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Sample records for rap1 mutant tissue

  1. Rap1 integrates tissue polarity, lumen formation, and tumorigenicpotential in human breast epithelial cells

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    Itoh, Masahiko; Nelson, Celeste M.; Myers, Connie A.; Bissell,Mina J.

    2006-09-29

    Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their non-malignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to formation of acinar structures with correct apico-basal polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing apical polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

  2. A novel interplay between Rap1 and PKA regulates induction of angiogenesis in prostate cancer.

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    Jyotsana Menon

    Full Text Available Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1 suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.

  3. Independent regulation of Rap1 and mitogen-activated protein kinase by the alpha chain of Go.

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    Bernasconi, Francesca; Malgaroli, Antonio; Vallar, Lucia

    Receptors coupled to G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) cascade. The intracellular pathways linking the alpha chains of these G proteins to MAPK activation are not completely understood. One of the signaling molecules which has been suggested to act downstream of Galpha(i/o) is the small G protein Rap1. We investigated the role of Rap1 in MAPK stimulation by Galpha(o) in Chinese hamster ovary (CHO) cells. Our previous results have shown that in this cell system activated Galpha(o) strongly potentiates the MAPK response to the epidermal growth factor (EGF) receptor. Rap1 regulation was examined in cells transfected with Rap1 and wild-type Galpha(o) or the activated mutant Galpha(o)-Q205L. Immunocytochemical analysis detected both Rap1 and the Galpha(o) subunit at the plasma membrane as well as on perinuclear cytoplasmic vesicles. Expression of wild-type Galpha(o) had no significant effect on the levels of activated Rap1. In contrast, Galpha(o)-Q205L virtually abolished the activation of Rap1 induced by EGF. Further experiments showed that MAPK stimulation by EGF was greatly inhibited by expression of activated Rap1, suggesting that Rap1 inhibition could mediate the effect of Galpha(o) on the MAPK cascade. However, Galpha(o)-Q205L efficiently inhibited the activation of Rap1 induced by fibroblast growth factor (FGF). We have previously found that the ability of FGF to activate MAPK is not modified by Galpha(o). In addition, expression of the GAP protein RAP1GAPII blocked Rap1 activation without affecting EGF- or FGF-dependent MAPK stimulation. These findings provide evidence for independent regulation of Rap1 and MAPK by the G(o )alpha chain.

  4. Identification of the functional domains of the telomere protein Rap1 in Schizosaccharomyces pombe.

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    Ikumi Fujita

    Full Text Available The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1(+ have revealed that the long N-terminal region (1-456 a.a. [amino acids] of Rap1 (full length: 693 a.a. is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457-693 a.a. containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.

  5. Human Rap1 modulates TRF2 attraction to telomeric DNA.

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    Janoušková, Eliška; Nečasová, Ivona; Pavloušková, Jana; Zimmermann, Michal; Hluchý, Milan; Marini, Victoria; Nováková, Monika; Hofr, Ctirad

    2015-03-11

    More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Modulation of angiotensin II-induced inflammatory cytokines by the Epac1-Rap1A-NHE3 pathway: implications in renal tubular pathobiology

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    Xie, Ping; Joladarashi, Darukeshwara; Dudeja, Pradeep; Sun, Lin

    2014-01-01

    Besides the glomerulus, the tubulointerstitium is often concomitantly affected in certain diseases, e.g., diabetic nephropathy, and activation of the renin-angiotensin system, to a certain extent, worsens its outcome because of perturbations in hemodynamics and possibly tubuloglomerular feedback. Certain studies suggest that pathobiology of the tubulointerstitium is influenced by small GTPases, e.g., Rap1. We investigated the effect of ANG II on inflammatory cytokines, while at the same time focusing on upstream effector of Rap1, i.e., Epac1, and some of the downstream tubular transport molecules, i.e., Na/H exchanger 3 (NHE3). ANG II treatment of LLC-PK1 cells decreased Rap1a GTPase activity in a time- and dose-dependent manner. ANG II treatment led to an increased membrane translocation of NHE3, which was reduced with Epac1 and PKA activators. ANG II-induced NHE3 translocation was notably reduced with the transfection of Rap1a dominant positive mutants, i.e., Rap1a-G12V or Rap1a-T35A. Transfection of cells with dominant negative Rap1a mutants, i.e., Rap1a-S17A, or Epac1 mutant, i.e., EPAC-ΔcAMP, normalized ANG II-induced translocation of NHE3. In addition, ANG II treatment led to an increased expression of inflammatory cytokines, i.e., IL-1β, IL-6, IL-8, and TNF-α, which was reduced with Rap1a-G12V or Rap1a-T35A transfection, while it reverted to previous comparable levels following transfection of Rap1a-S17A or EPAC-ΔcAMP. ANG II-induced expression of cytokines was reduced with the treatment with NHE3 inhibitor S3226 or with Epac1 and PKA activators. These data suggest that this novel Epac1-Rap1a-NHE3 pathway conceivably modulates ANG II-induced expression of inflammatory cytokines, and this information may yield the impetus for developing strategies to reduce tubulointertstitial inflammation in various renal diseases. PMID:24553435

  7. Human Rap1 modulates TRF2 attraction to telomeric DNA

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    Janoušková Eliška; Nečasová Ivona; Pavloušková Jana; Zimmermann Michal; Hluchý Milan; Marini Palomeque María Victoria; Nováková Monika; Hofr Ctirad

    2015-01-01

    More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed...

  8. Epac-Rap1-activated mesenchymal stem cells improve cardiac function in rat model of myocardial infarction.

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    Khan, Irfan; Ali, Anwar; Akhter, Muhammad Aleem; Naeem, Nadia; Chotani, Maqsood Ahmed; Iqbal, Hana'a; Kabir, Nurul; Atiq, Mehnaz; Salim, Asmat

    2017-04-01

    Rap1, a member of Ras superfamily of small GTP-binding proteins, is involved in cardiovascular biology in numerous ways. It is an evolutionary conserved regulator of adhesion, polarity, differentiation and growth. Our aim was to analyze Rap1-activated rat bone marrow mesenchymal stem cells (MSCs) for their potential role in adhesion and cardiac differentiation. Myocardial infarction (MI) was produced in Sprague Dawley (SD) rats through occlusion of the left anterior descending coronary artery. MSCs were treated with 8-pCPT-2'-O-Me-cAMP (CPT) to activate Rap1. Normal (untreated) and CPT-treated MSCs were transplanted through intramyocardial injection in respective groups. Cardiac function was assessed by echocardiography at 2 and 4 weeks after cell transplantation. Histological analysis was performed to observe changes at tissue level. Homing of CPT-treated MSCs was significantly (***Pinfarcted hearts. This may be due to increase in the gene expression of some of the cell adhesion molecules as evident by qRT-PCR analysis. Significant (***Ptherapy groups showed significant increase as compared to MI model. Our findings showed that pharmacological activation of Epac-Rap1 improves cardiac function through better survival, adhesion and differentiation of transplanted cells. Transplantation of these MSCs in the infarct area restored functional myocardium. © 2016 John Wiley & Sons Ltd.

  9. Subtelomeric repetitive elements determine TERRA regulation by Rap1/Rif and Rap1/Sir complexes in yeast

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    Iglesias, Nahid; Redon, Sophie; Pfeiffer, Verena; Dees, Martina; Lingner, Joachim; Luke, Brian

    2011-01-01

    Telomeric repeat-containing RNA (TERRA) has been implicated in the control of heterochromatin and telomerase. We demonstrate that yeast TERRA is regulated by telomere-binding proteins in a chromosome-end-specific manner that is dependent on subtelomeric repetitive DNA elements. At telomeres that contain only X-elements, the Rap1 carboxy-terminal domain recruits the Sir2/3/4 and Rif1/2 complexes to repress transcription in addition to promoting Rat1-nuclease-dependent TERRA degradation. At telomeres that contain Y′ elements, however, Rap1 represses TERRA through recruitment of Rif1 and Rif2. Our work emphasizes the importance of subtelomeric DNA in the control of telomeric protein composition and telomere transcription. PMID:21525956

  10. Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

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    Greicy H Goto

    2015-08-01

    Full Text Available Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

  11. Dynamics of Mutant Cells in Hierarchical Organized Tissues

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    Werner, Benjamin; Dingli, David; Lenaerts, Tom; Pacheco, Jorge M.; Traulsen, Arne

    2011-01-01

    Most tissues in multicellular organisms are maintained by continuous cell renewal processes. However, high turnover of many cells implies a large number of error-prone cell divisions. Hierarchical organized tissue structures with stem cell driven cell differentiation provide one way to prevent the accumulation of mutations, because only few stem cells are long lived. We investigate the deterministic dynamics of cells in such a hierarchical multi compartment model, where each compartment represents a certain stage of cell differentiation. The dynamics of the interacting system is described by ordinary differential equations coupled across compartments. We present analytical solutions for these equations, calculate the corresponding extinction times and compare our results to individual based stochastic simulations. Our general compartment structure can be applied to different tissues, as for example hematopoiesis, the epidermis, or colonic crypts. The solutions provide a description of the average time development of stem cell and non stem cell driven mutants and can be used to illustrate general and specific features of the dynamics of mutant cells in such hierarchically structured populations. We illustrate one possible application of this approach by discussing the origin and dynamics of PIG-A mutant clones that are found in the bloodstream of virtually every healthy adult human. From this it is apparent, that not only the occurrence of a mutant but also the compartment of origin is of importance. PMID:22144884

  12. Dynamics of mutant cells in hierarchical organized tissues.

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    Benjamin Werner

    2011-12-01

    Full Text Available Most tissues in multicellular organisms are maintained by continuous cell renewal processes. However, high turnover of many cells implies a large number of error-prone cell divisions. Hierarchical organized tissue structures with stem cell driven cell differentiation provide one way to prevent the accumulation of mutations, because only few stem cells are long lived. We investigate the deterministic dynamics of cells in such a hierarchical multi compartment model, where each compartment represents a certain stage of cell differentiation. The dynamics of the interacting system is described by ordinary differential equations coupled across compartments. We present analytical solutions for these equations, calculate the corresponding extinction times and compare our results to individual based stochastic simulations. Our general compartment structure can be applied to different tissues, as for example hematopoiesis, the epidermis, or colonic crypts. The solutions provide a description of the average time development of stem cell and non stem cell driven mutants and can be used to illustrate general and specific features of the dynamics of mutant cells in such hierarchically structured populations. We illustrate one possible application of this approach by discussing the origin and dynamics of PIG-A mutant clones that are found in the bloodstream of virtually every healthy adult human. From this it is apparent, that not only the occurrence of a mutant but also the compartment of origin is of importance.

  13. Human Rap1 interacts directly with telomeric DNA and regulates TRF2 localization at the telomere.

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    Arat, N Özlem; Griffith, Jack D

    2012-12-07

    The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3' ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection.

  14. Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere*

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    Arat, N. Özlem; Griffith, Jack D.

    2012-01-01

    The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3′ ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection. PMID:23086976

  15. A crucial role for DOK1 in PDGF-BB-stimulated glioma cell invasion through p130Cas and Rap1 signalling.

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    Barrett, Angela; Evans, Ian M; Frolov, Antonina; Britton, Gary; Pellet-Many, Caroline; Yamaji, Maiko; Mehta, Vedanta; Bandopadhyay, Rina; Li, Ningning; Brandner, Sebastian; Zachary, Ian C; Frankel, Paul

    2014-06-15

    DOK1 regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility. Mechanisms regulating tumour cell motility are essential for invasion and metastasis. We report here that PDGF-BB-mediated glioma cell invasion and migration are dependent on the adaptor protein downstream of kinase 1 (DOK1). DOK1 is expressed in several glioma cell lines and in tumour biopsies from high-grade gliomas. DOK1 becomes tyrosine phosphorylated upon PDGF-BB stimulation of human glioma cells. Knockdown of DOK1 or expression of a DOK1 mutant (DOK1FF) containing Phe in place of Tyr at residues 362 and 398, resulted in inhibition of both the PDGF-BB-induced tyrosine phosphorylation of p130Cas (also known as BCAR1) and the activation of Rap1. DOK1 colocalises with tyrosine phosphorylated p130Cas at the cell membrane of PDGF-BB-treated cells. Expression of a non-tyrosine-phosphorylatable substrate domain mutant of p130Cas (p130Cas15F) inhibited PDGF-BB-mediated Rap1 activation. Knockdown of DOK1 and Rap1 inhibited PDGF-BB-induced chemotactic cell migration, and knockdown of DOK1 and Rap1 and expression of DOK1FF inhibited PDGF-mediated three-dimensional (3D) spheroid invasion. These data show a crucial role for DOK1 in the regulation of PDGF-BB-mediated tumour cell motility through a p130Cas-Rap1 signalling pathway. [Corrected] © 2014. Published by The Company of Biologists Ltd.

  16. Epac1 and PDZ-GEF cooperate in Rap1 mediated endothelial junction control

    NARCIS (Netherlands)

    Pannekoek, W.J.; van Dijk, J.; Chan, O.Y.; Huveneers, S.; Linnemann, J.R.; Spanjaard, E.; Brouwer, P.E.M.; van der Meer, A.J.; Zwartkruis, F.J.; Rehmann, H.; de Rooij, J.; Bos, J.

    2011-01-01

    Epac1 and its effector Rap1 are important mediators of cAMP induced tightening of endothelial junctions and consequential increased barrier function. We have investigated the involvement of Rap1 signalling in basal, unstimulated, barrier function of a confluent monolayer of HUVEC using real time

  17. Direct Interaction between TalinB and Rap1 is necessary for adhesion of Dictyostelium cells

    NARCIS (Netherlands)

    Plak, Katarzyna; Pots, Henderikus; Van Haastert, Peter J M; Kortholt, Arjan

    2016-01-01

    BACKGROUND: The small G-protein Rap1 is an important regulator of cellular adhesion in Dictyostelium, however so far the downstream signalling pathways for cell adhesion are not completely characterized. In mammalian cells talin is crucial for adhesion and Rap1 was shown to be a key regulator of

  18. Sequential regulation of the small GTPase Rap1 in human platelets

    NARCIS (Netherlands)

    Franke, B; van Triest, M; de Bruijn, KMT; van Willligen, G; Nieuwenhuis, HK; Negrier, C; Akkerman, JWN; Bos, JL

    Rap1, a small GTPase of the Ras family, is ubiquitously expressed and particularly abundant in platelets. Previously we have shown that Rap1 is rapidly activated after stimulation of human platelets with alpha-thrombin. For this activation, a phospholipase C-mediated increase in intracellular

  19. Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5' ends.

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    Runnberg, Rikard; Narayanan, Saishyam; Cohn, Marita

    2017-08-18

    Telomere DNA ends with a single-stranded 3' overhang. Long 3' overhangs may cause aberrant DNA damage responses and accelerate telomere attrition, which is associated with cancer and aging, respectively. Genetic studies have indicated several important players in preventing 5' end hyper-resection, yet detailed knowledge about the molecular mechanism in which they act is still lacking. Here, we use an in vitro DNA 5' end protection assay, to study how N. castellii Cdc13 and Rap1 protect against 5' exonucleolytic degradation by λ-exonuclease. The homogeneous telomeric repeat sequence of N. castellii allows us to study their protection ability at exact binding sites relative to the 5' end. We find efficient protection by both Cdc13 and Rap1 when bound close to the 5' end. Notably, Rap1 provides protection when binding dsDNA at a distance from the 5' end. The DNA binding domain of Rap1 is sufficient for 5' end protection, and its wrapping loop region is essential. Intriguingly, Rap1 facilitates protection also when its binding site contains 2 nt of ssDNA, thus spanning across the ds-ss junction. These results highlight a role of Rap1 in 5' end protection and indicate that Cdc13 and Rap1 have complementary roles in maintaining proper 3' overhang length.

  20. Activation of Rac1 by paxillin-Crk-DOCK180 signaling complex is antagonized by Rap1 in migrating NBT-II cells.

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    Vallés, Ana M; Beuvin, Maud; Boyer, Brigitte

    2004-10-22

    Induction of epithelial cell motility is a fundamental morphogenetic event that is recapitulated during carcinoma metastasis. Random motility of NBT-II carcinoma cells on collagen critically depends on paxillin phosphorylation at Tyr-31 and Tyr-118, the binding sites for the adapter protein CrkII. Two constitutive partners of CrkII are the exchange factors DOCK180 and C3G. CrkII bound to DOCK180 formed a signaling complex with phosphorylated paxillin that was necessary for cell migration as inferred from the inhibition caused by a DOCK180-interfering mutant. DOCK180, which acts predominantly on the Rho family GTPase Rac1, restored cell locomotion in cells expressing Phe-31/118 paxillin mutants deficient in Rac1 GTP-loading, suggesting that formation of paxillin-Crk-DOCK180 signaling complex controls collagen-dependent migration mainly through Rac1 activation. In migrating cells, CrkII constitutive association with C3G was not sufficient to stimulate its GDP/GTP exchange activity toward the Ras family GTPase Rap1. However, when constitutively active RapV12 was overexpressed, it negatively regulated cell motility. Activation of the C3G/Rap1 signaling pathway resulted in down-regulation of the paxillin-Crk-DOCK180 complex and reduction of Rac1-GTP, suggesting that Rap1 activation could suppress the Rac1 signaling pathway in epithelial cells.

  1. New Variations in the Promoter Regions of Human DOCK4 and RAP1A Genes, and Coding Regions of RAP1A in Sporadic Breast Tumors

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    Jalali, Akram; Ebrahimi, Hassan; Ohadi, Mina; Karimloo, Masood; Shemirani, Atena Irani; Mohajer, Behrokh; Khorshid, Hamid Reza Khorram

    2009-01-01

    Breast cancer is the most common cancer among women in developed countries. The prevalence of the disease is increasing in the world. Its annual incidence among Iranian women is about 7000 cases. RAP1A, a tumor suppressor gene, is located at 1p13.3 and plays an important role in the cellular adhesion pathway and is involved in the pathogenesis of breast cancer. The DOCK4 gene, which is located at 7q31.1, specifically activates RAP1A gene. In the present study, DNA samples from 64 cases of spo...

  2. Neuronal Rap1 Regulates Energy Balance, Glucose Homeostasis, and Leptin Actions

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    Kaneko, Kentaro; Xu, Pingwen; Cordonier, Elizabeth L.; Chen, Siyu S.; Ng, Amy; Xu, Yong; Morozov, Alexei; Fukuda, Makoto

    2016-01-01

    The CNS contributes to obesity and metabolic disease; however, the underlying neurobiological pathways remain to be fully established. Here, we show that the small GTPase Rap1 is expressed in multiple hypothalamic nuclei that control whole-body metabolism and is activated in high-fat diet (HFD)-induced obesity. Genetic ablation of CNS Rap1 protects mice from dietary obesity, glucose imbalance, and insulin resistance in the periphery and from HFD-induced neuropathological changes in the hypoth...

  3. New Variations in the Promoter Regions of Human DOCK4 and RAP1A Genes, and Coding Regions of RAP1A in Sporadic Breast Tumors.

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    Jalali, Akram; Ebrahimi, Hassan; Ohadi, Mina; Karimloo, Masood; Shemirani, Atena Irani; Mohajer, Behrokh; Khorshid, Hamid Reza Khorram

    2009-07-01

    Breast cancer is the most common cancer among women in developed countries. The prevalence of the disease is increasing in the world. Its annual incidence among Iranian women is about 7000 cases. RAP1A, a tumor suppressor gene, is located at 1p13.3 and plays an important role in the cellular adhesion pathway and is involved in the pathogenesis of breast cancer. The DOCK4 gene, which is located at 7q31.1, specifically activates RAP1A gene. In the present study, DNA samples from 64 cases of sporadic breast tumors (referred to Mehrad Hospital in Tehran) were screened using PCR-SSCP method and the number of observed variations compared with the control group (100 normal women). Mutation detection for coding exons of RAP1A gene and the 500 bp upstream of transcription initiation site as promoters of both DOCK4 and RAP1A were carried out and compared with the control group. The promoter region of DOCK4 showed a heterozygous mutation with G>A transition at nucleotide -303 in a fibroadenoma case. With regard to RAP1A we found a heterozygous mutation, G>A transition in an adenoid cystic carcinoma case, and another heterozygous mutation, G>T transversion in an intraductal papilloma case both at nucleotide +45. A homozygous variation, T>A transversion was also found at nucleotide +29 of a fibroadenoma case. The differences in the frequency of variations mentioned above were not statistically significant. However Fisher's exact showed significant difference for T>A transversion. Although, the higher frequency of these mutations and variations may be related to the disease, a larger sample size is needed for the confirmation of our findings.

