

  17. Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction

    Science.gov (United States)

    Elmonem, Mohamed A.; Khalil, Ramzi; Khodaparast, Ladan; Khodaparast, Laleh; Arcolino, Fanny O.; Morgan, Joseph; Pastore, Anna; Tylzanowski, Przemko; Ny, Annelii; Lowe, Martin; de Witte, Peter A.; Baelde, Hans J.; van den Heuvel, Lambertus P.; Levtchenko, Elena

    2017-01-01

    The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis. PMID:28198397

  18. Conceptualising the lack of health insurance coverage.

    Science.gov (United States)

    Davis, J B

    2000-01-01

    This paper examines the lack of health insurance coverage in the US as a public policy issue. It first compares the problem of health insurance coverage to the problem of unemployment to show that in terms of the numbers of individuals affected lack of health insurance is a problem comparable in importance to the problem of unemployment. Secondly, the paper discusses the methodology involved in measuring health insurance coverage, and argues that the current method of estimation of the uninsured underestimates the extent that individuals go without health insurance. Third, the paper briefly introduces Amartya Sen's functioning and capabilities framework to suggest a way of representing the extent to which individuals are uninsured. Fourth, the paper sketches a means of operationalizing the Sen representation of the uninsured in terms of the disability-adjusted life year (DALY) measure.

  19. CMPD: cancer mutant proteome database.

    Science.gov (United States)

    Huang, Po-Jung; Lee, Chi-Ching; Tan, Bertrand Chin-Ming; Yeh, Yuan-Ming; Julie Chu, Lichieh; Chen, Ting-Wen; Chang, Kai-Ping; Lee, Cheng-Yang; Gan, Ruei-Chi; Liu, Hsuan; Tang, Petrus

    2015-01-01

    Whole-exome sequencing, which centres on the protein coding regions of disease/cancer associated genes, represents the most cost-effective method to-date for deciphering the association between genetic alterations and diseases. Large-scale whole exome/genome sequencing projects have been launched by various institutions, such as NCI, Broad Institute and TCGA, to provide a comprehensive catalogue of coding variants in diverse tissue samples and cell lines. Further functional and clinical interrogation of these sequence variations must rely on extensive cross-platforms integration of sequencing information and a proteome database that explicitly and comprehensively archives the corresponding mutated peptide sequences. While such data resource is a critical for the mass spectrometry-based proteomic analysis of exomic variants, no database is currently available for the collection of mutant protein sequences that correspond to recent large-scale genomic data. To address this issue and serve as bridge to integrate genomic and proteomics datasets, CMPD (http://cgbc.cgu.edu.tw/cmpd) collected over 2 millions genetic alterations, which not only facilitates the confirmation and examination of potential cancer biomarkers but also provides an invaluable resource for translational medicine research and opportunities to identify mutated proteins encoded by mutated genes.

  20. Study on culturing Trichodema mutants

    Institute of Scientific and Technical Information of China (English)

    CHEN Jian-ai; WANG Wei-ming

    2004-01-01

    @@ Trichodema mutants strains T5, T0803, T1010, T1003were cultured in different conditions and media, also in the presence of fungicides at 40 mg/kg (CK or procymidone + chlorothalonil, or maneb or phosethyl-Al) . The pH values of media were 5, 6, 7 and 8 and hyphae were grown at temperatures of 15, 20, 25 and 30 ℃. After being cultured for 3, 4, 5, or 6 days, the strains were transferred at a lower temperature to sporulate (20℃) Obtained data were analyzed statistically, with the orthogonal array and ranges (R) differing dependes on the treatments (R = 40.0,42.4, 48.0, 62.8,107.0). The results indicated that the most important factor was the nature of the strain (R =107.0), while the change in temperature and time of cultivation produced the lowest effect (R =40.0). Each factor variance was significant and A3B4C2D1E3 was the optimum combined condition, in which strain T1010 grew more quickly and sporulated most.

  1. Why does Colombia lack agricultural commodity futures?

    Directory of Open Access Journals (Sweden)

    Pablo Moreno-Alemay

    2015-11-01

    Full Text Available This article explores the reasons why futures contracts are not traded as an alternative to price hedging for agricultural goods in Colombia. Based on surveys, interviews and statistical analysis, this study identified that conceptual gaps in contract negotiation, lack of consensus in the agricultural sector regarding the use of financial mechanisms and the sector’s infrequent contact with Colombia’s financial institutions, are the main reasons why a futures contracts market has not emerged.

  2. Increased sensitivity to salt stress in tocopherol-deficient Arabidopsis mutants growing in a hydroponic system.

    Science.gov (United States)

    Ellouzi, Hasna; Hamed, Karim Ben; Cela, Jana; Müller, Maren; Abdelly, Chedly; Munné-Bosch, Sergi

    2013-02-01

    Recent studies suggest that tocopherols could play physiological roles in salt tolerance but the mechanisms are still unknown. In this study, we analyzed changes in growth, mineral and oxidative status in vte1 and vte4 Arabidopsis thaliana mutants exposed to salt stress. vte1 and vte4 mutants lack α-tocopherol, but only the vte1 mutant is additionally deficient in γ-tocopherol. Results showed that a deficiency in vitamin E leads to reduced growth and increased oxidative stress in hydroponically-grown plants. This effect was observed at early stages, not only in rosettes but also in roots. The vte1 mutant was more sensitive to salt-induced oxidative stress than the wild type and the vte4 mutant. Salt sensitivity was associated with (i) high contents of Na(+), (ii) reduced efficiency of PSII photochemistry (Fv/Fm ratio) and (iii) more pronounced oxidative stress as indicated by increased hydrogen peroxide and malondialdeyde levels. The vte 4 mutant, which accumulates γ- instead of α-tocopherol showed an intermediate sensitivity to salt stress between the wild type and the vte1 mutant. Contents of abscisic acid, jasmonic acid and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid were higher in the vte1 mutant than the vte4 mutant and wild type. It is concluded that vitamin E-deficient plants show an increased sensitivity to salt stress both in rosettes and roots, therefore indicating the positive role of tocopherols in stress tolerance, not only by minimizing oxidative stress, but also controlling Na(+)/K(+) homeostasis and hormonal balance.

  3. Quantum preparation uncertainty and lack of information

    Science.gov (United States)

    Rozpędek, Filip; Kaniewski, Jędrzej; Coles, Patrick J.; Wehner, Stephanie

    2017-02-01

    The quantum uncertainty principle famously predicts that there exist measurements that are inherently incompatible, in the sense that their outcomes cannot be predicted simultaneously. In contrast, no such uncertainty exists in the classical domain, where all uncertainty results from ignorance about the exact state of the physical system. Here, we critically examine the concept of preparation uncertainty and ask whether similarly in the quantum regime, some of the uncertainty that we observe can actually also be understood as a lack of information (LOI), albeit a lack of quantum information. We answer this question affirmatively by showing that for the well known measurements employed in BB84 quantum key distribution (Bennett and Brassard 1984 Int. Conf. on Computer System and Signal Processing), the amount of uncertainty can indeed be related to the amount of available information about additional registers determining the choice of the measurement. We proceed to show that also for other measurements the amount of uncertainty is in part connected to a LOI. Finally, we discuss the conceptual implications of our observation to the security of cryptographic protocols that make use of BB84 states.

  4. Lack of transplacental transmission of Bartonella bovis.

    Science.gov (United States)

    Chastant-Maillard, S; Boulouis, H-J; Reynaud, K; Thoumire, S; Gandoin, C; Bouillin, C; Cordonnier, N; Maillard, R

    2015-02-01

    Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue.

  5. Male-like sexual behavior of female mouse lacking fucose mutarotase

    Directory of Open Access Journals (Sweden)

    Lim Dae-sik

    2010-07-01

    Full Text Available Abstract Background Mutarotases are recently characterized family of enzymes that are involved in the anomeric conversions of monosaccharides. The mammalian fucose mutarotase (FucM was reported in cultured cells to facilitate fucose utilization and incorporation into protein by glycosylation. However, the role of this enzyme in animal has not been elucidated. Results We generated a mutant mouse specifically lacking the fucose mutarotase (FucM gene. The FucM knockout mice displayed an abnormal sexual receptivity with a drastic reduction in lordosis score, although the animals were fertile due to a rare and forced intromission by a typical male. We examined the anteroventral periventricular nucleus (AVPv of the preoptic region in brain and found that the mutant females showed a reduction in tyrosine hydoxylase positive neurons compared to that of a normal female. Furthermore, the mutant females exhibited a masculine behavior, such as mounting to a normal female partner as well as showing a preference to female urine. We found a reduction of fucosylated serum alpha-fetoprotein (AFP in a mutant embryo relative to that of a wild-type embryo. Conclusions The observation that FucM-/- female mouse exhibits a phenotypic similarity to a wild-type male in terms of its sexual behavior appears to be due to the neurodevelopmental changes in preoptic area of mutant brain resembling a wild-type male. Since the previous studies indicate that AFP plays a role in titrating estradiol that are required to consolidate sexual preference of female mice, we speculate that the reduced level of AFP in FucM-/- mouse, presumably resulting from the reduced fucosylation, is responsible for the male-like sexual behavior observed in the FucM knock-out mouse.

  6. Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

    Science.gov (United States)

    Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

    2015-07-01

    The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future.

  7. Amuvatinib has cytotoxic effects against NRAS- but not BRAF-mutant melanoma

    OpenAIRE

    Fedorenko, Inna V.; Fang, Bin; Koomen, John M; Gibney, Geoffrey T.; Smalley, Keiran S. M.

    2014-01-01

    Effective targeted therapy strategies are still lacking for the 15–20% of melanoma patients whose melanomas are driven by oncogenic NRAS. We here report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα and Rad51. An analysis of BRAF- and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively i...

  8. Mice Lacking Hbp1 Function Are Viable and Fertile

    Science.gov (United States)

    Wilhelm, Dagmar; Jans, David A.; Bowles, Josephine; Koopman, Peter

    2017-01-01

    Fetal germ cell development is tightly regulated by the somatic cell environment, and is characterised by cell cycle states that differ between XY and XX gonads. In the testis, gonocytes enter G1/G0 arrest from 12.5 days post coitum (dpc) in mice and maintain cell cycle arrest until after birth. Failure to correctly maintain G1/G0 arrest can result in loss of germ cells or, conversely, germ cell tumours. High mobility group box containing transcription factor 1 (HBP1) is a transcription factor that was previously identified in fetal male germ cells at the time of embryonic cell cycle arrest. In somatic cells, HBP1 is classified as a tumour suppressor protein, known to regulate proliferation and senescence. We therefore investigated the possible role of HBP1 in the initiation and maintenance of fetal germ cell G1/G0 arrest using the mouse model. We identified two splice variants of Hbp1, both of which are expressed in XY and XX fetal gonads, but only one of which is localised to the nucleus in in vitro assays. To investigate Hbp1 loss of function, we used embryonic stem (ES) cells carrying a Genetrap mutation for Hbp1 to generate mice lacking Hbp1 function. We found that Hbp1-genetrap mouse mutant germ cells proliferated correctly throughout development, and adult males were viable and fertile. Multiple Hbp1-LacZ reporter mouse lines were generated, unexpectedly revealing Hbp1 embryonic expression in hair follicles, eye and limbs. Lastly, in a model of defective germ cell G1/G0 arrest, the Rb1-knockout model, we found no evidence for Hbp1 mis-regulation, suggesting that the reported RB1-HBP1 interaction is not critical in the germline, despite co-expression. PMID:28107452

  9. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1-Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1-Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT...mutations found in breast cancer using both structural and cell based assays. We have now have evidence for the effects of the most recurrent

  10. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    Introduction Approximately 70% of ER+ breast cancers harbor expression of the estrogen receptor and are dependent upon its activity for various aspects of the...resistance to current FDA approved ER antagonists, but that more potent and selective estrogen receptor antagonists will be sufficiently active to...antagonists and their potency against ER mutants both in vitro and in vivo . Targeting ESR1-Mutant Breast Cancer W81XWH-14-1-0359 9 4. Impact A) Impact

  11. Energy brands lack vitality; Energiemerken ontberen vitaliteit

    Energy Technology Data Exchange (ETDEWEB)

    Godri, S.; Wilders, E. [CONSULT Brand Strategy, Y and R Advertising, Amsterdam (Netherlands)

    2004-06-01

    The three Dutch energy companies (Nuon, Essent and Eneco Energie) have relatively little brand strength. The brands are not perceived to be sufficiently different from one another and are not valued by consumers. With liberalisation imminent, this is hardly a strong starting point. How can you win over consumers if it is not clear what is on offer? In the business market, decision-makers are better placed to distinguish between brands. However, the brands lack vitality in this sector of the market too. The only consolation is that the situation is by no means exclusive to the Netherlands. [Dutch] De merksterkte van de drie Nederlandse energiebedrijven Nuon, Essent en Eneco Energie, is gering. De energiemerken onderscheiden zich niet genoeg en missen waardering. Met het oog op de aanstaande liberalisering geen goede uitgangspositie. Hoe kun je consumenten voor je winnen als onduidelijk is waarvoor ze moeten kiezen? Op de zakelijk markt zijn de beslissers beter in staat om de merken te onderscheiden. Maar ook hier missen de energiemerken vitaliteit. Een troost: Nederlandse energiebedrijven zijn niet de enige met dit probleem.

  12. Lack of universal scaling in magnetohydrodynamic turbulence

    Science.gov (United States)

    Pouquet, A.; Brachet, M.; Krstulovic, G.; Lee, E.; Mininni, P.; Rosenberg, D. L.

    2012-12-01

    Universality is often viewed as a hallmark of turbulent flows, with a search for scaling exponents that derive from intrinsic dynamics and do not depend on initial conditions or forcing, the Kolmogorov law for the energy spectrum of an incompressible fluid being the best known case. However, in the presence of waves due to an external agent such as rotation, stratification or a strong large-scale magnetic field B0, different regimes -- such as weak or strong turbulence, may arise and thus, different scaling behavior may arise as well. This is observed for example in the ocean, and it leads to different mixing and transport properties. In this talk, we shall first review, in the context of MHD turbulence, the phenomenological models that can be constructed using the following plausible dimensionless parameters: (i) RT, the ratio of characteristic time scales (here, the wave period Tw=L_0/B_0 and the eddy-turn-over time based on large-scale length and velocity, TNL=L0/U_0; (ii) RE, the ratio of magnetic to kinetic energy EM/E_V; and (iii) RA, the degree of alignment between the velocity and the magnetic field \\cos(v,b), or between the magnetic potential and magnetic induction, \\cos(A,b). Note that these ratios can also be defined at scale ℓ of velocity uℓ (as opposed to L0, U0), and thus one can consider as well the variation of such ratios across scales. We shall then contrast these models with data stemming from (mostly) solar observations that indicate a clear lack of universal scaling behavior. Similarly, a number of direct numerical simulations (DNS) including some at high resolution, in the spin-down of forcing case, in the presence of boundaries or not, and with or without an imposed strong external magnetic field B0, all point-out to different energy spectra, although the attainable Reynolds numbers in present-day DNS are still limited when contrasted with geophysical and astrophysical flows. In particular, we shall show that, when using as initial

  13. Lack of RNase L attenuates macrophage functions.

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    Xin Yi

    Full Text Available Macrophages are one of the major cell types in innate immunity against microbial infection. It is believed that the expression of proinflammatory genes such as tumor necrosis factor-α (TNF-α, interleukin (IL-1β, IL-6, and cyclooxygenase-2 (Cox-2 by macrophages is also crucial for activation of both innate and adaptive immunities. RNase L is an interferon (IFN inducible enzyme which is highly expressed in macrophages. It has been demonstrated that RNase L regulates the expression of certain inflammatory genes. However, its role in macrophage function is largely unknown.Bone marrow-derived macrophages (BMMs were generated from RNase L(+/+and (-/- mice. The migration of BMMs was analyzed by using Transwell migration assays. Endocytosis and phagocytosis of macrophages were assessed by using fluorescein isothiocyanate (FITC-Dextran 40,000 and FITC-E. coli bacteria, respectively. The expression of inflammatory genes was determined by Western Blot and ELISA. The promoter activity of Cox-2 was measured by luciferase reporter assays.Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2. Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition. RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF-β, IL-1β, IL-10, CCL2 and Cox-2. Furthermore, the functional analysis of the Cox-2 promoter revealed that RNase L regulated the expression of Cox-2 in macrophages at its transcriptional level. Taken together, our findings provide direct evidence showing that RNase L contributes to innate immunity through regulating macrophage functions.

  14. Mighty Dwarfs: Arabidopsis Autoimmune Mutants and Their Usages in Genetic Dissection of Plant Immunity

    Science.gov (United States)

    van Wersch, Rowan; Li, Xin; Zhang, Yuelin

    2016-01-01

    Plants lack the adaptive immune system possessed by mammals. Instead they rely on innate immunity to defend against pathogen attacks. Genomes of higher plants encode a large number of plant immune receptors belonging to different protein families, which are involved in the detection of pathogens and activation of downstream defense pathways. Plant immunity is tightly controlled to avoid activation of defense responses in the absence of pathogens, as failure to do so can lead to autoimmunity that compromises plant growth and development. Many autoimmune mutants have been reported, most of which are associated with dwarfism and often spontaneous cell death. In this review, we summarize previously reported Arabidopsis autoimmune mutants, categorizing them based on their functional groups. We also discuss how their obvious morphological phenotypes make them ideal tools for epistatic analysis and suppressor screens, and summarize genetic screens that have been carried out in various autoimmune mutant backgrounds. PMID:27909443

  15. Connexin mutant embryonic stem cells and human diseases

    Institute of Scientific and Technical Information of China (English)

    Kiyomasa; Nishii; Yosaburo; Shibata; Yasushi; Kobayashi

    2014-01-01

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  16. Constitutive Stringent Response Restores Viability of Bacillus subtilis Lacking Structural Maintenance of Chromosome Protein.

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    Camille Benoist

    Full Text Available Bacillus subtilis mutants lacking the SMC-ScpAB complex are severely impaired for chromosome condensation and partitioning, DNA repair, and cells are not viable under standard laboratory conditions. We isolated suppressor mutations that restored the capacity of a smc deletion mutant (Δsmc to grow under standard conditions. These suppressor mutations reduced chromosome segregation defects and abrogated hypersensitivity to gyrase inhibitors of Δsmc. Three suppressor mutations were mapped in genes involved in tRNA aminoacylation and maturation pathways. A transcriptomic survey of isolated suppressor mutations pointed to a potential link between suppression of Δsmc and induction of the stringent response. This link was confirmed by (pppGpp quantification which indicated a constitutive induction of the stringent response in multiple suppressor strains. Furthermore, sublethal concentrations of arginine hydroxamate (RHX, a potent inducer of stringent response, restored growth of Δsmc under non permissive conditions. We showed that production of (pppGpp alone was sufficient to suppress the thermosensitivity exhibited by the Δsmc mutant. Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

  17. Changes in Thermostability of Photosystem Ⅱ and Leaf Lipid Composition of Rice Mutant with Deficiency of Light-harvesting Chlorophyll Protein Complexes

    Institute of Scientific and Technical Information of China (English)

    Yunlai Tang; Mei Chen; Yinong Xu; Tingyun Kuang

    2007-01-01

    We studied the difference in thermostability of photosystem Ⅱ (PSⅡ) and leaf lipid composition between a T-DNA insertion mutant rice (Oryza sativa L.) VG28 and its wild type Zhonghua11. Native green gel and SDS-PAGE electrophoreses revealed that the mutant VG28 lacked all light-harvesting chlorophyll a/b protein complexes. Both the mutant and wild type were sensitive to high temperatures, and the maximal efficiency of PSⅡ photochemistry (Fv/Fm) and oxygen-evolving activity of PSⅡ in leaves significantly decreased with increasing temperature. However, the PSⅡ activity of the mutant was markedly more sensitive to high temperatures than that of the wild type. Lipid composition analysis showed that the mutant had less phosphatidylglycerol and sulfoquinovosyl diacylglycerol compared with the wild type. Fatty acid analysis revealed that the mutant had an obvious decrease in the content of unsaturation of membrane lipids on the thermostability of PSll are discussed.

  18. Boc modifies the holoprosencephaly spectrum of Cdo mutant mice

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    Wei Zhang

    2011-05-01

    Holoprosencephaly (HPE is caused by a failure to form the midline of the forebrain and/or midface. It is one of the most common human birth defects, but clinical expression is extremely variable. HPE is associated with mutations in the sonic hedgehog (SHH pathway. Mice lacking the Shh pathway regulator Cdo (also called Cdon display HPE with strain-dependent penetrance and expressivity, implicating silent modifier genes as one cause of the variability. However, the identities of potential HPE modifiers of this type are unknown. We report here that whereas mice lacking the Cdo paralog Boc do not have HPE, Cdo;Boc double mutants on a largely Cdo-resistant genetic background have lobar HPE with strong craniofacial anomalies and defects in Shh target gene expression in the developing forebrain. Boc is therefore a silent HPE modifier gene in mice. Furthermore, Cdo and Boc have specific, selective roles in Shh signaling in mammals, because Cdo;Boc double-mutant mice do not display the most severe HPE phenotype seen in Shh-null mice, nor do they have major defects in digit patterning or development of vertebrae, which are also Shh-dependent processes. This is in contrast to reported observations in Drosophila, where genetic removal of the Cdo and Boc orthologs Ihog and Boi results in a complete loss of response to the hedgehog ligand. Therefore, there is evolutionary divergence between mammals and insects in the requirement of the hedgehog pathway for Cdo/Ihog family members, with mammalian development involving additional factors and/or distinct mechanisms at this level of pathway regulation.

  19. Lack of efficacy of ergocalciferol repletion

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    Thomas Wasser

    2012-04-01

    Full Text Available Introduction: Vitamin D has become an area of intensive scrutiny, both in medical and lay literature. However, there are limited data to suggest proper repletion regimens for those patients who have hypovitaminosis D. Consequently, various methods are used in clinical practice. The aim of this study was to assess the efficacy of various treatment strategies for hypovitaminosis D in an ambulatory internal medicine practice. Methods: A retrospective chart review between October 2005 and June 2010 of a suburban internal medicine practice was performed via query of the electronic medical record (Centricity, General Electric Healthcare, UK. Patients with a 25-hydroxyvitamin D concentration less than 32 mg/dl were identified and treated. Treatment success was defined as 25-hydroxyvitamin D concentrations greater than 32 mg/dl. Statistical analysis to assess changes in vitamin D level controlling for season, comorbidities, and demographics were used. Results: A total of 607 treatment episodes were identified, with 395 excluded due to lack of follow-up vitamin D level within 16 weeks, no treatment documented, topical treatment, doxercalciferol treatment, or non-compliance. Of the remaining patients, there were 212 treatment instances on 178 patients. Ergocalciferol 50,000 international units (IU was used most frequently (71.4% of the time.. A higher initial vitamin D level was positively associated with treatment success (adjusted odds ratio = 1.11, p=0.002. Increased doses of ergocalciferol increased the likelihood of treatment success (p=0.0011. Seasonal variation was related to posttreatment 25-hydroxyvitamin D concentration as was body mass index (BMI (p=0.003 and p=0.044. Conclusion: Pretreatment levels of 25-hydroxyvitamin D, BMI, season, and vitamin D dose are predictors of successful hypovitaminosis D treatment. Our data suggest that patients with initial 25-hydroxyvitamin D concentrations of <20 should be treated with a higher total dose of

  20. Phanerochaete mutants with enhanced ligninolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  1. Tyrosinase degradation is prevented when EDEM1 lacks the intrinsically disordered region.

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    Marioara B Marin

    Full Text Available EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121-598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40-119, predicted to be intrinsically disordered (ID and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.

  2. Enhanced cellulase production in mutants of Thermomonospora

    Energy Technology Data Exchange (ETDEWEB)

    Fennington, G.; Lupo, D.; Stutzenberger, F.

    1982-01-01

    Thermomonospora curvata, a thermophilic actinomycete, secretes multiple forms of endo-beta, 1-4-glucanase (EG) when grown on cellulose-mineral salts liquid medium. The EG activity (measured as carboxymethyl cellulose hydrolysis) was separated by ion exchange chromatography into three distinct components which differed in their kinetic properties. Exposure of T. curvata to ultraviolet light, N-nitrosoguanidine, or ethane methyl sulfonate produced mutants with enhanced EG production. Selection of colonies which cleared cellulose agar plants containing 2-deoxyglucose or glycerol yielded mutants having 1.5 to 2.6 times the extracellular EG and saccharifying activity (measured by filter-paper and cotton-fiber hydrolysis). The secretion of extracellular protein was increased proportionally in mutant cultures. (Refs. 40).

  3. Aging Kit mutant mice develop cardiomyopathy.

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    Lei Ye

    Full Text Available Both bone marrow (BM and myocardium contain progenitor cells expressing the c-Kit tyrosine kinase. The aims of this study were to determine the effects of c-Kit mutations on: i. myocardial c-Kit(+ cells counts and ii. the stability of left ventricular (LV contractile function and structure during aging. LV structure and contractile function were evaluated (echocardiography in two groups of Kit mutant (W/Wv and W41/W42 and in wild type (WT mice at 4 and 12 months of age and the effects of the mutations on LV mass, vascular density and the numbers of proliferating cells were also determined. In 4 month old Kit mutant and WT mice, LV ejection fractions (EF and LV fractional shortening rates (FS were comparable. At 12 months of age EF and FS were significantly decreased and LV mass was significantly increased only in W41/W42 mice. Myocardial vascular densities and c-Kit(+ cell numbers were significantly reduced in both mutant groups when compared to WT hearts. Replacement of mutant BM with WT BM at 4 months of age did not prevent these abnormalities in either mutant group although they were somewhat attenuated in the W/Wv group. Notably BM transplantation did not prevent the development of cardiomyopathy in 12 month W41/W42 mice. The data suggest that decreased numbers and functional capacities of c-Kit(+ cardiac resident progenitor cells may be the basis of the cardiomyopathy in W41/W42 mice and although defects in mutant BM progenitor cells may prove to be contributory, they are not causal.