  4. Ubc9 Binds to ADAP and Is Required for Rap1 Membrane Recruitment, Rac1 Activation, and Integrin-Mediated T Cell Adhesion.

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    Xiong, Yiwei; Ye, Chengjin; Yang, Naiqi; Li, Madanqi; Liu, Hebin

    2017-11-10

    Although the immune adaptor adhesion and degranulation-promoting adaptor protein (ADAP) acts as a key mediator of integrin inside-out signaling leading to T cell adhesion, the regulation of this adaptor during integrin activation and clustering remains unclear. We now identify Ubc9, the sole small ubiquitin-related modifier E2 conjugase, as an essential regulator of ADAP where it is required for TCR-induced membrane recruitment of the small GTPase Rap1 and its effector protein RapL and for activation of the small GTPase Rac1 in T cell adhesion. We show that Ubc9 interacted directly with ADAP in vitro and in vivo, and the association was increased in response to anti-CD3 stimulation. The Ubc9-binding domain on ADAP was mapped to a nuclear localization sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by short hairpin RNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCR-induced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, although it had little effect on the TCR proximal signaling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 and RapL, but not Rap1-interacting adaptor molecule. Taken together, our data demonstrate for the first time, to our knowledge, that Ubc9 acts as a functional binding partner of ADAP and plays a selective role in integrin-mediated T cell adhesion via modulation of Rap1-RapL membrane recruitment and Rac1 activation. Copyright © 2017 by The American Association of Immunologists, Inc.

  5. Neuronal Rap1 Regulates Energy Balance, Glucose Homeostasis, and Leptin Actions

    Directory of Open Access Journals (Sweden)

    Kentaro Kaneko

    2016-09-01

    Full Text Available The CNS contributes to obesity and metabolic disease; however, the underlying neurobiological pathways remain to be fully established. Here, we show that the small GTPase Rap1 is expressed in multiple hypothalamic nuclei that control whole-body metabolism and is activated in high-fat diet (HFD-induced obesity. Genetic ablation of CNS Rap1 protects mice from dietary obesity, glucose imbalance, and insulin resistance in the periphery and from HFD-induced neuropathological changes in the hypothalamus, including diminished cellular leptin sensitivity and increased endoplasmic reticulum (ER stress and inflammation. Furthermore, pharmacological inhibition of CNS Rap1 signaling normalizes hypothalamic ER stress and inflammation, improves cellular leptin sensitivity, and reduces body weight in mice with dietary obesity. We also demonstrate that Rap1 mediates leptin resistance via interplay with ER stress. Thus, neuronal Rap1 critically regulates leptin sensitivity and mediates HFD-induced obesity and hypothalamic pathology and may represent a potential therapeutic target for obesity treatment.

  6. Neuronal Rap1 Regulates Energy Balance, Glucose Homeostasis, and Leptin Actions.

    Science.gov (United States)

    Kaneko, Kentaro; Xu, Pingwen; Cordonier, Elizabeth L; Chen, Siyu S; Ng, Amy; Xu, Yong; Morozov, Alexei; Fukuda, Makoto

    2016-09-13

    The CNS contributes to obesity and metabolic disease; however, the underlying neurobiological pathways remain to be fully established. Here, we show that the small GTPase Rap1 is expressed in multiple hypothalamic nuclei that control whole-body metabolism and is activated in high-fat diet (HFD)-induced obesity. Genetic ablation of CNS Rap1 protects mice from dietary obesity, glucose imbalance, and insulin resistance in the periphery and from HFD-induced neuropathological changes in the hypothalamus, including diminished cellular leptin sensitivity and increased endoplasmic reticulum (ER) stress and inflammation. Furthermore, pharmacological inhibition of CNS Rap1 signaling normalizes hypothalamic ER stress and inflammation, improves cellular leptin sensitivity, and reduces body weight in mice with dietary obesity. We also demonstrate that Rap1 mediates leptin resistance via interplay with ER stress. Thus, neuronal Rap1 critically regulates leptin sensitivity and mediates HFD-induced obesity and hypothalamic pathology and may represent a potential therapeutic target for obesity treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Rap1 is indispensable for TRF2 function in etoposide-induced DNA damage response in gastric cancer cell line.

    Science.gov (United States)

    Li, X; Liu, W; Wang, H; Yang, L; Li, Y; Wen, H; Ning, H; Wang, J; Zhang, L; Li, J; Fan, D

    2015-03-30

    The telomeric protein TRF2, involving in telomeric and extratelomeric DNA damage response, has been previously reported to facilitate multidrug resistance (MDR) in gastric cancer cells by interfering ATM-dependent DNA damage response induced by anticancer drugs. Rap1 is the TRF2-interacting protein in the shelterin complex. Complex formation between Rap1 and TRF2 is essential for their function in telomere and end protection. Here we focus on the effects of Rap1 on TRF2 function in DNA damage response induced by anticancer drugs. Both Rap1 and TRF2 expression were upregulated in SGC7901 and its MDR variant SGC7901/VCR after etoposide treatment, which was more marked in SGC7901/VCR than in SGC7901. Rap1 silencing by siRNA in SGC7901/VCR partially reversed the etoposide resistance. And Rap1 silencing partially reversed the TRF2-mediated resistance to etoposide in SGC7901. Rap1 silencing did not affect the TRF2 upregulation induced by etoposide, but eliminated the inhibition effect of TRF2 on ATM expression and ATM phosphorylation at serine 1981 (ATM pS1981). Furthermore, phosphorylation of ATM targets, including γH2AX and serine 15 (S15) on p53, were increased in Rap1 silencing cells in response to etoposide. Thus, we confirm that Rap1, interacting with TRF2 in the shelterin complex, also has an important role in TRF2-mediated DNA damage response in gastric cancer cells treated by etoposide.

  8. SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

    Science.gov (United States)

    Georgiadou, Maria; Jacquemet, Guillaume; De Franceschi, Nicola; Peuhu, Emilia; Hamidi, Hellyeh; Pouwels, Jeroen; Martens, Victoria; Nia, Fatemeh Hassani; Beifuss, Malte; Boeckers, Tobias; Kreienkamp, Hans-Juergen; Barsukov, Igor L; Ivaska, Johanna

    2017-01-01

    SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN-domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN-domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN-domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1/RIAM/talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes. PMID:28263956

  9. SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras.

    Science.gov (United States)

    Lilja, Johanna; Zacharchenko, Thomas; Georgiadou, Maria; Jacquemet, Guillaume; De Franceschi, Nicola; Peuhu, Emilia; Hamidi, Hellyeh; Pouwels, Jeroen; Martens, Victoria; Nia, Fatemeh Hassani; Beifuss, Malte; Boeckers, Tobias; Kreienkamp, Hans-Juergen; Barsukov, Igor L; Ivaska, Johanna

    2017-04-01

    SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.

  10. A higher-order entity formed by the flexible assembly of RAP1 with TRF2.

    Science.gov (United States)

    Gaullier, Guillaume; Miron, Simona; Pisano, Sabrina; Buisson, Rémi; Le Bihan, Yann-Vaï; Tellier-Lebègue, Carine; Messaoud, Wala; Roblin, Pierre; Guimarães, Beatriz G; Thai, Robert; Giraud-Panis, Marie-Josèphe; Gilson, Eric; Le Du, Marie-Hélène

    2016-02-29

    Telomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shelterin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Neuronal Rap1 regulates energy balance, glucose homeostasis, and leptin actions

    Science.gov (United States)

    The Central Nervous System (CNS) contributes to obesity and metabolic disease; however, the underlying neurobiological pathways remain to be fully established. Here, we show that the small GTPase Rap1 is expressed in multiple hypothalamic nuclei that control whole-body metabolism and is activated in...

  12. TRF2-RAP1 is required to protect telomeres from engaging in homologous recombination-mediated deletions and fusions.

    Science.gov (United States)

    Rai, Rekha; Chen, Yong; Lei, Ming; Chang, Sandy

    2016-03-04

    Repressor/activator protein 1 (RAP1) is a highly conserved telomere-interacting protein. Yeast Rap1 protects telomeres from non-homologous end joining (NHEJ), plays important roles in telomere length control and is involved in transcriptional gene regulation. However, a role for mammalian RAP1 in telomere end protection remains controversial. Here we present evidence that mammalian RAP1 is essential to protect telomere from homology directed repair (HDR) of telomeres. RAP1 cooperates with the basic domain of TRF2 (TRF2(B)) to repress PARP1 and SLX4 localization to telomeres. Without RAP1 and TRF2(B), PARP1 and SLX4 HR factors promote rapid telomere resection, resulting in catastrophic telomere loss and the generation of telomere-free chromosome fusions in both mouse and human cells. The RAP1 Myb domain is required to repress both telomere loss and formation of telomere-free fusions. Our results highlight the importance of the RAP1-TRF2 heterodimer in protecting telomeres from inappropriate processing by the HDR pathway.

  13. p53 increases caspase-6 expression and activation in muscle tissue expressing mutant huntingtin

    DEFF Research Database (Denmark)

    Ehrnhoefer, Dagmar E; Skotte, Niels H; Ladha, Safia

    2014-01-01

    a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well...... as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse......-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6...

  14. Loss of phosphodiesterase 4D mediates acquired triapine resistance via Epac-Rap1-Integrin signaling.

    Science.gov (United States)

    Miklos, Walter; Heffeter, Petra; Pirker, Christine; Hager, Sonja; Kowol, Christian R; van Schoonhoven, Sushilla; Stojanovic, Mirjana; Keppler, Bernhard K; Berger, Walter

    2016-12-20

    Triapine, an anticancer thiosemicarbazone, is currently under clinical investigation. Whereas promising results were obtained in hematological diseases, trials in solid tumors widely failed. To understand mechanisms causing triapine insensitivity, we have analysed genomic alterations in a triapine-resistant SW480 subline (SW480/tria). Only one distinct genomic loss was observed specifically in SW480/tria cells affecting the phosphodiesterase 4D (PDE4D) gene locus. Accordingly, pharmacological inhibition of PDE4D resulted in significant triapine resistance in SW480 cells. Hence, we concluded that enhanced cyclic AMP levels might confer protection against triapine. Indeed, hyperactivation of both major downstream pathways, namely the protein kinase A (PKA)-cAMP response element-binding protein (Creb) and the exchange protein activated by cAMP (Epac)-Ras-related protein 1 (Rap1) signaling axes, was observed in SW480/tria cells. Unexpectedly, inhibition of PKA did not re-sensitize SW480/tria cells against triapine. In contrast, Epac activation resulted in distinct triapine resistance in SW480 cells. Conversely, knock-down of Epac expression and pharmacological inhibition of Rap1 re-sensitized SW480/tria cells against triapine. Rap1 is a well-known regulator of integrins. Accordingly, SW480/tria cells displayed enhanced plasma membrane expression of several integrin subunits, enhanced adhesion especially to RGD-containing matrix components, and bolstered activation/expression of the integrin downstream effectors Src and RhoA/Rac. Accordingly, integrin and Src inhibition resulted in potent triapine re-sensitization especially of SW480/tria cells. In summary, we describe for the first time integrin activation based on cAMP-Epac-Rap1 signaling as acquired drug resistance mechanism. combinations of triapine with inhibitors of several steps in this resistance cascade might be feasible strategies to overcome triapine insensitivity of solid tumors.

  15. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

    LENUS (Irish Health Repository)

    McSherry, Elaine A

    2011-03-23

    ABSTRACT: INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF

  16. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

    LENUS (Irish Health Repository)

    McSherry, Elaine A

    2012-02-01

    INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and beta1-integrin, we examined activation of the beta1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and beta1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the beta1-integrin substrate fibronectin. This was accompanied by reduced protein expression of beta1-integrin and its binding partners alphaV- and alpha5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and beta1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between

  17. Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase

    LENUS (Irish Health Repository)

    McSherry, Elaine A

    2011-03-23

    Abstract Introduction The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. Methods MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. Results JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6

  18. Rap1 can bypass the FAK-Src-Paxillin cascade to induce cell spreading and focal adhesion formation.

    Directory of Open Access Journals (Sweden)

    Sarah H Ross

    Full Text Available We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.

  19. The Ras-related Protein, Rap1A, Mediates Thrombin-stimulated, Integrin-dependent Glioblastoma Cell Proliferation and Tumor Growth*

    Science.gov (United States)

    Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha; Quilliam, Lawrence A.; Stupack, Dwayne G.; Brown, Joan Heller

    2014-01-01

    Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo. PMID:24790104

  20. The Ras-related protein, Rap1A, mediates thrombin-stimulated, integrin-dependent glioblastoma cell proliferation and tumor growth.

    Science.gov (United States)

    Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha; Quilliam, Lawrence A; Stupack, Dwayne G; Brown, Joan Heller

    2014-06-20

    Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Overexpression of Rap-1A indicates a poor prognosis for oral cavity squamous cell carcinoma and promotes tumor cell invasion via Aurora-A modulation.

    Science.gov (United States)

    Chen, Chang-Han; Chuang, Hui-Ching; Huang, Chao-Cheng; Fang, Fu-Min; Huang, Hsuan-Ying; Tsai, Hsin-Ting; Su, Li-Jen; Shiu, Li-Yen; Leu, Steve; Chien, Chih-Yen

    2013-02-01

    The functions of Rap-1A in oral carcinogenesis are largely unexplored. In this study, we examined the expression of Rap-1A at different malignant stages of oral cavity squamous cell carcinoma (OCSCC). Semiquantitative RT-PCR, quantitative RT-PCR, and Western blotting were used to evaluate Rap-1A mRNA and protein expressions, respectively, in paired OCSCC patient specimens. To determine the possible correlation between Rap-1A expression and various clinical characteristics, 256 samples from patients with OCSCC were evaluated by immunohistochemical staining. Strong Rap-1A expression was a significant prognostic marker and predictor of aggressive OCSCC. The overall and disease-specific 5-year survival rates were significantly correlated with strong expression of Rap-1A (P oral cancer cell migration and invasion by Transwell chambers and wound healing assay. Conversely, the suppression of Rap-1A expression using Rap-1A-mediated siRNA was sufficient to decrease cell motility. Furthermore, our data also illustrated that Aurora-A could not only induce mRNA and protein expressions of Rap-1A for enhancing cancer cell motility but also co-localize and form a complex with Rap-1A in the oral cancer cell line. Finally, immunohistochemical staining, indirect immunofluorescence, and Western blotting analysis of human aggressive OCSCC specimens revealed a significantly positive correlation between Rap-1A and Aurora-A expression. Taken together, our results suggest that the Aurora-A/Rap-1A pathway is associated with survival, tumor progression, and metastasis of OCSCC patients. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids.

    Science.gov (United States)

    Nanavaty, Vishal; Sandhu, Ranjodh; Jehi, Sanaa E; Pandya, Unnati M; Li, Bibo

    2017-06-02

    Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. p53 increases caspase-6 expression and activation in muscle tissue expressing mutant huntingtin.

    Science.gov (United States)

    Ehrnhoefer, Dagmar E; Skotte, Niels H; Ladha, Safia; Nguyen, Yen T N; Qiu, Xiaofan; Deng, Yu; Huynh, Khuong T; Engemann, Sabine; Nielsen, Signe M; Becanovic, Kristina; Leavitt, Blair R; Hasholt, Lis; Hayden, Michael R

    2014-02-01

    Activation of caspase-6 in the striatum of both presymptomatic and affected persons with Huntington's disease (HD) is an early event in the disease pathogenesis. However, little is known about the role of caspase-6 outside the central nervous system (CNS) and whether caspase activation might play a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53's mitochondrial pro-apoptotic function. Remarkably, the p53-mediated increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6. Furthermore, these results suggest that this pathway is activated both within and outside the CNS in HD and may contribute to both loss of CNS neurons and muscle atrophy.

  4. Mutant p53 accumulates in cycling and proliferating cells in the normal tissues of p53 R172H mutant mice

    Science.gov (United States)

    Leushacke, Marc; Li, Ling; Wong, Julin S.; Chiam, Poh Cheang; Rahmat, Siti Aishah Binte; Mann, Michael B.; Mann, Karen M.; Barker, Nick; Lozano, Guillermina; Terzian, Tamara; Lane, David P.

    2015-01-01

    The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy. PMID:26255629

  5. Rap1 Can Bypass the FAK-Src-Paxillin Cascade to Induce Cell Spreading and Focal Adhesion Formation

    NARCIS (Netherlands)

    Ross, S.H.; Spanjaard, E.; Post, A.; Vliem, M.J.; Kristyanto, H.; Bos, J.L.; de Rooij, J.

    2012-01-01

    We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial

  6. Rap1 Binds Single-stranded DNA at Telomeric Double- and Single-stranded Junctions and Competes with Cdc13 Protein*

    Science.gov (United States)

    Gustafsson, Cecilia; Rhodin Edsö, Jenny; Cohn, Marita

    2011-01-01

    The ends of eukaryotic chromosomes are protected by specialized telomere chromatin structures. Rap1 and Cdc13 are essential for the formation of functional telomere chromatin in budding yeast by binding to the double-stranded part and the single-stranded 3′ overhang, respectively. We analyzed the binding properties of Saccharomyces castellii Rap1 and Cdc13 to partially single-stranded oligonucleotides, mimicking the junction of the double- and single-stranded DNA (ds-ss junction) at telomeres. We determined the optimal and the minimal DNA setup for a simultaneous binding of Rap1 and Cdc13 at the ds-ss junction. Remarkably, Rap1 is able to bind to a partially single-stranded binding site spanning the ds-ss junction. The binding over the ds-ss junction is anchored in a single double-stranded hemi-site and is stabilized by a sequence-independent interaction of Rap1 with the single-stranded 3′ overhang. Thus, Rap1 is able to switch between a sequence-specific and a nonspecific binding mode of one hemi-site. At a ds-ss junction configuration where the two binding sites partially overlap, Rap1 and Cdc13 are competing for the binding. These results shed light on the end protection mechanisms and suggest that Rap1 and Cdc13 act together to ensure the protection of both the 3′ and the 5′ DNA ends at telomeres. PMID:22075002

  7. Significance of Coronavirus Mutants in Feces and Diseased Tissues of Cats Suffering from Feline Infectious Peritonitis

    Directory of Open Access Journals (Sweden)

    Niels C. Pedersen

    2009-08-01

    Full Text Available The internal FECV→FIPV mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of FIP at geographically disparate regions. Coronavirus from feces and extraintestinal FIP lesions from the same cat were always >99% related in accessory and structural gene sequences. SNPs and deletions causing a truncation of the 3c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from FIP, whereas most, but not all fecal isolates from these same cats had intact 3c genes. Other accessory and structural genes appeared normal in both fecal and lesional viruses. Deliterious mutations in the 3c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. Compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. Although 3c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of FIP to its housemate. There was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. More than one variant could be identified in both diseased tissues and feces of the same cat. Laboratory cats inoculated with a mixture of two closely related variants from the same FIP cat developed disease from one or the other variant, but not both. Significant genetic drift existed between isolates from geographically distinct regions of the Western US.

  8. Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character

    Science.gov (United States)

    Fisher, Marilyn; Hirsch, Nicolas; Cox, Amanda; Reeder, Rollin; Carruthers, Samantha; Hall, Amanda; Stemple, Derek L.; Grainger, Robert M.

    2014-01-01

    SUMMARY The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant. PMID:25224223

  9. [The Expression of Pokemon in Endometrial Carcinoma Tissue and the Correlation with Mutant p53].

    Science.gov (United States)

    Yi, Tian-jin; Wang, Ping

    2016-05-01

    To detect the expression of Pokemon in endometrial carcinoma (EC), to provide preliminary theoretical basis for clarifying pathogenesis and searching for effective targets. Ninety-eight cases of endometrial tissue paraffin specimens form July 2012 to July 2014 in West China Second University Hospital, Sichuan University, were collected, including: EC group, consisting of adenocarcinoma 23 cases, adenosquamous 12 cases, serous 3 cases, mucinous 11 cases and clear cell 9 cases, and control group, consisting of atypical hyperplasia endometrium 20 cases and normal endometrium 20 cases (secretory 10 cases, hyperplasia 10 cases). Immunohistochemistry was used to detect the expression of Pokemonin each section, analyzing the correlation of Pokemon expression with clinicopathologic characteristics and p53 expression. The positive rate of Pokemon in normal endometrium was 25% (5/20), significantly lower than that in atypical hyperplasia endometrium (60.0%, 12/20) and EC (93.1%, 54/58) (P Pokemon in III-IV stage, type II and Ki-67 ≥ 50 EC tissue was much higher (P = 0.012, 0.023, 0.029). In type II EC tissue, the correlation index between Pokemon and p53 is 0.669 (P = 0.000). The over expression of Pokemon upregulates the expression of mutant p53, which may be one of the carcinogenesis modes in type II EC.

  10. Structural Basis for Small G Protein Effector Interaction of Ras-related Protein 1 (Rap1) and Adaptor Protein Krev Interaction Trapped 1 (KRIT1)

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaofeng; Zhang, Rong; Draheim, Kyle M.; Liu, Weizhi; Calderwood, David A.; Boggon, Titus J. (Yale-MED)

    2012-09-17

    Cerebral cavernous malformations (CCMs) affect 0.1-0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with {approx}40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 {angstrom} co-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain 'modules.'

  11. Genetic diversity and natural selection in the rhoptry-associated protein 1 (RAP-1) of recent Plasmodium knowlesi clinical isolates from Malaysia.

    Science.gov (United States)

    Rawa, Mira Syahfriena Amir; Fong, Mun-Yik; Lau, Yee-Ling

    2016-02-05

    The Plasmodium rhoptry-associated protein 1 (RAP-1) plays a role in the formation of the parasitophorous vacuole following the parasite's invasion of red blood cells. Although there is some evidence that the protein is recognized by the host's immune system, study of Plasmodium falciparum RAP-1 (PfRAP-1) suggests that it is not under immune pressure. A previous study on five old (1953-1962) P. knowlesi strains suggested that RAP-1 has limited genetic polymorphism and might be under negative selection. In the present study, 30 recent P. knowlesi isolates were studied to obtain a better insight into the polymorphism and natural selection of PkRAP-1. Blood samples from 30 knowlesi malaria patients were used. These samples were collected between 2010 and 2014. The PkRAP-1 gene, which contains two exons, was amplified by PCR, cloned into Escherichia coli and sequenced. Genetic diversity and phylogenetic analyses were performed using MEGA6 and DnaSP ver. 5.10.00 programs. Thirty PkRAP-1 sequences were obtained. The nucleotide diversity (π) of exons 1, 2 and the total coding region (0.00915, 0.01353 and 0.01298, respectively) were higher than those of the old strains. Further analysis revealed a lower rate of non-synonymous (dN) than synonymous (dS) mutations, suggesting negative (purifying) selection of PkRAP-1. Tajima's D test and Fu and Li's D test values were not significant. At the amino acid level, 22 haplotypes were established with haplotype H7 having the highest frequency (7/34, 20.5 %). In the phylogenetic analysis, two distinct haplotype groups were observed. The first group contained the majority of the haplotypes, whereas the second had fewer haplotypes. The present study found higher genetic polymorphism in the PkRAP-1 gene than the polymorphism level reported in a previous study. This observation may stem from the difference in sample size between the present (n = 30) and the previous (n = 5) study. Synonymous and non-synonymous mutation analysis indicated

  12. The signaling module cAMP/Epac/Rap1/PLCε/IP3 mobilizes acrosomal calcium during sperm exocytosis.

    Science.gov (United States)

    Lucchesi, Ornella; Ruete, María C; Bustos, Matías A; Quevedo, María F; Tomes, Claudia N

    2016-04-01

    Exocytosis of the sperm's single secretory granule, or acrosome, is a regulated exocytosis triggered by components of the egg's investments. In addition to external calcium, sperm exocytosis (termed the acrosome reaction) requires cAMP synthesized endogenously and calcium mobilized from the acrosome through IP3-sensitive channels. The relevant cAMP target is Epac. In the first part of this paper, we present a novel tool (the TAT-cAMP sponge) to investigate cAMP-related signaling pathways in response to progesterone as acrosome reaction trigger. The TAT-cAMP sponge consists of the cAMP-binding sites of protein kinase A regulatory subunit RIβ fused to the protein transduction domain TAT of the human immunodeficiency virus-1. The sponge permeated into sperm, sequestered endogenous cAMP, and blocked exocytosis. Progesterone increased the population of sperm with Rap1-GTP, Rab3-GTP, and Rab27-GTP in the acrosomal region; pretreatment with the TAT-cAMP sponge prevented the activation of all three GTPases. In the second part of this manuscript, we show that phospholipase Cε (PLCε) is required for the acrosome reaction downstream of Rap1 and upstream of intra-acrosomal calcium mobilization. Last, we present direct evidence that cAMP, Epac, Rap1, and PLCε are necessary for calcium mobilization from sperm's secretory granule. In summary, we describe here a pathway that connects cAMP to calcium mobilization from the acrosome during sperm exocytosis. Never before had direct evidence for each step of the cascade been put together in the same study. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. A novel approach to prevent endothelial hyperpermeability: the Crataegus extract WS® 1442 targets the cAMP/Rap1 pathway.

    Science.gov (United States)

    Bubik, Martin F; Willer, Elisabeth A; Bihari, Peter; Jürgenliemk, Guido; Ammer, Hermann; Krombach, Fritz; Zahler, Stefan; Vollmar, Angelika M; Fürst, Robert

    2012-01-01

    Endothelial hyperpermeability followed by edema formation is a hallmark of many severe disorders. Effective drugs directly targeting endothelial barrier function are widely lacking. We hypothesized that the hawthorn (Crataegus spp.) extract WS® 1442, a proven multi-component drug against moderate forms of heart failure, would prevent vascular leakage by affecting endothelial barrier-regulating systems. In vivo, WS® 1442 inhibited the histamine-evoked extravasation of FITC-dextran from mouse cremaster muscle venules. In cultured human endothelial cells, WS® 1442 blocked the thrombin-induced FITC-dextran permeability. By applying biochemical and microscopic techniques, we revealed that WS® 1442 abrogates detrimental effects of thrombin on adherens junctions (vascular endothelial-cadherin), the F-actin cytoskeleton, and the contractile apparatus (myosin light chain). Mechanistically, WS® 1442 inhibited the thrombin-induced rise of intracellular calcium (ratiometric measurement), followed by an inactivation of PKC and RhoA (pulldown assay). Moreover, WS® 1442 increased endothelial cAMP levels (ELISA), which consequently activated PKA and Rap1 (pulldown assay). Utilizing pharmacological inhibitors or siRNA, we found that PKA is not involved in barrier protection, whereas Epac1, Rap1, and Rac1 play a crucial role in the WS® 1442-induced activation of cortactin, which triggers a strong cortical actin rearrangement. In summary, WS® 1442 effectively protects against endothelial barrier dysfunction in vitro and in vivo. It specifically interacts with endothelial permeability-regulating systems by blocking the Ca(2+)/PKC/RhoA and activating the cAMP/Epac1/Rap1 pathway. As a proven safe herbal drug, WS® 1442 opens a novel pharmacological approach to treat hyperpermeability-associated diseases. This in-depth mechanistic work contributes to a better acceptance of this herbal remedy. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. miR-203a is involved in HBx-induced inflammation by targeting Rap1a

    Energy Technology Data Exchange (ETDEWEB)

    Wu, AiRong [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Chen, Huo [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Xu, ChunFang [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Zhou, Ji; Chen, Si [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Shi, YuQi [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Xu, Jie [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Gan, JianHe, E-mail: j_pzhang@suda.edu.cn [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Zhang, JinPing, E-mail: ganjianhe@aliyun.com [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China)

    2016-11-15

    Hepatitis B virus (HBV) causes acute and chronic hepatitis, and is one of the major causes of cirrhosis and hepatocellular carcinoma. Accumulating evidence suggests that inflammation is the key factor for liver cirrhosis and hepatocellular carcinoma. MicroRNAs play important roles in many biological processes. Here, we aim to explore the function of microRNAs in the HBX-induced inflammation. First, microarray experiment showed that HBV{sup +} liver samples expressed higher level of miR-203a compared to HBV{sup -} liver samples. To verify these alterations, HBx-coding plasmid was transfected into HepG2 cells to overexpress HBx protein. The real-time PCR results suggested that over-expression of HBx could induce up-regulation of miR-203a. To define how up-regulation of miR-203a can induce liver cells inflammation, we over-expressed miR-203a in HepG2 cells. Annexin V staining and BrdU staining suggested that overexpression of miR-203a significantly increased the cell apoptosis and proliferation, meanwhile, over-expression of miR-203a could lead to a decrease in G0/G1 phase cells and an increase in G2/M phase cells. Some cytokines production including IL-6 and IL-8 were significantly increased, but TGFβ and IFNγ were decreased in miR-203a over-expressed HepG2 cells. Luciferase reporter assay experiments, protein mass-spectrum assay and real-time PCR all together demonstrated that Rap1a was the target gene of miR-203a. Further experiments showed that these alterations were modulated through PI3K/ERK/p38/NFκB pathways. These data suggested that HBV-infection could up-regulate the expression of miR-203a, thus down regulated the expression of Rap1a and affected the PI3K/ERK/p38/NFκB pathways, finally induced the hepatitis inflammation. - Highlights: • HBX induces the over-expression of miR-203a in HepG2 cells. • miR-203a targets Rap1a to induce the inflammation in HepG2 cells. • miR-203a regulates the apoptosis and cell cycles of HepG2 cells. • miR-203a alters

  15. Basic domain of telomere guardian TRF2 reduces D-loop unwinding whereas Rap1 restores it.

    Science.gov (United States)

    Necasová, Ivona; Janoušková, Eliška; Klumpler, Tomáš; Hofr, Ctirad

    2017-09-13

    Telomeric repeat binding factor 2 (TRF2) folds human telomeres into loops to prevent unwanted DNA repair and chromosome end-joining. The N-terminal basic domain of TRF2 (B-domain) protects the telomeric displacement loop (D-loop) from cleavage by endonucleases. Repressor activator protein 1 (Rap1) binds TRF2 and improves telomeric DNA recognition. We found that the B-domain of TRF2 stabilized the D-loop and thus reduced unwinding by BLM and RPA, whereas the formation of the Rap1-TRF2 complex restored DNA unwinding. To understand how the B-domain of TRF2 affects DNA binding and D-loop processing, we analyzed DNA binding of full-length TRF2 and a truncated TRF2 construct lacking the B-domain. We quantified how the B-domain improves TRF2's interaction with DNA via enhanced long-range electrostatic interactions. We developed a structural envelope model of the B-domain bound on DNA. The model revealed that the B-domain is flexible in solution but becomes rigid upon binding to telomeric DNA. We proposed a mechanism for how the B-domain stabilizes the D-loop. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Ontogenetically-regulated male sterility in tissue culture - induced and spontaneous sorghum mutants

    Directory of Open Access Journals (Sweden)

    Elkonin L.A.

    2003-01-01

    Full Text Available Variability of male fertility expression in the AS-1 line, a somaclonal variant obtained from tissue culture of CMS-plant, and in the progeny of revenant '124-1' obtained from fertile tiller, which developed on CMS-plant transferred from the field to the greenhouse, was investigated. Both revertants were characterized by similar expression of male fertility during plant ontogenesis: the panicle on the main tiller was almost completely sterile whereas formation of fertile pollen grains and seed set were observed on the panicles of the shoot tillers. A clear basipetal gradient of male fertility was manifested on all panicles: the base had significantly higher per cent of fertile pollen grains in comparison with the middle part, while in the top the anthers were either absent or had few sterile pollen grains. Such an ontogenetically-regulated restoration of male fertility was controlled by nuclear genes and could be transferred through the pollen in crosses with progenitor CMS-line. Growing of AS-1 plants in the growth chambers simultaneously under a long (16/8 and a short (12/12 daylength conditions demonstrated that differences of fertility level in different tillers was not caused by change of photoperiod during plant ontogenesis and functioning of photoperiod-sensitive fertility restoring gene. Whereas, the ontogenetically-regulated expression of male fertility in both revenants was temperature-dependent and was clearly manifested under relatively cool conditions during 2-week period before the beginning of anthesis of the first panicle (average daily temperature 21°C. The increase of the average daily temperature by 2-3 С resulted in sharp increase of male fertility level. Possibility of using AS-1 line in a new "two-line system" of hybrid seed production, which require only two lines (sterile mutant and fertility restorer, is discussed.

  17. PI3-kinase γ promotes Rap1a-mediated activation of myeloid cell integrin α4β1, leading to tumor inflammation and growth.

    Directory of Open Access Journals (Sweden)

    Michael C Schmid

    Full Text Available Tumor inflammation, the recruitment of myeloid lineage cells into the tumor microenvironment, promotes angiogenesis, immunosuppression and metastasis. CD11b+Gr1lo monocytic lineage cells and CD11b+Gr1hi granulocytic lineage cells are recruited from the circulation by tumor-derived chemoattractants, which stimulate PI3-kinase γ (PI3Kγ-mediated integrin α4 activation and extravasation. We show here that PI3Kγ activates PLCγ, leading to RasGrp/CalDAG-GEF-I&II mediated, Rap1a-dependent activation of integrin α4β1, extravasation of monocytes and granulocytes, and inflammation-associated tumor progression. Genetic depletion of PLCγ, CalDAG-GEFI or II, Rap1a, or the Rap1 effector RIAM was sufficient to prevent integrin α4 activation by chemoattractants or activated PI3Kγ (p110γCAAX, while activated Rap (RapV12 promoted constitutive integrin activation and cell adhesion that could only be blocked by inhibition of RIAM or integrin α4β1. Similar to blockade of PI3Kγ or integrin α4β1, blockade of Rap1a suppressed both the recruitment of monocytes and granulocytes to tumors and tumor progression. These results demonstrate critical roles for a PI3Kγ-Rap1a-dependent pathway in integrin activation during tumor inflammation and suggest novel avenues for cancer therapy.

  18. [Expression of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia].

    Science.gov (United States)

    Wang, Yan; Xu, Rui-Rong; Zhou, Yan-Feng; Liu, Zhao-Xia

    2012-10-01

    This study was aimed to investigate the expression levels of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia, and to explore their relation with onset of acquired aplastic anemia. Peripheral blood mononuclear cells of 40 patients with acquired aplastic anemia and 20 normal subjects as control were collected to detect mRNA expression of TRF1, TRF2 and RAP1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of TRF1 and RAP1 in peripheral blood mononuclear cells of patients with acquired aplastic anemia were significantly higher than that in normal controls (P TRF2 was lower than that in normal controls (P TRF2 and RAP1 expressions level (r = 0.522, P = 0.001). It is concluded that the changes in expression levels of TRF1, TRF2 and RAP1 may play a role in the pathogenesis of acquired aplastic anemia.

  19. Tissue persistence and vaccine efficacy of tricarboxylic acid cycle and one-carbon metabolism mutant strains of Edwardsiella ictaluri.

    Science.gov (United States)

    Dahal, Neeti; Abdelhamed, Hossam; Karsi, Attila; Lawrence, Mark L

    2014-06-30

    Edwardsiella ictaluri causes enteric septicemia in fish. Recently, we reported construction of E. ictaluri mutants with single and double gene deletions in tricarboxylic acid cycle (TCA) and one-carbon (C-1) metabolism. Here, we report the tissue persistence, virulence, and vaccine efficacy of TCA cycle (EiΔsdhC, EiΔfrdA, and EiΔmdh), C-1 metabolism (EiΔgcvP and EiΔglyA), and combination mutants (EiΔfrdAΔsdhC, EiΔgcvPΔsdhC, EiΔmdhΔsdhC, and EiΔgcvPΔglyA) in channel catfish. The tissue persistence study showed that EiΔsdhC, EiΔfrdA, EiΔfrdAΔsdhC, and EiΔgcvPΔsdhC were able to invade catfish and persist until 11 days post-infection. Vaccination of catfish fingerlings with all nine mutants provided significant (P<0.05) protection against subsequent challenge with the virulent parental strain. Vaccinated catfish fingerlings had 100% survival when subsequently challenged by immersion with wild-type E. ictaluri except for EiΔgcvPΔglyA and EiΔgcvP. Mutant EiΔgcvPΔsdhC was found to be very good at protecting catfish fry, as evidenced by 10-fold higher survival compared to non-vaccinated fish. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. The role of Rap1 in cell-cell junction formation

    NARCIS (Netherlands)

    Kooistra, M.R.H.

    2008-01-01

    Both epithelial and endothelial cells form cell-cell junctions at the cell-cell contacts to maintain tissue integrity. Proper regulation of cell-cell junctions is required for the organisation of the tissue and to prevent leakage of blood vessels. In endothelial cells, the cell-cell junctions are

  1. High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.

    Directory of Open Access Journals (Sweden)

    Ehud Goldin

    Full Text Available Gaucher disease (GD, the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase. Previously, wildtype GCase was used for high throughput screening (HTS of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

  2. Antibody responses to Rhoptry-Associated Protein-1 (RAP-1) of Plasmodium falciparum parasites in humans from areas of different malaria endemicity

    DEFF Research Database (Denmark)

    Jakobsen, P H; Kurtzhals, J A; Riley, E M

    1997-01-01

    Plasma IgM and IgG antibody reactivities against the recombinant Plasmodium falciparum protein, Rhoptry Associated Protein-1 (rRAP-1) were measured by ELISA in individuals from Sudan, Indonesia, Kenya and The Gambia living in areas of different malaria endemicity. IgG and IgM reactivities to rRAP...

  3. Pituitary adenylate cyclase-activating polypeptide stimulates glial fibrillary acidic protein gene expression in cortical precursor cells by activating Ras and Rap1.

    Science.gov (United States)

    Lastres-Becker, Isabel; Fernández-Pérez, Antonio; Cebolla, Beatriz; Vallejo, Mario

    2008-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on cortical precursor cells to trigger glial fibrillary acidic protein (GFAP) gene expression and astrocyte differentiation by stimulation of intracellular cAMP production. Here, we show that as expected, PACAP activates cAMP-dependent protein kinase A. However, inhibition of protein kinase A does not prevent PACAP-induced GFAP gene expression or astrocytogenesis. PACAP also activates the small GTPases Rap1 and Ras, but either activation of Rap1 alone by selective stimulation of the guanine nucleotide exchange factor Epac, or expression of a constitutively active form of Ras, do not induce GFAP gene expression. Ras is activated by PACAP in a cAMP-dependent manner, and inhibition of Ras and/or Rap1 decreases PACAP-induced GFAP promoter stimulation. Thus, cAMP-dependent PACAP-induced GFAP expression during astrocytogenesis involves the coordinated activation of both Ras and Rap1, but activation of either one of them in isolation is not sufficient to trigger this response.

  4. Normalization of periodontal tissues in osteopetrotic mib mutant rats, treated with CSF-1

    Science.gov (United States)

    Wojtowicz, A.; Yamauchi, M.; Sotowski, R.; Ostrowski, K.

    1998-01-01

    The osteopetrotic mib mutation in rats causes defects in the skeletal bone tissue in young animals. These defects, i.e. slow bone remodelling, changes in both crystallinity and mineral content, are transient and undergo normalization, even without any treatment in 6-wk-old animals. Treatment with CSF-1 (colony stimulating factor-1) accelerates the normalization process in skeletal bones. The periodontal tissues around the apices of incisors show abnormalities caused by the slow remodelling process of the mandible bone tissue, the deficiency of osteoclasts and their abnormal morphology, as well as the disorganization of periodontal ligament fibres. In contrast to the skeletal tissues, these abnormalities would not undergo spontaneous normalization. Under treatment with colony stimulating factor 1 (CSF-1), the primitive bone trabeculae of mandible are resorbed and the normalization of the number of osteoclasts and their cytology occurs. The organization of the periodontal ligament fibres is partially restored, resembling the histological structure of the normal one.

  5. Expanding the body mass range: associations between BMR and tissue morphology in wild type and mutant dwarf mice (David mice).

    Science.gov (United States)

    Meyer, Carola W; Neubronner, Juliane; Rozman, Jan; Stumm, Gabi; Osanger, Andreas; Stoeger, Claudia; Augustin, Martin; Grosse, Johannes; Klingenspor, Martin; Heldmaier, Gerhard

    2007-02-01

    We sought to identify associations of basal metabolic rate (BMR) with morphological traits in laboratory mice. In order to expand the body mass (BM) range at the intra-strain level, and to minimize relevant genetic variation, we used male and female wild type mice (C3HeB/FeJ) and previously unpublished ENU-induced dwarf mutant littermates (David mice), covering a body mass range from 13.5 g through 32.3 g. BMR was measured at 30 degrees C, mice were killed by means of CO(2 )overdose, and body composition (fat mass and lean mass) was subsequently analyzed by dual X-ray absorptiometry (DEXA), after which mice were dissected into 12 (males) and 10 (females) components, respectively. Across the 44 individuals, 43% of the variation in the basal rates of metabolism was associated with BM. The latter explained 47% to 98% of the variability in morphology of the different tissues. Our results demonstrate that sex is a major determinant of body composition and BMR in mice: when adjusted for BM, females contained many larger organs, more fat mass, and less lean mass compared to males. This could be associated with a higher mass adjusted BMR in females. Once the dominant effects of sex and BM on BMR and tissue mass were removed, and after accounting for multiple comparisons, no further significant association between individual variation in BMR and tissue mass emerged.