  4. Behavioral characterization of system xc- mutant mice.

    Science.gov (United States)

    McCullagh, Elizabeth A; Featherstone, David E

    2014-05-15

    The slc7a11 gene encodes xCT, an essential component of 'system xc-', a plasma membrane exchanger that imports cystine and exports glutamate. Slc7a11 is expressed primarily in the brain, but its role there is not clear. We performed behavioral tests on two different strains of homozygous slc7a11 mutant mice ('sut' and 'xCT'), as well as heteroallelic offspring of these two strains ('xCT/sut') and their associated genetic backgrounds. Homozygous sut mutant males showed reduced spontaneous alternation in spontaneous alternation tasks as well as reduced movement in an open field maze, but xCT and xCT/sut strains did not show significant changes in these tasks compared to appropriate controls. Neither xCT nor sut mutants showed differences from controls in rotarod tests. Female behavioral phenotypes were independent of estrus cycle stage. To ensure that homozygous xCT, sut, and xCT/sut strains all represent protein null alleles, we measured whole brain xCT protein levels using immunoblots. xCT, sut and xCT/sut strains showed no detectable xCT protein expression, confirming them as null alleles. Previously published microdialysis experiments showed reduced striatal glutamate in xCT mutants. Using the same methods, we measured reduced interstitial glutamate levels in the striatum but not cerebellum of sut mutants. However, we detected no glutamate change in the striatum or cerebellum of sut/xCT mice. We detected no changes in whole brain EAAT-1, -2, or -3 expression. We conclude that the behavioral and chemical differences exist between slc7a11 mutant strains, but we were unable to definitively attribute any of these differences to loss of system xc-.

  5. Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

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    Fresno Manuel

    2011-07-01

    Full Text Available Abstract Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II, to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus, infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

  6. Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

    Science.gov (United States)

    Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

    2017-12-01

    The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr (a) ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr (a) has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr (a) significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

  7. Mutants with Enhanced Nitrogenase Activity in Hydroponic Azospirillum brasilense-Wheat Associations

    Science.gov (United States)

    Pereg Gerk, Lily; Gilchrist, Kate; Kennedy, Ivan R.

    2000-01-01

    The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism. PMID:10788397

  8. Nodulation gene mutants of Mesorhizobium loti R7A-nodZ and nolL mutants have host-specific phenotypes on Lotus spp.

    Science.gov (United States)

    Rodpothong, Patsarin; Sullivan, John T; Songsrirote, Kriangsak; Sumpton, David; Cheung, Kenneth W J-T; Thomas-Oates, Jane; Radutoiu, Simona; Stougaard, Jens; Ronson, Clive W

    2009-12-01

    Rhizobial Nod factors induce plant responses and facilitate bacterial infection, leading to the development of nitrogen-fixing root nodules on host legumes. Nodule initiation is highly dependent on Nod-factor structure and, hence, on at least some of the nodulation genes that encode Nod-factor production. Here, we report the effects of mutations in Mesorhizobium loti R7A nodulation genes on nodulation of four Lotus spp. and on Nod-factor structure. Most mutants, including a DeltanodSDeltanolO double mutant that produced Nod factors lacking the carbamoyl and possibly N-methyl groups on the nonreducing terminal residue, were unaffected for nodulation. R7ADeltanodZ and R7ADeltanolL mutants that produced Nod factors without the (acetyl)fucose on the reducing terminal residue had a host-specific phenotype, forming mainly uninfected nodule primordia on Lotus filicaulis and L. corniculatus and effective nodules with a delay on L. japonicus. The mutants also showed significantly reduced infection thread formation and Nin gene induction. In planta complementation experiments further suggested that the acetylfucose was important for balanced signaling in response to Nod factor by the L. japonicus NFR1/NFR5 receptors. Overall the results reveal differences in the sensitivity of plant perception with respect to signaling leading to root hair deformation and nodule primordium development versus infection thread formation and rhizobial entry.

  9. Analysis of the endocardium and cardiac jelly in truncal development in the cardiac lethal mutant axolotl Ambystoma mexicanum.

    Science.gov (United States)

    Lemanski, L F; Fitzharris, T P

    1989-05-01

    Recessive mutant gene c in axolotls results in a failure of the heart to function because of abnormal embryonic induction processes. The myocardium in this mutant lacks organized sarcomeric myofibrils. The present study was undertaken to determine if developmental abnormalities were evident in other areas of the heart besides the myocardium. A detailed comparative survey of the structure of developing normal and mutant hearts, including the endocardium, its cellular derivatives, and the extracellular matrix, known as cardiac jelly, showed that in the mutant there are fewer than the normal number of endocardial cells lining the heart lumen, the number of mesenchyme cells is reduced, and the cardiac jelly area is greatly enlarged in the posterior part of the truncus adjacent to the ventricle.

  10. The thick aleurone1 mutant defines a negative regulation of maize aleurone fate that functions downstream of dek1

    Science.gov (United States)

    The maize aleurone layer occupies the single outermost layer of the endosperm. The dek1 gene is a central regulator required for aleurone cell fate specification. Dek1 mutants have pleiotropic phenotypes including lack of aleurone cells, aborted embryos, carotenoid deficiency and a soft, floury endo...

  11. Preferential repair in Saccharomyces cerevisiae rad mutants after induction of interstrand cross-links by 8-methoxypsoralen plus UVA.

    Science.gov (United States)

    Meniel, V; Magaña-Schwencke, N; Averbeck, D

    1995-11-01

    The gene specific induction and the incision step of the removal of 8-methoxypsoralen (8-MOP) plus UVA-induced interstrand cross-links (ICL) was measured in repair mutants of Saccharomyces cerevisiae. Events were examined at the MAT alpha and HML alpha loci in mutants deficient in the repair of ICL, namely rad1, rad2 delta, rad52, pso2 and the rad16 mutant which is impaired in the removal of UV-induced pyrimidine dimers from the silent HML alpha locus. Previously, we observed in a wild-type strain (K107) preferential repair concerning the incision of 8-MOP photo-induced ICL. The present study indicates that the two mutants rad1 and rad2 delta show no repair in either locus, due presumably to their deficiency in the incision step of ICL repair. The rad52 mutant which is defective in recombination, is proficient in the preferential incision of ICL at the MAT alpha locus versus the HML alpha locus. The same is true for the pso2 mutant which also lacks the ability to perform complete repair of ICL. The rad16 mutant is unable to repair ICL in the silent locus HML alpha but is proficient in repair (i.e. the incision of ICL) in the transcriptionally active MAT alpha locus.

  12. A dominant semi dwarf mutant in rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ In the winter of 1997, a semi dwarf mutant was found in the F6 population of M9056/ R8018 xuan in Hainan Province. In the spring of 1998, the seeds were sown in Hefei, Anhui Province and the plant height of the population was measured at maturity.

  13. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    Science.gov (United States)

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  14. Colored HOMFLY polynomials can distinguish mutant knots

    CERN Document Server

    Nawata, Satoshi; Singh, Vivek Kumar

    2015-01-01

    We illustrate from the viewpoint of braiding operations on WZNW conformal blocks how colored HOMFLY polynomials with multiplicity structure can detect mutations. As an example, we explicitly evaluate the (2,1)-colored HOMFLY polynomials that distinguish a famous mutant pair, Kinoshita-Terasaka and Conway knot.

  15. GAMPMS: Genetic algorithm managed peptide mutant screening.

    Science.gov (United States)

    Long, Thomas; McDougal, Owen M; Andersen, Tim

    2015-06-30

    The prominence of endogenous peptide ligands targeted to receptors makes peptides with the desired binding activity good molecular scaffolds for drug development. Minor modifications to a peptide's primary sequence can significantly alter its binding properties with a receptor, and screening collections of peptide mutants is a useful technique for probing the receptor-ligand binding domain. Unfortunately, the combinatorial growth of such collections can limit the number of mutations which can be explored using structure-based molecular docking techniques. Genetic algorithm managed peptide mutant screening (GAMPMS) uses a genetic algorithm to conduct a heuristic search of the peptide's mutation space for peptides with optimal binding activity, significantly reducing the computational requirements of the virtual screening. The GAMPMS procedure was implemented and used to explore the binding domain of the nicotinic acetylcholine receptor (nAChR) α3β2-isoform with a library of 64,000 α-conotoxin (α-CTx) MII peptide mutants. To assess GAMPMS's performance, it was compared with a virtual screening procedure that used AutoDock to predict the binding affinity of each of the α-CTx MII peptide mutants with the α3β2-nAChR. The GAMPMS implementation performed AutoDock simulations for as few as 1140 of the 64,000 α-CTx MII peptide mutants and could consistently identify a set of 10 peptides with an aggregated binding energy that was at least 98% of the aggregated binding energy of the 10 top peptides from the exhaustive AutoDock screening.

  16. Partially Functional Outer-Arm Dynein in a Novel Chlamydomonas Mutant Expressing a Truncated γ Heavy Chain▿

    Science.gov (United States)

    Liu, Zhongmei; Takazaki, Hiroko; Nakazawa, Yuki; Sakato, Miho; Yagi, Toshiki; Yasunaga, Takuo; King, Stephen M.; Kamiya, Ritsu

    2008-01-01

    The outer dynein arm of Chlamydomonas flagella contains three heavy chains (α, β, and γ), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose γ heavy chain is truncated at about 30% of the sequence. While the previously isolated γ chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain α and β heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the γ heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the α heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the β heavy chain). Thus, the outer-arm dynein lacking the γ heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein. PMID:18487347

  17. Lack of malate valve capacities lead to improved N-assimilation and growth in transgenic A. thaliana plants.

    Science.gov (United States)

    Selinski, Jennifer; Scheibe, Renate

    2014-01-01

    In this study we analyzed the relationship between malate valve capacities, N-assimilation, and energy metabolism. We used transgenic plants either lacking the chloroplast NADP-dependent malate dehydrogenase or mutants with a decreased transcript level of the plastid-localized NAD-dependent malate dehydrogenase. Plants were grown on nitrate or ammonium, respectively, as the sole N-source and transcripts were analyzed by qRT-PCR. We could show that the lack of malate valve capacities enhances N-assimilation and plastidial glycolysis by increasing transcript levels of Fd-GOGATs or NADH-GOGAT and plastidic NAD-GAPDHs (GapCps), respectively. Based on our results, we conclude that the lack of malate valve capacities is balanced by an increase of the activity of plastid-localized glycolysis in order to cover the high demand for plastidial ATP, stressing the importance of the plastids for energy metabolism in plant cells.

  18. Mutations in rpoBC suppress the defects of a Sinorhizobium meliloti relA mutant.

    Science.gov (United States)

    Wells, Derek H; Long, Sharon R

    2003-09-01

    The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa requires complex physiological adaptation by both partners. One method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the ability of RNA polymerase (RNAP) to initiate and elongate transcription and affect the affinity of the core enzyme for various sigma factors. An S. meliloti relA mutant which cannot produce ppGpp was previously shown to be defective in the ability to form nodules. This mutant also overproduces a symbiotically necessary exopolysaccharide called succinoglycan. The work presented here encompasses the analysis of suppressor mutants, isolated from host plants, that suppress the symbiotic defects of the relA mutant. All suppressor mutations are extragenic and map to either rpoB or rpoC, which encode the beta and beta' subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal that suppressor mutants can be divided into two classes; one is specific in its effect on stringent response-regulated genes and shares striking similarity with suppressor mutants of Escherichia coli strains that lack ppGpp, and another reduces transcription of all genes tested in comparison to that in the relA parent strain. Our findings indicate that the ability to successfully establish symbiosis is tightly coupled with the bacteria's ability to undergo global physiological adjustment via the stringent response.

  19. A suite of Lotus japonicus starch mutants reveals both conserved and novel features of starch metabolism.

    Science.gov (United States)

    Vriet, Cécile; Welham, Tracey; Brachmann, Andreas; Pike, Marilyn; Pike, Jodie; Perry, Jillian; Parniske, Martin; Sato, Shusei; Tabata, Satoshi; Smith, Alison M; Wang, Trevor L

    2010-10-01

    The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

  20. Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

    Science.gov (United States)

    Fritzsch, B.; Signore, M.; Simeone, A.

    2001-01-01

    We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.

  1. Lack of SIRPα phosphorylation and concomitantly reduced SHP-2-PI3K-Akt2 signaling decrease osteoblast differentiation.

    Science.gov (United States)

    Holm, Cecilia Koskinen; Engman, Sara; Sulniute, Rima; Matozaki, Takashi; Oldenborg, Per-Arne; Lundberg, Pernilla

    2016-09-09

    Normal differentiation of bone forming osteoblasts is a prerequisite for maintenance of skeletal health and is dependent on intricate cellular signaling pathways, including the essential transcription factor Runx2. The cell surface glycoprotein CD47 and its receptor signal regulatory protein alpha (SIRPα) have both been suggested to regulate bone cell differentiation. Here we investigated osteoblastic differentiation of bone marrow stromal cells from SIRPα mutant mice lacking the cytoplasmic signaling domain of SIRPα. An impaired osteoblastogenesis in SIRPα-mutant cell cultures was demonstrated by lower alkaline phosphatase activity and less mineral formation compared to wild-type cultures. This reduced osteoblastic differentiation potential in SIRPα-mutant stromal cells was associated with a significantly reduced expression of Runx2, osterix, osteocalcin, and alkaline phosphatase mRNA, as well as a reduced phosphorylation of SHP-2 and Akt2, as compared with that in wild-type stromal cells. Addition of a PI3K-inhibitor to wild-type stromal cells could mimic the impaired osteoblastogenesis seen in SIRPα-mutant cells. In conclusion, our data suggest that SIRPα signaling through SHP-2-PI3K-Akt2 strongly influences osteoblast differentiation from bone marrow stromal cells.

  2. Impact of vitamin C on the cardiometabolic and inflammatory profiles of mice lacking a functional Werner syndrome protein helicase.

    Science.gov (United States)

    Aumailley, Lucie; Dubois, Marie Julie; Garand, Chantal; Marette, André; Lebel, Michel

    2015-12-01

    Werner syndrome (WS) is a premature aging disorder caused by mutations in a DNA helicase/exonuclease. Mice lacking the helicase domain of this protein exhibit metabolic abnormalities that are reversed by vitamin C. In this study, we used a targeted metabolomic approach to identify serum metabolites significantly altered in young mutant mice treated with or without vitamin C. We also measured several serum inflammatory and cardiometabolic factors. We show that young mutant mice exhibit an increase in serum hydroxyproline and plasminogen activator inhibitor-1 (PAI-1), markers of cardiovascular diseases and inflammation, before they exhibit morphological anomalies in different tissues. We also observed an increase in three very long chain lysophosphatidylcholines underlying peroxisome perturbation. Vitamin C reversed the concentrations of these metabolites and PAI-1 to wild type values. Transcriptomic analyses on the liver of mutant mice revealed a decrease in the expression of genes involved in fatty acid degradation compared to wild type animals. Vitamin C treatment increased the expression of genes involved in glutathione metabolism and the synthesis of unsaturated fatty acids in these mice. These results show that changes at the transcriptomic level concord with the alterations of several serum metabolites in these mice. Finally, we found that a mislocalization of the Wrn mutant protein in the liver endoplasmic reticulum fraction increased oxidative stress in that cellular compartment. Vitamin C reversed this oxidative stress. To conclude, this study provides novel potential predictive cardiometabolic biomarkers in WS that will allow the assessment of the impact of vitamin C on patients with WS.

  3. 1H, 15N and 13C backbone assignments of GDP-bound human H-Ras mutant G12V.

    Science.gov (United States)

    Amin, Nader; Chiarparin, Elisabetta; Coyle, Joe; Nietlispach, Daniel; Williams, Glyn

    2016-04-01

    Harvey Ras (H-Ras) is a membrane-associated GTPase with critical functions in cell proliferation and differentiation. The G12V mutant of H-Ras is one of the most commonly encountered oncoproteins in human cancer. This mutation disrupts the GTPase activity of H-Ras, leading to constitutive activation and aberrant downstream signalling. Here we report the backbone resonance assignments of human H-Ras mutant G12V lacking the C-terminal membrane attachment domain.

  4. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    disease-free survival, the majority of breast cancer patients will present recurrent antiestrogen resistant metastatic lesions following prolonged...exposure to these therapies. By investigating how these lesions become resistant to antiestrogen while maintaining expression of ERα, we found...Retreat. Invited oral presentation. C) Other products Nothing to report. Targeting  ESR1-­‐Mutant  Breast  Cancer   W81XWH-­‐14-­‐1-­‐0360

  5. Characterization of a Legionella micdadei mip mutant

    DEFF Research Database (Denmark)

    O'Connell, W A; Bangsborg, Jette Marie; Cianciotto, N P

    1995-01-01

    The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human m...... into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei....

  6. Lack of developmental redundancy between Unc45 proteins in zebrafish muscle development.

    Directory of Open Access Journals (Sweden)

    Sophie A Comyn

    Full Text Available Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b(-/-; unc45a(-/- mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b(-/- and unc45b(-/-; unc45a(-/- embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.

  7. Pterin and folate salvage. Plants and Escherichia coli lack capacity to reduce oxidized pterins.

    Science.gov (United States)

    Noiriel, Alexandre; Naponelli, Valeria; Gregory, Jesse F; Hanson, Andrew D

    2007-03-01

    Dihydropterins are intermediates of folate synthesis and products of folate breakdown that are readily oxidized to their aromatic forms. In trypanosomatid parasites, reduction of such oxidized pterins is crucial for pterin and folate salvage. We therefore sought evidence for this reaction in plants. Three lines of evidence indicated its absence. First, when pterin-6-aldehyde or 6-hydroxymethylpterin was supplied to Arabidopsis (Arabidopsis thaliana), pea (Pisum sativum), or tomato (Lycopersicon esculentum) tissues, no reduction of the pterin ring was seen after 15 h, although reduction and oxidation of the side chain of pterin-6-aldehyde were readily detected. Second, no label was incorporated into folates when 6-[(3)H]hydroxymethylpterin was fed to cultured Arabidopsis plantlets for 7 d, whereas [(3)H]folate synthesis from p-[(3)H]aminobenzoate was extensive. Third, no NAD(P)H-dependent pterin ring reduction was found in tissue extracts. Genetic evidence showed a similar situation in Escherichia coli: a GTP cyclohydrolase I (folE) mutant, deficient in pterin synthesis, was rescued by dihydropterins but not by the corresponding oxidized forms. Expression of a trypanosomatid pterin reductase (PTR1) enabled rescue of the mutant by oxidized pterins, establishing that E. coli can take up oxidized pterins but cannot reduce them. Similarly, a GTP cyclohydrolase I (fol2) mutant of yeast (Saccharomyces cerevisiae) was rescued by dihydropterins but not by most oxidized pterins, 6-hydroxymethylpterin being an exception. These results show that the capacity to reduce oxidized pterins is not ubiquitous in folate-synthesizing organisms. If it is lacking, folate precursors or breakdown products that become oxidized will permanently exit the metabolically active pterin pool.

  8. Cell sorting enriches Escherichia coli mutants that rely on peptidoglycan endopeptidases to suppress highly aberrant morphologies.

    Science.gov (United States)

    Laubacher, Mary E; Melquist, Amy L; Chandramohan, Lakshmi; Young, Kevin D

    2013-02-01

    Bacterial morphology imparts physiological advantages to cells in different environments and, judging by the fidelity with which shape is passed to daughter cells, is a tightly regulated characteristic. Surprisingly, only in the past 10 to 15 years has significant headway been made in identifying the mechanisms by which cells create and maintain particular shapes. One reason for this is that the relevant discoveries have relied heavily on the arduous, somewhat subjective process of manual microscopy. Here, we show that flow cytometry, coupled with the sorting capability of fluorescence-activated cell sorting (FACS), can detect, quantify, and enrich bacteria with morphological alterations. The light scattering properties of several highly aberrant morphological mutants of Escherichia coli were characterized by flow cytometry. Cells from a region that overlapped the distribution of normal rod-shaped cells were collected by FACS and reincubated. After 4 to 15 iterations of this enrichment process, suppressor mutants were isolated that returned almost all the population to a near-normal shape. Suppressors were successfully isolated from strains lacking three or four penicillin binding proteins (PBPs) but not from a mutant lacking a total of seven PBPs. The peptidoglycan endopeptidase, AmpH, was identified as being important for the suppression process, as was a related endopeptidase, MepA. The results validate the use of cell sorting as a means for studying bacterial morphology and identify at least one new class of enzymes required for the suppression of cell shape defects.

  9. Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue.

    Science.gov (United States)

    Pinto, Ana F; Romão, Célia V; Pinto, Liliana C; Huber, Harald; Saraiva, Lígia M; Todorovic, Smilja; Cabelli, Diane; Teixeira, Miguel

    2015-01-01

    Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the -EKHVP- motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic Crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (-E23T24HVP-), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild-type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.

  10. Newly generated cells are increased in hippocampus of adult mice lacking a serine protease inhibitor

    Directory of Open Access Journals (Sweden)

    Sticker Melanie

    2010-06-01

    Full Text Available Abstract Background Neurogenesis in the hippocampal dentate gyrus and the subventricular zone occurs throughout the life of mammals and newly generated neurons can integrate functionally into established neuronal circuits. Neurogenesis levels in the dentate gyrus are modulated by changes in the environment (enrichment, exercise, hippocampal-dependent tasks, NMDA receptor (NMDAR activity, sonic hedgehog (SHH and/or other factors. Results previously, we showed that Protease Nexin-1 (PN-1, a potent serine protease inhibitor, regulates the NMDAR availability and activity as well as SHH signaling. Compared with wild-type (WT, we detected a significant increase in BrdU-labeled cells in the dentate gyrus of mice lacking PN-1 (PN-1 -/- both in controls and after running exercise. Patched homologue 1 (Ptc1 and Gli1 mRNA levels were higher and Gli3 down-regulated in mutant mice under standard conditions and to a lesser extent after running exercise. However, the number of surviving BrdU-positive cells did not differ between WT and PN-1 -/- animals. NMDAR availability was altered in the hippocampus of mutant animals after exercise. Conclusion All together our results indicate that PN-1 controls progenitors proliferation through an effect on the SHH pathway and suggest an influence of the serpin on the survival of newly generated neurons through modulation of NMDAR availability.

  11. Endocytosis of Ubiquitylation-Deficient EGFR Mutants via Clathrin-Coated Pits is Mediated by Ubiquitylation.

    Science.gov (United States)

    Fortian, Arola; Dionne, Lai K; Hong, Sun H; Kim, Woong; Gygi, Steven P; Watkins, Simon C; Sorkin, Alexander

    2015-11-01

    Signaling by epidermal growth factor receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here, we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP-2-binding sites (21KRΔAP2) was internalized at relatively high rates via the clathrin-dependent pathway in human duodenal adenocarcinoma HuTu-80 cells. RNA interference analysis revealed that this residual internalization is strongly inhibited by depletion of Grb2 and the E2 ubiquitin-conjugating enzyme UbcH5b/c, and partially affected by depletion of the E3 ubiquitin ligase Cbl and ubiquitin-binding adaptors, indicating that an ubiquitylation process is involved. Several new ubiquitin conjugation sites were identified by mass spectrometry in the 21KRΔAP2 mutant, suggesting that cryptic ubiquitylation may mediate endocytosis of this mutant. Total internal reflection fluorescence microscopy imaging of HuTu-80 cells transfected with labeled ubiquitin adaptor epsin1 demonstrated that the ubiquitylation-deficient EGFR mutant was endocytosed through a limited population of epsin-enriched clathrin-coated pits (CCPs), although with a prolonged CCP lifetime. Native EGFR was recruited with the same efficiency into CCPs containing either AP-2 or epsin1 that were tagged with fluorescent proteins by genome editing of MDA-MD-231 cells. We propose that two redundant mechanisms, ubiquitylation and interaction with AP-2, contribute to EGFR endocytosis via CCPs in a stochastic fashion.

  12. Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Deltaomp25 deletion mutant.

    Science.gov (United States)

    Edmonds, M D; Cloeckaert, A; Hagius, S D; Samartino, L E; Fulton, W T; Walker, J V; Enright, F M; Booth, N J; Elzer, P H

    2002-06-01

    The Brucella melitensis mutant BM 25, which lacks the major 25 kDa outer membrane protein Omp25, has previously been found to be attenuated in the murine brucellosis model. In the present study, the capacity of the Deltaomp25 mutant to colonise and cause abortions in the caprine host was evaluated. The vaccine potential of BM 25 was also investigated in goats. Inoculation of nine pregnant goats in late gestation with the B. melitensis mutant resulted in 0/9 abortions, while the virulent parental strain, B. melitensis 16M, induced 6/6 dams to abort (Pgoats for two weeks post-infection. Owing to the ability of BM 25 to colonise both non-pregnant and pregnant adults without inducing abortions, a vaccine efficacy study was performed. Vaccination of goats prior to breeding with either BM 25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in 100 per cent protection against abortion following challenge in late gestation with virulent strain 16M (Pmelitensis Deltaomp25 mutant, BM 25, may be a safe and efficacious alternative to strain Rev. 1 when dealing with goat herds of mixed age and pregnancy status.

  13. Systematic Triple-Mutant Analysis Uncovers Functional Connectivity between Pathways Involved in Chromosome Regulation

    Directory of Open Access Journals (Sweden)

    James E. Haber

    2013-06-01

    Full Text Available Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed triple-mutant analysis (TMA. We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae, including ASF1 and CAC1, two histone chaperones. After subjecting asf1Δ cac1Δ to TMA, we found that the Swi/Snf Rdh54 protein compensates for the absence of Asf1 and Cac1. Rdh54 more strongly associates with the chromatin apparatus and the pericentromeric region in the double mutant. Moreover, Asf1 is responsible for the synthetic lethality observed in cac1Δ strains lacking the HIRA-like proteins. A similar TMA was carried out after deleting both CLB5 and CLB6, cyclins that regulate DNA replication, revealing a strong functional connection to chromosome segregation. This approach can reveal functional redundancies that cannot be uncovered through traditional double-mutant analyses.