  6. Tissue and protoplast culture studies in Lycopersicon esculentum miller var. flammatum lehm. cv. 'Bonner Beste' and its mutant chloronerva.

    Science.gov (United States)

    Koblitz, H; Koblitz, D

    1983-08-01

    Methods of shoot and plant development from cotyledon explants of the tomato cultivar 'Bonner Beste' and its mutant chloronerva are described. Cotyledon mesophyll protoplasts of the mutant chloronerva have been isolated and cultured, and induced to regenerate shoots and plants. By grafting regenerated shoots of the chloronerva mutant on rootstocks of the cultivar 'Bonner Beste' plants were obtained producing fruits and seeds. Plants derived from these seeds show all the typical characteristics of the chloronerva mutant.

  7. Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1).

    Science.gov (United States)

    Fang, Zhenhao; Marshall, Christopher B; Yin, Jiani C; Mazhab-Jafari, Mohammad T; Gasmi-Seabrook, Geneviève M C; Smith, Matthew J; Nishikawa, Tadateru; Xu, Yang; Neel, Benjamin G; Ikura, Mitsuhiko

    2016-07-22

    RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Design of High-Affinity Stapled Peptides To Target the Repressor Activator Protein 1 (RAP1)/Telomeric Repeat-Binding Factor 2 (TRF2) Protein-Protein Interaction in the Shelterin Complex.

    Science.gov (United States)

    Ran, Xu; Liu, Liu; Yang, Chao-Yie; Lu, Jianfeng; Chen, Yong; Lei, Ming; Wang, Shaomeng

    2016-01-14

    Shelterin, a six-protein complex, plays a fundamental role in protecting both the length and the stability of telomeres. Repressor activator protein 1 (RAP1) and telomeric repeat-binding factor 2 (TRF2) are two subunits in shelterin that interact with each other. Small-molecule inhibitors that block the RAP1/TRF2 protein-protein interaction can disrupt the structure of shelterin and may be employed as pharmacological tools to investigate the biology of shelterin. On the basis of the cocrystal structure of RAP1/TRF2 complex, we have developed first-in-class triazole-stapled peptides that block the protein-protein interaction between RAP1 and TRF2. Our most potent stapled peptide binds to RAP1 protein with a Ki value of 7 nM and is >100 times more potent than the corresponding wild-type TRF2 peptide. On the basis of our high-affinity peptides, we have developed and optimized a competitive, fluorescence polarization (FP) assay for accurate and rapid determination of the binding affinities of our designed compounds and this assay may also assist in the discovery of non-peptide, small-molecule inhibitors capable of blocking the RAP1/TRF2 protein-protein interaction.

  9. Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

    Science.gov (United States)

    Chantreau, Maxime; Portelette, Antoine; Dauwe, Rebecca; Kiyoto, Shingo; Crônier, David; Morreel, Kris; Arribat, Sandrine; Neutelings, Godfrey; Chabi, Malika; Boerjan, Wout; Yoshinaga, Arata; Mesnard, François; Grec, Sebastien; Chabbert, Brigitte; Hawkins, Simon

    2014-11-01

    Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply. © 2014 American Society of Plant Biologists. All rights reserved.

  10. Ectopic Lignification in the Flax lignified bast fiber1 Mutant Stem Is Associated with Tissue-Specific Modifications in Gene Expression and Cell Wall Composition[C][W

    Science.gov (United States)

    Chantreau, Maxime; Portelette, Antoine; Dauwe, Rebecca; Kiyoto, Shingo; Crônier, David; Morreel, Kris; Arribat, Sandrine; Neutelings, Godfrey; Chabi, Malika; Boerjan, Wout; Yoshinaga, Arata; Mesnard, François; Grec, Sebastien; Chabbert, Brigitte; Hawkins, Simon

    2014-01-01

    Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply. PMID:25381351

  11. Rapgef2, a guanine nucleotide exchange factor for Rap1 small GTPases, plays a crucial role in adherence junction (AJ) formation in radial glial cells through ERK-mediated upregulation of the AJ-constituent protein expression.

    Science.gov (United States)

    Farag, Maged Ibrahim; Yoshikawa, Yoko; Maeta, Kazuhiro; Kataoka, Tohru

    2017-11-04

    Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap1, characterized by possession of the Ras/Rap-associating domains and implicated in the etiology of schizophrenia. We previously found that dorsal telencephalon-specific Rapgef2 conditional knockout mice exhibits severe defects in formation of apical surface adherence junctions (AJs) and localization of radial glial cells (RGCs). In this study, we analyze the underlying molecular mechanism by using primary cultures of RGCs established from the developing cerebral cortex. The results show that Rapgef2-deficient RGCs exhibit a decreased ability of neurosphere formation, morphological changes represented by regression of radial glial (RG) fibers and reduced expression of AJ-constituent proteins such as N-cadherin, zonula occludens-1, E-cadherin and β-catenin. Moreover, siRNA-mediated knockdown of Rapgef2 or Rap1A inhibits the AJ protein expression and RG fiber formation while overexpression of Rapgef2, Rapgef6, Rap1AG12V or Rap1BG12V in Rapgef2-deficient RGCs restores them. Furthermore, Rapgef2-deficient RGCs exhibit a reduction in phosphorylation of extracellular signal-regulated kinase (ERK) leading to downregulation of the expression of c-jun, which is implicated in the AJ protein expression. These results indicate a crucial role of the Rapgef2-Rap1A-ERK-c-jun pathway in regulation of the AJ formation in RGCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Alzheimer’s Disease Mutant Mice Exhibit Reduced Brain Tissue Stiffness Compared to Wild-type Mice in both Normoxia and following Intermittent Hypoxia Mimicking Sleep Apnea

    Directory of Open Access Journals (Sweden)

    Maria José Menal

    2018-01-01

    Full Text Available BackgroundEvidence from patients and animal models suggests that obstructive sleep apnea (OSA may increase the risk of Alzheimer’s disease (AD and that AD is associated with reduced brain tissue stiffness.AimTo investigate whether intermittent hypoxia (IH alters brain cortex tissue stiffness in AD mutant mice exposed to IH mimicking OSA.MethodsSix-eight month old (B6C3-Tg(APPswe,PSEN1dE985Dbo/J AD mutant mice and wild-type (WT littermates were subjected to IH (21% O2 40 s to 5% O2 20 s; 6 h/day or normoxia for 8 weeks. After euthanasia, the stiffness (E of 200-μm brain cortex slices was measured by atomic force microscopy.ResultsTwo-way ANOVA indicated significant cortical softening and weight increase in AD mice compared to WT littermates, but no significant effects of IH on cortical stiffness and weight were detected. In addition, reduced myelin was apparent in AD (vs. WT, but no significant differences emerged in the cortex extracellular matrix components laminin and glycosaminoglycans when comparing baseline AD and WT mice.ConclusionAD mutant mice exhibit reduced brain tissue stiffness following both normoxia and IH mimicking sleep apnea, and such differences are commensurate with increased edema and demyelination in AD.

  13. Specific maceration and induction of PR-3 gene in potato tuber tissue by Pectobacterium carotovorum subsp. atrosepticum type III secretion system mutants.

    Science.gov (United States)

    Aghabozorgy, Sohrab; Niakan, Mohammad

    2009-12-15

    The exact function of type III secretion system in some phytopathogenes including Pectobacterium carotovorum subsp. atrosepticum (Pca) is not understood and is a matter of debate. The aim of this study were to determine specific effect of type III secretion system on potato tubers and to reveal the connection of this system with potato resistant genes such as PR-3. A Pca hrpW fragment was subcloned into a low-copy-number cloning vector (pZH448). The resulting plasmid (pAS19) was then conjugated into the wild-type and mutant strains of Pca by type III secretion system. The virulence property of different Pca strains was studied and the influence of over expression of hrpW on maceration activity was also investigated. Furthermore, the effect of mentioned mutation on the maceration of carrot-root was evaluated. Finally, using real-time PCR, the copy-number of PR-3 gene in potato tuber tissue was assessed. In conclusion, for type III secretion system mutant strains, in contrast with the wild-type, the maceration amount of potato tuber tissue decreased after over expression of hrpW while inoculation of tubers by mutants, increased this amount. In the case of potato, HrpN and DspE proteins appeared to be avirulent factors. Compared with the wild-type strains, Pca nominated mutants significantly reduced potato PR-3 expression thus, PR-3 expression level in potato tuber tissue in answer to infiltration by Pca, depends on functional type III secretion system in the bacterium.

  14. Sensitive detection of KRAS mutations in archived formalin-fixed paraffin-embedded tissue using mutant-enriched PCR and reverse-hybridization.

    Science.gov (United States)

    Ausch, Christoph; Buxhofer-Ausch, Veronika; Oberkanins, Christian; Holzer, Barbara; Minai-Pour, Michael; Jahn, Stephan; Dandachi, Nadia; Zeillinger, Robert; Kriegshäuser, Gernot

    2009-11-01

    Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.

  15. Kinetic characterization of tissue-type plasminogen activator (t-PA) and t-PA deletion mutants

    NARCIS (Netherlands)

    de Vries, C. [=Carlie J. M.; Veerman, H.; Nesheim, M. E.; Pannekoek, H.

    1991-01-01

    The binding of t-PA to fibrin is mediated both by its "finger" (F) and its "kringle 2" (K2) domain. In addition, these domains are involved in the stimulation of t-PA activity by fibrin. We analyzed the kinetic characteristics of Glu-plasminogen activation by t-PA and a set of t-PA deletion mutants

  16. Mutant Wars2 gene in spontaneously hypertensive rats impairs brown adipose tissue function and predisposes to visceral obesity

    Czech Academy of Sciences Publication Activity Database

    Pravenec, Michal; Zídek, Václav; Landa, Vladimír; Mlejnek, Petr; Šilhavý, Jan; Šimáková, Miroslava; Trnovská, J.; Škop, V.; Marková, I.; Malínská, H.; Hüttl, M.; Kazdová, L.; Bardová, Kristina; Tauchmannová, Kateřina; Vrbacký, Marek; Nůsková, Hana; Mráček, Tomáš; Kopecký, Jan; Houštěk, Josef

    2017-01-01

    Roč. 66, č. 6 (2017), s. 917-924 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA13-04420S Institutional support: RVO:67985823 Keywords : brown adipose tissue * spontaneously hypertensive rat * quantitative trait loci * transgenic * Wars2 gene * mitochondrial proteosynthesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.461, year: 2016

  17. Performance of peanut mutants and their offspring generated from mixed high-energy particle field radiation and tissue culture.

    Science.gov (United States)

    Wang, J S; Qiao, L X; Zhao, L S; Wang, P; Guo, B T; Liu, L X; Sui, J M

    2015-09-09

    To develop new ways to breed peanut, we irradiated seeds of the Luhua 11 cultivar with a mixed high-energy particle field at different doses. The embryonic leaflets were extracted as explants and incubated on somatic embryo induction medium and then on somatic embryo germination and regeneration medium. After being grafted, the M1-generation plants were transplanted, and seeds from each M1-generation plant were harvested. In the following year, the M2-generation seeds were planted separately. Some M2-generation plants showed distinct character segregation relative to the mutagenic parent in terms of vigor, fertility, plant height, branch number, and pod size and shape. M2-generation plants that had a high pod weight per plant tended to produce M3-generation offspring that also had a high pod weight per plant, much higher than that of the mutagenic parent, Luhua 11. M4-generation seeds varied greatly in quality, and 35 individuals with an increased fat content (>55%) were obtained. Overall, the results indicate that the combination of mutagenesis via mixed high-energy particle field exposure and tissue culture is promising for peanut breeding.

  18. ATM mutants

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. ATM mutants. ATM (Ataxia Telangiectasia Mutated). AT2BE and AT5B1 cells – fibroblast cell lines from Ataxia telangiectasia patients. Deletion mutants expressing truncated ATM protein which is inactive. Have been used in studies looking at the role of ATM in DNA damage ...

  19. Genome-wide identification and expression profiling reveal tissue-specific expression and differentially-regulated genes involved in gibberellin metabolism between Williams banana and its dwarf mutant.

    Science.gov (United States)

    Chen, Jingjing; Xie, Jianghui; Duan, Yajie; Hu, Huigang; Hu, Yulin; Li, Weiming

    2016-05-27

    Dwarfism is one of the most valuable traits in banana breeding because semi-dwarf cultivars show good resistance to damage by wind and rain. Moreover, these cultivars present advantages of convenient cultivation, management, and so on. We obtained a dwarf mutant '8818-1' through EMS (ethyl methane sulphonate) mutagenesis of Williams banana 8818 (Musa spp. AAA group). Our research have shown that gibberellins (GAs) content in 8818-1 false stems was significantly lower than that in its parent 8818 and the dwarf type of 8818-1 could be restored by application of exogenous GA3. Although GA exerts important impacts on the 8818-1 dwarf type, our understanding of the regulation of GA metabolism during banana dwarf mutant development remains limited. Genome-wide screening revealed 36 candidate GA metabolism genes were systematically identified for the first time; these genes included 3 MaCPS, 2 MaKS, 1 MaKO, 2 MaKAO, 10 MaGA20ox, 4 MaGA3ox, and 14 MaGA2ox genes. Phylogenetic tree and conserved protein domain analyses showed sequence conservation and divergence. GA metabolism genes exhibited tissue-specific expression patterns. Early GA biosynthesis genes were constitutively expressed but presented differential regulation in different tissues in Williams banana. GA oxidase family genes were mainly transcribed in young fruits, thus suggesting that young fruits were the most active tissue involved in GA metabolism, followed by leaves, bracts, and finally approximately mature fruits. Expression patterns between 8818 and 8818-1 revealed that MaGA20ox4, MaGA20ox5, and MaGA20ox7 of the MaGA20ox gene family and MaGA2ox7, MaGA2ox12, and MaGA2ox14 of the MaGA2ox gene family exhibited significant differential expression and high-expression levels in false stems. These genes are likely to be responsible for the regulation of GAs content in 8818-1 false stems. Overall, phylogenetic evolution, tissue specificity and differential expression analyses of GA metabolism genes can provide a

  20. Null mutants of Drosophila B-type lamin Dm(0) show aberrant tissue differentiation rather than obvious nuclear shape distortion or specific defects during cell proliferation.

    Science.gov (United States)

    Osouda, Shinichi; Nakamura, Yoshihiro; de Saint Phalle, Brigitte; McConnell, Maeve; Horigome, Tsuneyoshi; Sugiyama, Shin; Fisher, Paul A; Furukawa, Kazuhiro

    2005-08-01

    To elucidate the function of metazoan B-type lamins during development, new null mutations of the Drosophila B-type lamin gene, lamDm(0), were analyzed in parallel with the misg(sz18) mutation, a lamDm(0) allele reported previously. Although in all these mutants, lamin Dm(0) protein was undetectable in neuroblasts and imaginal disc cells from the second instar larval stage onward, cells continued to proliferate. In contrast to the embryonic lethality of another Drosophila lamDm(0) allele, lam(PM15), reported previously, lethality did not occur until late pupal stages. Chromosomal structure and the overall nuclear shape remained normal even at these late pupal stages, although obviously abnormal nuclear pore complex distribution was observed concomitant with the loss of lamin Dm(0) protein. Compensating expression of lamin C was not induced in the absence of lamin Dm(0). Thus, no lamin-containing nuclear structures were found in proliferating larval neuroblasts. We did find that developmental abnormalities appeared in specific organs during the late pupal stage, preceding lethality. Surprisingly, coordinated size increase (hypertrophy) of the ventriculus was observed accompanied by cell division and muscle layer formation. Hypertrophy of the ventriculus correlated with a decrease in ecdysteroid hormone receptor B1 (EcRB1) protein, and furthermore could be suppressed by a heat-inducible EcRB1 transgene. In contrast, both gonadal and CNS tissues exhibited underdevelopment.

  1. 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.

  2. Activation of G protein-coupled estrogen receptor 1 induces coronary artery relaxation via Epac/Rap1-mediated inhibition of RhoA/Rho kinase pathway in parallel with PKA.

    Directory of Open Access Journals (Sweden)

    Xuan Yu

    Full Text Available Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 μM, or ESI-09 (20 μM, or CE3F4 (100 μM, all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3-3 μM; while concurrent administration of BFA and PKI (5 μM, a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1-100 μM, induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1 was up regulated by G-1 (1 μM treatment of porcine coronary artery smooth muscle cells (CASMCs. Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP was elevated by G-1 (1 μM treatment, but not by 007 (50 μM; and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases.

  3. Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation.

    Science.gov (United States)

    Komaru, Keiichi; Ishida, Yoko; Amaya, Yoshihiro; Goseki-Sone, Masae; Orimo, Hideo; Oda, Kimimitsu

    2005-04-01

    In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).

  4. TP53 mutation p.R337H in gastric cancer tissues of a 12-year-old male child - evidence for chimerism involving a common mutant founder haplotype: case report

    Science.gov (United States)

    2011-01-01

    Background Gastric adenocarcinoma is rare in children and adolescents, with about 17 cases under age 21 in the world's literature. We report a case of invasive well-differentiated metastatic gastric cancer in a Brazilian 12-year-old boy without documented familial history of cancer. Case presentation The patient, diagnosed with metastatic disease, died seven months after surgery. DNA from intra-surgical specimens revealed a TP53 mutation at codon 337 (p.R337H) in samples with neoplastic cells (dysplasia, tumor and metastasis) but not in non-transformed cells (incomplete intestinal metaplasia and non-involved celiac lymph node). In all mutation-positive tissues, p.R337H occurred on the same background, a founder allele identified by a specific haplotype previously described in Brazilian Li-Fraumeni syndrome patients. The same mutant haplotype, corresponding to a founder mutation present in 0.3% of the general population in Southern Brazil, was found in the genome of the father. Presence of this inherited haplotype in the tumor as well as in the father's germline, suggests a rare case of microchimerism in this patient, who may have harbored a small number of mutant cells originating in another individual, perhaps a dizygotic twin that died early in gestation. Conclusion This case represents one of the earliest ages at diagnosis of gastric cancer ever reported. It shows that cancer inheritance can occur in the absence of an obvious germline mutation, calling for caution in assessing early cancers in populations with common founder mutations such as p.R337H in Southern Brazil. PMID:22004116

  5. TP53 mutation p.R337H in gastric cancer tissues of a 12-year-old male child - evidence for chimerism involving a common mutant founder haplotype: case report

    Directory of Open Access Journals (Sweden)

    Prolla Patricia A

    2011-10-01

    Full Text Available Abstract Background Gastric adenocarcinoma is rare in children and adolescents, with about 17 cases under age 21 in the world's literature. We report a case of invasive well-differentiated metastatic gastric cancer in a Brazilian 12-year-old boy without documented familial history of cancer. Case presentation The patient, diagnosed with metastatic disease, died seven months after surgery. DNA from intra-surgical specimens revealed a TP53 mutation at codon 337 (p.R337H in samples with neoplastic cells (dysplasia, tumor and metastasis but not in non-transformed cells (incomplete intestinal metaplasia and non-involved celiac lymph node. In all mutation-positive tissues, p.R337H occurred on the same background, a founder allele identified by a specific haplotype previously described in Brazilian Li-Fraumeni syndrome patients. The same mutant haplotype, corresponding to a founder mutation present in 0.3% of the general population in Southern Brazil, was found in the genome of the father. Presence of this inherited haplotype in the tumor as well as in the father's germline, suggests a rare case of microchimerism in this patient, who may have harbored a small number of mutant cells originating in another individual, perhaps a dizygotic twin that died early in gestation. Conclusion This case represents one of the earliest ages at diagnosis of gastric cancer ever reported. It shows that cancer inheritance can occur in the absence of an obvious germline mutation, calling for caution in assessing early cancers in populations with common founder mutations such as p.R337H in Southern Brazil.

  6. TP53 mutation p.R337H in gastric cancer tissues of a 12-year-old male child: evidence for chimerism involving a common mutant founder haplotype: case report.