  14. New Rice Mutants(NR676 and NR827)Low in Nitrate Reduction

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Mutants deficient in nitrate reductase(NR) were classfied into two groups,nia types are deficient in apoprotein and cnx types are lack of Mo- Co. This paper reports the characteristics of low NR mutants,which are not deficient in NR activity but 40%~60% of the NR activity of the wild type,Nipponbare. Mutants NR676 and NR827 were selected as seedings showing poor growth with nitrate as sole mitrogen source from M2 population. They exhibited chlorate resistance. Genetic analysis in the F1 and F2 indicated that chlorate resistance in the mutants was transmitted by a single recessive nuclear gene and that NR676 and NR827 were induced by a mutation at a single locus. In the F2 population,all of the seedings showing yellow green leaves were resistant to chlorate and low in NR activity. Photosynthetic rate and mRNA expression of NR676 and NR827 were lower than wild type. These results suggested that low NR activity and chlorate resistance of NR676 and NR827 were caused by a defect in photosynthetic process.

  15. The aurea mutant of tomato is deficient in spectrophotometrically and immunochemically detectable phytochrome.

    Science.gov (United States)

    Parks, B M; Jones, A M; Adamse, P; Koornneef, M; Kendrick, R E; Quail, P H

    1987-03-01

    The aurea locus mutant (au (w)) of tomato contains less than 5% of the level of phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. Immunoblot analysis using antibodies directed against etiolated-oat phytochrome demonstrates that crude extracts of etiolated mutant tissue are deficient in a major immunodetectable protein (116 kDa) normally present in the parent wild type. Analyses of wild-type tissue extracts strongly indicate that the 116-kDa protein is phytochrome by showing that this protein: a) is degraded more rapidly in vitro after a brief far-red irradiation than after a brief red irradiation (Vierstra RD, Quail PH, Planta 156: 158-165, 1982); b) contains a covalently bound chromophore as detected by Zn-chromophore fluorescence on nitrocellulose blots; and c) has an apparent molecular mass comparable to phytochrome from other species on size exclusion chromatography under non-denaturing conditions. The demonstration that the aurea mutant is deficient in this 116-kDa phytochrome indicates that the lack of spectrally detectable phytochrome in this mutant is the result of a lesion which affects the abundance of the phytochrome molecule as opposed to its spectral integrity.

  16. Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

    Science.gov (United States)

    Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

    2016-01-01

    We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

  17. Mutants of Streptomyces cattleya defective in the synthesis of a factor required for thienamycin production.

    Science.gov (United States)

    Buchan, T; Roach, C; Ruby, C; Taylor, D; Preisig, C; Reeves, C

    1994-09-01

    Thienamycin non-producing mutants of Streptomydes cattleya were identified that displayed a cross-feeding relationship. A diffusible product from one of these mutants (RK-11) resulted in restoration of thienamycin production when fed to cultures of another mutant (RK-4). In vivo radiolabeling experiments were conducted to test whether the RK-11 mutant produced a late biosynthetic intermediate which contained a carbapenem ring and a cysteaminyl and/or a hydroxyethyl side chain. Both [35S]cystine and [methyl-3H]methionine were used to label the RK-11 product which was then fed to RK-4 cultures. None of the thienamycin subsequently produced by RK-4 converter cells was labeled, implying the lack of either side chain of the thienamycin molecule in the RK-11 product. Further stability studies suggested that the RK-11 product does not contain a carbapenem ring. Additional feeding experiments with RK-4 cells also ruled out the possibility that the RK-11 product is a co-factor necessary for thienamycin production. It is concluded that the RK-11 product may regulate expression of the thienamycin gene cluster.

  18. Generation of mice lacking DUF1220 protein domains

    DEFF Research Database (Denmark)

    Keeney, J G; O'Bleness, M S; Anderson, N

    2015-01-01

    , fasting glucose levels, reduce sodium levels in male mutants, increased levels of the liver functional indicator ALAT/GPT in males, levels of alkaline phosphatase (also an indicator of liver function), mean R and SR amplitude by electrocardiography, elevated IgG3 levels, a reduced ratio of CD4:CD8 cells...

  19. MASTR: A Technique for Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination Using Conditional Floxed Alleles in Mice

    Directory of Open Access Journals (Sweden)

    Zhimin Lao

    2012-08-01

    Full Text Available Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination, which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26MASTR was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26MASTR mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

  20. Monitoring of bcr/abl Fusion Gene by Interphase-Dual-Color and Dual-Fusion Fluorescence in situ Hybridization in CML after Allo-HSCT%间期双色双融合荧光原位杂交监测慢性髓系白血病异基因造血干细胞移植后bcr/abl融合基因

    Institute of Scientific and Technical Information of China (English)

    钱思轩; 李建勇; 张闰; 洪鸣; 仇海荣; 李丽; 徐卫; 盛瑞兰; 吴汉新

    2006-01-01

    本研究探讨bcr/abl双色双融合荧光原位杂交(DD-FISH)的敏感性及临床应用价值.对19例慢性髓细胞白血病(CML)异基因造血干细胞移植(allo-HSCT)后微小残留病灶(MRD)用DD-FISH进行监测,同时与常规细胞遗传学(CC)、逆转录-聚合酶链反应(RT-PCR)结果相比较.样本取自骨髓,少数来源于骨髓片或外周血.结果表明:14例CML患者在Allo-HSCT后CC显示持续正常供者核型,RT-PCR转为阴性,移植2月后均为完全供者嵌合(DC),DD-FISH检测结果持续为阴性,平均随访11.25月,MRD无增加.1例CC及RT-PCR阴性,而性染色体FISH为混合嵌合,DD-FISH阳性,监测无MRD增加,临床未治疗,疾病稳定.3例骨髓复发患者的DD-FISH及性染色体FISH均提示MRD明显增加,RT-PCR转为阳性,却只有1例CC异常,供者淋巴细胞输注(DLI)及甲磺酸伊马替尼治疗后均为DC,DD-FISH阴性,RT-PCR阴性.1例骨活检证实髓外复发患者骨髓或外周血样本的DD-FISH、CC及PCR均阴性,供受者完全嵌合.结论:间期双色双融合FISH可应用于CML异基因造血干细胞移植后MRD的监测,其操作简易快速,灵敏度高,且骨髓或外周血均可采用.动态监测能及时发现扩大的白血病细胞克隆.

  1. Role of the three-dimensional distribution of abl and bcr genes in the formation of bcr/abl fusion gene in interphase neucleus%间期细胞核中abl和bcr基因的三维空间分布在bcr/abl融合基因形成中的作用

    Institute of Scientific and Technical Information of China (English)

    张青; 周淑芸; 刘晓力; 牛超; 徐岚; 陈赛娟

    2002-01-01

    目的从生物体视学角度分析bcr/abl融合基因的形成机制.方法应用荧光原位杂交和激光共聚焦扫描显微镜技术,观察γ-射线对IM9细胞中的bcr和abl基因在间期核内三维空间分布的影响.结果abl和bcr基因在间期核内有其特定的分布区域,且该分布区域在细胞周期中呈规律性的动态变化.放射性照射会使abl和bcr基因之间的距离缩短.结论abl和bcr基因在间期细胞核内的易接近性是bcr/abl融合基因形成的因素之一.

  2. Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export.

    Directory of Open Access Journals (Sweden)

    David González

    Full Text Available BACKGROUND: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. METHODOLOGY/PRINCIPAL FINDINGS: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that

  3. An Arabidopsis thaliana knock-out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilisation is abolished

    DEFF Research Database (Denmark)

    Schneider, Anja; Häusler, Rainer E; Kolukisaoglu, Uner

    2002-01-01

    The Arabidopsis thaliana tpt-1 mutant which is defective in the chloroplast triose phosphate/phosphate translocator (TPT) was isolated by reverse genetics. It contains a T-DNA insertion 24 bp upstream of the start ATG of the TPT gene. The mutant lacks TPT transcripts and triose phosphate (TP......,6-bisphosphatase. Despite its regulatory role in the feed-forward control of sucrose biosynthesis, variations in the fructose 2,6-bisphosphate content upon illumination were similar in the mutant and the wild type. Crosses of tpt-1 with mutants unable to mobilise starch (sex1) or to synthesise starch (adg1......-1) revealed that growth and photosynthesis of the double mutants was severely impaired only when starch biosynthesis, but not its mobilisation, was affected. For tpt-1/sex1 combining a lack in the TPT with a deficiency in starch mobilisation, an additional compensatory mechanism emerged, i.e. the formation...

  4. Characterisation of cuticular mutants in Arabidopsis thaliana

    OpenAIRE

    Faust, Andrea

    2006-01-01

    Plants are protected by the extracellular cuticle, which is made up of cutin, cutan and waxes. The cutin composition of a variety of plants has been known and models of the biosynthesis of cutin monomers exist but not many enzymes have been identified. It is generally accepted that a defect in the cuticle leads to an organ fusion phenotype. In the model plant A. thaliana many fusion mutants have been identified but the identification of genes involved have not lead to a complete picture of th...

  5. Why Art Education Lacks Social Relevance: A Contextual Analysis.

    Science.gov (United States)

    Bersson, Robert

    1987-01-01

    This article examines why both the individual-centered and discipline-centered approaches to art education lack social relevance. Maintains that the lack of a social orientation in these two approaches is due to our social context and our capitalistic system. Proposes that democratic principles serve as the basis of a socially relevant,…

  6. 29 CFR 18.602 - Lack of personal knowledge.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Lack of personal knowledge. 18.602 Section 18.602 Labor... OFFICE OF ADMINISTRATIVE LAW JUDGES Rules of Evidence Witnesses § 18.602 Lack of personal knowledge. A... witness has personal knowledge of the matter. Evidence to prove personal knowledge may, but need...

  7. Using of AFLP to evaluate gamma-irradiated amaranth mutants

    Directory of Open Access Journals (Sweden)

    Labajová Mária

    2013-01-01

    Full Text Available To determine which of several gamma-irradiated mutants of amaranth Ficha cultivar and K-433 hybrid are most genetically similar to their non-irradiated control genotypes, we performed amplified fragment length polymorphism (AFLP based analysis. A total of 40 selective primer combinations were used in reported analyses. First analyses of gamma-irradiated amaranth mutant lines were done used the AFLP. In the study, primers with the differentiation ability for all analysed mutant lines are reported. The very specific changes in the mutant lines´ non-coding regions based on AFLP length polymorphism were analysed. Mutant lines of the Ficha cultivar (C15, C26, C27, C82, C236 shared a genetic dissimilarity of 0,11 and their ISSR profiles are more similar to the Ficha than those of K-433 hybrid mutant lines. The K-433 mutant lines (D54, D279, D282 shared genetic dissimilarity of 0,534 but are more distinct to their control plant as a whole, as those of the Ficha mutant lines. Different AFLP fingerprints patters of the mutant lines when compared to the Ficha cultivar and K-433 hybrid AFLP profiles may be a consequence of the complex response of the intergenic space of mutant lines to the gamma-radiance. Although a genetic polymorphism was detected within accessions, the AFLP markers successfully identified all the accessions. The AFLP results are discussed by a combination of biochemical characteristics of mutant lines and their control genotypes.

  8. Distribution of soluble amino acids in maize endosperm mutants

    Directory of Open Access Journals (Sweden)

    Toro Alejandro Alberto

    2003-01-01

    Full Text Available For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperm of wild-type, opaque and floury maize (Zea mays L. mutants were determined by HPLC. The total absolute concentration of soluble amino acids among the mutants varied depending on the mutant. The o11 and o13 mutants exhibited the highest average content, whereas o10, fl3 and fl1 exhibited the lowest average content. In general, the mutants exhibited similar concentrations of total soluble amino acids when compared to the wild-type lines, with the clear exception of mutants o11 and fl1, with the o11 mutant exhibiting a higher concentration of total soluble amino acids when compared to its wild-type counterpart W22 and the fl1 mutant a lower concentration when compared to its wild-type counterpart Oh43. For methionine, the mutants o2 and o11 and wild-type Oh43 exhibited the highest concentrations of this amino acid. Significant differences were not observed between mutants for other amino acids such as lysine and threonine. The high lysine concentrations obtained originally for these mutants may be due to the amino acids incorporated into storage proteins, but not those present in the soluble form.

  9. Phenol removal from refinery wastewater by mutant recombinant horseradish peroxidase.

    Science.gov (United States)

    Asad, Sedigheh; Dabirmanesh, Bahareh; Khajeh, Khosro

    2014-01-01

    Application of mutated recombinant horseradish peroxidase (HRP) for phenol removal from refinery effluents is reported. Recombinant HRP produced in Escherichia coli suffers from the disadvantage of lacking glycosylation, which affects its catalytic efficiency and stability toward inactivating parameters such as increased temperature and enhanced amounts of hydrogen peroxide. In the present study, the previously reported variant (in which Asn268 was substituted with Asp, N268D) with improved stability characteristics and catalytic efficiency was used to remove phenol from a petroleum refinery effluent. The presence and removal of phenol was studied by high-performance liquid chromatography; the precipitated oxidized phenol was also observed and removed from the sample by centrifugation. Results showed that the N268D variant can remove 61%, 67%, and 81% of phenol from effluent in 1, 2, and 16 H, respectively. By exploiting the N268D mutant, removal of 50% phenol could be achieved in 42 Min, which was more than 22 times less than the treatment time required by native recombinant enzyme.

  10. The thick aleurone1 mutant defines a negative regulation of maize aleurone cell fate that functions downstream of defective kernel1.

    Science.gov (United States)

    Yi, Gibum; Lauter, Adrienne M; Scott, M Paul; Becraft, Philip W

    2011-08-01

    The maize (Zea mays) aleurone layer occupies the single outermost layer of the endosperm. The defective kernel1 (dek1) gene is a central regulator required for aleurone cell fate specification. dek1 mutants have pleiotropic phenotypes including lack of aleurone cells, aborted embryos, carotenoid deficiency, and a soft, floury endosperm deficient in zeins. Here we describe the thick aleurone1 (thk1) mutant that defines a novel negative function in the regulation of aleurone differentiation. Mutants possess multiple layers of aleurone cells as well as aborted embryos. Clonal sectors of thk1 mutant tissue in otherwise normal endosperm showed localized expression of the phenotype with sharp boundaries, indicating a localized cellular function for the gene. Sectors in leaves showed expanded epidermal cell morphology but the mutant epidermis generally remained in a single cell layer. Double mutant analysis indicated that the thk1 mutant is epistatic to dek1 for several aspects of the pleiotropic dek1 phenotype. dek1 mutant endosperm that was mosaic for thk1 mutant sectors showed localized patches of multilayered aleurone. Localized sectors were surrounded by halos of carotenoid pigments and double mutant kernels had restored zein profiles. In sum, loss of thk1 function restored the ability of dek1 mutant endosperm to accumulate carotenoids and zeins and to differentiate aleurone. Therefore the thk1 mutation defines a negative regulator that functions downstream of dek1 in the signaling system that controls aleurone specification and other aspects of endosperm development. The thk1 mutation was found to be caused by a deletion of approximately 2 megabases.

  11. Quantitative proteomic analysis of Burkholderia pseudomallei Bsa type III secretion system effectors using hypersecreting mutants.

    Science.gov (United States)

    Vander Broek, Charles W; Chalmers, Kevin J; Stevens, Mark P; Stevens, Joanne M

    2015-04-01

    Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS "gatekeeper" family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei.

  12. Indy mutants: live long and prosper

    Directory of Open Access Journals (Sweden)

    Stewart eFrankel

    2012-02-01

    Full Text Available Indy encodes the fly homologue of a mammalian transporter of di and tricarboxylatecomponents of the Krebs cycle. Reduced expression of fly Indy or two of the C. elegansIndy homologs leads to an increase in life span. Fly and worm tissues that play key roles inintermediary metabolism are also the places where Indy genes are expressed. One of themouse homologs of Indy (mIndy is mainly expressed in the liver. It has been hypothesizedthat decreased INDY activity creates a state similar to caloric restriction (CR. Thishypothesis is supported by the physiological similarities between Indy mutant flies on highcalorie food and control flies on CR, such as increased physical activity and decreases inweight, egg production, triglyceride levels, starvation resistance, and insulin signaling. Inaddition, Indy mutant flies undergo changes in mitochondrial biogenesis also observed inCR animals. Recent findings with mIndy knockout mice support and extend the findingsfrom flies. mIndy-/- mice display an increase in hepatic mitochondrial biogenesis, lipidoxidation and decreased hepatic lipogenesis. When mIndy-/- mice are fed high calorie foodthey are protected from adiposity and insulin resistance. These findings point to INDY as apotential drug target for the treatment of metabolic syndrome, type 2 diabetes and obesity.

  13. An Epstein-Barr virus mutant produces immunogenic defective particles devoid of viral DNA.

    Science.gov (United States)

    Pavlova, Sophia; Feederle, Regina; Gärtner, Kathrin; Fuchs, Walter; Granzow, Harald; Delecluse, Henri-Jacques

    2013-02-01

    Virus-like particles (VLPs) from hepatitis B and human papillomaviruses have been successfully used as preventative vaccines against these infectious agents. These VLPs consist of a self-associating capsid polymer formed from a single structure protein and are devoid of viral DNA. Since virions from herpesviruses consist of a large number of molecules of viral and cellular origin, generating VLPs from a subset of these would be a particularly arduous task. Therefore, we have adopted an alternative strategy that consists of producing DNA-free defective virus particles in a cell line infected by a herpesvirus mutant incapable of packaging DNA. We previously reported that an Epstein-Barr virus (EBV) mutant devoid of the terminal repeats (ΔTR) that act as packaging signals in herpesviruses produces substantial amounts of VLPs and of light particles (LPs). However, ΔTR virions retained some infectious genomes, and although these mutants had lost their transforming abilities, this poses potential concerns for clinical applications. Therefore, we have constructed a series of mutants that lack proteins involved in maturation and assessed their ability to produce viral DNA-free VLP/LPs. Some of the introduced mutations were deleterious for capsid maturation and virus production. However, deletion of BFLF1/BFRF1A or of BBRF1 resulted in the production of DNA-free VLPs/LPs. The ΔBFLF1/BFRF1A viruses elicited a potent CD4(+) T-cell response that was indistinguishable from the one obtained with wild-type controls. In summary, the defective particles produced by the ΔBFLF1/BFRF1A mutant fulfill the criteria of efficacy and safety expected from a preventative vaccine.

  14. A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect.

    Science.gov (United States)

    Abdullaev, Ziedulla Kh; Bodrova, Marina E; Chernyak, Boris V; Dolgikh, Dmitry A; Kluck, Ruth M; Pereverzev, Mikhail O; Arseniev, Alexander S; Efremov, Roman G; Kirpichnikov, Mikhail P; Mokhova, Elena N; Newmeyer, Donald D; Roder, Heinrich; Skulachev, Vladimir P

    2002-01-01

    A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12. PMID:11879204

  15. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

    Directory of Open Access Journals (Sweden)

    Nour Hammoudeh

    2014-12-01

    Full Text Available Leishmania Homologue of receptors for Activated C Kinase (LACK antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica.The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR technique.The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed.Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.

  16. Premature aging in skeletal muscle lacking serum response factor.

    Directory of Open Access Journals (Sweden)

    Charlotte Lahoute

    Full Text Available Aging is associated with a progressive loss of muscle mass, increased adiposity and fibrosis that leads to sarcopenia. At the molecular level, muscle aging is known to alter the expression of a variety of genes but very little is known about the molecular effectors involved. SRF (Serum Response Factor is a crucial transcription factor for muscle-specific gene expression and for post-natal skeletal muscle growth. To assess its role in adult skeletal muscle physiology, we developed a post-mitotic myofiber-specific and tamoxifen-inducible SRF knockout model. Five months after SRF loss, no obvious muscle phenotype was observed suggesting that SRF is not crucial for myofiber maintenance. However, mutant mice progressively developed IIB myofiber-specific atrophy accompanied by a metabolic switch towards a more oxidative phenotype, muscular lipid accumulation, sarcomere disorganization and fibrosis. After injury, mutant muscles exhibited an altered regeneration process, showing smaller regenerated fibers and persistent fibrosis. All of these features are strongly reminiscent of abnormalities encountered in aging skeletal muscle. Interestingly, we also observed an important age associated decrease in SRF expression in mice and human muscles. Altogether, these results suggest that a naturally occurring SRF down-regulation precedes and contributes to the muscle aging process. Indeed, triggering SRF loss in the muscles of mutant mice results in an accelerated aging process.

  17. Mutants of Saccharomyces cerevisiae with defects in acetate metabolism: isolation and characterization of Acn- mutants.

    Science.gov (United States)

    McCammon, M T

    1996-09-01

    The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn- ("ACetate Nonutilizing") mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism.

  18. Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice.

    Directory of Open Access Journals (Sweden)

    Lucie Aumailley

    Full Text Available Werner syndrome (WS is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles.

  19. Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice.

    Science.gov (United States)

    Aumailley, Lucie; Garand, Chantal; Dubois, Marie Julie; Johnson, F Brad; Marette, André; Lebel, Michel

    2015-01-01

    Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice) do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles.

  20. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    Energy Technology Data Exchange (ETDEWEB)

    Cook, Melloni N. [University of Memphis; Dunning, Jonathan P [University of Memphis; Wiley, Ronald G [Vanderbilt University and Veterans Administration, Nashville, TN; Chesler, Elissa J [ORNL; Johnson, Dabney K [ORNL; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  1. Mutants of downy mildew resistance in Lactuca sativa (lettuce).

    Science.gov (United States)

    Okubara, P A; Anderson, P A; Ochoa, O E; Michelmore, R W

    1994-07-01

    As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

  2. UROKINASE MUTANT WITH BETTER FIBRIN SPECIFICITY

    Institute of Scientific and Technical Information of China (English)

    MaZhong; YuRuirong; HuaZichun; ZhuDexu

    1997-01-01

    Urokinsae (UK),one of the principal activators of plasminogen, has been widely used in the clinical therapy of thrombosis. However ,it lacks fibrin specificity ,and can lead to extensive systemic activation of plasminogen when injected into veins in large doses. On the other hand,

  3. Chlamydomonas fla mutants reveal a link between deflagellation and intraflagellar transport

    Directory of Open Access Journals (Sweden)

    Quarmby Lynne

    2003-08-01

    Full Text Available Abstract Background Cilia and flagella are often lost in anticipation of mitosis or in response to stress. There are two ways that a cell can lose its flagella: resorption or deflagellation. Deflagellation involves active severing of the axoneme at the base of the flagellum; this process is defective in Chlamydomonas fa mutants. In contrast, resorption has been thought to occur as a consequence of constitutive disassembly at the tip in the absence of continued assembly, which requires intraflagellar transport (IFT. Chlamydomonas fla mutants are unable to build and maintain flagella due to defects in IFT. Results fla10 cells, which are defective in kinesin-II, the anterograde IFT motor, resorb their flagella at the restrictive temperature (33°C, as previously reported. We find that in standard media containing ~300 microM calcium, fla10 cells lose flagella by deflagellation at 33°C. This temperature-induced deflagellation of a fla mutant is not predicted by the IFT-based model for flagellar length control. Other fla mutants behave similarly, losing their flagella by deflagellation instead of resorption, if adequate calcium is available. These data suggest a new model whereby flagellar resorption involves active disassembly at the base of the flagellum via a mechanism with components in common with the severing machinery of deflagellation. As predicted by this model, we discovered that deflagellation stimuli induce resorption if deflagellation is blocked either by mutation in a FA gene or by lack of calcium. Further support for this model comes from our discovery that fla10-fa double mutants resorb their flagella more slowly than fla10 mutants. Conclusions Deflagellation of the fla10 mutant at the restrictive temperature is indicative of an active disassembly signal, which can manifest as either resorption or deflagellation. We propose that when IFT is halted by either an inactivating mutation or a cellular signal, active flagellar disassembly

  4. Spectroscopic studies of Synechococcus sp PCC 7002 phycobilisome core mutants

    Energy Technology Data Exchange (ETDEWEB)

    Gindt, Y.M.

    1993-04-01

    The role of the L[sub cm] (I), [beta][sup 18] (II), and [alpha][sup AP-B] (III) chromoproteins in the phycobilisome (PBS) core was investigated using genetically engineered strains of Synechococcus missing different polypeptides. Intact cells, isolated PBS, and subcore preparations for each mutant were studied to determine the effect of that mutation on energy transfer within the PBS core and to the reaction centers. Three mutants lacked the II and/or III polypeptides, while the I chromophore was altered in others. A lower energy absorbing chromophore, A[sub max] = 695 nm, was substituted for the I chromophore. The deletion of the II and III subunits had no discernible effect on energy transfer from the PBS to PSII. In cells and isolated PBS, the altered I chromophore acts to quench the PBS complex and to redirect the energy which would be transferred to PSII. In the PBS and subcore preparations, deletion of the III subunit did not alter energy transfer within the core. The deletion of the II subunit from the PBS caused a small decrease in the excited state lifetimes of the final emitters indicating more disorder within the core. The I chromophore was found to absorb at 670nm and to emit at 683nm within the intact PBS. The II chromophore emits at 679nm while the III chromophore emits at 682nm. A strong interaction exists between the I chromophore and the II subunit. Upon deletion of the II subunit from the PBS core, the I chromophore emits at a higher energy. The II subunit could act to stabilize the I chromophore-binding pocket, or exciton coupling could be occurring between the two. The role of the III chromophore is still unclear at this time. The III chromophore does contribute to the RT emission of the isolated PBS, but it transfers energy to I at 77 K. One can conclude that the III subunit is adjacent to the trimer containing the I polypeptide.

  5. Spectroscopic studies of Synechococcus sp PCC 7002 phycobilisome core mutants

    Energy Technology Data Exchange (ETDEWEB)

    Gindt, Y.M.

    1993-04-01

    The role of the L{sub cm} (I), {beta}{sup 18} (II), and {alpha}{sup AP-B} (III) chromoproteins in the phycobilisome (PBS) core was investigated using genetically engineered strains of Synechococcus missing different polypeptides. Intact cells, isolated PBS, and subcore preparations for each mutant were studied to determine the effect of that mutation on energy transfer within the PBS core and to the reaction centers. Three mutants lacked the II and/or III polypeptides, while the I chromophore was altered in others. A lower energy absorbing chromophore, A{sub max} = 695 nm, was substituted for the I chromophore. The deletion of the II and III subunits had no discernible effect on energy transfer from the PBS to PSII. In cells and isolated PBS, the altered I chromophore acts to quench the PBS complex and to redirect the energy which would be transferred to PSII. In the PBS and subcore preparations, deletion of the III subunit did not alter energy transfer within the core. The deletion of the II subunit from the PBS caused a small decrease in the excited state lifetimes of the final emitters indicating more disorder within the core. The I chromophore was found to absorb at 670nm and to emit at 683nm within the intact PBS. The II chromophore emits at 679nm while the III chromophore emits at 682nm. A strong interaction exists between the I chromophore and the II subunit. Upon deletion of the II subunit from the PBS core, the I chromophore emits at a higher energy. The II subunit could act to stabilize the I chromophore-binding pocket, or exciton coupling could be occurring between the two. The role of the III chromophore is still unclear at this time. The III chromophore does contribute to the RT emission of the isolated PBS, but it transfers energy to I at 77 K. One can conclude that the III subunit is adjacent to the trimer containing the I polypeptide.

  6. Google: a narrativa de uma marca mutante

    Directory of Open Access Journals (Sweden)

    Elizete de Azevedo Kreutz

    2010-01-01

    Full Text Available As marcas mutantes já fazem parte de nossa realidade, embora ainda não totalmente percebidas e/ou aceitas como tal. O presente artigo busca refletir sobre a relevância dessas novas estratégias de comunicação e branding, identificando suas principais características. Para isso, utilizamos o método de estudo de caso, o Google, ancorado nos métodos de pesquisa bibliográfica e de internet. A escolha foi intencional, posto que a organização é referência em sua categoria, mecanismo de busca, e reflete essa estratégia comunicacional contemporânea. Como resultado, as informações obtidas nos possibilitam compreender essa tendência de comportamento de marca que busca a interação com seus públicos.

  7. Colony mutants of compatible nocardiae displaying variations in recombining capacity.

    Science.gov (United States)

    Brownell, G H; Walsh, R S

    1972-03-01

    Colonial morphology mutants of Nocardia erythropolis were isolated following ultraviolet (UV) irradiation. The alleles rou-1/smo-1 were located by recombinant analysis and found to be linked to previously mapped characters. On the basis of recombinant class type patterns obtained from various selective characters it was postulated that the rou-1 allele may span a region of unique nucleotides in the Mat-Ce genome. Recombination frequencies of rou-1 and smo-2 bearing mutants of the Mat-Ce mating type were found to differ by over 1000 fold. Attempts to demonstrate that low recombination frequencies produced by the Smo mutants were due to Rec(-) genes were unsuccessful. No increased sensitivity to either UV or X irradiation was observed by the Smo mutants. Acriflavine treatment of either Rou or Smo colony mutants failed to accelerate reversion or to alter the recombining potentials of the mutants.

  8. Screening and identification of mutants of Magnaporthe grisea by REMI

    Institute of Scientific and Technical Information of China (English)

    XIONG Ruyi; LIU Juan; ZHOU Yijun; FAN Yongjian; ZHENG Xiaobo

    2007-01-01

    The plasmid pUCATPH was used to establish a transformation system in wild-type isolate M131 of Magnaporthe grisea.Six hundred and thirty-nine transformants were obtained by restriction enzyme-mediated integration (REMI) with hygromycin B (hyg B) resistance as a tag.Morphological analysis of two of the REMI mutants confirmed that they produced little melanin under black light and continued for three generations.Pathogenicity identification of six mutants screened proved that they made pathogenicity changes on three sets of differential varieties with different resistance genes.Rep-PCR analyses showed that two morphological mutants and two pathogenicity mutants differed from wild-type isolate M131 at the molecular level.RFLP analyses were performed to study the four mutants at the molecular level and the integration sites of the plasmid DNA.The results showed that the plasmid was inserted into all four mutants and that the insertion sites were random.

  9. Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity

    Energy Technology Data Exchange (ETDEWEB)

    Upchurch, R.G.; Walker, D.C.; Rollins, J.A.; Ehrenshaft, M.; Daub, M.E. (North Carolina State Univ., Raleigh (United States))

    1991-10-01

    The authors have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

  10. Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity.

    Science.gov (United States)

    Upchurch, R G; Walker, D C; Rollins, J A; Ehrenshaft, M; Daub, M E

    1991-10-01

    We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

  11. Effects of Chlorophyll Availability on Fluorescence Components of Photosystems in the ORF469-Deletion Mutant of Cyanobacterium

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    PCR-amplified ORF469 fragment from Synechocystis sp.PCC 6803 was cloned into pUC118 and a construct was made in which part of ORF469 was deleted and replaced by erythromycin resistance cassette.Transformation of wild type strain of Synechocystis sp.PCC 6803 with this construct yielded a mutant in which ORF469 was deleted.In the resulting mutant, the light-independent pathway of chlorophyll biosynthesis was inactivated and availability of chlorophyll was fully dependent on light.When propagated the mutant in dark, the chlorophyll was non-detectable and protochlorophyllide with 645 nm fluorescence emission peak was accumulated.Meanwhile, the fluorescence emission peaks (excited at 435 nm) of thylakoids at 685 nm, 695 nm and 725 nm, which represented relative chlorophyll-binding proteins, disappeared.Upon return of dark-grown ORF469 mutant to the light, greening occurred and chlorophyll was synthesized to assembly fluorescence emission components in photosystems.Newly synthesized chlorophyll combined the fluorescence component of 685 nm at first, then 725 nm and 695 nm at last, which indicates a pecking order for biogenesis of chlorophyll-binding proteins when availability of chlorophyll is limited.The mutant lacking ORF469 in Synechocystis sp.PCC 6803 was suggested as an excellent cyanobacterial system for studies on the interactions between chlorophyll and chlorophyll-binding proteins in photosystems.

  12. Behavioral evidence for photophobia and stress-related ipsilateral head pain in transgenic Cacna1a mutant mice.

    Science.gov (United States)

    Chanda, Mona Lisa; Tuttle, Alexander H; Baran, Inna; Atlin, Cori; Guindi, Daniella; Hathaway, Georgia; Israelian, Nyrie; Levenstadt, Jeremy; Low, Daniel; Macrae, Lynn; O'Shea, Louise; Silver, Alex; Zendegui, Elaina; Mariette Lenselink, A; Spijker, Sabine; Ferrari, Michel D; van den Maagdenberg, Arn M J M; Mogil, Jeffrey S

    2013-08-01

    Migraine is a highly prevalent, disabling and complex episodic brain disorder whose pathogenesis is poorly understood, due in part to the lack of valid animal models. Here we report behavioral evidence of hallmark migraine features, photophobia and unilateral head pain, in transgenic knock-in mice bearing human familial hemiplegic migraine, type 1 (FHM-1) gain-of-function missense mutations (R192Q or S218L) in the Cacna1a gene encoding the CaV2.1 calcium channel α1 subunit. Photophobia was demonstrated using a modified elevated plus maze in which the safe closed arms were brightly illuminated; mutant mice avoided the light despite showing no differences in the standard (anxiety) version of the test. Multiple behavioral measures suggestive of spontaneous head pain were found in 192Q mutants subjected to novelty and/or restraint stress. These behaviors were: (1) more frequent in mutant versus wildtype mice; (2) lateralized in mutant but not in wildtype mice; (3) more frequent in females versus males; and (4) dose-dependently normalized by systemic administration of 2 different acute analgesics, rizatriptan and morphine. Furthermore, some of these behaviors were found to be more frequent and severe in 218L compared to 192Q mutants, consistent with the clinical presentation in humans. We suggest that Cacna1a transgenic mice can experience migraine-related head pain and can thus serve as unique tools to study the pathogenesis of migraine and test novel antimigraine agents.

  13. Graviresponsiveness and abscisic-acid content of roots of carotenoid-deficient mutants of Zea mays L

    Science.gov (United States)

    Moore, R.; Smith, J. D.

    1985-01-01

    The abscisic-acid (ABA) content of roots of the carotenoid-deficient w-3, vp-5, and vp-7 mutants of Z. mays was analyzed using gas chromatography-mass spectrometry with an analysis sensitivity of 6 ng ABA g-1 fresh weight (FW). Roots of normal seedlings of the same lines were characterized by the following amounts of ABA (as ng ABA g-1 FW, +/- standard deviation): w-3, 279 +/- 43; vp-5, 237 +/- 26; vp-7, 338 +/- 61. We did not detect any ABA in roots of any of the mutants. Thus, the lack of carotenoids in these mutants correlated positively with the apparent absence of ABA. Primary roots of normal and mutant seedlings were positively gravitropic, with no significant differences in the curvatures of roots of normal as compared with mutant seedlings. These results indicate that ABA 1) is synthesized in maize roots via the carotenoid pathway, and 2) is not necessary for positive gravitropism by primary roots of Z. mays.

  14. Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

    Directory of Open Access Journals (Sweden)

    Qi Jia

    2012-01-01

    Full Text Available In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

  15. Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant.

    Science.gov (United States)

    Rouws, Luc F M; Simões-Araújo, Jean L; Hemerly, Adriana S; Baldani, José I

    2008-04-01

    Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5Tnp Transposome (Epicentre). Up to 1 x 10(6) mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool.

  16. Mouse mutants for the nicotinic acetylcholine receptor ß2 subunit display changes in cell adhesion and neurodegeneration response genes.

    Directory of Open Access Journals (Sweden)

    Carol M Rubin

    Full Text Available Mice lacking expression of the ß2 subunit of the neuronal nicotinic acetylcholine receptor (CHRNB2 display abnormal retinal waves and a dispersed projection of retinal ganglion cell (RGC axons to their dorsal lateral geniculate nuclei (dLGNs. Transcriptomes of LGN tissue from two independently generated Chrnb2-/- mutants and from wildtype mice were obtained at postnatal day 4 (P4, during the normal period of segregation of eye-specific afferents to the LGN. Microarray analysis reveals reduced expression of genes located on the cell membrane or in extracellular space, and of genes active in cell adhesion and calcium signaling. In particular, mRNA for cadherin 1 (Cdh1, a known axon growth regulator, is reduced to nearly undetectable levels in the LGN of P4 mutant mice and Lypd2 mRNA is similarly suppressed. Similar analysis of retinal tissue shows increased expression of crumbs 1 (Crb1 and chemokine (C-C motif ligand 21 (Ccl21 mRNAs in Chrnb2-/- mutant animals. Mutations in these genes are associated with retinal neuronal degeneration. The retinas of Chrnb2-/- mutants are normal in appearance, but the increased expression of these genes may also be involved in the abnormal projection patterns of RGC to the LGN. These data may provide the tools to distinguish the interplay between neural activity and molecular expression. Finally, comparison of the transcriptomes of the two different Chrnb2-/- mutant strains reveals the effects of genetic background upon gene expression.

  17. Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/beta-catenin signaling.

    Science.gov (United States)

    Scholer-Dahirel, Alix; Schlabach, Michael R; Loo, Alice; Bagdasarian, Linda; Meyer, Ronald; Guo, Ribo; Woolfenden, Steve; Yu, Kristine K; Markovits, Judit; Killary, Karen; Sonkin, Dmitry; Yao, Yung-Mae; Warmuth, Markus; Sellers, William R; Schlegel, Robert; Stegmeier, Frank; Mosher, Rebecca E; McLaughlin, Margaret E

    2011-10-11

    Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible β-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/β-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon β-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/β-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear β-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/β-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/β-catenin pathway.

  18. [Eremothecium ashbyii mutants resistant to 2,6-diaminopurine].

    Science.gov (United States)

    Stepanov, A I; Beburov, M Iu; Zhdanov, V G

    1975-01-01

    3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.

  19. Preliminary study on a gravity-insensitive rice mutant

    Institute of Scientific and Technical Information of China (English)

    金静; 朱诚; 张红心; 孙宗修

    2004-01-01

    A gravity-insensitive mutant was isolated from rice (Oryza sativa L. Cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism.Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type

  20. Preliminary study on a gravity-insensitive rice mutant

    Institute of Scientific and Technical Information of China (English)

    金静; 朱诚; 张红心; 孙宗修

    2004-01-01

    A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

  1. Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

    Science.gov (United States)

    Goodrich-Tanrikulu, M; Stafford, A E; Lin, J T; Makapugay, M I; Fuller, G; McKeon, T A

    1994-10-01

    New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

  2. Thogoto virus lacking interferon-antagonistic protein ML is strongly attenuated in newborn Mx1-positive but not Mx1-negative mice.

    Science.gov (United States)

    Pichlmair, Andreas; Buse, Johanna; Jennings, Stephanie; Haller, Otto; Kochs, Georg; Staeheli, Peter

    2004-10-01

    The Thogoto virus ML protein suppresses interferon synthesis in infected cells. Nevertheless, a virus mutant lacking ML remained highly pathogenic in standard laboratory mice. It was strongly attenuated, however, in mice carrying the interferon-responsive Mx1 gene found in wild mice, demonstrating that enhanced interferon synthesis is protective only if appropriate antiviral effector molecules are present. Our study shows that the virulence-enhancing effects of some viral interferon antagonists may escape detection in conventional animal models.

  3. Induced mutants from dihaploid potatoes after pollen mother cell treatment.

    Science.gov (United States)

    Przewoźny, T; Schieder, O; Wenzel, G

    1980-05-01

    Microspore mother cells of dihaploid Solanum tuberosum plants were mutagenically treated during the stage of meiosis. Mutagenesis was performed either by irradiation with x- or γ-rays or by the application of nitrosomethylurethane or methylnitronitrosoguanidine. Then, by use of the anther culture technique, 913 functional plants and 442 untreated control plants were regenerated. From the exposed plants seven distinct mutants could be isolated, predominantly chlorophyll deficient lines, while from the controls no clear-cut mutants arose. One mutant turned out to be photomorphogenetic in addition to having a chlorophyll defect. In addition to the production of mutants the treatments significantly increased the frequency of multicellular structure formation from microspores.

  4. plenty, a novel hypernodulation mutant in Lotus japonicus.

    Science.gov (United States)

    Yoshida, Chie; Funayama-Noguchi, Sachiko; Kawaguchi, Masayoshi

    2010-09-01

    Nitrogen fixation in nodules that contain symbiotic rhizobial bacteria enables legumes to thrive in nitrogen-poor soils. However, this symbiosis is energy consuming. Therefore, legumes strictly control nodulation at both local and systemic levels. Mutants deficient in such controls exhibit a range of phenotypes from non-nodulation to hypernodulation. Here, we isolated a novel hypernodulation mutant from the M(2) progeny derived from Lotus japonicus MG-20 seeds mutagenized by irradiation with a carbon ion beam. We named the mutant 'plenty' because it formed more nodules than the wild-type MG-20. The nodulation zone in the plenty mutant was wider than that in the wild type, but not as enhanced as those in other previously reported hypernodulation mutants such as har1, klv or tml of L. japonicus. Unlike these hypernodulation mutants, the plenty mutant developed nodules of the same size as MG-20. Overall, the plenty mutant exhibited a unique phenotype of moderate hypernodulation. However, a biomass assay indicated that this unique pattern of hypernodulation was a hindrance to host plant growth. The plenty mutant displayed some tolerance to external nitrates and a normal triple response to ethylene. Grafting experiments demonstrated that the root of plenty was responsible for its hypernodulation phenotype. Genetic mapping indicated that the PLENTY gene was located on chromosome 2.

  5. Characterization of host-range mutants of cyanophage N-1.

    Science.gov (United States)

    Sarma, T A; Kaur, B

    1997-10-01

    Fifteen host-range (h) mutants of cyanophage N-1 were characterized with reference to their efficiency of plating, time of appearance, morphology and size of plaques on Nostoc muscorum and its three phage-resistant (Nm 1/N-1, Nm 2/N-1 and Nm 8/N-1) mutants. While phage N-1 did not adsorb to the three phage-resistant mutants, the h mutants differed one from the other in having lower or higher adsorption rate constants on N. muscorum or the phage-resistant mutants. The inability of majority of h mutants isolated on Nm 1/N-1 to grow in Nm 8/N-1 was shown to be due to a failure of adsorption. The h mutants also differed one from the other in their reversion (back mutation) frequencies. The lethal doses (LD37) required to kill 37% of free phage particles after UV-irradiation, heating and ethylenediamine tetraacetate (EDTA) treatment greatly varied. Most of the h mutants were found to be considerably more sensitive to UV and thermic inactivation than N-1 while they were resistant to EDTA. The h mutants except five of them were unable to multiply at 40 degrees C. The significance of these features is discussed.

  6. Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1 Mutant Zebrafish.

    Directory of Open Access Journals (Sweden)

    Brian P Grone

    Full Text Available Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1, are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.

  7. Deafness and permanently reduced potassium channel gene expression and function in hypothyroid Pit1dw mutants.

    Science.gov (United States)

    Mustapha, Mirna; Fang, Qing; Gong, Tzy-Wen; Dolan, David F; Raphael, Yehoash; Camper, Sally A; Duncan, R Keith

    2009-01-28

    The absence of thyroid hormone (TH) during late gestation and early infancy can cause irreparable deafness in both humans and rodents. A variety of rodent models have been used in an effort to identify the underlying molecular mechanism. Here, we characterize a mouse model of secondary hypothyroidism, pituitary transcription factor 1 (Pit1(dw)), which has profound, congenital deafness that is rescued by oral TH replacement. These mutants have tectorial membrane abnormalities, including a prominent Hensen's stripe, elevated beta-tectorin composition, and disrupted striated-sheet matrix. They lack distortion product otoacoustic emissions and cochlear microphonic responses, and exhibit reduced endocochlear potentials, suggesting defects in outer hair cell function and potassium recycling. Auditory system and hair cell physiology, histology, and anatomy studies reveal novel defects of hormone deficiency related to deafness: (1) permanently impaired expression of KCNJ10 in the stria vascularis of Pit1(dw) mice, which likely contributes to the reduced endocochlear potential, (2) significant outer hair cell loss in the mutants, which may result from cellular stress induced by the lower KCNQ4 expression and current levels in Pit1(dw) mutant outer hair cells, and (3) sensory and strial cell deterioration, which may have implications for thyroid hormone dysregulation in age-related hearing impairment. In summary, we suggest that these defects in outer hair cell and strial cell function are important contributors to the hearing impairment in Pit1(dw) mice.

  8. Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants.

    Science.gov (United States)

    Brodehl, Andreas; Hedde, Per Niklas; Dieding, Mareike; Fatima, Azra; Walhorn, Volker; Gayda, Susan; Šarić, Tomo; Klauke, Bärbel; Gummert, Jan; Anselmetti, Dario; Heilemann, Mike; Nienhaus, Gerd Ulrich; Milting, Hendrik

    2012-05-01

    Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.

  9. Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants*

    Science.gov (United States)

    Brodehl, Andreas; Hedde, Per Niklas; Dieding, Mareike; Fatima, Azra; Walhorn, Volker; Gayda, Susan; Šarić, Tomo; Klauke, Bärbel; Gummert, Jan; Anselmetti, Dario; Heilemann, Mike; Nienhaus, Gerd Ulrich; Milting, Hendrik

    2012-01-01

    Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies. PMID:22403400

  10. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

    Science.gov (United States)

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-12-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  11. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

    Directory of Open Access Journals (Sweden)

    Eliton da Silva Vasconcelos

    2013-12-01

    Full Text Available Clavulanic acid (CA is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064. The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  12. Differential binding of prohibitin-2 to estrogen receptor α and to drug-resistant ERα mutants

    Energy Technology Data Exchange (ETDEWEB)

    Chigira, Takeru, E-mail: 8120661875@mail.ecc.u-tokyo.ac.jp [Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Nagatoishi, Satoru, E-mail: nagatoishi@bioeng.t.u-tokyo.ac.jp [Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan); Tsumoto, Kouhei, E-mail: tsumoto@bioeng.t.u-tokyo.ac.jp [Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan)

    2015-08-07

    Endocrine resistance is one of the most challenging problems in estrogen receptor alpha (ERα)-positive breast cancer. The transcriptional activity of ERα is controlled by several coregulators, including prohibitin-2 (PHB2). Because of its ability to repress the transcriptional activity of activated ERα, PHB2 is a promising antiproliferative agent. In this study, were analyzed the interaction of PHB2 with ERα and three mutants (Y537S, D538G, and E380Q) that are frequently associated with a lack of sensitivity to hormonal treatments, to help advance novel drug discovery. PHB2 bound to ERα wild-type (WT), Y537S, and D538G, but did not bind to E380Q. The binding thermodynamics of Y537S and D538G to PHB2 were favorably altered entropically compared with those of WT to PHB2. Our results show that PHB2 binds to the ligand binding domain of ERα with a conformational change in the helix 12 of ERα. - Highlights: • Molten globule-likeness of an ERα repressor Prohibitin-2 (PHB2) is identified. • The thermodynamics is validated for the interaction between ERα and PHB2. • PHB2 binds to Y537S and D538G mutants of ERα commonly found in breast cancer. • ERα WT and mutants showed different thermodynamic parameters in the binding to PHB2. • ERα binds to PHB2 with conformational change involving packing of helix 12.

  13. Characterization of the growth and auxin physiology of roots of the tomato mutant, diageotropica

    Science.gov (United States)

    Muday, G. K.; Lomax, T. L.; Rayle, D. L.

    1995-01-01

    Roots of the tomato (Lycopersicon esculentum, Mill.) mutant (diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([3H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [3H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.

  14. Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Distefano, M.D.; Au, K.G.; Walsh, C.T. (Harvard Medical School, Boston, MA (USA))

    1989-02-07

    Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys{sub 135}Cys{sub 140}, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys{sub 135} (Ala{sub 135}Cys{sub 140}), Cys{sub 14} (Cys{sub 135}Ala{sub 140}), or both (Ala{sub 135}Ala{sub 140}). Additionally, they have made double mutants that lack Cys{sub 135} (Ala{sub 135}Cys{sub 139}Cys{sub 140}) or Cys{sub 140} (Cys{sub 135}Cys{sub 139}Ala{sub 140}) but introduce a new Cys in place of Gly{sub 139} with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH{sub 2} redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala{sub 135}Cys{sub 139}Cys{sub 14} mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala{sub 135}Cys{sub 140} protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys{sub 135} and Cys{sub 140} thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.

  15. Chemotyping of yeast mutants using robotics.

    Science.gov (United States)

    Rieger, K J; El-Alama, M; Stein, G; Bradshaw, C; Slonimski, P P; Maundrell, K

    1999-07-01

    By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed. Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories. Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547-1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics. The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors. Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae. In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype. The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper-pleiotropic phenotype with sensitivities to a dozen or more compounds. Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in-depth functional analysis and a more precise definition of molecular targets.

  16. [Breeding of Actinobacillus succiniogenes mutants with improved succinate production based on metabolic flux analysis].

    Science.gov (United States)

    Pan, Lijun; Li, Xingjiang; Jiang, Shaotong; Wei, Zhaojun; Chen, Xiaohui; Cai, Licheng; Wang, Hefeng; Jiang, Jijun

    2008-09-01

    It is very important to obtain high yield mutant strains on the base of metabolic flux analysis of Actinobacillus succinogenes S.JST for the industrial bioconversion of succinic acid. The metabolic pathway was analized at first and the flux of the metabolic networks was calculated by matrix. In order to decrease acetic acid flux, the strains mutated by soft X-ray of synchronous radiation were screened on the plates with high concentration of fluoroacetic acid. For decreasing the metabolic flux of ethanol the site-directed mutagenesis was carried out for the reduction of alcohol dehydrogenase(Adh) specific activity. Then the enzyme activity determination and the gene sequence analysis of the mutant strain was compared with those of the parent strain. Metabolic flux analysis of the parent strain indicated that the flux of succinic acid was 1.78(mmol/g/h) and that the flux of acetic acid and ethanol were 0.60 (mmol/g/h) and 1.04( mmol/g/h), respectively. Meanwhile the metabolic pathway analysis showed that the ethanol metabolism enhanced the lacking of H electron donor during the synthesis of succinic acid and that the succinic acid flux was weakened by the metabolism of byproducts ethanol and acetic acid. Compared with the parent strain, the acetic acid flux of anti-fluoroacetic mutant strain S.JST1 was 0.024 (mmol/g/h), decreasing by 96%. Then the enzyme determination showed that the specific activity unit of phosphotransacetylase(Pta) decreased from 602 to 74 and a mutated site was founded in the pta gene of the mutant strain S.JST1. Compared with that of the parent strain S.JST1 the ethanol flux of adh-site-directed mutant strain S.JST2 was 0.020 (mmol/g/h), decreasing by 98%. Then the enzyme determination showed that the specific activity unit of Adh decreased from 585 to 62 and the yield of end product succinic acid was 65.7 (g/L). The interdiction of Adh and Pta decreased the metabolism of byproducts and the H electron donor was well balanced, thus the succinic

  17. klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

    Directory of Open Access Journals (Sweden)

    Peter Novodvorsky

    Full Text Available The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2 transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development.Using Transcription Activator-Like Effector Nucleases (TALEN we generated a klf2a mutant (klf2ash317 with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl, a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17 in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants.The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

  18. Absence of Pneumocystis dihydropteroate synthase mutants in Brittany, France.

    Science.gov (United States)

    Le Gal, Solène; Robert-Gangneux, Florence; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Damiani, Céline; Totet, Anne; Gangneux, Jean-Pierre; Nevez, Gilles

    2013-05-01

    Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.