    Science.gov (United States)

    da Silva, Edaise M; Achatz, Maria Isabel W; Martel-Planche, Ghyslaine; Montagnini, André L; Olivier, Magali; Prolla, Patricia A; Hainaut, Pierre; Soares, Fernando A

    2011-10-17

    Gastric adenocarcinoma is rare in children and adolescents, with about 17 cases under age 21 in the world's literature. We report a case of invasive well-differentiated metastatic gastric cancer in a Brazilian 12-year-old boy without documented familial history of cancer. The patient, diagnosed with metastatic disease, died seven months after surgery. DNA from intra-surgical specimens revealed a TP53 mutation at codon 337 (p.R337H) in samples with neoplastic cells (dysplasia, tumor and metastasis) but not in non-transformed cells (incomplete intestinal metaplasia and non-involved celiac lymph node). In all mutation-positive tissues, p.R337H occurred on the same background, a founder allele identified by a specific haplotype previously described in Brazilian Li-Fraumeni syndrome patients. The same mutant haplotype, corresponding to a founder mutation present in 0.3% of the general population in Southern Brazil, was found in the genome of the father. Presence of this inherited haplotype in the tumor as well as in the father's germline, suggests a rare case of microchimerism in this patient, who may have harbored a small number of mutant cells originating in another individual, perhaps a dizygotic twin that died early in gestation. This case represents one of the earliest ages at diagnosis of gastric cancer ever reported. It shows that cancer inheritance can occur in the absence of an obvious germline mutation, calling for caution in assessing early cancers in populations with common founder mutations such as p.R337H in Southern Brazil.

  7. Clearance of mutant huntingtin.

    Science.gov (United States)

    Li, Xiao-Jiang; Li, He; Li, Shihua

    2010-07-01

    Mutant huntingtin (htt) carries an expanded polyglutamine (polyQ) repeat (> 36 glutamines) in its N-terminal region, which leads htt to become misfolded and kill neuronal cells in Huntington disease (HD). The cytotoxicity of N-terminal mutant htt fragments is evident by severe neurological phenotypes of transgenic mice that express these htt fragments. Clearance of mutant htt is primarily mediated by the ubiquitin-proteasomal sysmtem (UPS) and autophagy. However, the relative efficiency of these two systems to remove toxic forms of mutant htt has not been rigorously compared. Using cellular and mouse models of HD, we found that inhibiting the UPS leads to a greater accumulation of mutant htt than inhibiting autophagy. Moreover, N-terminal mutant htt fragments, but not full-length mutant htt, accumulate in the HD mouse brains after inhibiting the UPS. These findings suggest that the UPS is more efficient than autophagy to remove N-terminal mutant htt.

  8. Cadmium-Sensitive Mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Howden, R; Cobbett, C S

    1992-09-01

    A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (gamma-glutamylcysteine)(n)-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd.

  9. Saccharomyces cerevisiae aldolase mutants.

    OpenAIRE

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldol...

  10. Copper-sensitive mutant of Arabidopsis thaliana.

    Science.gov (United States)

    van Vliet, C; Anderson, C R; Cobbett, C S

    1995-11-01

    A Cu-sensitive mutant, cup1-1, of Arabidopsis thaliana has a pattern of heavy-metal sensitivity different from that of the cad1 and cad2 mutants, which are deficient in phytochelatin biosynthesis. The latter are significantly sensitive to Cd and Hg and only slightly sensitive to Cu, whereas the cup1-1 mutant is significantly sensitive to Cu, slightly sensitive to Cd, and not more sensitive to Hg, compared to the wild type. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus, which has been mapped to chromosome 1. Genetic and biochemical studies demonstrate that the cup1-1 mutant is not affected in phytochelatin biosynthesis or function. The sensitive phenotype of the cup1-1 mutant is associated with, and probably due to, increased accumulation of higher levels of Cd and Cu compared with the wild type. Consistent with this, a Cu-inducible, root-specific metallothionein gene, MT2a, is expressed in cup1-1 roots under conditions in which it is not expressed in the wild type. Undifferentiated cup1-1 callus tissue did not show the Cu-sensitive phenotype, suggesting that the mutant phenotype, in contrast to cad1 and cad2, is not expressed at the cellular level.

  11. Morphological mutants of garlic

    Energy Technology Data Exchange (ETDEWEB)

    Choudhary, A.D.; Dnyansagar, V.R. (Nagpur Univ. (India). Dept. of Botany)

    1982-01-01

    Cloves of garlic (Allium sativuum Linn.) were exposed to gamma rays with various doses and different concentrations of ethylmethane sulphonate (EMS), diethyl sulphate (dES) and ethylene imine (EI). In the second and third generations, 16 types of morphological mutants were recorded with varied frequencies. Of all the mutagens used, gamma rays were found to be the most effective in inducing the maximum number of mutations followed EI, EMS and dES in that order.

  12. Cadmium-Sensitive Mutants of Arabidopsis thaliana1

    Science.gov (United States)

    Howden, Ross; Cobbett, Christopher S.

    1992-01-01

    A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (γ-glutamylcysteine)n-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd. Images Figure 1 Figure 7 PMID:16652930

  13. The pineapple AcMADS1 promoter confers high level expression in tomato and Arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant.

    Science.gov (United States)

    Moyle, Richard L; Koia, Jonni H; Vrebalov, Julia; Giovannoni, James; Botella, Jose R

    2014-11-01

    A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of ethylene in climacteric tomato. LeMADS-RIN has been proposed to be a global ripening regulator shared among climacteric and non-climacteric species, although few functional homologs of LeMADS-RIN have been identified in non-climacteric species. AcMADS1 shares 67 % protein sequence similarity and a similar expression pattern in ripening fruits as LeMADS-RIN. However, in this study AcMADS1 was not able to complement the tomato rin mutant phenotype, indicating AcMADS1 may not be a functionally conserved homolog of LeMADS-RIN or has sufficiently diverged to be unable to act in the context of the tomato network of interacting proteins. The AcMADS1 promoter directed strong expression of the GUS reporter gene to fruits and developing floral organs in tomato and Arabidopsis thaliana, suggesting AcMADS1 may play a role in flower development as well as fruitlet ripening. The AcMADS1 promoter provides a useful molecular tool for directing transgene expression, particularly where up-regulation in developing flowers and fruits is desirable.

  14. Rap1-dependent pathways coordinate cytokinesis in Dictyostelium

    NARCIS (Netherlands)

    Plak, Katarzyna; Keizer-Gunnink, Ineke; van Haastert, Peter J. M.; Kortholt, Arjan

    2014-01-01

    Cytokinesis is the final step of mitosis when a mother cell is separated into two daughter cells. Major cytoskeletal changes are essential for cytokinesis; it is, however, not well understood how the microtubules and actomyosin cytoskeleton are exactly regulated in time and space. In this paper, we

  15. Aequorin mutants with increased thermostability.

    Science.gov (United States)

    Qu, Xiaoge; Rowe, Laura; Dikici, Emre; Ensor, Mark; Daunert, Sylvia

    2014-09-01

    Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent-based studies. To improve the thermostability of the bioluminescent protein aequorin, we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild-type aequorin during thermostability studies at 37 °C. Moreover, the quadruple aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable aequorin mutant identified thus far.

  16. Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

    Science.gov (United States)

    Saji, Shoko; Bathula, Srinivas; Kubo, Akihiro; Tamaoki, Masanori; Aono, Mitsuko; Sano, Tomoharu; Tobe, Kazuo; Timm, Stefan; Bauwe, Hermann; Nakajima, Nobuyoshi; Saji, Hikaru

    2017-05-01

    An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  17. Altered protein dynamics of disease-associated lamin A mutants

    Directory of Open Access Journals (Sweden)

    Worman Howard J

    2004-12-01

    Full Text Available Abstract Background Recent interest in the function of the nuclear lamina has been provoked by the discovery of lamin A/C mutations in the laminopathy diseases. However, it is not understood why mutations in lamin A give such a range of tissue-specific phenotypes. Part of the problem in rationalising genotype-phenotype correlations in the laminopathies is our lack of understanding of the function of normal and mutant lamin A. To investigate this we have used photobleaching in human cells to analyse the dynamics of wild-type and mutant lamin A protein at the nuclear periphery. Results We have found that a large proportion of wild-type lamin A at the nuclear periphery is immobile, but that there is some slow movement of lamin A within the nuclear lamina. The mobility of an R482W mutant lamin A was indistinguishable from wild-type, but increased mobility of L85R and L530P mutant proteins within the nuclear lamina was found. However, the N195K mutant shows the most enhanced protein mobility, both within the nucleoplasm and within the lamina. Conclusion The slow kinetics of lamin A movement is compatible with its incorporation into a stable polymer that only exchanges subunits very slowly. All of the myopathy-associated lamin A mutants that we have studied show increased protein movement compared with wild-type. In contrast, the dynamic behaviour of the lipodystrophy-associated lamin A mutant was indistinguishable from wild-type. This supports the hypothesis that the underlying defect in lamin A function is quite distinct in the laminopathies that affect striated muscle, compared to the diseases that affect adipose tissue. Our data are consistent with an alteration in the stability of the lamin A molecules within the higher-order polymer at the nuclear lamina in myopathies.

  18. 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.

  19. 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 and Miy...

  20. Effects of cold acclimation on the energetic metabolism of the staggerer mutant mouse.

    Science.gov (United States)

    Bertin, R; Guastavino, J M; Portet, R

    1990-02-01

    Staggerer mutant mice are lean despite their hyperphagia. Brown adipose tissue activity may be implicated in this phenomenon. The aim of this work is to determine the energetic metabolism and to detail some characteristics of the brown adipose tissue of Staggerer mutant mice born and reared either at 28 degrees C (within the thermoneutral zone) or 22 degrees C (cold temperature) compared to nonmutant control mice. In mutant mice reared at thermoneutrality the resting metabolism was found to be higher than that of controls, and further the activity of the brown adipose tissue increased as indicated in relative mass, composition and cytochrome oxydase activity. A stimulatory effect of cold exposure was observed in both mutant and nonmutant mice. It is suggested that Staggerer mice may provide a good model for the study of the cold-induced or diet-induced mechanisms of brown fat stimulation.

  1. 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 wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function mutation

  2. Mutant p53 promotes cell spreading and migration via ARHGAP44.

    Science.gov (United States)

    Xu, Jinjin; Jiao, Jian; Xu, Wei; Ji, Lei; Jiang, Dongjie; Xie, Shaofang; Kubra, Syeda; Li, Xiaotao; Fu, Junjiang; Xiao, Jianru; Zhang, Bianhong

    2017-09-01

    The tumor suppressor p53 protein is either lost or mutated in about half of all human cancers. Loss of p53 function is well known to influence cell spreading, migration and invasion. While expression of mutant p53 is not equivalent to p53 loss, mutant p53 can acquire new functions to drive cell spreading and migration via different mechanisms. In our study, we found that mutant p53 significantly increased cell spreading and migration when comparing with p53-null cells. RNA-Seq analysis suggested that Rho GTPase activating protein 44 (ARHGAP44) is a new target of mutant p53, which suppressed ARHGAP44 transcription. ARHGAP44 has GAP activity and catalyze GTP hydrolysis on Cdc42. Higher level of GTP-Cdc42 was correlated with increase expression of mutant p53 and reduced ARHGAP44. Importantly, wt-ARHGAP44 but not mutant ARHGAP44 (R291A) suppressed mutant p53 mediated cell spreading and migration. Bioinformatics analysis indicated lower expression of ARHGAP44 in lung carcinoma compared with normal tissues, which was verified by RT-qPCR using specimens from patients. More interestingly, ARHGAP44 mRNA level was lower in tumors with mutant p53 than those with normal p53. Collectively, our results disclose a new mechanism by which mutant p53 stimulates cell spreading and migration.

  3. 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…

  4. Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

    Science.gov (United States)

    Grygoryev, Dmytro; Dan, Cristian; Gauny, Stacey; Eckelmann, Bradley; Ohlrich, Anna P; Connolly, Marissa; Lasarev, Michael; Grossi, Gianfranco; Kronenberg, Amy; Turker, Mitchell S

    2014-05-01

    High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

  5. Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA.

    Science.gov (United States)

    Malena, Adriana; Pantic, Boris; Borgia, Doriana; Sgarbi, Gianluca; Solaini, Giancarlo; Holt, Ian J; Spinazzola, Antonella; Perissinotto, Egle; Sandri, Marco; Baracca, Alessandra; Vergani, Lodovica

    2016-11-01

    Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.

  6. Temperature regulates expression of the Drosophila vestigial gene only in mutant wing discs.

    Science.gov (United States)

    Silber, J; Flagiello, D; Cossard, R; Zider, A; Becker, J L

    1997-10-01

    All Vestigial mutants in Drosophila melanogaster display a thermosensitive phenotype, with the exception of two which disrupt an intronic wing-specific enhancer element. Here we report a very unusual transcriptional regulation; temperature changes are associated with alterations in the level of vg expression only in the wing disc of thermosensitive mutant flies and not in the brain. No effect is observed in the wild-type strain. The tissue specificity of the temperature effect indicates an involvement of the intronic wing-specific enhancer element in determining the thermosensitivity of mutants.

  7. Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

    Science.gov (United States)

    Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

    2018-02-02

    There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

  8. Lactate dehydrogenase A silencing in IDH mutant gliomas.

    Science.gov (United States)

    Chesnelong, Charles; Chaumeil, Myriam M; Blough, Michael D; Al-Najjar, Mohammad; Stechishin, Owen D; Chan, Jennifer A; Pieper, Russell O; Ronen, Sabrina M; Weiss, Samuel; Luchman, H Artee; Cairncross, J Gregory

    2014-05-01

    Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant ((mt)) and IDH wild-type ((wt)) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDH(mt) gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDH(mt) derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDH(wt)), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDH(mt) glioblastomas. To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDH(mt) gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis.

  9. Incomplete flagellar structures in Escherichia coli mutants.

    OpenAIRE

    Suzuki, T; Komeda, Y

    1981-01-01

    Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative ...

  10. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    The induced mutagenesis method for deriving pigment mutants of a green microalga, Chlamydomonas reinhardtii CC-124 and their pigment composition as well as ability to assess mutability of contaminated aquatic ecosystems were studied. In the present study, 14086 mutants (colonies) were obtained by exposure of the ...

  11. Induced High Lysine Mutants in Barley

    DEFF Research Database (Denmark)

    Doll, Hans; Køie, B.; Eggum, B. O.

    1974-01-01

    Screening of mutagenically treated materials by combined Kjeldahl nitrogen and dye-binding capacity determinations disclosed fourteen barley mutants, which have from a few to about 40 per cent more lysine in the protein and one mutant with 10 per cent less lysine in the protein than the parent...

  12. Isolation and characterization of Arabidopsis mutants defective in the induction of ethylene biosynthesis by cytokinin

    Science.gov (United States)

    Vogel, J. P.; Schuerman, P.; Woeste, K.; Brandstatter, I.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Cytokinins elevate ethylene biosynthesis in etiolated Arabidopsis seedlings via a post-transcriptional modification of one isoform of the key biosynthetic enzyme ACC synthase. In order to begin to dissect the signaling events leading from cytokinin perception to this modification, we have isolated a series of mutants that lack the ethylene-mediated triple response in the presence of cytokinin due to their failure to increase ethylene biosynthesis. Analysis of genetic complementation and mapping revealed that these Cin mutants (cytokinin-insensitive) represent four distinct complementation groups, one of which, cin4, is allelic to the constitutive photomorphogenic mutant fus9/cop10. The Cin mutants have subtle effects on the morphology of adult plants. We further characterized the Cin mutants by analyzing ethylene biosynthesis in response to various other inducers and in adult tissues, as well as by assaying additional cytokinin responses. The cin3 mutant did not disrupt ethylene biosynthesis under any other conditions, nor did it disrupt any other cytokinin responses. Only cin2 disrupted ethylene biosynthesis in multiple circumstances. cin1 and cin2 made less anthocyanin in response to cytokinin. cin1 also displayed reduced shoot initiation in tissue culture in response to cytokinin, suggesting that it affects a cytokinin signaling element.

  13. Los mutantes de la escuela

    Directory of Open Access Journals (Sweden)

    Diego Armando Jaramillo-Ocampo

    2013-01-01

    Full Text Available El presente artículo muestra los resultados parciales del estudio “Juegos en el recreo escolar: un escenario para la formación ciudadana”, cuya pretensión fue comprender los imaginarios sociales de juego en el recreo escolar y su relación con la convivencia social desde la proximidad del enfoque de complementariedad y el diseño de investigación emergente, planteado por Murcia y Jaramillo (2008. Se presentan los desarrollos logrados en dos categorías centrales del estudio: el patio y el cuerpo; dos categorías que mutan constantemente como entidades vivas en la escuela, hacia la configuración de sujetos que reconocen en el otro y lo otro su posibilidad. La escuela viva, donde es posible “ser en relación con”… se reduce a un espacio temporal y físico, limitado por la campana, “el recreo”. El texto muestra, desde la voz de los actores, esa vida que se da y se quita en la escuela y que se posiciona como una más de las imposiciones normalizadas para controlar. Reconoce, finalmente, una propuesta desde la posibilidad que estos dos mutantes propician para una escuela libre y dinámica.

  14. Histological Characterization of the Dicer1 Mutant Zebrafish Retina

    Directory of Open Access Journals (Sweden)

    Saeed Akhtar

    2015-01-01

    Full Text Available DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA and RNA-interference (RNAi functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1W1457Ter/W1457Ter have an arrest in early growth with significantly smaller eyes and are dead at 14–18 dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE cells are normal with no sign of degeneration at the age of 20 months.

  15. Generation and Performance of R132H Mutant IDH1 Rabbit Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Juliet Rashidian

    2017-12-01

    Full Text Available Isocitrate dehydrogenase 1 (IDH1 gene mutations have been observed in a majority of diffuse astrocytomas, oligodendrogliomas, and secondary glioblastomas, and the mutant IDH1 R132H is detectable in most of these lesions. By specifically targeting the R132H mutation through B-cell cloning, a novel rabbit monoclonal antibody, MRQ-67, was produced that can recognize mutant IDH1 R132H and does not react with the wild type protein as demonstrated by Enzyme-linked immunosorbent assay (ELISA and Western blotting. Through immunohistochemistry, the antibody is able to highlight neoplastic cells in glioma tissue specimens, and can be used as a tool in glioma subtyping. Immunohistochemistry (IHC detection of IDH1 mutant protein may also be used to visualize single infiltrating tumor cells in surrounding brain tissue with an otherwise normal appearance.

  16. Flavonoid accumulation patterns of transparent testa mutants of arabidopsis

    Science.gov (United States)

    Peer, W. A.; Brown, D. E.; Tague, B. W.; Muday, G. K.; Taiz, L.; Murphy, A. S.

    2001-01-01

    Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.

  17. Responses to novelty in staggerer mutant mice.

    Science.gov (United States)

    Misslin, R; Cigrang, M; Guastavino, J M

    1986-01-01

    Responses to novelty in normal C57BL/6 and staggerer mutant mice were recorded. The normal mice confronted a novel object in their familiar environment showed avoidance and burying responses while the staggerer mutant mice contacted it. When given the opportunity to move around freely in simultaneously presented novel and familiar environments, the mutant mice more quickly entered the novel areas than normal animals. these data reveal a significant decrease in the neophobic components of the neotic behaviour in the staggerer mice. However, since the mutant mice did not show a locomotor deficit, the impairment of neophobia seems not to be due to the gait abnormalities of these animals. The results support the view that the cerebellum may contribute to the organization of complex behaviours. Copyright © 1986. Published by Elsevier B.V.

  18. Robust mutant strain design by pessimistic optimization.

    Science.gov (United States)

    Apaydin, Meltem; Xu, Liang; Zeng, Bo; Qian, Xiaoning

    2017-10-03

    Flux Balance Analysis (FBA) based mathematical modeling enables in silico prediction of systems behavior for genome-scale metabolic networks. Computational methods have been derived in the FBA framework to solve bi-level optimization for deriving "optimal" mutant microbial strains with targeted biochemical overproduction. The common inherent assumption of these methods is that the surviving mutants will always cooperate with the engineering objective by overproducing the maximum desired biochemicals. However, it has been shown that this optimistic assumption may not be valid in practice. We study the validity and robustness of existing bi-level methods for strain optimization under uncertainty and non-cooperative environment. More importantly, we propose new pessimistic optimization formulations: P-ROOM and P-OptKnock, aiming to derive robust mutants with the desired overproduction under two different mutant cell survival models: (1) ROOM assuming mutants have the minimum changes in reaction fluxes from wild-type flux values, and (2) the one considered by OptKnock maximizing the biomass production yield. When optimizing for desired overproduction, our pessimistic formulations derive more robust mutant strains by considering the uncertainty of the cell survival models at the inner level and the cooperation between the outer- and inner-level decision makers. For both P-ROOM and P-OptKnock, by converting multi-level formulations into single-level Mixed Integer Programming (MIP) problems based on the strong duality theorem, we can derive exact optimal solutions that are highly scalable with large networks. Our robust formulations P-ROOM and P-OptKnock are tested with a small E. coli core metabolic network and a large-scale E. coli iAF1260 network. We demonstrate that the original bi-level formulations (ROOM and OptKnock) derive mutants that may not achieve the predicted overproduction under uncertainty and non-cooperative environment. The knockouts obtained by the

  19. Characterization of MarR Superrepressor Mutants

    OpenAIRE

    Alekshun, Michael N.; Levy, Stuart B.