  19. A Mutant Hunt Using the C-Fern (Ceratopteris Richardii)

    Science.gov (United States)

    Calie, Patrick J.

    2005-01-01

    A modification of the popular C-Fern system, the tropical fern Ceratopteris richardii is developed in which students plate out a genetically mixed set of fern spores and then select for specific mutants. This exercise can provide students with an experience in plant mutant selection and can be used as a platform to expose students to a diverse…

  20. Characterization of Gibberellin Receptor Mutants of Barley (Hordeum vulgare L.)

    Institute of Scientific and Technical Information of China (English)

    Peter M.Chandler; Carol A.Harding; Anthony R.Ashton; Mark D.Mulcair; Nicholas E.Dixon; Lewis N.Mander

    2008-01-01

    The sequence of Gidl (a gene for a gibberellin (GA) receptor from rice) was used to identify a putative orthoIogue from barley.This was expressed in E.coil,and produced a protein that was able to bind GA in vitro with both structural specificity and saturability.Its potential role in GA responses was investigated using barley mutants with reduced GA sensitivity (gsel mutants).Sixteen different gsel mutants each carried a unique nucleotide substitution in this sequence.In all but one case,these changes resulted in single amino acid substitutions,and,for the remaining mutant,a substitution in the 5' untranslated region of the mRNA is proposed to interfere with translation initiation.There was perfect linkage in segregating populations between new mutant alleles and the gsel phenotype,leading to the conclusion that the putative GID1 GA receptor sequence in barley corresponds to the Gsel locus.Determination of endogenous GA contents in one of the mutants revealed enhanced accumulation of bioactive GA1,and a deficit of C20 GA precursors.All of the gsel mutants had reduced sensitivity to exogenous GA3,and to AC94377 (a GA analogue) at concentrations that are normally 'saturating',but,at much higher concentrations,there was often a considerable response.The comparison between barley and rice mutants reveals interesting differences between these two cereal species in GA hormonal physiology.

  1. New types of Escherichia coli recombination-deficient mutants.

    Science.gov (United States)

    Freifelder, D

    1976-11-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

  2. Retention of Photoinduction of Cytosolic Enzymes in aurea Mutant of Tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Goud, K. V.; Sharma, R.

    1994-06-01

    The tomato (Lycopersicon esculentum Mill.) aurea (au) mutant has been characterized as a phytochrome-deficient mutant lacking spectrally detectable phytochrome A in etiolated seedlings. Seedlings of au grown under red light (RL) lack phytochrome regulation of nuclear genes encoding plastidic proteins, possess ill-developed chloroplasts, and are slow to de-etiolate. In the present study, the effect of phytochrome deficiency on photoinduction of enzymes in etiolated au seedlings was investigated. The photoinduction of the cytosolic enzymes amylase and nitrate reductase (NR) and of the plastidic enzyme nitrite reductase (NiR) in au was compared with that in the isogenic wild-type (WT) tomato and the high-pigment (hp) mutant with exaggerated phytochrome response. In WT and hp, both brief RL pulses and continuous RL induced amylase, NR, and NiR activities, whereas in au no photoinduction of enzymes was observed with brief RL pulses, and continuous RL induced only amylase and NR activities. The time courses of photoinduction of NR and amylase in au under continuous RL followed patterns qualitatively similar to hp and WT. A blue-light pretreatment prior to continuous RL exposure was ineffective in inducing NiR activity in au. Only continuous white light could elicit a photoinduction of NiR in au seedlings. The norflurazon-triggered loss of photoinduction of NiR in WT and hp indicated that NiR photoinduction depended on chloroplast biogenesis. The results indicate that observed photoinduction of NR and amylase in au may be mediated by a residual phytochrome pool.

  3. Protective properties of rifampin-resistant rough mutants of Brucella melitensis.

    Science.gov (United States)

    Adone, R; Ciuchini, F; Marianelli, C; Tarantino, M; Pistoia, C; Marcon, G; Petrucci, P; Francia, M; Riccardi, G; Pasquali, P

    2005-07-01

    Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.

  4. Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants.

    Science.gov (United States)

    Tedde, Vittorio; Rosini, Roberto; Galeotti, Cesira L

    2016-01-01

    An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized null deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn2+ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and null mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the null adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

  5. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    Science.gov (United States)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  6. Special Relativity in Week One: 4) Lack of Simultaneity

    Science.gov (United States)

    Huggins, Elisha

    2011-01-01

    This is our final article on teaching special relativity in the first week of an introductory physics course. One of the profound changes in our view of the world was Einstein's discovery of the lack of simultaneity. He illustrated this result with a thought experiment in which we observe a railroad car passing by us. We see the two ends of the…

  7. Garlic exhibits lack of control over gastrointestinal nematodes in goats

    Science.gov (United States)

    Gastrointestinal nematodes (GIN) continue to hinder small ruminant production because of anthelmintic resistance and lack of effective products for GIN control in organic production. The objective of this study was to examine the effectiveness of a commercially available certified organic garlic pr...

  8. Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin.

    Science.gov (United States)

    Williams, Margaret M; Sen, Kathryn; Weigand, Michael R; Skoff, Tami H; Cunningham, Victoria A; Halse, Tanya A; Tondella, M Lucia

    2016-02-01

    A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin-deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.

  9. Siim Nestor soovitab : lack of Eoins / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2008-01-01

    Väikefirma Seksound annab sel nädalavahetusel välja Viljandi indiebändi Lack of Eoins esikplaadi "Echo Group" (plaadiesitlused 11. dets. Tallinnas Von Krahlis ja 12. dets. Tartus Genialistide klubis, esinevad ka Ans. Andur ja Popidiot, plaate keerutavad Hannes Praks ja Taavi Laatsit)

  10. Impaired intestinal proglucagon processing in mice lacking prohormone convertase 1

    DEFF Research Database (Denmark)

    Ugleholdt, Randi; Zhu, Xiaorong; Deacon, Carolyn F;

    2003-01-01

    The neuroendocrine prohormone convertases 1 and 2 (PC1 and PC2) are expressed in endocrine intestinal L cells and pancreatic A cells, respectively, and colocalize with proglucagon in secretory granules. Mice lacking PC2 have multiple endocrinopathies and cannot process proglucagon to mature gluca...

  11. Lack of peaceful resolution with Israel: economic cost for Palestinians

    NARCIS (Netherlands)

    P.M.C. de Boer (Paul)

    2008-01-01

    textabstractWe propose to estimate the economic cost for Palestine and for Palestinian residents due to the lack of peaceful resolution with Israel. Thereto we make use of the consensus estimates of the International Monetary Fund (IMF) and the World Bank (WB) of real growth rates of economic variab

  12. Lack of Clarity in University Teaching: A Case Study.

    Science.gov (United States)

    Hativa, Nira

    1998-01-01

    A study used qualitative methods to examine lack of clarity and need for inference in teaching of an undergraduate physics course for nonscience majors. Evidence from several data sources converge, revealing very low levels of student understanding of material presented and strong dissatisfaction with instruction, and also insights into teacher…

  13. Phenotypic Characterization of a Female Sterile Mutant in Rice

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A female sterile mutant, derived from a spontaneous mutation, wasfirst discovered in rice (Oryza sativa L. ssp.indica) restorer line 202R. With normal flowering, the mutant exhibits an extremely Iow seed-setting rate. When the mutant is crossed as a pollen donor, the seeds set normally; whereas when it is used as a pollen receiver,no seeds are obtained even with mixed pollen grains of different varieties sprinkled over the stigmas. The floret of the mutant, consisting of six stamens and one pistil, looks the same as that of the wild type in the malefemale organs, except that less than 10% of the mutant florets have three stigmas on the ovary. Although the mutant has a low seed-setting rate, Its pollen fertility is approximately 87.1%, which is equal to that of the wild type. In addition, more than 90% of the mature embryo sacs of the mutant have complete inner structures. At every stage after pollination, the sperm, embryo, and endosperm are not found in the mutant embryo sac,whereas the disintegration of the egg cell that does not accomplish fertilization is visible. Through observations with a fluorescence microscope, we have found that the pollen grains germinate normally, whereas the pollen tube abnormally elongates in the style-transmitting tissue. The mutant pollen tubes display various defects in the style, such as slower elongation, conversed elongation, distorted elongation, swollen tips, or branched tips. As a result, the growth of the pollen tubes ceases in the style, and, therefore, the pollen tubes cannot reach the embryo sac and the process of double fertilization is blocked. Based on these observations,we conclude that this mutant, designated as fs-202R, is a novel type of female sterile mutation in rice, which causes the arrest of the elongation of the pollen tube.

  14. Early Neurobehavioral Development of Mice Lacking Endogenous PACAP.

    Science.gov (United States)

    Farkas, Jozsef; Sandor, Balazs; Tamas, Andrea; Kiss, Peter; Hashimoto, Hitoshi; Nagy, Andras D; Fulop, Balazs D; Juhasz, Tamas; Manavalan, Sridharan; Reglodi, Dora

    2017-04-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide. In addition to its diverse physiological roles, PACAP has important functions in the embryonic development of various tissues, and it is also considered as a trophic factor during development and in the case of neuronal injuries. Data suggest that the development of the nervous system is severely affected by the lack of endogenous PACAP. Short-term neurofunctional outcome correlates with long-term functional deficits; however, the early neurobehavioral development of PACAP-deficient mice has not yet been evaluated. Therefore, the aim of the present study was to describe the postnatal development of physical signs and neurological reflexes in mice partially or completely lacking PACAP. We examined developmental hallmarks during the first 3 weeks of the postnatal period, during which period most neurological reflexes and motor coordination show most intensive development, and we describe the neurobehavioral development using a complex battery of tests. In the present study, we found that PACAP-deficient mice had slower weight gain throughout the observation period. Interestingly, mice partially lacking PACAP weighed significantly less than homozygous mice. There was no difference between male and female mice during the first 3 weeks. Some other signs were also more severely affected in the heterozygous mice than in the homozygous mice, such as air righting, grasp, and gait initiation reflexes. Interestingly, incisor teeth erupted earlier in mice lacking PACAP. Motor coordination, shown by the number of foot-faults on an elevated grid, was also less developed in PACAP-deficient mice. In summary, our results show that mice lacking endogenous PACAP have slower weight gain during the first weeks of development and slower neurobehavioral development regarding a few developmental hallmarks.

  15. Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station.

    Science.gov (United States)

    Scherer, G F E; Pietrzyk, P

    2014-01-01

    Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity.

  16. Cerebellar deficits and hyperactivity in mice lacking Smad4.

    Science.gov (United States)

    Zhou, Yong-Xing; Zhao, Mingrui; Li, Dan; Shimazu, Kazuhiro; Sakata, Kazuko; Deng, Chu-Xia; Lu, Bai

    2003-10-24

    Smad4 is a central mediator of TGF-beta signals, which are known to play essential roles in many biological processes. Using a Cre-loxP approach to overcome early embryonic lethality, we have studied functions of TGF-beta/Smad4 signals in the central nervous system (CNS). No obvious deficits were detected in mice carrying the targeted disruption of Smad4 in the CNS. The overall morphology of the hippocampus appeared normal. There was no change in the proliferation of neuronal precursor cells, nor in several forms of synaptic plasticity. In contrast, deletion of Smad4 resulted in a marked decrease in the number of cerebellar Purkinje cells and parvalbumin-positive interneurons. Accompanied by the abnormality in the cerebellum, mutant mice also exhibited significantly increased vertical activity. Thus, our study reveals an unexpected role for Smad4 in cerebellar development and in the control of motor function.

  17. The lack of autophagy triggers precocious activation of Notch signaling during Drosophila oogenesis

    Directory of Open Access Journals (Sweden)

    Barth Julia MI

    2012-12-01

    Full Text Available Abstract Background The proper balance of autophagy, a lysosome-mediated degradation process, is indispensable for oogenesis in Drosophila. We recently demonstrated that egg development depends on autophagy in the somatic follicle cells (FC, but not in the germline cells (GCs. However, the lack of autophagy only affects oogenesis when FCs are autophagy-deficient but GCs are wild type, indicating that a dysfunctional signaling between soma and germline may be responsible for the oogenesis defects. Thus, autophagy could play an essential role in modulating signal transduction pathways during egg development. Results Here, we provide further evidence for the necessity of autophagy during oogenesis and demonstrate that autophagy is especially required in subsets of FCs. Generation of autophagy-deficient FCs leads to a wide range of phenotypes that are similar to mutants with defects in the classical cell-cell signaling pathways in the ovary. Interestingly, we observe that loss of autophagy leads to a precocious activation of the Notch pathway in the FCs as monitored by the expression of Cut and Hindsight, two downstream effectors of Notch signaling. Conclusion Our findings point to an unexpected function for autophagy in the modulation of the Notch signaling pathway during Drosophila oogenesis and suggest a function for autophagy in proper receptor activation. Egg development is affected by an imbalance of autophagy between signal sending (germline and signal receiving cell (FC, thus the lack of autophagy in the germline is likely to decrease the amount of active ligand and accordingly compensates for increased signaling in autophagy-defective follicle cells.

  18. Identification and Targeting of Tyrosine Kinase Activity in Prostate Cancer Initiation, Progression, and Metastasis

    Science.gov (United States)

    2012-10-01

    metastatic spread via bioluminescence imaging (Figure 4B). Over time, these mice developed lung and lymph node metastasis from subcutaneous prostate tumors...subcutaneously into the left flank of SCID mice and tumor growth was monitored using bioluminescence imaging. After 31 days post injection, the...2006) Phosphorylation of the ATP -binding loop directs oncogenicity of drug-resistant BCR-ABL mutants. Proc Natl Acad Sci USA 103:19466–19471. 58

  19. A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

    Directory of Open Access Journals (Sweden)

    S.C. Silva

    2010-02-01

    Full Text Available Bovine herpesvirus 5 (BoHV-5, the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99. The recombinants are defective in glycoprotein E (BoHV-5gEΔ, thymidine kinase (BoHV-5TKΔ and both proteins (BoHV-5gEΔTKΔ. Rabbits inoculated with the parental virus (N = 8 developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi. Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEΔ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKΔ (N = 8 or BoHV-5gEΔTKΔ (N = 8 remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKΔ and BoHV-5gEΔTKΔ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

  20. Ethanol production using nuclear petite yeast mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hutter, A.; Oliver, S.G. [Department of Biomolecular Sciences, UMIST, Manchester (United Kingdom)

    1998-12-31

    Two respiratory-deficient nuclear petites, FY23{Delta}pet191 and FY23{Delta}cox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23{rho}{sup 0}. Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant. K{sub i}, of 2.3% (w/v) and a specific rate of ethanol production, q{sub p}, of 0.90 g ethanol g dry cells{sup -1} h{sup -1}. FY23{rho}{sup 0} was the most sensitive to ethanol, exhibiting a K{sub i} of 1.71% (w/v) and q{sub p} of 0.87 g ethanol g dry cells{sup -1} h{sup -1}. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23{Delta}pet191, having a K{sub i} of 2.14% (w/v) and the 85% respiratory-deficient FY23{Delta}cox5a, having a K{sub i} of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23{rho}{sup 0} is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject of the Pasteur effect and so exhibit higher rates of fermentation. (orig.)

  1. Mapping pathological phenotypes in Reelin mutant mice

    Directory of Open Access Journals (Sweden)

    Caterina eMichetti

    2014-09-01

    Full Text Available Autism Spectrum Disorders (ASD are neurodevelopmental disorders with multifactorial origin characterized by social communication and behavioural perseveration deficits. Several studies showed an association between the reelin gene mutation and increased risk of ASD and a reduced reelin expression in some brain regions of ASD subjects, suggesting a role for reelin deficiency in ASD etiology. Reelin is a large extracellular matrix glycoprotein playing important roles during development of the central nervous system. To deeply investigate the role of reelin dysfunction as vulnerability factor in ASD, we investigated the behavioural, neurochemical and brain morphological features of reeler male mice. We recently reported a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reeler pups. We now report that adult male heterozygous reeler mice did not show social behaviour and communication deficits during male-female social interactions. Wildtype and heterozygous mice also showed a typical light/dark locomotor activity profile, with a peak during the central interval of the dark phase. However, when faced with a mild stressful stimulus (a saline injection only heterozygous mice showed an over response to stress. At the end of the behavioural studies, we conducted high performance liquid chromatography and magnetic resonance imaging and spectroscopy to investigate whether reelin mutation influences brain monoamine and metabolites levels in regions involved in ASD. Low levels of dopamine in cortex and high levels of glutamate and taurine in hippocampus were detected in heterozygous mice, in line with clinical data collected on ASD children. Altogether, our data detected subtle but relevant neurochemical abnormalities in reeler mice supporting this mutant line, particularly male subjects, as a valid experimental model to estimate the contribution played by reelin deficiency in the global ASD

  2. Brn3c null mutant mice show long-term, incomplete retention of some afferent inner ear innervation

    Directory of Open Access Journals (Sweden)

    Pirvola Ulla

    2003-01-01

    Full Text Available Abstract Background Ears of Brn3c null mutants develop immature hair cells, identifiable only by certain molecular markers, and undergo apoptosis in neonates. This partial development of hair cells could lead to enough neurotrophin expression to sustain sensory neurons through embryonic development. We have therefore investigated in these mutants the patterns of innervation and of expression of known neurotrophins. Results At birth there is a limited expression of BDNF and NT-3 in the mutant sensory epithelia and DiI tracing shows no specific reduction of afferents or efferents that resembles neurotrophin null mutations. At postnatal day 7/8 (P7/8, innervation is severely reduced both qualitatively and quantitatively. 1% of myosin VIIa-positive immature hair cells are present in the mutant cochlea, concentrated in the base. Around 20% of immature hair cells exist in the mutant vestibular sensory epithelia. Despite more severe loss of hair cells (1% compared to 20%, the cochlea retains many more sensory neurons (46% compared to 15% than vestibular epithelia. Even 6 months old mutant mice have some fibers to all vestibular sensory epithelia and many more to the cochlear apex which lacks MyoVIIa positive hair cells. Topologically organized central cochlea projections exist at least until P8, suggesting that functional hair cells are not required to establish such projections. Conclusion The limited expression of neurotrophins in the cochlea of Brn3c null mice suffices to support many sensory neurons, particularly in the cochlea, until birth. The molecular nature of the long term survival of apical spiral neurons remains unclear.

  3. Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene

    Science.gov (United States)

    Czerwinska, Areta M.; Grabowska, Iwona; Archacka, Karolina; Bem, Joanna; Swierczek, Barbara; Helinska, Anita; Streminska, Wladyslawa; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Koblowska, Marta

    2016-01-01

    The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs. PMID:26649785

  4. Effect of different immunosuppressive drugs on calcineurin and its mutants

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis.Their phosphatase activity and the corresponding solution conformation were examined.Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immunosuppressive drugs with CN.The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN.Furthermore,circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein.Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN,and had no effects on the phosphatase activity of mutants in Loop7 region,which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN.Examination of the activities of these mutants resulted in the presence of immunosuppressive component from traditional Chinese drugs.The component of Chinese drug,ZIP1,could directly inhibit both CN and CN mutants without drug binding protein.These results suggest that the Loop7 region is an important structural area involved in the inhibition by CyP-CsA.It is valuable to further study the inhibition by ZIP1.

  5. Methods of producing protoporphyrin IX and bacterial mutants therefor

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  6. Epigenetic Suppression of T-DNA Insertion Mutants in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Yangbin Gao; Yunde Zhao

    2013-01-01

    T-DNA insertion mutants have been widely used to define gene functions in Arabidopsis and in other plants.Here,we report an unexpected phenomenon of epigenetic suppression of T-DNA insertion mutants in Arabidopsis.When the two T-DNA insertion mutants,yucl-1 and ag-TD,were crossed together,the defects in all of the ag-TD plants in the F2 population were partially suppressed regardless of the presence of yucl-1.Conversion of ag-TD to the suppressed ag-TD (named as ag-TD*) did not follow the laws of Mendelian genetics.The ag-TD* could be stably transmitted for many generations without reverting to ag-TD,and ag-TD* had the capacity to convert ag-TD to ag-TD*.We show that epigenetic suppression of T-DNA mutants is not a rare event,but certain structural features in the T-DNA mutants are needed in order for the suppression to take place.The suppressed T-DNA mutants we observed were all intronic T-DNA mutants and the T-DNA fragments in both the trigger T-DNA as well as in the suppressed T-DNA shared stretches of identical sequences.We demonstrate that the suppression of intronic T-DNA mutants is mediated by trans-interactions between two ToDNA insertions.This work shows that caution is needed when intronic T-DNA mutants are used.

  7. Unresolved questions from the analysis of mice lacking MCU expression.

    Science.gov (United States)

    Murphy, Elizabeth; Pan, Xin; Nguyen, Tiffany; Liu, Jie; Holmström, Kira M; Finkel, Toren

    2014-07-11

    Entry of mitochondrial calcium is believed to play an essential role in regulating bioenergetics and initiating cell death pathways. We have recently described a mouse model lacking MCU expression. Surprisingly, these mice are viable and the cells and tissues from these animals do not exhibit any marked protection from cell death. Here, we discuss our findings as well as potential explanations for some of the more unexpected results.

  8. LACK OF VITAMIN B12 AFTER ROUX Y GASTRIC BYPASS

    OpenAIRE

    Reginaldo, Greise Janaina; Silva, Alice Freitas da

    2014-01-01

    Obesity is a public health issue, with several factors as its cause. It may be related to several co-morbidities such as diabetes mellitus II, systemic arterial pressure, dyslipidemia, cancer and others. Current research verified aspects of bariatric surgery with special focus on alterations caused by the lack of vitamin B12. The bibliographical review evaluated scientific papers, particularly recent ones, to obtain data on the theme. Obesity is defined as a disease characterized by the accum...

  9. Importance Of Business Planning In An Environment Lacking Economic Predictability

    OpenAIRE

    Ciumara Tudor

    2010-01-01

    The importance of business planning in an environment that lacks economic predictability is enhanced because it increases the ability of firms to be proactive. It is however necessary to accept the fundamental assumption that planning activity is conducted in a turbulent environment, where unpredictable events occur frequently. Planning is possible even when changes are common and difficult to predict, by adopting a flexible model to address all significant aspects.

  10. REPROBATION AND LACK OF INTEREST IN MECHATRONICS ENGINEERING STUDENTS

    Directory of Open Access Journals (Sweden)

    César Humberto Guzmán Valdivia

    2013-07-01

    Full Text Available Engineering education in mechatronics is an attractive field of research because it is a new multidisciplinary career. However, a potential problem is the reprobation rate. In the period from January to April 2012 at the Universidad Politécnica de Zacatecas a 53% regular students of a total of 197 were registered. To find the causes of this problem, a survey was conducted to determine the causes of reprobation, lack of motivation and interest to a population of 96 students, of which 40 were the first training cycle, 32 the second and 24 the third. The surveys yielded three main results. The first indicates that the lack of interest is proportional to the time spent in college. The second shows that the reprobation rate is linked to the laziness and the excess of courses. And the last shows a lack of motivation and low expectations of student due to the monotony of the theoretical courses. In conclusion, more research is needed to have a motivated student in an engineering career in mechatronics.

  11. The subjetivacion of the lack: between Lacan and Hegel

    Directory of Open Access Journals (Sweden)

    Lorena Souyris Oportot

    2014-05-01

    Full Text Available The present article develops a reflection concerning the figure of the subjectivation and the statute of the lack  in relation to Jacques Lacan y Hegel's thought . The analysis will be addressed from a philosophical approach as and with a psychoanalytic perspective, to show the need to understand the subjectivity, not already as a "work" of duel, but ligature to the loss and the split. The idea is that the above mentioned significances make possible deconstruir and to rethink the duel in lack, that he structures to the subject in an experience "escripturaire" (escriptural and, for the same thing, of dispossession. So that the figure of the subjetivación "in" lack  will allow to grant an important place to the non-place while I spread where the unthinkable thing and the "Autre" registers.  Once exposed this, the reflection will focus on the tragic exigences behind experience “escripturaire” expressed in the image of Antigone

  12. Sensorimotor learning in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Lalonde, R; Strazielle, C

    2011-04-15

    Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex and neocortex were compared with non-ataxic controls on two tests of motor coordination: rotorod and grid climbing. Even at the minimal speed of 4 rpm and unlike controls, none of the Dab1(scm) mutants reached criterion on the constant speed rotorod. In contrast, Dab1(scm) mutants improved their performances on the vertical grid over the course of the same number of trials. Thus, despite massive cerebellar degeneration, sensorimotor learning for equilibrium is still possible, indicating the potential usefulness of the grid-climbing test in determining residual functions in mice with massive cerebellar damage.

  13. DCP-LA neutralizes mutant amyloid beta peptide-induced impairment of long-term potentiation and spatial learning.

    Science.gov (United States)

    Nagata, Tetsu; Tomiyama, Takami; Tominaga, Takemi; Mori, Hiroshi; Yaguchi, Takahiro; Nishizaki, Tomoyuki

    2010-01-01

    Long-term potentiation (LTP) was monitored from the CA1 region of the intact rat hippocampus by delivering high frequency stimulation (HFS) to the Schaffer collateral commissural pathway. Intraventricular injection with mutant amyloid beta(1-42) peptide lacking glutamate-22 (Abeta(1-42)E22Delta), favoring oligomerization, 10 min prior to HFS, inhibited expression of LTP, with the potency more than wild-type amyloid beta(1-42) peptide. Intraperitoneal injection with the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) 70 min prior to HFS neutralized mutant Abeta(1-42)E22Delta peptide-induced LTP inhibition. In the water maze test, continuous intraventricular injection with mutant Abeta(1-42)E22Delta peptide for 14 days prolonged the acquisition latency as compared with that for control, with the potency similar to wild-type Abeta(1-42) peptide, and intraperitoneal injection with DCP-LA shortened the prolonged latency to control levels. The results of the present study indicate that DCP-LA neutralizes mutant Abeta(1-42)E22Delta peptide-induced impairment of LTP and spatial learning.