    1999-01-01

    MarR negatively regulates expression of the multiple antibiotic resistance (mar) locus in Escherichia coli. Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant pr...

  20. Patulin degradation in saccharomyces cerevisiae: Sensitive mutants.

    Science.gov (United States)

    Thonart, P; Sumbu, Z L; Bechet, J

    1985-03-01

    The present experiments (sensitive mutants and transient inhibition of growth) are compatible with the synthesis of an inductible detoxifying substance in the wild type strain. This substance could be glutathione because glutathione detoxification scheme essentially involves properties of the SH group and it is well known that patulin reacts with sulfhy dril groups.Studies are presently being carried out with sensitive mutants to establish definitively the relation between intracellular pool of glutathone and the resistance mechanism of a yeast to patulin.

  1. Genetic Analysis and Mapping of TWH Gene in Rice Twisted Hull Mutant

    Directory of Open Access Journals (Sweden)

    Jin-bo LI

    2009-03-01

    Full Text Available A mutant with twisted hulls was found in a breeding population of rice (Oryza sativa L.. The mutant shows less grain weight and inferior grain quality in addition to twisted hulls. Genetic analysis indicated that the phenotype of mutant was controlled by a single recessive gene (temporarily designated as TWH. To map the TWH gene, an F2 population was generated by crossing the twh mutant to R725, an indica rice variety with normal hulls. For bulked segregant analysis, the bulk of mutant plants was prepared by mixing equal amount of plant tissue from 10 twisted-hull plants and the bulk of normal plants was obtained by pooling equal amount tissue of 10 normal-hull plants. Two hundred and seven pairs of simple sequence repeat (SSR primers, which are distributed on 12 rice chromosomes, were used for polymorphism analysis of the parents and the two bulks. The TWH locus was initially mapped close to the SSR marker RM526 on chromosome 2. Therefore, further mapping was performed using 50 pairs of SSR primers around the marker RM526. The TWH was delimited between the SSR markers RM14128 and RM208 on the long arm of chromosome 2 at the genetic distances of 1.4 cM and 2.7 cM, respectively. These results provide the foundation for further fine mapping, cloning and functional analysis of the TWH gene.

  2. A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana.

    Science.gov (United States)

    Howden, R; Andersen, C R; Goldsbrough, P B; Cobbett, C S

    1995-04-01

    The roots of the cadmium-sensitive mutant of Arabidopsis thaliana, cad1-1, become brown in the presence of cadmium. A new cadmium-sensitive mutant affected at a second locus, cad2, has been identified using this phenotype. Genetic analysis has grown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Assays of cadmium accumulation by intact plants indicated that the mutant is deficient in its ability to sequester cadmium. Undifferentiated callus tissue was also cadmium sensitive, suggesting that the mutant phenotype is expressed at the cellular level. The level of cadmium-binding complexes formed in vivo was decreased compared with the wild type and accumulation of phytochelatins was about 10% of that in the wild type. The level of glutathione, the substrate for phytochelatin biosynthesis, in tissues of the mutant was decreased to about 15 to 30% of that in the wild type. Thus, the deficiency in phytochelatin biosynthesis can be explained by a deficiency in glutathione.

  3. Genetic dissection of leukemia-associated IDH1 and IDH2 mutants and D-2-hydroxyglutarate in Drosophila

    Science.gov (United States)

    Reitman, Zachary J.; Sinenko, Sergey A.; Spana, Eric P.

    2015-01-01

    Gain-of-function mutations in nicotinamide adenine dinucleotide phosphate–dependent isocitrate dehydrogenase (IDH)1 and IDH2 frequently arise in human leukemias and other cancers and produce high levels of D-2-hydroxyglutarate (D-2HG). We expressed the R195H mutant of Drosophila Idh (CG7176), which is equivalent to the human cancer-associated IDH1-R132H mutant, in fly tissues using the UAS-Gal4 binary expression system. Idh-R195H caused a >25-fold elevation of D-2HG when expressed ubiquitously in flies. Expression of mutant Idh in larval blood cells (hemocytes) resulted in higher numbers of circulating blood cells. Mutant Idh expression in fly neurons resulted in neurologic and wing-expansion defects, and these phenotypes were rescued by genetic modulation of superoxide dismutase 2, p53, and apoptotic caspase cascade mediators. Idh-R163Q, which is homologous to the common leukemia-associated IDH2-R140Q mutant, resulted in moderately elevated D-2HG and milder phenotypes. We identified the fly homolog of D-2-hydroxyglutaric acid dehydrogenase (CG3835), which metabolizes D-2HG, and showed that coexpression of this enzyme with mutant Idh abolishes mutant Idh-associated phenotypes. These results provide a flexible model system to interrogate a cancer-related genetic and metabolic pathway and offer insights into the impact of IDH mutation and D-2HG on metazoan tissues. PMID:25398939

  4. Tissue tests

    NARCIS (Netherlands)

    Sonneveld, C.; Voogt, W.

    2009-01-01

    Tissue tests are widely used in horticulture practice and have in comparison with soil or substrate testing advantages as well disadvantages in comparison with soil testing. One of the main advantages of tissue tests is the certainty that analysed nutrients in plant tissues are really present in the

  5. Combinatorial synthetic peptide vaccine strategy protects against hypervirulent CovR/S mutant streptococci

    DEFF Research Database (Denmark)

    Pandey, Manisha; Mortensen, Rasmus; Calcutt, Ainslie

    2016-01-01

    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 protease Streptococcus pyogenes cell 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 theM protein) and a recombinant SpyCEP fragment protects against CovR/S mutants. To enhance the vaccine's safety profile, we...

  6. Effect of plant growth regulators on leaf anatomy of the has mutant of Arabidopsis thaliana.

    Science.gov (United States)

    Janosević, D; Uzelac, B; Budimir, S

    2008-12-01

    In this study, the effect of plant growth regulators on leaf morphogenesis of the recessive T-DNA insertion mutant of Arabidopsis thaliana was analyzed. The morpho-anatomical analysis revealed that leaves of the has mutant are small and narrow, with lobed blades and disrupted tissue organization. When has plants were grown on the medium supplied with plant growth regulators: benzylaminopurine (BAP) or ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), the leaf anatomy was partially restored to the wild type, although plants still exhibited morphological abnormalities.

  7. Plasmodium berghei: in vivo generation and selection of karyotype mutants and non-gametocyte producer mutants

    NARCIS (Netherlands)

    Janse, C. J.; Ramesar, J.; van den Berg, F. M.; Mons, B.

    1992-01-01

    We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals

  8. Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

    OpenAIRE

    Lorowitz, W; CLARK, D.

    1982-01-01

    Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

  9. Targeting mutant NRAS signaling pathways in melanoma.

    Science.gov (United States)

    Vu, Ha Linh; Aplin, Andrew E

    2016-05-01

    Cutaneous melanoma is a devastating form of skin cancer and its incidence is increasing faster than any other preventable cancer in the United States. The mutant NRAS subset of melanoma is more aggressive and associated with poorer outcomes compared to non-NRAS mutant melanoma. The aggressive nature and complex molecular signaling conferred by this transformation has evaded clinically effective treatment options. This review examines the major downstream effectors of NRAS relevant in melanoma and the associated advances made in targeted therapies that focus on these effector pathways. We outline the history of MEK inhibition in mutant NRAS melanoma and recent advances with newer MEK inhibitors. Since MEK inhibitors will likely be optimized when combined with other targeted therapies, we focus on recently identified targets that can be used in combination with MEK inhibitors. Published by Elsevier Ltd.

  10. High Persister Mutants in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Heather L Torrey

    Full Text Available Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection.

  11. Native Mutant Huntingtin in Human Brain

    Science.gov (United States)

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  12. Aging Kit mutant mice develop cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    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.

  13. Ovarian abnormalities in the staggerer mutant mouse.

    Science.gov (United States)

    Guastavino, Jean-Marie; Boufares, Salima; Crusio, Wim E

    2005-08-24

    Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rora(sg) gene underlying the cerebellar abnormalities of the staggerer mutant.

  14. Ovarian Abnormalities in the Staggerer Mutant Mouse

    Directory of Open Access Journals (Sweden)

    Jean-Marie Guastavino

    2005-01-01

    Full Text Available Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rorasg gene underlying the cerebellar abnormalities of the staggerer mutant.

  15. A Landscape of Therapeutic Cooperativity in KRAS Mutant Cancers Reveals Principles for Controlling Tumor Evolution

    Directory of Open Access Journals (Sweden)

    Grace R. Anderson

    2017-07-01

    Full Text Available Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tissue-specific sensitizing combinations involving inhibitors of cell cycle, metabolism, growth signaling, chromatin regulation, and transcription. Furthermore, these screens revealed secondary genetic modifiers of sensitivity, yielding a SRC inhibitor-based combination therapy for KRAS/PIK3CA double-mutant colorectal cancers (CRCs with clinical potential. Surprisingly, acquired resistance to combinations of growth signaling pathway inhibitors develops rapidly following treatment, but by targeting signaling feedback or apoptotic priming, it is possible to construct three-drug combinations that greatly delay its emergence.

  16. Tissue types (image)

    Science.gov (United States)

    ... are 4 basic types of tissue: connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Connective tissue supports ... binds them together (bone, blood, and lymph tissues). Epithelial tissue provides a covering (skin, the linings of the ...

  17. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

    Science.gov (United States)

    Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

    2016-01-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

  18. 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.

  19. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    acer

    The result of bio-test, using the resulting pigment mutant of C. reinhardtii 124y-1 showed that mutagenic activity was observed significantly in both Tekeli River and Pavlodar Oil Refinery in Kazakhstan; the waste water of the. Pavlodar Oil Refinery had high-toxicity while the water of the Tekeli River had medium-toxicity.

  20. Avirulent mutants of Macrophomina phaseolina and Aspergillus ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 25; Issue 1. Avirulent mutants of Macrophomina phaseolina and Aspergillus fumigatus initiate infection in Phaseolus mungo in the presence of phaseo-linone; levamisole gives protection. Suchandra Sett Santosh K Mishra Kazia I Siddiqui. Articles Volume 25 Issue 1 March ...

  1. Ethanol production using engineered mutant E. coli

    Science.gov (United States)

    Ingram, Lonnie O.; Clark, David P.

    1991-01-01

    The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

  2. Flocculation phenomenon of a mutant flocculent Saccharomyces ...

    African Journals Online (AJOL)

    Flocculation phenomenon of a mutant flocculent Saccharomyces cerevisiae strain: Effects of metal ions, sugars, temperature, pH, protein-denaturants and ... was in the early stationary growth phase, which coincided with glucose depletion in the batch fermentation for the production of ethanol from kitchen refuse medium.

  3. Mutant PTEN in Cancer : Worse Than Nothing

    NARCIS (Netherlands)

    Leslie, Nick R; den Hertog, Jeroen

    2014-01-01

    Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant

  4. Iron uptake from plasma transferrin by a transferrin receptor 2 mutant mouse model of haemochromatosis.

    Science.gov (United States)

    Chua, Anita C G; Delima, Roheeth D; Morgan, Evan H; Herbison, Carly E; Tirnitz-Parker, Janina E E; Graham, Ross M; Fleming, Robert E; Britton, Robert S; Bacon, Bruce R; Olynyk, John K; Trinder, Debbie

    2010-03-01

    Hereditary haemochromatosis type 3 is caused by mutations in transferrin receptor (TFR) 2. TFR2 has been shown to mediate iron transport in vitro and regulate iron homeostasis. The aim of this study was to determine the role of Tfr2 in iron transport in vivo using a Tfr2 mutant mouse. Tfr2 mutant and wild-type mice were injected intravenously with (59)Fe-transferrin and tissue (59)Fe uptake was measured. Tfr1, Tfr2 and ferroportin expression was measured by real-time PCR and Western blot. Cellular localisation of ferroportin was determined by immunohistochemistry. Transferrin-bound iron uptake by the liver and spleen in Tfr2 mutant mice was reduced by 20% and 65%, respectively, whilst duodenal and renal uptake was unchanged compared with iron-loaded wild-type mice. In Tfr2 mutant mice, liver Tfr2 protein was absent, whilst ferroportin protein was increased in non-parenchymal cells and there was a low level of expression in hepatocytes. Tfr1 expression was unchanged compared with iron-loaded wild-type mice. Splenic Tfr2 protein expression was absent whilst Tfr1 and ferroportin protein expression was increased in Tfr2 mutant mice compared with iron-loaded wild-type mice. A small reduction in hepatic transferrin-bound iron uptake in Tfr2 mutant mice suggests that Tfr2 plays a minor role in liver iron transport and its primary role is to regulate iron metabolism. Increased ferroportin expression due to decreased hepcidin mRNA levels is likely to be responsible for impaired splenic iron uptake in Tfr2 mutant mice. Copyright (c) 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  5. Changes of mandibular incisor in Fgfr2 S252W mutant mice

    Directory of Open Access Journals (Sweden)

    Xia ZHOU

    2013-11-01

    Full Text Available Objective To compare the phenotypic differences of mandibular incisor between the wild-type mice and fibroblast growth factor receptor 2 (Fgfr2 gene S252W mutant mice, and explore the influence of gain-of-function mutation in Fgfr2 gene on mandibular incisors in mice. Methods The male EⅡa-Cre mice were mated with Fgfr2S252W-neo/+ females to obtain the Fgfr2 S252W mutant mice. On the 56th day after offspring's birth (P56, samples were taken for Micro-CT, HE staining and calcein double fluorescent labeling to observe the gross appearance, tissue morphology and mineral apposition rate of mandibular incisors, respectively. Results The newborn mutant mice showed short cranial deformity, which became more obvious on P56. Micro-CT showed a significant elongation and cross-bite deformity of mandibular incisors. HE staining showed that there were more ameloblasts and odontoblasts in the mutant mice, mostly with irregular appearance; epithelial diaphragm composed of inner and outer enamel epithelium shrank. Calcein double fluorescent labeling showed that the mineral apposition rate of dentin in mutant mice was significantly higher than that in controls. Conclusion Fgfr2 S252W mutation accelerates the growth of mandibular incisors in mice, resulting in the elongation and cross-bite deformity of mandibular incisors. DOI: 10.11855/j.issn.0577-7402.2013.10.005

  6. Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways

    Science.gov (United States)

    Padavano, Julianna; Henkhaus, Rebecca S; Chen, Hwudaurw; Skovan, Bethany A; Cui, Haiyan; Ignatenko, Natalia A

    2015-01-01

    Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis. PMID:26512205

  7. The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients.

    Directory of Open Access Journals (Sweden)

    Eva K Roth

    Full Text Available BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF. Previous studies demonstrated that the K⁺ channel opener 1-ethyl-2-benzimidazolone (1-EBIO potentiates CFTR-mediated Cl⁻ secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl⁻ secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl⁻ secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl⁻ secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl⁻ secretion by 39.2±6.7% (P<0.001 via activation of basolateral Ca²⁺-activated and clotrimazole-sensitive KCNN4 K⁺ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl⁻ secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001, but had no effect on tissues lacking CFTR-mediated Cl⁻ conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl⁻secretion in native CF tissues expressing CFTR mutants with residual Cl⁻ channel function by activation of basolateral KCNN4 K⁺ channels that increase the driving force for luminal Cl⁻ exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF.

  8. Tissue Classification

    DEFF Research Database (Denmark)

    Van Leemput, Koen; Puonti, Oula

    2015-01-01

    Computational methods for automatically segmenting magnetic resonance images of the brain have seen tremendous advances in recent years. So-called tissue classification techniques, aimed at extracting the three main brain tissue classes (white matter, gray matter, and cerebrospinal fluid), are now...... well established. In their simplest form, these methods classify voxels independently based on their intensity alone, although much more sophisticated models are typically used in practice. This article aims to give an overview of often-used computational techniques for brain tissue classification...

  9. Standardized, systemic phenotypic analysis of Slc12a1I299F mutant mice.

    Science.gov (United States)

    Kemter, Elisabeth; Rathkolb, Birgit; Becker, Lore; Bolle, Ines; Busch, Dirk H; Dalke, Claudia; Elvert, Ralf; Favor, Jack; Graw, Jochen; Hans, Wolfgang; Ivandic, Boris; Kalaydjiev, Svetoslav; Klopstock, Thomas; Rácz, Ildikó; Rozman, Jan; Schrewe, Anja; Schulz, Holger; Zimmer, Andreas; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabe de Angelis, Martin; Wolf, Eckhard; Aigner, Bernhard

    2014-08-02

    Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl- cotransporter NKCC2. We recently established the mutant mouse line Slc12a1I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye. To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1I299F in the German Mouse Clinic. Slc12a1I299F homozygous mutant mice and Slc12a1I299F heterozygous mutant littermates as controls were tested at the age of 4-6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed. In this systemic analysis no clear primary effects of the Slc12a1I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.

  10. Fibrinolytic Activity of Recombinant Mutant Streptokinase

    Directory of Open Access Journals (Sweden)

    Mahboobeh Mobarrez

    2015-04-01

    Full Text Available Background: Streptokinase is a bacterial protein produced by different beta hemolytic streptococci and widely used in thrombolytic treatment. The main disadvantage of using streptokinase is antibody formation which causes allergic reaction to neutralize effects of streptokinase therapy. Aim of this study was investigate of recombinant mutant streptokinase fibrinolytic activity.Materials and Methods: In this study recombinant mutant streptokinase without 42 amino acids from the C terminal region was purified by affinity S-Tag column chromatography and its fibrinolytic activity was studied.Results: The concentration of expressed and purified protein was 10 mg/ml. Its enzyme activity was assayed using zymography, radial caseinolytic activity and fibrin plate test methods and estimated quantitatively by casein digestion method compared to a commercial form.Conclusion: It was found that this product had the more volume and more enzymatic activity.

  11. 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...... Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip- organism displayed a 24-fold reduction in survivability immediately after its entry...... 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....

  12. A new neurological rat mutant "mutilated foot".

    OpenAIRE

    Jacobs, J M; Scaravilli, F; Duchen, L W; Mertin, J

    1981-01-01

    A new autosomal recessive mutant rat (mutilated foot) with a neurological disorder is described. Affected animals become ataxic and the feet, generally of the hind limbs, are mutilated. Quantitative studies show a severe reduction in numbers of sensory ganglion cells and fibres, including unmyelinated fibres. The numbers of ventral root fibres, particularly those of small diameter, are also reduced. Markedly decreased numbers of spindles are found in the limb muscles. These quantitative abnor...

  13. Analysis of vascular development in the hydra sterol biosynthetic mutants of Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Margaret Pullen

    2010-08-01

    Full Text Available The control of vascular tissue development in plants is influenced by diverse hormonal signals, but their interactions during this process are not well understood. Wild-type sterol profiles are essential for growth, tissue patterning and signalling processes in plant development, and are required for regulated vascular patterning.Here we investigate the roles of sterols in vascular tissue development, through an analysis of the Arabidopsis mutants hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase respectively. We show that defective vascular patterning in the shoot is associated with ectopic cell divisions. Expression of the auxin-regulated AtHB8 homeobox gene is disrupted in mutant embryos and seedlings, associated with variably incomplete vascular strand formation and duplication of the longitudinal axis. Misexpression of the auxin reporter proIAA2ratioGUS and mislocalization of PIN proteins occurs in the mutants. Introduction of the ethylene-insensitive ein2 mutation partially rescues defective cell division, localization of PIN proteins, and vascular strand development.The results support a model in which sterols are required for correct auxin and ethylene crosstalk to regulate PIN localization, auxin distribution and AtHB8 expression, necessary for correct vascular development.