  14. Phenotype, virulence and immunogenicity of Edwardsiella ictaluri cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants in catfish host.

    Science.gov (United States)

    Santander, Javier; Mitra, Arindam; Curtiss, Roy

    2011-12-01

    Edwardsiella ictaluri is an Enterobacteriaceae that causes lethal enteric septicemia in catfish. Being a mucosal facultative intracellular pathogen, this bacterium is an excellent candidate to develop immersion-oral live attenuated vaccines for the catfish aquaculture industry. Deletion of the cyclic 3',5'-adenosine monophosphate (cAMP) receptor protein (crp) gene in several Enterobacteriaceae has been utilized in live attenuated vaccines for mammals and birds. Here we characterize the crp gene and report the effect of a crp deletion in E. ictaluri. The E. ictaluri crp gene and encoded protein are similar to other Enterobacteriaceae family members, complementing Salmonella enterica Δcrp mutants in a cAMP-dependent fashion. The E. ictaluri Δcrp-10 in-frame deletion mutant demonstrated growth defects, loss of maltose utilization, and lack of flagella synthesis. We found that the E. ictaluri Δcrp-10 mutant was attenuated, colonized lymphoid tissues, and conferred immune protection against E. ictaluri infection to zebrafish (Danio rerio) and catfish (Ictalurus punctatus). Evaluation of the IgM titers indicated that bath immunization with the E. ictaluri Δcrp-10 mutant triggered systemic and skin immune responses in catfish. We propose that deletion of the crp gene in E. ictaluri is an effective strategy to develop immersion live attenuated antibiotic-sensitive vaccines for the catfish aquaculture industry.

  15. Hydrogen production by draTGB hupL double mutant of Rhodospirillum rubrum under different light conditions

    Institute of Scientific and Technical Information of China (English)

    ZHU Ruiyan; WANG Di; ZHANG Yaoping; LI Jilun

    2006-01-01

    To increase H2 yield of Rhodospirillum rubrum in two-stage hydrogen production process,two deletion mutants were constructed. One is single mutant designated R. rubrum UR801 that deleted hupL gene encoding the large subunit of uptake hydrogenase, and the other is a double mutant designated R. rubrum UR805 lacked both draTGB encoding regulators for the activity of nitrogenase and hupL.Comparing H2 yields of two mutants with R. rubrum UR2 (wild type) and UR472 (△draTGB) under different light conditions, the results showed that the H2 yield of R. rubrum UR801 under continuous light is the highest (5700 mL of H2 per liter culture), and it is 1.56, 2.24 and 2.32-fold that of R. rubrum UR2,UR472 and UR805, respectively. However, the total H2 yield of R. rubrum UR805 in two-stage hydrogen production process is the highest (4303 mL/L), and it is 1.35, 1.21 and 1.04-fold that of R. rubrum UR2,UR801 and UR472, respectively. Thus, R. rubrum UR805 might be a valuable strain to produce a large amount of hydrogen in two-stage hydrogen production process.

  16. Mutant γPKC that causes spinocerebellar ataxia type 14 upregulates Hsp70, which protects cells from the mutant's cytotoxicity.

    Science.gov (United States)

    Ogawa, Kota; Seki, Takahiro; Onji, Tomoya; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2013-10-11

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is misfolded, susceptible to aggregation and cytotoxic. Molecular chaperones assist the refolding and degradation of misfolded proteins and prevention of the proteins' aggregation. In the present study, we found that the expression of mutant γPKC-GFP increased the levels of heat-shock protein 70 (Hsp70) in SH-SY5Y cells. To elucidate the role of this elevation, we investigated the effect of siRNA-mediated knockdown of Hsp70 on the aggregation and cytotoxicity of mutant γPKC. Knockdown of Hsp70 exacerbated the aggregation and cytotoxicity of mutant γPKC-GFP by inhibiting this mutant's degradation. These findings suggest that mutant γPKC increases the level of Hsp70, which protects cells from the mutant's cytotoxicity by enhancing its degradation.

  17. Blue light is required for survival of the tomato phytochrome-deficient aurea mutant and the expression of four nuclear genes coding for plastidic proteins.

    Science.gov (United States)

    Oelmüller, R; Kendrick, R E

    1991-02-01

    When dark-grown aurea mutant tomato seedlings which lack more than 95% of the phytochrome present in isogenic wild-type seedlings are kept in white or blue light, four nuclear-encoded transcripts coding for plastidic proteins (the light-harvesting chlorophyll a/b-binding protein of photosystem I and II [cab-PSII], plastocyanin and subunit 2 of photosystem I) are present in comparable amounts. These transcript levels in red light are strongly reduced in aurea seedlings when compared with those of wild type. Thus, blue light is required for normal expression of these genes in the mutant, while red light alone is not sufficient. Red light-grown aurea seedlings are very sensitive to blue light, even 10 minutes of blue light every day suffices to cause a measurable increase in cab-PSII transcript level. The action of blue light on the expression of cab-PSII in the mutant is under phytochrome control. After 8 days of blue light, phytochrome is almost as effective in inducing cab-PSII mRNA as in the isogenic wild type, whereas after 8 days of red light, only a small phytochrome response was observed in the mutant. It is concluded that blue light sensitizes the mutant to the residual phytochrome which allows normal gene expression and survival of the mutant under daylight conditions.

  18. Infection and transmission of Rift Valley fever viruses lacking the NSs and/or NSm genes in mosquitoes: potential role for NSm in mosquito infection.

    Directory of Open Access Journals (Sweden)

    Mary B Crabtree

    Full Text Available BACKGROUND: Rift Valley fever virus is an arthropod-borne human and animal pathogen responsible for large outbreaks of acute and febrile illness throughout Africa and the Arabian Peninsula. Reverse genetics technology has been used to develop deletion mutants of the virus that lack the NSs and/or NSm virulence genes and have been shown to be stable, immunogenic and protective against Rift Valley fever virus infection in animals. We assessed the potential for these deletion mutant viruses to infect and be transmitted by Aedes mosquitoes, which are the principal vectors for maintenance of the virus in nature and emergence of virus initiating disease outbreaks, and by Culex mosquitoes which are important amplification vectors. METHODOLOGY AND PRINCIPAL FINDINGS: Aedes aegypti and Culex quinquefasciatus mosquitoes were fed bloodmeals containing the deletion mutant viruses. Two weeks post-exposure mosquitoes were assayed for infection, dissemination, and transmission. In Ae. aegypti, infection and transmission rates of the NSs deletion virus were similar to wild type virus while dissemination rates were significantly reduced. Infection and dissemination rates for the NSm deletion virus were lower compared to wild type. Virus lacking both NSs and NSm failed to infect Ae. aegypti. In Cx. quinquefasciatus, infection rates for viruses lacking NSm or both NSs and NSm were lower than for wild type virus. CONCLUSIONS/SIGNIFICANCE: In both species, deletion of NSm or both NSs and NSm reduced the infection and transmission potential of the virus. Deletion of both NSs and NSm resulted in the highest level of attenuation of virus replication. Deletion of NSm alone was sufficient to nearly abolish infection in Aedes aegypti mosquitoes, indicating an important role for this protein. The double deleted viruses represent an ideal vaccine profile in terms of environmental containment due to lack of ability to efficiently infect and be transmitted by mosquitoes.

  19. Azotobacter vinelandii lacking the Na(+)-NQR activity: a potential source for producing alginates with improved properties and at high yield.

    Science.gov (United States)

    Gaytán, Itzel; Peña, Carlos; Núñez, Cinthia; Córdova, María S; Espín, Guadalupe; Galindo, Enrique

    2012-08-01

    The mutant ATCN4 strain of Azotobacter vinelandii, which lacks the Na(+)-NQR activity and results in an alginate overproduction (highly mucoid phenotype), was cultured in shake flasks in minimal and rich medium, and the chemical composition and rheological properties of the alginate were determined. Mutant ATCN4 exhibited a high efficiency for sucrose conversion to alginate and PHB accumulation, reaching yields that were 3.6- and 1.6-fold higher than those obtained from the wildtype cultures in minimal medium (Burk's sucrose, BS). The alginate produced by ATCN4 in the minimal medium had a high degree of acetylation (≥4 %) and a low G/M ratio (=2) with respect to the polymer synthesised in the rich medium (BS with yeast extract) (degree of acetylation = 0 % and G/M ratio of 4.5). The alginate produced in the minimal medium exhibited a pronounced pseudoplastic behaviour and a higher G* module in comparison to that observed in the alginate obtained in the cultures using a rich medium. The ATCN4 mutant culture in the minimal medium promoted the synthesis of a polymer of improved rheological quality in terms of its mechanical properties. These characteristics make this mutant a valuable source for producing alginates with improved or special properties.

  20. Characterization of Glutamine-Requiring Mutants of Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Janssen, Dick B.; Joosten, Han M.L.J.; Herst, Patricia M.; Drift, Chris van der

    1982-01-01

    Revertants were isolated from a glutamine-requiring mutant of Pseudomonas aeruginosa PAO. One strain showed thermosensitive glutamine requirement and formed thermolabile glutamine synthetase, suggesting the presence of a mutation in the structural gene for glutamine synthetase. The mutation conferri

  1. Status and Perspectives on the Researches of Rice Glutelin Mutants

    Institute of Scientific and Technical Information of China (English)

    ZHENG Tian-qing; SHEN Wen-biao; ZHU Su-song; ZHAI Hu-qu; WAN Jian-min

    2003-01-01

    Rice (Oryza sativa L. ) is one of the model plants for genomics research. As the raising offunctional rice breeding for special usage, glutelin mutants play a more and more important role in the func-tional rice breeding as well as eukaryotic gene expression and regulation research materials. For example, therice cultivar special for the patients suffering from kidney disease and diabetes could be developed from the riceglutelin mutants. In this paper, current researches on characterization, mutation mechanism and breeding us-age of various rice glutelin mutants, especially the low glutelin content cultivars, were all discussed with per-spectives on the trends of the glutelin mutant researches in the era of post-genomics.

  2. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib....... Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate...

  3. Selection of mutants of capsicum annuum induced by gamma ray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Y. I.; Lee, Y. B. [Korea Atomic Energy Research Institute, Taejeon (Korea, Republic of); Lee, E. K. [Chungnam National Univ., Taejeon (Korea, Republic of)

    1998-06-01

    For induction and selection of mutations of Capsicum annuum L., dry seeds of pure lines No.1 and No.2 were irradiated with gamma ray of 150Gy, 200Gy and 250Gy. Various mutants were selected such as showing early maturity, short plant height, long fruit and chlorophyll mutations. Mutation frequency of No.1 line was 3.4% in the dose of 150Gy, while the frequency of No.2 line was 2.7% in the dose of 250Gy. For selection of resistant mutant to amino acid analog, the optimum concentration of 5-methyltryptophan (5-MT) and S-(2-aminoethyl)-L-cysteine were 25 ppm and 30 ppm, respectively. Four resistant mutant lines to 5-MT were selected among 400 mutant lines.

  4. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.;

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosine......-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5...... phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5...

  5. Resistant mechanism study of benzalkonium chloride selected Salmonella Typhimurium mutants.

    Science.gov (United States)

    Guo, Wei; Cui, Shenghui; Xu, Xiao; Wang, Haoyan

    2014-02-01

    Benzalkonium chloride is one of the invaluable biocides that is extensively used in healthcare settings as well as in the food processing industry. After exposing wild-type Salmonella Typhimurium 14028s or its AcrAB inactivation mutant to gradually increasing levels of benzalkonium chloride, resistance mutants S-41, S-150, S-AB-23, S-AB-38, and S-AB-73 were selected and these mutants also showed a 2-64-fold stable minimum inhibitory concentration (MIC) increase to chloramphenicol, ciprofloxacin, nalidixic acid, and tetracycline. In S-41 and S-150, the expression of acrB was increased 2.7- and 7.6-fold, and ΔtolC or ΔacrAB mutants of S-41 and S-150 showed the same MICs to all tested antimicrobials as the equivalent Salmonella Typhimurium 14028s mutants. However, in S-AB-23, S-AB-38, and S-AB-73, the expression of acrF was increased 96-, 230-, and 267-fold, respectively, and ΔtolC or ΔacrEF mutants of S-AB-23, S-AB-38, and S-AB-73 showed the similar MICs to all tested antimicrobials as the ΔtolC mutant of Salmonella Typhimurium 14028s. Our data showed that constitutively over-expressed AcrAB working through TolC was the main resistance mechanism in ST14028s benzalkonium chloride resistance mutants. However, after AcrAB had been inactivated, benzalkonium chloride-resistant mutants could still be selected and constitutively over-expressed, AcrEF became the dominant efflux pump working through TolC and being responsible for the increasing antimicrobial resistance. These data indicated that different mechanisms existed for acrB and acrF constitutive over-expression. Since exposure to benzalkonium chloride may lead to Salmonella mutants with a decreased susceptibility to quinolones, which is currently one of the drugs of choice for the treatment of life-threatening salmonelosis, research into the pathogenesis and epidemiology of the benzalkonium chloride resistance mutants will be of increasing importance.

  6. Effects of salt stress on wild type and vte4 mutant Arabidopsis thaliana: Model plant to engineer tolerance towards salinity

    Directory of Open Access Journals (Sweden)

    Khalatbari Amir Ali

    2013-01-01

    Full Text Available One of the major environmental constraints impairing plant distribution and yield is believed to be salt stress. Additionally, engineered abiotic stress resistance or/and tolerance is considered as an indispensable target in order to enhance plant productivity. In this study, the effects of salinity on physiological and morphological of wild type (Columbia-0 and vte4 mutant Arabidopsis thaliana were investigated under different NaCl concentrations. These salt treatments, including control condition, 50mM and 100mM NaCl were imposed on the plants. Each salt treatment was replicated three times in a complete randomized design with factorial arrangement. Wild type and mutant A.thaliana plants were subjected to the abiotic stress (salinity for up to 11 days to evaluate the parameters of growth, development and water relations. As a result, the performance of wild type plants was stronger than vte4 mutant under different salt treatments. Under control condition, rosette dry weight, maximum quantum efficiency (PSII and specific leaf area obtained the highest values of 13.85 mg, considered, wild type A.thaliana recorded higher value of 0.82 gW/gFW for relative water content (RWC under 50mM NaCl whereas mutant plants gained the value of 0.78 gW/gFW under the same condition. However, root mass fraction indicated an increase for both wild type and vte4 mutant plants after 11 days of salt stress onset. The reduction of water potential was observed for wild type and mutant A.thaliana where it scored -1.3 MPa and -1.4, respectively. As a conclusion, these findings implied that under different salt treatments morphological and physiological responses of wild type and vte4 mutant were affected in which wild type plants showed more tolerance. Lack of γ-tocopherol methyltransferase (γ -TMT gene in vte4 seemed to impair defence mechanism of this mutant against salinity.

  7. A relaxed mutant with an altered ribosomal protein L11.

    Science.gov (United States)

    Parker, J; Watson, R J; Friesen, J D

    1976-02-27

    Relaxed mutants of Escherichia coli have been isolated which have an altered electrophoretic mobility of ribosomal protein L11. It can be shown that reversion to stringency in one of these mutants occurs simultaneously with a reversion of L11 protein to tis normal mobility. The L11 structural gene, rplK, maping near rif, is carried by the bacteriophage lambdacI857S7drifd18, and is most likely identical with relC.

  8. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    Science.gov (United States)

    2014-08-01

    regulation of synaptic and neuronal functions.17 A point mutation in DHHC21 was identified in the depilated (dep) mouse mutant, resulting in hair follicle ...and hair follicle differentiation. PLoS Genet. 5, e1000748. (19) Mansilla, F., Birkenkamp-Demtroder, K., Kruhoffer, M., Sorensen, F. B., Andersen, C...AWARD NUMBER: W81XWH-13-1-0203 TITLE: Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma PRINCIPAL INVESTIGATOR: Xu Wu

  9. LACK OF NOTIFICATION OF COMPULSORY NOTIFICATION DISEASES IN HOSPITAL SETTINGS

    Directory of Open Access Journals (Sweden)

    Rubens Griep

    2004-04-01

    Full Text Available This research encompasses the diseases of compulsory lack of notification inhospital settings and its interface with the Health Information System (Sistema de Informaçãode Saúde – SIS developed and implemented by the Welfare System in Brazil (Sistema Únicode Saúde – SUS. It tries to identify the elements epidemiology is based on, referring to itsaspects as basis for the development of epidemiologic monitoring actions of contagiousdiseases in the country. It focuses on the following question: what are the factors thatcontribute and/or determine the flaws in the process of notification for compulsory notificationdiseases? The results obtained through a questionnaire presented to the personnel responsiblefor the Hospital Infection Control Service (Serviço de Controle de Infecção Hospitalar showtheir lack of preparedness and technical knowledge, as well as their team’s, in relation to thedynamic functioning of the Epidemiologic Vigilance Service (Serviço de VigilânciaEpidemiológica. It points to the flaws due to the influence exerted by the public and/or privatecharacter of the institutions and considers the possibility of lack of commitment and responsibility of the multi-professional team in the maintenance of the preestablished flow. As aproposal, we present an adoption of continuous educational actions through the implementationof a Long Distance Post Graduation course, aiming for the development of new possibilities forthe teaching-learning process, characterized by the ongoing quest for new knowledge and focuson the student. The implementation of a local and municipal Permanent Habilitation Programmay complement the need for updating, as well as make the discussion of the cases and dataof the reality possible, thus aiming to adopt joined measures in order to cope with the presentedepidemiologic situations.

  10. Lack of Syneresis during Gelation of Dense Colloidal Suspensions.

    Science.gov (United States)

    Helbig; Hütter; Schönholzer

    2000-02-01

    This study reports experimental results about the shrinkage of particle networks produced by pH-induced destabilization of dense colloidal suspensions. The resulting solid networks exhibit no syneresis effects, at least prior to aging of the gel. From this lack of syneresis it is concluded that the solidification in wet particle systems either is not purely determined by energy (but is also influenced by entropic effects) or cannot be explained within the framework of (static) equilibrium thermodynamics at all. Copyright 2000 Academic Press.

  11. Increased sensitivity to kindling in mice lacking TSP1.

    Science.gov (United States)

    Mendus, D; Rankin-Gee, E K; Mustapha, M; Porter, B E

    2015-10-01

    The development of a hyperexcitable neuronal network is thought to be a critical event in epilepsy. Thrombospondins (TSPs) regulate synaptogenesis by binding the neuronal α2δ subunit of the voltage-gated calcium channel. TSPs regulate synapse formation during development and in the mature brain following injury. It is unclear if TSPs are involved in hyperexcitability that contributes to the development of epilepsy. Here we explore the development of epilepsy using a pentylenetetrazole (PTZ) kindling model in mice lacking TSP1 and TSP2. Unexpectedly, we found increased sensitivity to PTZ kindling in mice lacking TSP1, while mice lacking TSP2 kindled similar to wild-type. We found that the increased seizure susceptibility in the TSP1 knockout (KO) mice was not due to a compensatory increase in TSP2 mRNA as TSP1/2 KO mice were sensitive to PTZ, similar to the TSP1 KO mice. Furthermore, there were similar levels of TGF-B signal activation during kindling in the TSP1 KO mice compared to wild-type. We observed decreased expression of voltage-dependent calcium channel subunit CACNA2D1 mRNA in TSP1, TSP2, and TSP1/2 KO mice. Decreased CACNA2D2 mRNA was only detected in mice that lacked TSP1 and α2δ-1/2 protein levels in the cortex were lower in the TSP 1/2 KO mice. CACNA2D2 knockout mice have spontaneous seizures and increased PTZ seizure susceptibility. Here we report similar findings, TSP1, and TSP1/2 KO mice have low levels of CACNA2D2 mRNA expression and α2δ-1/2 receptor level in the cortex, and are more susceptible to seizures. CACNA2D2 mutations in mice and humans can cause epilepsy. Our data suggest TSP1 in particular may control CACNA2D2 levels and could be a modifier of seizure susceptibility.

  12. Molecular analysis of mutants of the Neurospora adenylosuccinate synthetase locus

    Indian Academy of Sciences (India)

    A. Wiest; A. J. McCarthy; R. Schnittker; K. McCluskey

    2012-08-01

    The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5′ end of the gene and no mutants were identified in the 3′ untranslated region of this gene. We additionally analysed ad-8+ alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.

  13. Isolation of New Gravitropic Mutants under Hypergravity Conditions

    Science.gov (United States)

    Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T.

    2016-01-01

    Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis.

  14. Differential contribution of the proline and glutamine pathways to glutamate biosynthesis and nitrogen assimilation in yeast lacking glutamate dehydrogenase.

    Science.gov (United States)

    Sieg, Alex G; Trotter, Pamela J

    2014-01-01

    In Saccharomyces cerevisiae, the glutamate dehydrogenase (GDH) enzymes play a pivotal role in glutamate biosynthesis and nitrogen assimilation. It has been proposed that, in GDH-deficient yeast, either the proline utilization (PUT) or the glutamine synthetase-glutamate synthase (GS/GOGAT) pathway serves as the alternative pathway for glutamate production and nitrogen assimilation to the exclusion of the other. Using a gdh-null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway-specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). The GDH-null mutant was viable on all nitrogen sources tested, confirming that alternate pathways for nitrogen assimilation exist in the gdh-null strain. Enzyme assays point to GS/GOGAT as the primary alternative pathway on the preferred nitrogen sources ammonia and glutamine, whereas growth on proline required both the PUT and GS/GOGAT pathways. In contrast, growth on glucose-urea media elicited a decrease in GOGAT activity along with an increase in activity of the PUT pathway specific enzyme Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH). Together, these results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH-dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serves as the sole compensatory pathway.

  15. Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism

    Science.gov (United States)

    Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

    2017-01-01

    Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis. PMID:28241048

  16. Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

    Science.gov (United States)

    Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

    2013-10-01

    Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters.

  17. Expansion of the lateral ventricles and ependymal deficits underlie the hydrocephalus evident in mice lacking the transcription factor NFIX.

    Science.gov (United States)

    Vidovic, Diana; Harris, Lachlan; Harvey, Tracey J; Evelyn Heng, Yee Hsieh; Smith, Aaron G; Osinski, Jason; Hughes, James; Thomas, Paul; Gronostajski, Richard M; Bailey, Timothy L; Piper, Michael

    2015-08-01

    Nuclear factor one X (NFIX) has been shown to play a pivotal role during the development of many regions of the brain, including the neocortex, the hippocampus and the cerebellum. Mechanistically, NFIX has been shown to promote neural stem cell differentiation through the activation of astrocyte-specific genes and via the repression of genes central to progenitor cell self-renewal. Interestingly, mice lacking Nfix also exhibit other phenotypes with respect to development of the central nervous system, and whose underlying causes have yet to be determined. Here we examine one of the phenotypes displayed by Nfix(-/-) mice, namely hydrocephalus. Through the examination of embryonic and postnatal Nfix(-/-) mice we reveal that hydrocephalus is first seen at around postnatal day (P) 10 in mice lacking Nfix, and is fully penetrant by P20. Furthermore, we examined the subcommissural organ (SCO), the Sylvian aqueduct and the ependymal layer of the lateral ventricles, regions that when malformed and functionally perturbed have previously been implicated in the development of hydrocephalus. SOX3 is a factor known to regulate SCO development. Although we revealed that NFIX could repress Sox3-promoter-driven transcriptional activity in vitro, SOX3 expression within the SCO was normal within Nfix(-/-) mice, and Nfix mutant mice showed no abnormalities in the structure or function of the SCO. Moreover, these mutant mice exhibited no overt blockage of the Sylvian aqueduct. However, the ependymal layer of the lateral ventricles was frequently absent in Nfix(-/-) mice, suggesting that this phenotype may underlie the development of hydrocephalus within these knockout mice.

  18. Isolation and characterization of selenate resistant mutants of Acremonium chrysogenum

    Directory of Open Access Journals (Sweden)

    Airton Vialta

    1999-01-01

    Full Text Available Mutants unable to convert exogenous sulfate to sulfite were isolated using the toxic analogue selenate. Three of twenty-eight isolated mutants were chromate sensitive. They showed a possible lesion in the gene that codes the ATP sulfurylase. The others were chromate resistant, and probably had a lesion in one or both of the genes that code the sulfate permease. Methionine increased the resistance levels to selenate. In addition, the frequency of spontaneous mutants obtained in a medium containing methionine was higher (between 2.4 x 10-6 and 18.0 x 10-6 than that obtained using a medium without any intentional source of sulfur (between 0.7 x 10-6 and 5.0 x 10-6. The original strain, as well as the mutants, were able to grow in a sulfur-free liquid medium even after 4 consecutive inoculation procedures. These results indicated the existence of sulfur traces in the medium and/or an efficient intracellular storage system. There was no significant difference between cephalosporin C production in mutants and the original strain.Mutantes incapazes de converter o sulfato extracelular em sulfito foram isolados utilizando o análogo tóxico selenato. De 28 mutantes isolados, apenas 3 foram sensíveis ao cromato, provavelmente apresentando lesão no gene que codifica a ATP sulfurilase. Os demais foram resistentes ao cromato e devem conter lesão no gene sB ou também no gene sC. A metionina elevou os níveis de resistência ao selenato e a freqüência de mutantes espontâneos obtida em meio contendo este aminoácido foi maior (entre 2,42 x 10-6 e 18,04 x 10-6 do que a obtida no meio sem a adição de qualquer fonte intencional de enxofre (entre 0,71 x 10-6 e 5,0 x 10-6. A linhagem original e os mutantes foram capazes de crescer, mesmo depois de quatro etapas de inóculo, fato que pode ser explicado pela existência de traços do referido elemento no meio e/ou a presença de um sistema eficiente de estocagem intracelular. A produção de cefalosporina C

  19. Spontaneous chlorophyll mutants of Pennisetum americanum: Genetics and chlorophyll quantities.

    Science.gov (United States)

    Koduru, P R; Rao, M K

    1980-05-01

    Thirteen spontaneously occurring chlorophyll deficient phenotypes have been described and their genetic basis was established. Ten of these - 'white', 'white tipped green', 'patchy white', 'white virescent', 'white striping 1', 'white striping 2', 'white striping 4', 'fine striping', 'chlorina' and 'yellow virescent' showed monogenic recessive inheritance and the remaining three - 'yellow striping', 'yellow green' and 'light green' seedling phenotypes showed digenic recessive inheritance. The genes for (i) 'white tipped green' (wr) and 'yellow virescent' (yv) and (ii) 'patchy white' (pw) and 'white striping 1' (wst 1) showed independent assortment. Further, the genes for 'white' (w), 'white tipped green' (wr) and 'yellow virescent' (yv) were inherited independently of the gene for hairy leaf margin (Hm).In the mutants - 'white tipped green', 'patchy white', 'white striping 1', 'white striping 2', 'fine striping', 'chlorina', 'yellow virescent', 'yellow striping', 'yellow green' and 'light green' phenotypes total quantity of chlorophyll was significantly less than that in the corresponding controls, while in 'white virescent' there was no reduction in the mature stage. For nine of the mutants the quantity of chlorophyll was also estimated in F1's (mutant x control green). In F1's of six of the mutants - 'white tip', 'patchy white', 'chlorina', 'yellow virescent', 'fine striping' and 'yellow striping' the quantity of chlorophyll was almost equal to the wild type. In the F1's of three of the mutants - 'white striping 1', 'white striping 2' and 'light green' an intermediate value between the mutant and wild types was observed. In 'yellow virescent' retarded synthesis of chlorophyll, particularly chlorophyll a was observed in the juvenile stage. Reduced quantity of chlorophyll was associated with defective chloroplasts. In the mutants - 'white tipped green, 'white virescent', 'fine striping', 'chlorina', 'yellow striping', 'yellow green' and 'light green' defective

  20. Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutants.

    Directory of Open Access Journals (Sweden)

    Kailiang Sun

    2012-02-01

    Full Text Available miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology.

  1. Combinatorial Synthetic Peptide Vaccine Strategy Protects against Hypervirulent CovR/S Mutant Streptococci.

    Science.gov (United States)

    Pandey, Manisha; Mortensen, Rasmus; Calcutt, Ainslie; Powell, Jessica; Batzloff, Michael R; Dietrich, Jes; Good, Michael F

    2016-04-15

    Cluster of virulence responder/sensor (CovR/S) mutant group A streptococci (GAS) are serious human pathogens of multiple M protein strains that upregulate expression of virulence factors, including the IL-8 proteaseStreptococcus pyogenescell envelope proteinase (SpyCEP), thus blunting neutrophil-mediated killing and enabling ingress of bacteria from a superficial wound to deep tissue. We previously showed that a combination vaccine incorporating J8-DT (conserved peptide vaccine from the M protein) and a recombinant SpyCEP fragment protects against CovR/S mutants. To enhance the vaccine's safety profile, we identified a minimal epitope (S2) that was the target for anti-SpyCEP Abs that could protect IL-8 from SpyCEP-mediated proteolysis. Abs from healthy humans and from mice experimentally infected with GAS also recognized S2, albeit at low titers. Native SpyCEP may be poorly immunogenic (cryptic or subdominant), and it would be to the organism's advantage if the host did not induce a strong Ab response against it. However, S2 conjugated to diphtheria toxoid is highly immunogenic and induces Abs that recognize and neutralize SpyCEP. Hence, we describe a two-component peptide vaccine that induces Abs (anti-S2) that protect IL-8 from proteolysis and other Abs (anti-J8) that cause strain-independent killing in the presence of neutrophils. We show that either component alone is ineffectual in preventing skin infection and bacteremia due to CovR/S mutants but that the combination induces complete protection. This protection correlated with a significant influx of neutrophils to the infection site. The data strongly suggest that the lack of natural immunity to hypervirulent GAS strains in humans could be rectified by this combination vaccine.

  2. Alopecia in a viable phospholipase C delta 1 and phospholipase C delta 3 double mutant.

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    Fabian Runkel

    Full Text Available BACKGROUND: Inositol 1,4,5trisphosphate (IP(3 and diacylglycerol (DAG are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia, whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab that resulted from the insertion of an intracisternal A particle (IAP into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab alleles are phenotypically normal. However, the presence of one Plcd3(mNab allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9olt1Pas and the Plcd3(mNab mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface.

  3. Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling

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    Dandekar Thomas

    2010-10-01

    Full Text Available Abstract Background The human pathogen Listeria monocytogenes resides and proliferates within the cytoplasm of epithelial cells. While the virulence factors essentially contributing to this step of the infection cycle are well characterized, the set of listerial genes contributing to intracellular replication remains to be defined on a genome-wide level. Results A comprehensive library of L. monocytogenes strain EGD knockout mutants was constructed upon insertion-duplication mutagenesis, and 1491 mutants were tested for their phenotypes in rich medium and in a Caco-2 cell culture assay. Following sequencing of the plasmid insertion site, 141 different genes required for invasion of and replication in Caco-2 cells were identified. Ten in-frame deletion mutants were constructed that confirmed the data. The genes with known functions are mainly involved in cellular processes including transport, in the intermediary metabolism of sugars, nucleotides and lipids, and in information pathways such as regulatory functions. No function could be ascribed to 18 genes, and a counterpart of eight genes is missing in the apathogenic species L. innocua. Mice infection studies revealed the in vivo requirement of IspE (Lmo0190 involved in mevalonate synthesis, and of the novel ABC transporter Lmo0135-0137 associated with cysteine transport. Based on the data of this genome-scale screening, an extreme pathway and elementary mode analysis was applied that demonstrates the critical role of glycerol and purine metabolism, of fucose utilization, and of the synthesis of glutathione, aspartate semialdehyde, serine and branched chain amino acids during intracellular replication of L. monocytogenes. Conclusion The combination of a genetic screening and a modelling approach revealed that a series of transporters help L. monocytogenes to overcome a putative lack of nutrients within cells, and that a high metabolic flexibility contributes to the intracellular replication of

  4. Characterization of neonatal vocal and motor repertoire of reelin mutant mice.

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    Emilia Romano

    Full Text Available Reelin is a large secreted extracellular matrix glycoprotein playing an important role in early neurodevelopment. Several genetic studies found an association between RELN gene and increased risk of autism suggesting that reelin deficiency may be a vulnerability factor in its etiology. Moreover, a reduced reelin expression has been observed in several brain regions of subjects with Autism Spectrum Disorders. Since a number of reports have documented presence of vocal and neuromotor abnormalities in patients with autism and suggested that these dysfunctions predate the onset of the syndrome, we performed a fine-grain characterization of the neonatal vocal and motor repertoire in reelin mutant mice to explore the developmental precursors of the disorder. Our findings evidence a general delay in motor and vocal development in heterozygous (50% reduced reelin and reeler (lacking reelin gene mutant mice. As a whole, an increased number of calls characterized heterozygous pup's emission. Furthermore, the typical ontogenetic peak in the number of calls characterizing wild-type pups on postnatal day 4 appeared slightly delayed in heterozygous pups (to day 6 and was quite absent in reeler littermates, which exhibited a flat profile during development. We also detected a preferential use of a specific call category (two-components by heterozygous and reeler mice at postnatal days 6 and 8 as compared to their wild-type littermates. With regard to the analysis of spontaneous movements, a differential profile emerged early in development among the three genotypes. While only slight coordination difficulties are exhibited by heterozygous pups, all indices of motor development appear delayed in reeler mice. Overall, our results evidence a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reelin mutant pups.

  5. Aurora kinase inhibitors: which role in the treatment of chronic myelogenous leukemia patients resistant to imatinib?

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    Giovanni Martinelli

    2009-02-01

    Full Text Available At present, there are no compounds in clinical development in the field of chronic myeloid leukemia (CML or Philadelphia-positive (Ph+ acute lymphoblastic leukemia (ALL that have been documented to harbor significant activity against the imatinib-resistant T315I mutation. Recent reports on the pre-clinical activity of some emerging tyrosine kinase inhibitors such as ON012380, VX-680 and PHA-739358 promise possible clinical efficacy against this specific Bcr-Abl mutant form. Here, we focus on the role of aurora kinase inhibitor VX-680 and PHA-739358 in blocking the leukemogenic pathways driven by wild-type and T315I-Bcr-Abl in CML or Ph+ ALL by reviewing recent research evidence. We also discuss the possibility of employing aurora kinase inhibitors as a promising new therapeutic approach in the treatment of CML and Ph+ ALL patients resistant to first and second generation TK inhibitors.

  6. Analysis of Candida albicans mutants defective in the Cdk8 module of mediator reveal links between metabolism and biofilm formation.

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    Allia K Lindsay

    2014-10-01

    Full Text Available Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO, a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module-Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development.

  7. Effects of altered catecholamine metabolism on pigmentation and physical properties of sclerotized regions in the silkworm melanism mutant.

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    Liang Qiao

    Full Text Available Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln silkworm strain as a model. Injection of β-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant and Dazao (wild-type silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of Bm-iAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao-mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research.

  8. Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern.

    Science.gov (United States)

    Decker, Amanda R; McNeill, Matthew S; Lambert, Aaron M; Overton, Jeffrey D; Chen, Yu-Chia; Lorca, Ramón A; Johnson, Nicolas A; Brockerhoff, Susan E; Mohapatra, Durga P; MacArthur, Heather; Panula, Pertti; Masino, Mark A; Runnels, Loren W; Cornell, Robert A

    2014-02-15

    Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation.

  9. Isolation of Escherichia coli mutants defective in uptake of molybdate.

    Science.gov (United States)

    Hemschemeier, S; Grund, M; Keuntje, B; Eichenlaub, R

    1991-10-01

    For the study of molybdenum uptake by Escherichia coli, we generated Tn5lac transposition mutants, which were screened for the pleiotropic loss of molybdoenzyme activities. Three mutants A1, A4, and M22 were finally selected for further analysis. Even in the presence of 100 microM molybdate in the growth medium, no active nitrate reductase, formate dehydrogenase, and trimethylamine-N-oxide reductase were detected in these mutants, indicating that the intracellular supply of molybdenum was not sufficient. This was also supported by the observation that introduction of plasmid pWK225 carrying the complete nif regulon of Klebsiella pneumoniae did not lead to a functional expression of nitrogenase. Finally, molybdenum determination by induced coupled plasma mass spectroscopy confirmed a significant reduction of cell-bound molybdenum in the mutants compared with that in wild-type E. coli, even at high molybdate concentrations in the medium. A genomic library established with the plasmid mini-F-derived cop(ts) vector pJE258 allowed the isolation of cosmid pBK229 complementing the molybdate uptake deficiency of the chlD mutant and the Tn5lac-induced mutants. Certain subfragments of pBK229 which do not contain the chlD gene are still able to complement the Tn5lac mutants. Mapping experiments showed that the Tn5lac insertions did not occur within the chromosomal region present in pBK229 but did occur very close to that region. We assume that the Tn5lac insertions have a polar effect, thus preventing the expression of transport genes, or that a positively acting regulatory element was inactivated.

  10. Auditory development in progressive motor neuronopathy mouse mutants.

    Science.gov (United States)

    Volkenstein, Stefan; Brors, Dominik; Hansen, Stefan; Berend, Achim; Mlynski, Robert; Aletsee, Christoph; Dazert, Stefan

    2009-11-06

    The present study was performed to elucidate the hearing development in the progressive motor neuronopathy (pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance.

  11. Mutant Enpp1asj mice as a model for generalized arterial calcification of infancy.

    Science.gov (United States)

    Li, Qiaoli; Guo, Haitao; Chou, David W; Berndt, Annerose; Sundberg, John P; Uitto, Jouni

    2013-09-01

    Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis - those mice developed stiffening of the joints, hence the mutant mouse was named 'ages with stiffened joints' (asj). These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi) levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

  12. The Binding Ability Analysis of the Normal VLDL Receptor and Its Mutant

    Institute of Scientific and Technical Information of China (English)

    QU Shen; FENG Ning; LIU Zhiguo; ZHOU Hua; DENG Yaozu; FENG Zongchen

    2001-01-01

    The ligand-binding domain of VLDL receptor contains eight imperfectly similar repeats.To discuss the contribution of each repeat to ligand binding, the RT-PCR technique was used to clone the VLDLR-cDNA from the heart muscle of Chinese people. Two recombinants were further constructed, which contained the full-length cDNA of VLDLR and the mutant lacking repeats 1-5.CHO cell line was transfected with two recombinants. The expression of VLDLR gene could be detected by RT-PCR from the CHO cells transfected with pCD-VR. The results of binding experiments showed that the ability of the CHO cells transfected with the full-length cDNA of VLDL-R binding DiI-labeled β-VLDL was higher than that of the CHO cells transfected with the mutant. Our findings indicated that human VLDL-R gene could be expressed effectively on CHO cells, and the receptor was almost inactivated when repeats1-5 were deleted.

  13. aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant

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    Sebastian Felgner

    2016-09-01

    Full Text Available Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine.

  14. Systematic generation of in vivo G protein-coupled receptor mutants in the rat.

    Science.gov (United States)

    van Boxtel, R; Vroling, B; Toonen, P; Nijman, I J; van Roekel, H; Verheul, M; Baakman, C; Guryev, V; Vriend, G; Cuppen, E

    2011-10-01

    G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

  15. Analysis of a Ferric Uptake Regulator (Fur) Mutant ofDesulfovibrio vulgaris Hildenborough

    Energy Technology Data Exchange (ETDEWEB)

    Bender, Kelly S.; Yen, Huei-Che Bill; Hemme, Christopher L.; Yang, Zamin K.; He, Zhili; He, Qiang; Zhou, Jizhong; Huang, Katherine H.; Alm, Eric J.; Hazen, Terry C.; Arkin, Adam P.; Wall, Judy D.

    2007-09-21

    Previous experiments examining the transcriptional profileof the anaerobe Desulfovibrio vulgaris demonstrated up-regulation of theFur regulon in response to various environmental stressors. To test theinvolvement of Fur in the growth response and transcriptional regulationof D. vulgaris, a targeted mutagenesis procedure was used for deletingthe fur gene. Growth of the resulting ?fur mutant (JW707) was notaffected by iron availability, but the mutant did exhibit increasedsensitivity to nitrite and osmotic stresses compared to the wild type.Transcriptional profiling of JW707 indicated that iron-bound Fur acts asa traditional repressor for ferrous iron uptake genes (feoAB) and othergenes containing a predicted Fur binding site within their promoter.Despite the apparent lack of siderophore biosynthesis genes within the D.vulgaris genome, a large 12-gene operon encoding orthologs to TonB andTolQR also appeared to be repressed by iron-bound Fur. While other genespredicted to be involved in iron homeostasis were unaffected by thepresence or absence of Fur, alternative expression patterns that could beinterpreted as repression or activation by iron-free Fur were observed.Both the physiological and transcriptional data implicate a globalregulatory role for Fur in the sulfate-reducing bacterium D.vulgaris.

  16. Salmonella Enteritidis flagellar mutants have a colonization benefit in the chicken oviduct.

    Science.gov (United States)

    Kilroy, Sofie; Raspoet, Ruth; Martel, An; Bosseler, Leslie; Appia-Ayme, Corinne; Thompson, Arthur; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip

    2017-02-01

    Egg borne Salmonella Enteritidis is still a major cause of human food poisoning. Eggs can become internally contaminated following colonization of the hen's oviduct. In this paper we aimed to analyze the role of flagella of Salmonella Enteritidis in colonization of the hen's oviduct. Using a transposon library screen we showed that mutants lacking functional flagella are significantly more efficient in colonizing the hen's oviduct in vivo. A micro-array analysis proved that transcription of a number of flagellar genes is down-regulated inside chicken oviduct cells. Flagella contain flagellin, a pathogen associated molecular pattern known to bind to Toll-like receptor 5, activating a pro-inflammatory cascade. In vitro tests using primary oviduct cells showed that flagellin is not involved in invasion. Using a ligated loop model, a diminished inflammatory reaction was seen in the oviduct resulting from injection of an aflagellated mutant compared to the wild-type. It is hypothesized that Salmonella Enteritidis downregulates flagellar gene expression in the oviduct and consequently prevents a flagellin-induced inflammatory response, thereby increasing its oviduct colonization efficiency.

  17. Acquirement and characterization of a carotenoid mutant (GM309) of Rhodobacter sphaeroides 601

    Institute of Scientific and Technical Information of China (English)

    LIU Yuan; ZHANG Wei; WU Yongqiang; XU Chunhe

    2004-01-01

    A green mutant was obtained among the chemically induced mutants of Rhodobacter sphaeroides 601 (RS601) and named GM309. A blue shift of 20 nm of the carotenoid absorption spectrum was found in the light-harvesting complex II (LH2) of GM309. Different from LH2 of RS601, it was found that the carotenoids in GM309-LH2 changed to be neurosporene by mutation. Neurosporene lacks a conjugate double bond, compared with the spheroidene in RS601-LH2 which has ten conjugate double bonds. As shown by absorption and circular dichroism spectroscopy, the overall structure of GM309-LH2 is little affected by this change. From fluorescence emission spectra, it is found that GM309-LH2 can transfer energy from carotenoids to Bchl-B850 without any change in efficiency. But the efficiency of energy transfer from B800 to B850 in GM309-LH2 is decreased to be 42% of that of the native. This work would provide a novel method to investigate the mechanism of excitation energy transfer in LH2.

  18. Mutant Enpp1asj mice as a model for generalized arterial calcification of infancy

    Directory of Open Access Journals (Sweden)

    Qiaoli Li

    2013-09-01

    Generalized arterial calcification of infancy (GACI, an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis – those mice developed stiffening of the joints, hence the mutant mouse was named ‘ages with stiffened joints’ (asj. These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

  19. Hydroxyproline O-arabinosyltransferase mutants oppositely alter tip growth in Arabidopsis thaliana and Physcomitrella patens.

    Science.gov (United States)

    MacAlister, Cora A; Ortiz-Ramírez, Carlos; Becker, Jörg D; Feijó, José A; Lippman, Zachary B

    2016-01-01

    Hydroxyproline O-arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant-specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall-associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co-opted to function in diverse species-specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell-wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall-associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants.

  20. Anomalies of autogamy in Paramecium tetraurelia temperature sensitive mutant induced by heat treatment.

    Science.gov (United States)

    Prajer, M; Iftode, F

    2001-01-01

    The run of autogamy in temperature sensitive mutant ts401, carrying also the mutation of trichocysts nd3a, at permissive (26 degrees C) and non-permissive temperature (35 degrees C) was studied and compared with this process in wild d4-2 stock of Paramecium tetraurelia. The effect of heat treatment on programmed nuclear and cortical events was investigated using cytological silver impregnation Fernandez-Galiano method and immunofluorescence technique with the application of two anti-alpha-tubulin antibodies. The appearance of cells with some large macronuclear fragments and an excess number of micronuclei suggests that nuclear differentiation is inhibited at restrictive temperature and that macronuclear regeneration takes place in this thermosensitive mutant. In such cells impaired oral reorganization was observed. After slight refeeding at the late autogamy, cells stopped at first postautogamous division were induced by heat treatment. The most striking feature of these abnormal divisions was the lack of a basal body duplication wave, which suggests that production of gamma-tubulin, structural protein being the essential component of the microtubule organizing centres, is disturbed at high temperature.

  1. Genetics and complementation of Haemophilus influenzae mutants deficient in adenosine 5'-triphosphate-dependent nuclease

    Energy Technology Data Exchange (ETDEWEB)

    Kooistra, J.; Small, G.D.; Setlow, J.K.; Shapanka, R.

    1976-04-01

    Eight different mutations in Haemophilus influenzae leading to deficiency in adenosine 5'-triphosphate (ATP)-dependent nuclease have been investigated in strains in which the mutations of the originally mutagenized strains have been transferred into the wild type. Sensitivity to mitomycin C and deoxycholate and complementation between extracts and deoxyribonucleic acid (DNA)-dependent ATPase activity have been measured. Genetic crosses have provided information on the relative position of the mutations on the genome. There are three complementation groups, corresponding to three genetic groups. The strains most sensitive to mitomycin and deoxycholate, derived from mutants originally selected on the basis of sensitivity to mitomycin C or methyl methanesulfonate, are in one group. Apparently all these sensitive strains lack DNA-dependent ATPase activity, as does a strain intermediate in sensitivity to deoxycholate, which is the sole representative of another group. There are four strains that are relatively resistant to deoxycholate and mitomycin C, and all of these contain the ATPase activity.

  2. Nonadherence is Associated with Lack of HIV-Related Knowledge

    DEFF Research Database (Denmark)

    Dyrehave, Charlotte; Rasmussen, Dlama Nggida; Hønge, Bo Langhoff

    2016-01-01

    -sectional study included 494 HIV-infected individuals from the Bissau HIV Cohort in Guinea-Bissau. They completed a questionnaire designed for assessment of adherence and HIV-related knowledge. RESULTS: A majority were female, 41% were illiterate, 25% did not take the medicine during the last 4 days, and 23......BACKGROUND: Poor treatment adherence is a main barrier for effective antiretroviral therapy (ART) globally. HIV-related knowledge may affect understanding and utilization of HIV medical information, hence limited health literacy is a known barrier to treatment adherence. DESIGN AND METHODS: A cross......% skipped their medicine during weekends. The most frequent reasons for not taking medicine were simply forgetting, side effects, lack of food, and being too ill to attend the clinic. Nonadherent patients had a lower level of HIV-related knowledge. CONCLUSION: Main barriers for nonadherence were side...

  3. Lack of time management as a psychosocial work risk

    Directory of Open Access Journals (Sweden)

    Ramon Cladellas

    2008-10-01

    Full Text Available This paper is aimed to explore the possible relationship between workers' lack of time management and several psychosocial risks. The psychosocial risks were assessed by means of the ISTAS21 Questionnaire, the Spanish version of the CoPsoQ (Copenhagen Psychological Questionnaire. More specifically, nine dimensions, which are directly related with time management, satisfaction, health and stress, were selected for evaluation. Time management was measured through the following variables: quantitative demands, influences and control of the time. Drawing on a sample of 142 workers from four departments (development, implantation, support and administration, the research results show that the employees who belong to a department that offers few opportunities for individual time management are less satisfied, have worse general and mental health, and experience more behavioral, symptomatic and cognitive stress than those who can manage their work schedule.

  4. The lack of carbon stars in the Galactic bulge

    Institute of Scientific and Technical Information of China (English)

    Zhu Chun-Hua; Lv Guo-Liang; Wang Zhao-Jun; Zhang Jun

    2008-01-01

    In order to explain the lack of carbon stars in the Galactic bulge, we have made a detailed study of thermal pulseasymptotic giant branch (TP-AGB) stars by using a population synthesis code. The effects of the oxygen overabundance and the mass loss rate on the ratio of the number of carbon stars to that of oxygen stars in the Galactic bulge are discussed. We find that the oxygen overabundance which is about twice as large as that in the solar neighbourhood (close to the present observations) is insufficient to explain the rareness of carbon stars in the bulge. We suggest that the large mass loss rate may serve as a controlling factor in the ratio of the number of carbon stars to that of oxygen stars.

  5. Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart

    Directory of Open Access Journals (Sweden)

    Wenjing Xu

    2015-10-01

    Full Text Available Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1 might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.

  6. When innovative instructional designs are too innovative: lack of schema

    DEFF Research Database (Denmark)

    Kjærgaard, Thomas; Wahl, Christian

    2015-01-01

    and it was developed to motivate and encourage the students to engage in more situated learning processes. The course is infamous for low attendance and for demotivating the students; hence the new instructional design should motivate students to attend the lessons and to participate. The new instructional design...... utilized teacher-produced video clips to qualify the students learning in the preparation for the lessons and new pedagogical activities during lessons to make the learning process more situated. However, the outcome was not as planned. The students didn’t recognize the video clips as a significant part...... of the preparation for the lessons and therefore they were not prepared for the activities that the teacher had planned for the lessons. The paper investigates the students’ lack of ‘schema’ and suggests ways of developing schemata to learn in a situated learning environment....

  7. Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart.

    Science.gov (United States)

    Xu, Wenjing; Barrientos, Tomasa; Mao, Lan; Rockman, Howard A; Sauve, Anthony A; Andrews, Nancy C

    2015-10-20

    Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.

  8. Pathogenesis of herpes simplex virus type 2 virion host shutoff (vhs) mutants.

    Science.gov (United States)

    Smith, Tracy J; Morrison, Lynda A; Leib, David A

    2002-03-01

    During lytic infection, the virion host shutoff (vhs) protein mediates the rapid degradation of mRNA and the shutoff of host protein synthesis. In vivo, herpes simplex virus type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. Homologs of vhs exist in all of the neurotropic herpesviruses, and the goal of this study was to determine the virulence of HSV-2 mutants lacking vhs. Two HSV-2 recombinants were used in this study: 333-vhsB, which has a lacZ cassette inserted into the N terminus of vhs, and 333d41, which has a 939-bp deletion in vhs. As expected, both 333-vhsB and 333d41 failed to induce the cellular RNA degradation characteristic of HSV. Corneal, vaginal, and intracerebral routes of infection were used to study pathogenesis. Both viruses grew to significantly lower titers in the corneas, trigeminal ganglia, vaginas, dorsal root ganglia, spinal cords, and brains of mice than wild-type and rescue viruses, with a correspondingly reduced induction of disease. Both viruses, however, reactivated efficiently from explanted trigeminal ganglia, showing that vhs is dispensable for reactivation. The lethality of 333d41 following peripheral infection of mice, however, was significantly higher than that of 333-vhsB, suggesting that some of the attenuation of 333-vhsB may be due to the presence of a lacZ cassette in the vhs locus. Taken together, these data show that vhs represents an important determinant of HSV-2 pathogenesis and have implications for the design of HSV-2 recombinants and vaccines.