  14. Courtship song analysis of Drosophila muscle mutants.

    Science.gov (United States)

    Chakravorty, Samya; Wajda, Mathew P; Vigoreaux, Jim O

    2012-01-01

    As part of the mating ritual, males of Drosophila species produce species-specific courtship songs through wing vibrations generated by the thoracic musculature. While previous studies have shown that indirect flight muscles (IFM) are neurally activated during courtship song production, the precise role of these muscles in song production has not been investigated. Fortunately, IFM mutants abound in Drosophila melanogaster and studies spanning several decades have shed light on the role of muscle proteins in IFM-powered flight. Analysis of courtship songs in these mutants offers the opportunity to uncover the role of the IFM in a behavior distinct than flight and subject to different evolutionary selection regimes. Here, we describe protocols for the recording and analysis of courtship behavior and mating song of D. melanogaster muscle transgenic and mutant strains. To record faint acoustic signal of courtship songs, an insulated mating compartment was used inside a recording device (INSECTAVOX) equipped with a modified electret microphone, a low-noise power supply, and noise filters. Songs recorded in the INSECTAVOX are digitized using Goldwave, whose several features enable extraction of critical song parameters, including carrier frequencies for pulse song and sine song. We demonstrate the utility of this approach by showing that deletion of the N-terminal region of the myosin regulatory light chain, a mutation known to decrease wing beat frequency and flight power, affects courtship song parameters. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Cellular differentiation regulated by gibberellin in the Arabidopsis thaliana pickle mutant

    Energy Technology Data Exchange (ETDEWEB)

    Ogas, J.; Somerville, C. [Carnegie Institution of Washington, Stanford, CA (United States); Cheng, Jin-Chen; Sung, R. [Univ. of California, Berkeley, CA (United States)

    1997-07-04

    The plant growth regulator gibberellin (GA) has a profound effect on shoot development and promotes developmental transitions such as flowering. Little is known about any analogous effect GA might have on root development. In a screen for mutants, Arabi-dopsis plants carrying a mutation designated pickle (pkl) were isolated in which the primary root meristem retained characteristics of embryonic tissue. Expression of this aberrant differentiation state was suppressed by GA. Root tissue from plants carrying the pkl mutation spontaneously regenerated new embryos and plants. 19 refs., 3 figs., 1 tab.

  16. Agrobacterium rhizogenes mutants that fail to bind to plant cells.

    OpenAIRE

    Crews, J L; Colby, S; Matthysse, A G

    1990-01-01

    Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria ...

  17. Mutant huntingtin activates Nrf2-responsive genes and impairs dopamine synthesis in a PC12 model of Huntington's disease

    NARCIS (Netherlands)

    van Roon-Mom, W.M.C.; Pepers, B.A.; 't Hoen, P.A.C.; Verwijmeren, C.A.C.M.; den Dunnen, J.T.; Dorsman, J.C.; van Ommen, G.B.

    2008-01-01

    Background: Huntington's disease is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion in the HD or Huntington's disease gene. Although micro array studies on patient and animal tissue provide valuable information, the primary effect of mutant

  18. Water relations of GA- and ABA-deficient tomato mutants during seed and fruit development and their influence on germination

    NARCIS (Netherlands)

    Liu, Y.; Bino, R.J.; Karssen, C.M.; Hilhorst, H.W.M.

    1996-01-01

    To explain the differing germination behaviour of seeds of wild type, gibberellin-deficient (gib1) or abscisic acid-deficient (sitw) mutants of tomato (Lycopersicon esculentum Mill. cv. Moneymaker), growth and water relations of fruit tissues, seeds and embryos were determined during development.

  19. Isolation and Characterization of Sexual Sporulation Mutants of Aspergillus nidulans

    NARCIS (Netherlands)

    Swart, K.; Heemst, van D.; Slakhorst, M.; Debets, A.J.M.; Heyting, C.

    2001-01-01

    For the genetic dissection of sexual sporulation in Aspergillus nidulans, we started a collection of ascosporeless mutants. After mutagenization of conidiospores with high doses of UV, we isolated 20 mutants with defects in ascospore formation. We crossed these mutants in two successive rounds with

  20. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants

    NARCIS (Netherlands)

    García-Contreras, R; Lira-Silva, E; Jasso-Chávez, R; Hernández-González, I.L.; Maeda, T.; Hashimoto, T.; Boogerd, F.C.; Sheng, L; Wood, TK; Moreno-Sánchez, R

    2013-01-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed

  1. Strain improvement in dye decolourising mutants of Mucor mucedo ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... The amounts of protoplasts obtained in the developed mutants of M. mucedo MMM1 (U.V. irradiated mutant) and MMM2 (ethyl methyl sulfonate treated mutant) which are very effective decolourisers were. 5.23 x 106 and 5.65 x 106 protoplasts/ml respectively. Among the 385 colonies isolated after ...

  2. Characterization of a novel curled-cotyledons mutant in soybean ...

    African Journals Online (AJOL)

    ... some organelles degradation, and membranous multilamellar appear at different stages. Protein and amino acid contents in seeds of mutant are higher than those of the wild type, especially methionine and cysteine. These results suggest that the curled-cotyledons mutant is a novel cotyledon development mutant, which ...

  3. Erwinia amylovora pyrC mutant causes fire blight despite pyrimidine auxotrophy.

    Science.gov (United States)

    Ramos, L S; Sinn, J P; Lehman, B L; Pfeufer, E E; Peter, K A; McNellis, T W

    2015-06-01

    Erwinia amylovora bacteria cause fire blight disease, which affects apple and pear production worldwide. The Erw. amylovora pyrC gene encodes a predicted dihydroorotase enzyme involved in pyrimidine biosynthesis. Here, we discovered that the Erw. amylovora pyrC244::Tn5 mutant was a uracil auxotroph. Unexpectedly, the Erw. amylovora pyrC244::Tn5 mutant grew as well as the wild-type in detached immature apple and pear fruits. Fire blight symptoms caused by the pyrC244::Tn5 mutant in immature apple and pear fruits were attenuated compared to those caused by the wild-type. The pyrC244::Tn5 mutant also caused severe fire blight symptoms in apple tree shoots. A plasmid-borne copy of the wild-type pyrC gene restored prototrophy and symptom induction in apple and pear fruit to the pyrC244::Tn5 mutant. These results suggest that Erw. amylovora can obtain sufficient pyrimidine from the host to support bacterial growth and fire blight disease development, although de novo pyrimidine synthesis by Erw. amylovora is required for full symptom development in fruits. Significance and impact of the study: This study provides information about the fire blight host-pathogen interaction. Although the Erwinia amylovora pyrC mutant was strictly auxotrophic for pyrimidine, it grew as well as the wild-type in immature pear and apple fruits and caused severe fire blight disease in apple trees. This suggests that Erw. amylovora can obtain sufficient pyrimidines from host tissue to support growth and fire blight disease development. This situation contrasts with findings in some human bacterial pathogens, which require de novo pyrimidine synthesis for growth in host blood, for example. © 2015 The Society for Applied Microbiology.

  4. Effects of Light and Wounding on Jasmonates in Rice phyAphyC Mutants

    Directory of Open Access Journals (Sweden)

    Rita Brendel

    2014-03-01

    Full Text Available Jasmonates (JA are lipid-derived plant hormones. They have been shown to be important regulators of photomorphogenesis, a developmental program in plants, which is activated by light through different red and blue light sensitive photoreceptors. In rice, inhibition of coleoptile growth by light is a central event in photomorphogenesis. This growth inhibition is impaired, when jasmonate biosynthesis is knocked out. Previously, we found that JASMONATE RESISTANT 1 (OsJAR1 transcripts were not induced in the phytochrome (phy mutant phyAphyC. Therefore, in the current study we investigated the regulation of JA and its highly bioactive derivative (+-7-iso-jasmonoyl-L-isoleucine (JA-Ile, as well as the transcriptional regulation of several JA-dependent genes both in wild type and phyAphyC mutant. JA and JA-Ile levels increased in the mutant seedlings in response to blue light. However, in phyAphyC mutant leaves, which were continuously wounded, JA and JA-Ile levels were lower compared to those in the wild type. Hence, the mutation of phyA and phyC has differential effects on jasmonate levels depending on the tissue and developmental stage. Our results suggest that the contribution of JA-Ile to signaling during photomorphogenesis of rice is minor, as coleoptile phenotypes of phyAphyC mutants resemble those of jasmonate-deficient mutants despite the fact that induction by blue light leads to higher levels of JA-Ile compared to the wild type. We postulate that phyA and phyC could control the activity of specific enzymes metabolizing JA to active derivatives.

  5. [Establishment of a mutant Lumican transgenic mouse model].

    Science.gov (United States)

    Song, Yanzheng; Zhao, Yanyan; Zhang, Fengju; Yu, Yanqiu; Ma, Ling

    2014-01-01

    Pathological myopia (PM) is a hereditary ocular disease leading to severe loss of visual acuity and blindness. Lumican gene (LUM) is one of those candidate genes of PM. The purpose of this study was to establish a mutant Lumican transgenic mouse model, and to prepare for the further study of the pathogenesis of PM. Experimental study. Mutation of LUM gene was created by site-directed mutagenesis. Recombinant DNA techniques were used for the construction of the pRP. EX3d-EF1A>LUM/flag>IRES/hrGFP transgene. The gene fragments were microinjected into the zygote male pronuclei of BDF1 mice, and then the zygote cells alive were transplanted into the oviduct of acceptor pregnant female ICR mice. The F0 generation transgenic mice obtained were named C57-TgN (LUM)CCMU. Genome DNA from mice tail was detected by PCR and Western blotting. Six of 31 F0 generation mice were positive transgenic mice. The western blotting study showed that the flag-tag was expressed in the mouse tail tissue. Sixty-eight of 128 mice (F1 to F3 generation) were positive transgenic mice, the positive rate is 53.13%. The mutant Lumican (cDNA 596T>C) transgenic mouse model has been established. This model will provide fundamental conditions for studies of the pathogenesis of PM. Also it will be the basis of further studies about the effect of Lumican mutation on the development of PM and structure and function of the extra cellular matrix.

  6. Tissue Classification

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, David Gerald [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-01-01

    The project began as a e ort to support InLight and Lumidigm. With the sale of the companies to a non-New Mexico entity, the project then focused on supporting a new company Medici Technologies. The Small Business (SB) is attempting to quantify glucose in tissue using a series of short interferometer scans of the nger. Each scan is produced from a novel presentation of the nger to the device. The intent of the project is to identify and, if possible, implement improved methods for classi cation, feature selection, and training to improve the performance of predictive algorithms used for tissue classi cation.

  7. Arabinose Kinase-Deficient Mutant of Arabidopsis thaliana.

    Science.gov (United States)

    Dolezal, O; Cobbett, C S

    1991-08-01

    A mutant of Arabidopsis thaliana that is sensitive to exogenous l-arabinose has been isolated. Comparisons of growth of the wild type, mutant, and F1 and F2 progeny of crosses showed the arabinose-sensitive phenotype is semidominant and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed the mutation is linked to the eceriferum-2 locus on chromosome 4. In vivo incorporation of exogenous labeled l-arabinose into ethanol-insoluble polysaccharides was greatly reduced in the mutant with a concomitant accumulation of free labeled arabinose. Enzyme assays of crude plant extracts demonstrated a defect in arabinose kinase activity in the mutant.

  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. 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.

  10. Ribosylurea accumulates in yeast urc4 mutants.

    Science.gov (United States)

    Björnberg, O; Vodnala, M; Domkin, V; Hofer, A; Rasmussen, A; Andersen, G; Piskur, J

    2010-06-01

    Yeast Saccharomyces (Lachancea) kluyveri urc4 mutants, unable to grow on uracil, biotransformed (14)C(2)-uracil into two labeled compounds, as detected by high performance liquid chromatography (HPLC). These two compounds could also be obtained following organic synthesis of ribosylurea. This finding demonstrates that in the URC pyrimidine degradation pathway, the opening of the uracil ring takes place when uracil is attached to the ribose moiety. Ribosylurea has not been reported in the cell metabolism before and the two observed compounds likely represent an equilibrium mixture of the pyranosyl and furanosyl forms.

  11. Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains.

    Science.gov (United States)

    Najdenski, Hristo; Golkocheva, Elica; Kussovski, Veselin; Ivanova, Ekaterina; Manov, Vasil; Iliev, Mihail; Vesselinova, Anna; Bengoechea, José Antonio; Skurnik, Mikael

    2006-08-01

    An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.

  12. Identification and quantification of flavonoids in yellow grain mutant of rice (Oryza sativa L.).

    Science.gov (United States)

    Kim, Backki; Woo, Sunmin; Kim, Mi-Jung; Kwon, Soon-Wook; Lee, Joohyun; Sung, Sang Hyun; Koh, Hee-Jong

    2018-02-15

    Flavonoids are naturally occurring phenolic compounds with potential health-promoting activities. Although anthocyanins and phenolic acids in coloured rice have been investigated, few studies have focused on flavonoids. Herein, we analysed flavonoids in a yellow grain rice mutant using UHPLC-DAD-ESI-Q-TOF-MS, and identified 19 flavonoids by comparing retention times and accurate mass measurements. Among them, six flavonoids, isoorientin, isoorientin 2″-O-glucoside, vitexin 2″-O-glucoside, isovitexin, isoscoparin 2″-O-glucoside and isoscoparin, were isolated and fully identified from the yellow grain rice mutant, and the levels were significantly higher than wild-type, with isoorientin particularly abundant in mutant embryo. Significant differences in total phenolic compounds and antioxidant activity were observed in mutant rice by DPPH, FRAP and TEAC assays. The results suggest that the representative six flavonoids may play an important role in colouration and antioxidant activity of embryo and endosperm tissue. The findings provide insight into flavonoid biosynthesis and the possibility of improving functionality in rice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Isolation and partial characterization of a lead-accumulating Brassica juncea mutant.

    Science.gov (United States)

    Schulman, R N; Salt, D E; Raskin, I

    1999-08-01

    A new screening method for non-destructive, high-sensitivity, high-throughput isolation of plant mutants capable of accumulating large amounts of heavy metals has been developed. This method is based on incubating seedlings in a solution containing radioisotopes of the metals of interest and visualizing the tissue accumulation of these metals with a phosphorimager. We used this technique to isolate mutants of Brassica juncea (L.) Czern with increased accumulation of Cd and Pb for use in phytoremediation, an emerging technology using plants to remediate polluted soil and water. Approximately 50,000 M2 seedlings were screened and 21 mutants were recovered that retained increased accumulation through the third generation. Mutant 7/15-1 is characterized by enhanced Pb accumulation per unit of root fresh weight, stunted root growth, and decreased root cell size. Data indicate that roots of 7/15-1 contain more cell-wall material on a fresh-weight basis than roots of the wild-type, which may at least partially explain its ability to accumulate more Pb.

  14. Primary Cilia in the Murine Cerebellum and in Mutant Models of Medulloblastoma.

    Science.gov (United States)

    Di Pietro, Chiara; Marazziti, Daniela; La Sala, Gina; Abbaszadeh, Zeinab; Golini, Elisabetta; Matteoni, Rafaele; Tocchini-Valentini, Glauco P

    2017-01-01

    Cellular primary cilia crucially sense and transduce extracellular physicochemical stimuli. Cilium-mediated developmental signaling is tissue and cell type specific. Primary cilia are required for cerebellar differentiation and sonic hedgehog (Shh)-dependent proliferation of neuronal granule precursors. The mammalian G-protein-coupled receptor 37-like 1 is specifically expressed in cerebellar Bergmann glia astrocytes and participates in regulating postnatal cerebellar granule neuron proliferation/differentiation and Bergmann glia and Purkinje neuron maturation. The mouse receptor protein interacts with the patched 1 component of the cilium-associated Shh receptor complex. Mice heterozygous for patched homolog 1 mutations, like heterozygous patched 1 humans, have a higher incidence of Shh subgroup medulloblastoma (MB) and other tumors. Cerebellar cells bearing primary cilia were identified during postnatal development and in adulthood in two mouse strains with altered Shh signaling: a G-protein-coupled receptor 37-like 1 null mutant and an MB-susceptible, heterozygous patched homolog 1 mutant. In addition to granule and Purkinje neurons, primary cilia were also expressed by Bergmann glia astrocytes in both wild-type and mutant animals, from birth to adulthood. Variations in ciliary number and length were related to the different levels of neuronal and glial cell proliferation and maturation, during postnatal cerebellar development. Primary cilia were also detected in pre-neoplastic MB lesions in heterozygous patched homolog 1 mutant mice and they could represent specific markers for the development and analysis of novel cerebellar oncogenic models.

  15. Isolation of an Escherichia coli K4 kfoC mutant over-producing capsular chondroitin

    Directory of Open Access Journals (Sweden)

    De Rosa Mario

    2010-05-01

    Full Text Available Abstract Background Chondroitin sulphate is a complex polysaccharide having important structural and protective functions in animal tissues. Extracted from animals, this compound is used as a human anti-inflammatory drug. Among bacteria, Escherichia coli K4 produces a capsule containing a non-sulphate chondroitin and its development may provide an efficient and cheap fermentative production of the polysaccharide. Results A random N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis was performed on E. coli K4 to isolate mutants showing an increased production of chondroitin. Several mutants were isolated, one of which, here named VZ15, produced about 80% more chondroitin than the wild type E. coli. We found that the mutant has a missense mutation in the codon 313 of kfoC, the gene encoding chondroitin polymerase (K4CP, with a change from arginine to glutamine. A docking analysis to explain the increased productivity of the K4CP enzyme is presented. Conclusion The enhanced chondroitin production by the E. coli K4 mutant reported here shows the validity of the strain improvement strategy for more cost-friendly fermentative processes in the production of this pharmaceutically important but so-far expensive polysaccharide.

  16. Effects of ion beam irradiation on size of mutant sector and genetic damage in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Hase, Yoshihiro, E-mail: hase.yoshihiro@qst.go.jp [Takasaki Advanced Radiation Research Institute, National Institutes for Quantum and Radiological Science and Technology (QST), 1233 Watanuki, Takasaki, Gunma 370-1292 (Japan); Nozawa, Shigeki [Takasaki Advanced Radiation Research Institute, National Institutes for Quantum and Radiological Science and Technology (QST), 1233 Watanuki, Takasaki, Gunma 370-1292 (Japan); Narumi, Issay [Faculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura, Gunma 374-0193 (Japan); Oono, Yutaka [Takasaki Advanced Radiation Research Institute, National Institutes for Quantum and Radiological Science and Technology (QST), 1233 Watanuki, Takasaki, Gunma 370-1292 (Japan)

    2017-01-15

    Size of mutant sector and genetic damage were evaluated in Arabidopsis to further our understanding of effective ion beam use in plant mutation breeding. Arabidopsis seeds, heterozygous for the GLABRA1 (GL1) gene (GL1/gl1-1), were irradiated with 15.8 MeV/u neon ions (mean linear energy transfer (LET): 352 keV/μm), 17.3 MeV/u carbon ions (113 keV/μm), or {sup 60}Co gamma rays. The frequency and size of glabrous sectors generated because of inactivation of the GL1 allele were examined. The frequency and overall size of large deletions were evaluated based on the loss of heterozygosity of DNA markers using DNA isolated from glabrous tissue. Irrespective of the radiation properties, plants with mutant sectors were obtained at similar frequencies at the same effective dosage necessary for survival reduction. Ion beams tended to induce larger mutant sectors than gamma rays. The frequency of large deletions (>several kbp) increased as the LET value increased, with chromosome regions larger than 100 kbp lost in most large deletions. The distorted segregation ratio of glabrous plants in the progenies of irradiated GL1/gl1-1 plants suggested frequent occurrence of chromosome rearrangement, especially those subjected to neon ions. Exposure to ion beams with moderate LET values (30–110 keV/μm) is thought effective for inducing mutant sectors without causing extensive genetic damage.

  17. Genetic Analysis of Streaked and Abnormal Floret Mutant st-fon

    Directory of Open Access Journals (Sweden)

    De-xi CHEN

    2013-07-01

    Full Text Available A double mutant with streaked leaf and abnormal floret was found and temporarily named streaked leaf and floral organ number mutant (st-fon. For this mutant, besides white streak appeared on culm, leaves and panicles, the number of floral organs increased and florets cracked. The extreme phenotype was that several small florets grew from one floret or branch rachis in small florets extended and developed into panicles. By using transmission electron microscope to observe the ultrastructure of white histocytes of leaves at the seedling stage, the white tissues which showed abnormal plastids, lamellas and thylakoids could not develop into normal chloroplast, and the development of chloroplast was blocked at the early growth stage of plastid. Scanning electron microscope and paraffin section were also used to observe the development of floral organs, and the results indicated that the development of floral meristem was out of order and unlimited, whereas in the twisty leaves, vascular bundle sheath cells grew excessively, or some bubbly cells increased. Genetic analyses carried out by means of cross and backcross with four normal-leaf-color materials revealed that the mutant is of cytoplasm inheritance.

  18. Chitin synthase-deficient mutant of Fusarium oxysporum elicits tomato plant defence response and protects against wild-type infection.