  9. Lack of bcr and abr promotes hypoxia-induced pulmonary hypertension in mice.

    Directory of Open Access Journals (Sweden)

    Min Yu

    Full Text Available BACKGROUND: Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined. METHODOLOGY/PRINCIPAL FINDINGS: Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr-/- and abr-/- macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia. CONCLUSIONS: Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.

  10. Lack of fructose 2,6-bisphosphate compromises photosynthesis and growth in Arabidopsis in fluctuating environments.

    Science.gov (United States)

    McCormick, Alistair J; Kruger, Nicholas J

    2015-03-01

    The balance between carbon assimilation, storage and utilisation during photosynthesis is dependent on partitioning of photoassimilate between starch and sucrose, and varies in response to changes in the environment. However, the extent to which the capacity to modulate carbon partitioning rapidly through short-term allosteric regulation may contribute to plant performance is unknown. Here we examine the physiological role of fructose 2,6-bisphosphate (Fru-2,6-P2 ) during photosynthesis, growth and reproduction in Arabidopsis thaliana (L.). In leaves this signal metabolite contributes to coordination of carbon assimilation and partitioning during photosynthesis by allosterically modulating the activity of cytosolic fructose-1,6-bisphosphatase. Three independent T-DNA insertional mutant lines deficient in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP), the bifunctional enzyme responsible for both the synthesis and degradation of Fru-2,6-P2 , lack Fru-2,6-P2 . These plants have normal steady-state rates of photosynthesis, but exhibit increased partitioning of photoassimilate into sucrose and have delayed photosynthetic induction kinetics. The F2KP-deficient plants grow normally in constant environments, but show reduced growth and seed yields relative to wildtype plants in fluctuating light and/or temperature. We conclude that Fru-2,6-P2 is required for optimum regulation of photosynthetic carbon metabolism under variable growth conditions. These analyses suggest that the capacity of Fru-2,6-P2 to modulate partitioning of photoassimilate is an important determinant of growth and fitness in natural environments.

  11. Transcriptional responses to glucose in Saccharomyces cerevisiae strains lacking a functional protein kinase A

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    Livas Daniela

    2011-08-01

    Full Text Available Abstract Background The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA, triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4. Results We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA. Conclusions In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in

  12. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    Science.gov (United States)

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

  13. Normal aging modulates the neurotoxicity of mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Elsa Diguet

    Full Text Available Aging likely plays a role in neurodegenerative disorders. In Huntington's disease (HD, a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt, the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or "normal" aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a beta-Galactosidase (beta-Gal reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week and old (15 month rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that "normal" aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.

  14. Mutant alpha-synuclein and autophagy in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Kangyong Liu; Chunfeng Liu; Chuancheng Ren; Yaping Yang; Liwei Shen; Xuezhong Li; Fen Wang; Zhenghong Qin

    2011-01-01

    Several studies have demonstrated that overexpression of mutant α-synuclein in PC12 cells is related to occurrence of autophagy.The present study established mutant a-synuclein (A30P)-transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively.Results demonstrated that mutant o-synuclein resulted in cell death via autophagy and involved α-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity.Mutant α-synuclein (A30P) also mediated toxicity of1-methyl-4-phenylpyridinium ion.Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted o-synuclein aggregation and cell death.To further determine the role of autophagy due to mutant a-synuclein, the present study measured expression of microtubule-associated protein light chain 3.Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3,while rapamycin promoted its expression.These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.

  15. Effect of different immunosuppressive drugs on calcineurin and its mutants

    Institute of Scientific and Technical Information of China (English)

    阎力君; 于翠娟; 张丽芳; 魏群

    2000-01-01

    Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis. Their phosphatase activity and the corresponding solution conformation were examined. Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immuno-suppressive drugs with CN. The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN. Furthermore, circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein. Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN, and had no effects on the phosphatase activity of mutants in Loop7 region, which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN. Examination of the activities of these

  16. Complementation of a Clostridium perfringens spo0A mutant with wild-type spo0A from other Clostridium species.

    Science.gov (United States)

    Huang, I-Hsiu; Sarker, Mahfuzur R

    2006-09-01

    To evaluate whether C. perfringens can be used as a model organism for studying the sporulation process in other clostridia, C. perfringens spo0A mutant IH101 was complemented with wild-type spo0A from four different Clostridium species. Wild-type spo0A from C. acetobutylicum or C. tetani, but not from C. botulinum or C. difficile, restored sporulation and enterotoxin production in IH101. The ability of spo0A from C. botulinum or C. difficile to complement the lack of spore formation in IH101 might be due, at least in part, to the low levels of spo0A transcription and Spo0A production.

  17. The Ctf18RFC clamp loader is essential for telomere stability in telomerase-negative and mre11 mutant alleles.

    Directory of Open Access Journals (Sweden)

    Honghai Gao

    Full Text Available The function of the replication clamp loaders in the semi-conservative telomere replication and their relationship to telomerase- and recombination mechanisms of telomere addition remains ambiguous. We have investigated the variant clamp loader Ctf18 RFC (Replication Factor C. To understand the role of Ctf18 at the telomere, we first investigated genetic interactions after loss of Ctf18 and TLC1 (the yeast telomerase RNA. We find that the tlc1Δ ctf18Δ double mutant confers a rapid >1000-fold decrease in viability. The rate of loss was similar to the kinetics of cell death in rad52Δ tlc1Δ cells. However, the Ctf18 pathway is distinct from Rad52, required for the repair of DSBs, as demonstrated by the synthetic lethality of rad52▵ tlc1Δ ctf18Δ triple mutants. These data suggest that each mutant elicits non-redundant defects acting on the same substrate. Second, interactions of the yeast hyper-recombinational mutant, mre11A470T, with ctf18▵ confer a synergistic cold sensitivity. The phenotype of these double mutants ultimately results in telomere loss and the generation of recombinational survivors. We observed a similar synergism between single mutants that led to hypersensitivity to the DNA alkylating agent, methane methyl sulphonate (MMS, the replication fork inhibitor hydroxyurea (HU, and to a failure to separate telomeres of sister chromatids. Hence, ctf18Δ and mre11A470T act in different pathways on telomere substrates for multiple phenotypes. The mre11A470T cells also displayed a DNA damage response (DDR at 15°C but not at 30°C while ctf18Δ mutants conferred a constitutive DDR activity. Both the 15°C DDR pattern and growth rate were reversible at 30°C and displayed telomerase activity in vivo. We hypothesize that Ctf18 confers protection against stalling and/or breaks at the replication fork in cells that either lack, or are compromised for, telomerase activity. This Ctf18-based function is likely to contribute another level

  18. Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species

    DEFF Research Database (Denmark)

    Yao, Shuo; Brandt, Anders Bøving; Egsgaard, Helge

    2012-01-01

    mutants of Chlamydomonas reinhardtii and Chlorella vulgaris, nine out of fourty-one mutants in C. reinhardtii and eleven out of fifty-three mutants in C. vulgaris contained increased amounts of neutral lipids, predominantly as triacylglycerols. Upon temperature induced cell-cycle arrest, these mutants...

  19. Candida albicans mutant construction and characterization of selected virulence determinants.

    Science.gov (United States)

    Motaung, T E; Albertyn, J; Pohl, C H; Köhler, Gerwald

    2015-08-01

    Candida albicans is a diploid, polymorphic yeast, associated with humans, where it mostly causes no harm. However, under certain conditions it can cause infections ranging from superficial to life threatening. This ability to become pathogenic is often linked to the immune status of the host as well as the expression of certain virulence factors by the yeast. Due to the importance of C. albicans as a pathogen, determination of the molecular mechanisms that allow this yeast to cause disease is important. These studies rely on the ability of researchers to create deletion mutants of specific genes in order to study their function. This article provides a critical review of the important techniques used to create deletion mutants in C. albicans and highlights how these deletion mutants can be used to determine the role of genes in the expression of virulence factors in vitro.

  20. Human mutant huntingtin disrupts vocal learning in transgenic songbirds.

    Science.gov (United States)

    Liu, Wan-Chun; Kohn, Jessica; Szwed, Sarah K; Pariser, Eben; Sepe, Sharon; Haripal, Bhagwattie; Oshimori, Naoki; Marsala, Martin; Miyanohara, Atsushi; Lee, Ramee

    2015-11-01

    Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

  1. Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

    DEFF Research Database (Denmark)

    Jensen, Anders Bøgh; Raventos, D.; Mundy, John Williams

    2002-01-01

    Jasmonates induce plant-defence responses and act to regulate defence-related genes including positive feedback of the lipoxygenase 2 (LOX2) gene involved in jasmonate synthesis. To identify jasmonate-signalling mutants, we used a fusion genetic strategy in which the firefly luciferase (FLUC......) and Escherichia coliß-glucuronidase (GUS) reporters were expressed under control of the jasmonate-responsive LOX2 promoter. Spatial and temporal patterns of reporter expression were determined initially, and revealed that JA-responsive expression from the LOX2 promoter required de novo protein synthesis. Reporter...... as two recessive mutants, designated joe1 and 2, that overexpress the reporter. Genetic analysis indicated that reporter overexpression in the joe mutants requires COI. joe1 responded to MeJA with increased anthocyanin accumulation, while joe2 responded with decreased root growth inhibition. In addition...

  2. How Life History Can Sway the Fixation Probability of Mutants.

    Science.gov (United States)

    Li, Xiang-Yi; Kurokawa, Shun; Giaimo, Stefano; Traulsen, Arne

    2016-07-01

    In this work, we study the effects of demographic structure on evolutionary dynamics when selection acts on reproduction, survival, or both. In contrast to the previously discovered pattern that the fixation probability of a neutral mutant decreases while the population becomes younger, we show that a mutant with a constant selective advantage may have a maximum or a minimum of the fixation probability in populations with an intermediate fraction of young individuals. This highlights the importance of life history and demographic structure in studying evolutionary dynamics. We also illustrate the fundamental differences between selection on reproduction and selection on survival when age structure is present. In addition, we evaluate the relative importance of size and structure of the population in determining the fixation probability of the mutant. Our work lays the foundation for also studying density- and frequency-dependent effects in populations when demographic structures cannot be neglected.

  3. The use of mutants to probe models of gravitropism.

    Science.gov (United States)

    Firn, R D; Wagstaff, C; Digby, J

    2000-08-01

    It has been widely believed for more than 70 years that auxin plays a central role in the induction of differential growth which causes gravitropic curvature. However, this long-standing consensus about a role for auxin in gravitropism has only been achieved by allowing several mutually exclusive models to coexist. Furthermore, because there is no detailed model which is unchallenged by evidence, consensus is now centred on ill-defined models which have a low predictive value, hence are harder to challenge experimentally. An increasing number of mutants with abnormal gravitropic behaviour are becoming available. Such mutants should be very helpful in challenging existing models of gravitropism and in providing new evidence on which to build improved, more precise models. However, to date, most studies of mutants with abnormal gravitropism have been guided, experimentally and conceptually, by the old inadequate and vague models. Consequently, the full potential of modern molecular analysis in aiding our understanding of gravitropism has yet to be realized.

  4. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M;

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...... technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras...... sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could...

  5. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.;

    2003-01-01

    of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. Methods NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...... or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...

  6. Callus cultures of tomato mutants: I. Nutritional requirements.

    Science.gov (United States)

    Ulrich, J M; Mackinney, G

    1969-01-01

    Callus from hypocotyl, stem, and fruit tissue of tomato mutants was grown on a complex pea extract medium. The genotypes responded differently to the levels of nutrients and stimulators or inhibitors in the medium. Hypocotyl callus of yellow (r) tomato required K(2) SO(4) for quick establishment and continued steady growth for several months; callus of this mutant could also grow with 0.5 % dimethyl sulfoxide in the medium, although growth was less than the control. The red ghost (r(+) gh) mutant is sensitive to a toxic component in the pea extract, and makes its best growth with the standard minerals and vitamins, but in 1/2 concentration pea extract plus 5 % coconut water. Tangerine (t), red lutescent stem (r(+) l(2) ), and r(+) gh are mutants which respond differently to thiourea: t grows about the same at all concentrations, r(+) gh grows best at low thiourea, and r(+) l(2) grows best at the specific level of 20 mg/l thiourea. The recent active t or r(+) l(1) and r(+) l(2) isolates require supplementary auxin to which the older, slow-growing isolates do not respond. However, there is variation in growth response of different isolates of the same mutant. The several red (r(+) ) cultures are similar in their slow growth, but somewhat different in responses to specific nutrients. The recent (+) isolate is one of the most active cultures, in comparison to the slow growth of t callus isolated in 1964. It is therefore concluded that growth is affected both by the specific requirements of the mutant and by the age and vigor of isolates.

  7. Nanoformulated cell-penetrating survivin mutant and its dual actions

    Directory of Open Access Journals (Sweden)

    Sriramoju B

    2014-07-01

    Full Text Available Bhasker Sriramoju, Rupinder K Kanwar, Jagat R Kanwar Nanomedicine Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine, Faculty of Health, Deakin University, Geelong, Australia Abstract: In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells, and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. Keywords: survivin mutant, neurological disorders, protein therapeutics, inhibitor of apoptosis protein family, poly(lactic-co-glycolic acid

  8. Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

    Directory of Open Access Journals (Sweden)

    Roland Züst

    Full Text Available Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

  9. Gain of oncogenic function of p53 mutants regulates E-cadherin expression uncoupled from cell invasion in colon cancer cells.

    Science.gov (United States)

    Roger, Lauréline; Jullien, Laurent; Gire, Véronique; Roux, Pierre

    2010-04-15

    Mutations in the p53 tumour suppressor gene are associated clinically with tumour progression and metastasis. Downregulation of the E-cadherin cell-cell adhesion molecule is a key event for epithelial to mesenchymal transition (EMT) in tumour progression. Here, we show that wild-type p53 induced to adopt a mutant conformation, and hot-spot p53 mutants, which are both transcriptionally inactive, downregulate E-cadherin expression in the colon carcinoma cell line HCT116. Downregulation of E-cadherin occurred concomitantly with the upregulation of Slug and Zeb-1, transcriptional factors known to repress E-cadherin gene expression. In addition, knockdown of Slug and Zeb-1 expression diminished p53-mediated E-cadherin repression. Knocking down endogenous mutant p53 in MDA-MB-231 and SW620 cancer cell lines lacking E-cadherin protein restored the expression of E-cadherin. Complete loss of E-cadherin expression in HCT116 cells induced morphological alterations along with upregulation of vimentin, a mesenchymal marker. These changes characteristic of the EMT phenotype were, however, not sufficient to confer invasiveness in a three-dimensional matrix. Downregulation of E-cadherin by mutant p53 was not required to promote the invasive phenotype induced by inactivation of p53. These findings indicate that independent control of E-cadherin expression and cell motility could be essential molecular events in p53 mutant-induced invasive phenotypes.

  10. Clinical errors as a lack of context responsiveness.

    Science.gov (United States)

    Bugatti, Matteo; Boswell, James F

    2016-09-01

    Although standardized treatments have the potential to decrease clinical errors, within-session responsiveness is complicated and complementary frameworks may be needed to foster enhanced responsiveness in the context of evidence-based treatments. Recent efforts have targeted the enhancement of flexibility and responsiveness in the delivery of manualized treatments, including the development of transdiagnostic treatments (i.e., protocols that are designed to be used across different diagnoses) intended to tailor intervention principles to the needs of individual patients. Context-Responsive Psychotherapy Integration (Constantino, Boswell, Bernecker, & Castonguay, 2013) offers an framework that supports the utilization of evidence-based clinical strategies in response to the identification of specific process markers. Failure to identify or appropriately respond to such markers may result in negative therapeutic process as well as outcomes. This case study uses the context-response psychotherapy integration framework to understand critical moments of clinical decision-making through examining an individual treatment case that unilaterally terminated after seven sessions of transdiagnostic treatment. This illustrative empirical case analysis focuses on three potential clinical errors, as indicated by a lack of responsiveness to three candidate process markers: (a) low outcome expectations, (b) self-strivings, and (c) outcome monitoring. For each clinical error, alternative clinical strategies are discussed. (PsycINFO Database Record

  11. Altered food consumption in mice lacking lysophosphatidic acid receptor-1.

    Science.gov (United States)

    Dusaulcy, R; Daviaud, D; Pradère, J P; Grès, S; Valet, Ph; Saulnier-Blache, J S

    2009-12-01

    The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.

  12. Lack of a Benign Interpretation Bias in Social Anxiety Disorder

    Science.gov (United States)

    Amir, Nader; Prouvost, Caroline; Kuckertz, Jennie M.

    2013-01-01

    Cognitive models of social anxiety posit that recurrent interpretation of ambiguous information as threatening maintains symptoms (e.g. Clark & Wells, 1995, pp. 69–93, Social phobia: Diagnosis, assessment, and treatment. New York: Guilford Press; Rapee & Heimberg, 1997, pp. 741–756, Behavior Research and Therapy, 35). However, biased interpretation may also be represented as a failure to make a benign interpretation of the ambiguous event. Furthermore, interpretation bias can be characterized by both an online (automatic) component and an offline (effortful) component (Hirsch & Clark, 2004, pp. 799–825, Clinical Psychology Review, 24). To measure both benign and threat biases, as well as examine the effect of social anxiety on offline versus online interpretations, Beard and Amir (2009, pp. 1135–1141, Behaviour Research and Therapy, 46) developed the Word Sentence Association Paradigm (WSAP). In the current study, we administered the WSAP to a group of participants diagnosed with social anxiety disorder (SAD) as well as to a group of non-anxious control (NAC) participants. We found that participants with SAD demonstrated a lack of benign online bias, but not an online threat bias when compared to NACs. However, when examining offline biases, SAD patients endorsed social threat interpretations and rejected benign social interpretations to a greater degree than non-anxious individuals. Our results, when taken together, clearly implicate the role of reduced bias toward benign information in SAD. PMID:22545788

  13. Vulnerability to schizophrenia and lack of common sense.

    Science.gov (United States)

    Stanghellini, G

    2000-01-01

    This article explores the hypothesis that the relational deficit in schizophrenia is not a consequence of acute symptoms and course but instead is a fundamental aspect of schizophrenic vulnerability. This basic relational deficit could be better understood as disconnectedness from common sense. Common sense is a tool for adaptation whose main scope is establishing cause-and-effect and motivational relationships in the physical and social realms. The common sense deficit appears to involve a lack of intuitive attunement (impaired capacity to accurately typify the mental states of other persons because of the incapacity to be involved in their mental lives) and a damaged social knowledge network (disorders of the background of knowledge useful for organizing everyday experiences). Three dimensions of schizophrenic vulnerability can be distinguished: the sensory, conceptualization, and attitudinal dimensions. Sensory disorders are aberrations of self, body, and world perceptions. Conceptualization disorders are disturbances in the attribution of meanings and intentions. Attitudinal disorders consist of eccentricities in the individual's structure of values and beliefs, characterized by distrust toward conventional knowledge and attunement. This article describes the present state and possible future directions of qualitative analyses and empirical investigations relevant to assessing the interplay between vulnerability dimensions and disorders of common sense.

  14. New graduate nurses' experiences about lack of professional confidence.

    Science.gov (United States)

    Ortiz, Jennifer

    2016-07-01

    Professional confidence is an essential trait for new graduate nurses to possess in order to provide quality patient care in today's complex hospital setting. However, many new graduates are entering the workforce without it and this remains to be explored. This study describes how new graduate nurses accounted for their lack of professional confidence upon entry into professional practice and how it developed during their first year of practice in the hospital setting. Two face-to-face, individual interviews of 12 participants were utilized to capture the lived experiences of new graduate nurses to gain an understanding of this phenomenon. After manual content analysis seven themes emerged: communication is huge, making mistakes, disconnect between school and practice, independence, relationship building, positive feedback is important, and gaining experience. The findings indicate that the development of professional confidence is a dynamic process that occurs throughout the first year of practice. New graduate nurses must experience both positive and negative circumstances in order to move toward the attainment of professional confidence. Knowing this, nurse educators in academia as well as in the hospital setting may better support the development of professional confidence both before and during the first year of practice.

  15. Lack of fiscal discipline: Challenges and possible solutions

    Directory of Open Access Journals (Sweden)

    Ćirović Nikola

    2013-01-01

    Full Text Available The lack of fiscal discipline is one of the most pressing issues in the framework of various economic systems today. The consequences of the global economic crisis have shown all the weaknesses of measures and mechanisms in terms of decision-making in fiscal policy. This problem can be seen now in almost every country. The problem of inadequate implementation of fiscal policy and the maintenance of fiscal discipline is reflected in the fact that their consequences are not visible right away, but tend to show their negative effects much later. Today, we have a situation where a certain current government suffers badly implemented prior fiscal discipline of a previous government. The paper identified causes of fiscal indiscipline, which is most easily seen in the budget process. In addition to determining the causes of fiscal indiscipline, the author puts forward a set of different possible solutions that could positively affect the stabilization of the growing public debt and eliminate the cause of fiscal indiscipline. Possible solutions are analyzed, as well as the positive and negative aspects of their use, and possible implementation problems of specific solutions.

  16. New constitutive latex osmotin-like proteins lacking antifungal activity.

    Science.gov (United States)

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events.

  17. Lack of global epigenetic methylation defects in CBS deficient mice.

    Science.gov (United States)

    Lee, Hyung-Ok; Wang, Liqun; Kuo, Yin-Ming; Gupta, Sapna; Slifker, Michael J; Li, Yue-Sheng; Andrews, Andrew J; Kruger, Warren D

    2017-01-01

    Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.

  18. Association between friction and wear in diarthrodial joints lacking lubricin

    Science.gov (United States)

    Jay, Gregory D; Torres, Jahn R; Rhee, David K; Helminen, Heikki J; Hytinnen, Mika M; Cha, Chung-Ja; Elsaid, Khaled; Kim, Kyung-Suk; Cui, Yajun; Warman, Matthew L

    2007-01-01

    Objective The glycoprotein lubricin (encoded by the gene Prg4) is secreted by surface chondrocytes and synovial cells, and has been shown to reduce friction in vitro. In contrast to man-made bearings, mammalian diarthrodial joints must endogenously produce friction-reducing agents. This study was undertaken to investigate whether friction is associated with wear. Methods The lubricating ability of synovial fluid (SF) samples from humans with genetic lubricin deficiency was tested in vitro. The coefficient of friction in the knee joints of normal and lubricin-null mice was measured ex vivo; these joints were also studied by light and electron microscopy. Atomic force microscopy was used to image and measure how lubricin reduces friction in vitro. Results SF lacking lubricin failed to reduce friction in the boundary mode. Joints of lubricin-null mice showed early wear and higher friction than joints from their wild-type counterparts. Lubricin self-organized and reduced the work of adhesion between apposing asperities. Conclusion These data show that friction is coupled with wear at the cartilage surface in vivo. They imply that acquired lubricin degradation occurring in inflammatory joint diseases predisposes the cartilage to damage. Lastly, they suggest that lubricin, or similar biomolecules, will have applications in man-made devices in which reducing friction is essential. PMID:17968947

  19. Lack of Galanin 3 Receptor Aggravates Murine Autoimmune Arthritis.

    Science.gov (United States)

    Botz, Bálint; Kemény, Ágnes; Brunner, Susanne M; Locker, Felix; Csepregi, Janka; Mócsai, Attila; Pintér, Erika; McDougall, Jason J; Kofler, Barbara; Helyes, Zsuzsanna

    2016-06-01

    Neurogenic inflammation mediated by peptidergic sensory nerves has a crucial impact on the pathogenesis of various joint diseases. Galanin is a regulatory sensory neuropeptide, which has been shown to attenuate neurogenic inflammation, modulate neutrophil activation, and be involved in the development of adjuvant arthritis, but our current understanding about its targets and physiological importance is incomplete. Among the receptors of galanin (GAL1-3), GAL3 has been found to be the most abundantly expressed in the vasculature and on the surface of some immune cells. However, since there are minimal in vivo data on the role of GAL3 in joint diseases, we analyzed its involvement in different inflammatory mechanisms of the K/BxN serum transfer-model of autoimmune arthritis employing GAL 3 gene-deficient mice. After arthritis induction, GAL3 knockouts demonstrated increased clinical disease severity and earlier hindlimb edema than wild types. Vascular hyperpermeability determined by in vivo fluorescence imaging was also elevated compared to the wild-type controls. However, neutrophil accumulation detected by in vivo luminescence imaging or arthritic mechanical hyperalgesia was not altered by the lack of the GAL3 receptor. Our findings suggest that GAL3 has anti-inflammatory properties in joints by inhibiting vascular hyperpermeability and consequent edema formation.

  20. Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization.

    Science.gov (United States)

    Bellemann, P; Geider, K

    1992-05-01

    Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

  1. Starch and Prolamin Level in Single and Double High-Lysine Barley Mutants

    DEFF Research Database (Denmark)

    Kreis, M.; Doll, Hans

    1980-01-01

    At maturity the high-lysine barley (Hordeum vulgare L.) Ris0 mutants 1508, 527 and 29 kernels contained about 20% less starch and twice as much free sugars as the parent varieties Bomi and Carlsberg II. An enhanched effect on starch reduction and free sugar accumulation was observed during kernel...... development when the single mutants 527 and 29 are combined with the mutant 1508. At maturity, kernels of the double mutants 527/1508 and 29/1508 contained, respectively, 68 and 43% less starch than Bomi. The double mutant 29/1508 kernel had a slightly lower prolamin content than mutant 1508 which is the most...... prolamin-deficient single mutant. In the double mutant 527/1508, however, an almost complete suppression of prolamin synthesis was observed during kernel development. The percentage of lysine in the seed proteins of the double mutants was about the same as in the most extreme single mutant 1508. Based...

  2. Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, Susan I.

    2009-06-08

    Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affect sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wil