    Science.gov (United States)

    Pareja-Jaime, Yolanda; Martín-Urdíroz, Magdalena; Roncero, María Isabel González; González-Reyes, José Antonio; Roldán, María Del Carmen Ruiz

    2010-07-01

    A mutant of the root pathogen Fusarium oxysporum f. sp. lycopersici, deficient in class V chitin synthase, has been shown previously to be nonvirulent. In this study, we tested the hypothesis that the cause of its avirulence could be the elicitation of the induced plant defence response, leading to the restriction of fungal infection. Co-inoculation of tomato plants with the wild-type strain and the DeltachsV mutant resulted in a significant reduction in symptom development, supporting a protective mechanism exerted by the mutant. The ability of the mutant to penetrate and colonize plant tissues was determined by scanning and transmission electron microscopy, as well as fluorescence microscopy using green fluorescent protein- or cherry fluorescent protein-labelled fungal strains. The extent of wild-type strain colonization in co-inoculated plants decreased steadily throughout the infection process, as shown by the quantification of fungal biomass using real-time polymerase chain reaction. The hypothesis that defence responses are activated by the DeltachsV mutant was confirmed by the analysis of plant pathogenesis-related genes using real-time reverse transcriptase-polymerase chain reaction. Tomato plants inoculated with the DeltachsV mutant showed a three fold increase in endochitinase activity in comparison with wild-type inoculated plants. Taken together, these results suggest that the perturbation of fungal cell wall biosynthesis results in elicitation of the plant defence response during the infection process.

  19. Uric Acid Accumulation in an Arabidopsis Urate Oxidase Mutant Impairs Seedling Establishment by Blocking Peroxisome Maintenance[W

    Science.gov (United States)

    Hauck, Oliver K.; Scharnberg, Jana; Escobar, Nieves Medina; Wanner, Gerhard; Giavalisco, Patrick; Witte, Claus-Peter

    2014-01-01

    Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants. PMID:25052714

  20. 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.

  1. pH dependency of sclerotial development and pathogenicity revealed by using genetically defined oxalate-minus mutants of Sclerotinia sclerotiorum.

    Science.gov (United States)

    Xu, Liangsheng; Xiang, Meichun; White, David; Chen, Weidong

    2015-08-01

    The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV-induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate-minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate-minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate-minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate-minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase beta genes.

    Science.gov (United States)

    Last, R L; Bissinger, P H; Mahoney, D J; Radwanski, E R; Fink, G R

    1991-01-01

    The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase beta genes (TSB1 and TSB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although TSB1 produces greater than 90% of tryptophan synthase beta mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase beta activity. The trp2-1 mutation is complemented by the TSB1 transgene and is linked genetically to a polymorphism in the TSB1 gene, strongly suggesting that trp2-1 is a mutation in TSB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, TSB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (TRP1 and TRP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis. PMID:1840915

  3. The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.

    Science.gov (United States)

    Hsieh, Wei-Yu; Liao, Jo-Chien; Wang, Hsin-Tzu; Hung, Tzu-Huan; Tseng, Ching-Chih; Chung, Tsui-Yun; Hsieh, Ming-Hsiun

    2017-07-01

    Thiamin diphosphate (TPP, vitamin B1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  4. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A.

    2017-03-01

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  5. 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.

  6. 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.

  7. 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.

  8. Studies on mutant breeding of Hibiscus syriacus

    Energy Technology Data Exchange (ETDEWEB)

    Song, Hi Sup; Kim, Jin Kyu; Lee, Ki Un; Kim, Young Taik

    1997-01-01

    Hibiscus has been known as a national flower of Korea. Hibiscus has such a characteristic of self-incompatibility that all the plant exist as natural hybrids and have heterogeneous genes. Many domestic 91 varieties of Hibiscus syriacus were collected. Radiosensitivity of H. Syriacus irradiated with {gamma}-ray was investigated in plant cuttings. The plant height was reduced by 45% in 5KR irradiated group, compared to control group. The radiation dose of 5KR could be recommended for mutation breeding of Hibiscus cuttings. Radiosensitivity of {gamma}-ray irradiated Hibiscus seed were investigated. The germination rate, survival rate and plant height was better in the 4KR irradiation plot than control. The radiation dose of 10{approx}12KR are recommended for mutation breeding of Hibiscus. Promising mutant lines were selected form the varieties of Hwarang, Wolsan no. 176, Ilpyondansim, Emille, Hanol, Yongkwang, Saeyongkwang, Chungmu, Imjinhong, Arang, Hungdansim-1 and Hongdansim-2. (author). 66 refs., 16 tabs., 13 figs.

  9. NAT10 is upregulated in hepatocellular carcinoma and enhances mutant p53 activity.

    Science.gov (United States)

    Li, Qijiong; Liu, Xiaofeng; Jin, Kemin; Lu, Min; Zhang, Chunfeng; Du, Xiaojuan; Xing, Baocai

    2017-08-31

    N-acetyltransferase 10 (NAT10) is a histone acetyltransferase which is involved in a wide range of cellular processes. Recent evidences indicate that NAT10 is involved in the development of human cancers. Previous study showed that NAT10 acetylates the tumor suppressor p53 and regulates p53 activation. As Tp53 gene is frequently mutated in hepatocellular carcinoma (HCC) and associates with the occurrence and development of HCC, the relationship between NAT10 and HCC was investigated in this study. Immunohistochemistry (IHC) and western blot analysis were performed to evaluate the NAT10 expression in HCC. Immunoprecipitation experiments were performed to verify the interaction of NAT10 with mutant p53 and Mdm2. RNA interference and Western blot were applied to determine the effect of NAT10 on mutant p53. Cell growth curve was used to examine the effect of NAT10 on HCC cell proliferation. NAT10 was upregulated in HCC and increased NAT10 expression was correlated with poor overall survival of the patients. NAT10 protein levels were significantly correlated with p53 levels in human HCC tissues. Furthermore, NAT10 increased mutant p53 levels by counteracting Mdm2 action in HCC cells and promoted proliferation in cells carrying p53 mutation. Increased NAT10 expression levels are associated with shortened patient survival and correlated with mutant p53 levels. NAT10 upregulates mutant p53 level and might enhance its tumorigenic activity. Hence, we propose that NAT10 is a potential prognostic and therapeutic candidate for p53-mutated HCC.

  10. Exchange Protein Directly Activated by cAMP (EPAC) Regulates Neuronal Polarization through Rap1B

    NARCIS (Netherlands)

    Munoz-Llancao, Pablo; Henriquez, Daniel R.; Wilson, Carlos; Bodaleo, Felipe; Boddeke, Erik W.; Lezoualc'h, Frank; Schmidt, Martina; Gonzalez-Billault, Christian

    2015-01-01

    Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cAMP is involved in axonal specification through activation of protein kinase A. However, an alternative cAMP-dependent mechanism involves the exchange protein directly activated by

  11. Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

    Science.gov (United States)

    2016-11-01

    AWARD NUMBER: W81XWH-14-1-0177 TITLE: Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Katerina...5a. CONTRACT NUMBER Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer 5b. GRANT NUMBER W81XWH-14-1-0177 5c. PROGRAM ELEMENT NUMBER...epigenomic landscape of EGFR mutant SCLCs and their corresponding pre- treatment LUADs. These are very rare specimens. Through our Yale rebiopsy program

  12. Mutant-specific gene programs in the zebrafish

    OpenAIRE

    Weber, Gerhard J.; Choe, Sung E; Dooley, Kimberly A.; Paffett-Lugassy, Noëlle N.; Zhou, Yi; Zon, Leonard I.

    2005-01-01

    Hematopoiesis involves the production of stem cells, followed by the orchestrated differentiation of the blood lineages. Genetic screens in zebrafish have identified mutants with defects that disrupt specific stages of hematopoiesis and vasculogenesis, including the cloche, spadetail (tbx16), moonshine (tif1g), bloodless, and vlad tepes (gata1) mutants. To better characterize the blood program, gene expression profiling was carried out in these mutants and in scl-morphants (scl mo). Distinct ...

  13. Mutant p53 in Cancer: New Functions and Therapeutic Opportunities

    Science.gov (United States)

    Muller, Patricia A.J.; Vousden, Karen H.

    2014-01-01

    Many different types of cancer show a high incidence of TP53 mutations, leading to the expression of mutant p53 proteins. There is growing evidence that these mutant p53s have both lost wild-type p53 tumor suppressor activity and gained functions that help to contribute to malignant progression. Understanding the functions of mutant p53 will help in the development of new therapeutic approaches that may be useful in a broad range of cancer types. PMID:24651012

  14. Growth and development of maize that contains mutant tubulin genes

    Energy Technology Data Exchange (ETDEWEB)

    Susan M. Wick

    2004-07-23

    Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.

  15. Sphingolipid synthesis deficiency in a mutant of Bacteroides levii

    Energy Technology Data Exchange (ETDEWEB)

    Brumleve, B.; Lev, M.

    1986-05-01

    Bacteroides levii, an anaerobic bacterium, synthesizes two sphingolipids; the sphingomyelin analogue, ceramide phosphorylethanolamine (CPE), and also ceramide phosphorylglycerol (CPG). The first enzyme in the sphingolipid pathway, 3-ketodihydro-sphingosine (3KDS) synthase, has been partially purified previously. To study subsequent steps in the pathways, mutants defective in sphingolipid synthesis were derived by ethyl methanesulfonate and nitrosoguanidine mutagenesis. Extracts of the mutant, 1075BB, show synthase activity although the cells do not synthesize CPE or CPG. The mutant differs from the wild type in that: (1) synthase activity was much diminished in the mutant, (2) sphingolipid synthesis does not occur in the mutant as evidenced by the absence of spots at sites where CPE and CPG migrate following two-dimensional thin layer chromatography, (3) incorporation of uniformly-labelled (/sup 14/C)serine carbon or (/sup 14/C)3KDS into sphingolipids was not observed in the mutant, (4) following incubation with (/sup 14/C)3KDS, radioactivity corresponding to dihydrosphingosine (DHS) and ceramide were observed in the mutant; no (/sup 14/C)DHS was detected in the wild type, and (5) enhanced incorporation of (/sup 14/C)serine carbon into two lipids not containing phosphorus was found in the mutant. The authors conclude, therefore, that this mutant, 1075BB, has a metabolic block at the terminal biosynthetic steps of sphingolipid synthesis.

  16. Therapeutic targeting of p53: all mutants are equal, but some mutants are more equal than others.

    Science.gov (United States)

    Sabapathy, Kanaga; Lane, David P

    2017-09-26

    TP53, which encodes the tumour-suppressor protein p53, is the most frequently mutated gene across all cancer types. The presence of mutant p53 predisposes to cancer development, promotes the survival of cancer cells, and is associated with ineffective therapeutic responses and unfavourable prognoses. Despite these effects, no drug that abrogates the oncogenic functions of mutant p53 has yet been approved for the treatment of cancer. Current investigational therapeutic strategies are mostly aimed at restoring the wild-type activity of mutant p53, based on the assumption that all p53 mutants are functionally equal. Our increasing knowledge of mutant forms of p53, however, supports the antithetical hypothesis that not all p53 mutants have equivalent cellular effects; hence, a judicious approach to therapeutic targeting of mutant p53 is required. In this Review, we propose a categorization of the major classes of p53 mutants based on their functionality in tumour suppression and response to therapy. The emerging picture is that the mutations across TP53 form a 'rainbow of mutants', with varying degrees of functionality and different pathobiological consequences, necessitating the use of diverse therapeutic strategies to selectively target specific classes of mutation. The utility of this knowledge of TP53 mutations in developing selective therapeutic options, and in facilitating clinical decision-making is discussed.

  17. Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae.

    Science.gov (United States)

    Hedman, Jamie M; Eggleston, Matthew D; Attryde, Amanda L; Marshall, Pamela A

    2007-10-01

    Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morphology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na(+)/H(+) exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.

  18. A Mutation in fat2 Uncouples Tissue Elongation from Global Tissue Rotation.

    Science.gov (United States)

    Aurich, Franziska; Dahmann, Christian

    2016-03-22

    Global tissue rotation was proposed as a morphogenetic mechanism controlling tissue elongation. In Drosophila ovaries, global tissue rotation of egg chambers coincides with egg chamber elongation. Egg chamber rotation was put forward to result in circumferential alignment of extracellular fibers. These fibers serve as molecular corsets to restrain growth of egg chambers perpendicular to the anteroposterior axis, thereby leading to the preferential egg chamber elongation along this axis. The atypical cadherin Fat2 is required for egg chamber elongation, rotation, and the circumferential alignment of extracellular fibers. Here, we have generated a truncated form of Fat2 that lacks the entire intracellular region. fat2 mutant egg chambers expressing this truncated protein fail to rotate yet display normal extracellular fiber alignment and properly elongate. Our data suggest that global tissue rotation, even though coinciding with tissue elongation, is not a necessary prerequisite for elongation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Identification of key uric acid synthesis pathway in a unique mutant silkworm Bombyx mori model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Hiroko Tabunoki

    Full Text Available Plasma uric acid (UA levels decrease following clinical progression and stage development of Parkinson's disease (PD. However, the molecular mechanisms underlying decreases in plasma UA levels remain unclear, and the potential to apply mutagenesis to a PD model has not previously been discovered. We identified a unique mutant of the silkworm Bombyx mori (B.mori op. Initially, we investigated the causality of the phenotypic "op" by microarray analysis using our constructed KAIKO functional annotation pipeline. Consequently, we found a novel UA synthesis-modulating pathway, from DJ-1 to xanthine oxidase, and established methods for large-scale analysis of gene expression in B. mori. We found that the mRNA levels of genes in this pathway were significantly lower in B. mori op mutants, indicating that downstream events in the signal transduction cascade might be prevented. Additionally, levels of B.mori tyrosine hydroxylase (TH and DJ-1 mRNA were significantly lower in the brain of B. mori op mutants. UA content was significantly lower in the B. mori op mutant tissues and hemolymph. The possibility that the B. mori op mutant might be due to loss of DJ-1 function was supported by the observed vulnerability to oxidative stress. These results suggest that UA synthesis, transport, elimination and accumulation are decreased by environmental oxidative stress in the B. mori op mutant. In the case of B. mori op mutants, the relatively low availability of UA appears to be due both to the oxidation of DJ-1 and to its expenditure to mitigate the effects of environmental oxidative stress. Our findings are expected to provide information needed to elucidate the molecular mechanism of decreased plasma UA levels in the clinical stage progression of PD.

  20. Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea.

    Science.gov (United States)

    Kawakami-Schulz, Sharolyn V; Verdoni, Angela M; Sattler, Shannon G; Jessen, Erik; Kao, Winston W-Y; Ikeda, Akihiro; Ikeda, Sakae

    2014-04-15

    Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstn(corn1) mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstn(corn1) mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstn(corn1) cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstn(corn1) cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstn(corn1) mice. Comparison of gene expression values from WT, Dstn(corn1) mutant, and Dstn(corn1) rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstn(corn1) mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstn(corn1) mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstn(corn1) mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis.

  1. Mutant spastin proteins promote deficits in axonal transport through an isoform-specific mechanism involving casein kinase 2 activation.

    Science.gov (United States)

    Leo, Lanfranco; Weissmann, Carina; Burns, Matthew; Kang, Minsu; Song, Yuyu; Qiang, Liang; Brady, Scott T; Baas, Peter W; Morfini, Gerardo

    2017-06-15

    Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Reliable diagnosis of IDH-mutant glioblastoma by 2-hydroxyglutarate detection: a study by 3-T magnetic resonance spectroscopy.

    Science.gov (United States)

    Natsumeda, Manabu; Motohashi, Kunio; Igarashi, Hironaka; Nozawa, Takanori; Abe, Hideaki; Tsukamoto, Yoshihiro; Ogura, Ryosuke; Okada, Masayasu; Kobayashi, Tsutomu; Aoki, Hiroshi; Takahashi, Hitoshi; Kakita, Akiyoshi; Okamoto, Kouichirou; Nakada, Tsutomu; Fujii, Yukihiko

    2017-09-27

    We have previously reported that reliable detection of 2-hydroxyglutarate (2HG) in isocitrate dehydrogenase (IDH)-mutant WHO grade 2 and 3 gliomas is possible utilizing 3.0-T single-voxel magnetic resonance spectroscopy (SVMRS). We set out to determine whether the same method could be applied to detect 2HG in IDH-mutant glioblastoma. Forty-four patients harboring glioblastoma underwent pre-operative MRS evaluation to detect 2HG and other metabolites. Presence of IDH-mutations was determined by IDH1 R132H immunohistochemical analysis and DNA sequencing of surgically obtained tissues. Six out of 44 (13.6%) glioblastomas were IDH-mutant. IDH-mutant glioblastoma exhibited significantly higher accumulation of 2HG (median 3.191 vs. 0.000 mM, p IDH-mutation in glioblastoma. Glioblastoma with high 2HG accumulation did not have significantly longer overall survival than glioblastoma with low 2HG accumulation (p = 0.107, log-rank test). Non-invasive and reliable detection of 2HG in IDH-mutant glioblastoma was possible by 3.0-T SVMRS.

  3. Isolation and characterization of stable mutants of Streptomyces ...

    Indian Academy of Sciences (India)

    Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer ...

  4. Differentially expressed genes in white egg 2 mutant of silkworm ...

    African Journals Online (AJOL)

    These pathways were related to amino acid metabolism, sugar metabolism, and series of major physiological metabolism. Our results hopefully shed light on the further study of molecular mechanism of white egg 2 mutant. Key words: Bombyx mori, white egg 2 mutant, microarray, embryo, differentially expressed gene.

  5. Genomic diversity among Basmati rice ( Oryza sativa L) mutants ...

    African Journals Online (AJOL)

    Genomic diversity among Basmati rice ( Oryza sativa L) mutants obtained through 60 Co gamma radiations using AFLP markers. ... In order to obtain new varieties of rice with improved agronomic and grain characteristics, gamma radiation (60Co) has been used to generate novel mutants of the Basmati rice. In this study ...

  6. Enhanced production of glucose oxidase from UV- mutant of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-01-19

    Jan 19, 2009 ... UV rays were used as mutagen in wild type strain of Aspergillus niger for enhanced production of glucose oxidase. After mutangenization and selection, mutant A. niger strains, resistant to 2-deoxy-D- glucose were obtained. The mutants showed 1.57 and 1.98 fold increase in activities of extra and intra.

  7. Comparison of lignin deposition in three ectopic lignification mutants.

    Science.gov (United States)

    Rogers, Louisa A; Dubos, Christian; Surman, Christine; Willment, Janet; Cullis, Ian F; Mansfield, Shawn D; Campbell, Malcolm M

    2005-10-01

    The Arabidopsis thaliana mutants de-etiolated3 (det3), pom-pom1 (pom1) and ectopic lignification1 (eli1) all deposit lignins in cells where these polymers would not normally be found. Comparison of these mutants provides an opportunity to determine if the shared mutant phenotype arose by perturbing a common regulatory mechanism in each of the mutants. The mutants were compared using a combination of genetics, histochemistry, chemical profiling, transcript profiling using both Northern blots and microarrays, and bioinformatics. The subset of cells that ectopically lignified was shared between all three mutants, but clear differences in cell wall chemistry were evident between the mutants. Northern blot analysis of lignin biosynthetic genes over diurnal and circadian cycles revealed that transcript abundance of several key genes was clearly altered in all three mutants. Microarray analysis suggests that changes in the expression of specific members of the R2R3-MYB and Dof transcription factor families may contribute to the ectopic lignification phenotypes. This comparative analysis provides a suite of hypotheses that can be tested to examine the control of lignin biosynthesis.

  8. Molecularly targeted therapies for p53-mutant cancers.

    Science.gov (United States)

    Zhao, Dekuang; Tahaney, William M; Mazumdar, Abhijit; Savage, Michelle I; Brown, Powel H

    2017-11-01

    The tumor suppressor p53 is lost or mutated in approximately half of human cancers. Mutant p53 not only loses its anti-tumor transcriptional activity, but also often acquires oncogenic functions to promote tumor proliferation, invasion, and drug resistance. Traditional strategies have been taken to directly target p53 mutants through identifying small molecular compounds to deplete mutant p53, or to restore its tumor suppressive function. Accumulating evidence suggest that cancer cells with mutated p53 often exhibit specific functional dependencies on secondary genes or pathways to survive, providing alternative targets to indirectly treat p53-mutant cancers. Targeting these genes or pathways, critical for survival in the presence of p53 mutations, holds great promise for cancer treatment. In addition, mutant p53 often exhibits novel gain-of-functions to promote tumor growth and metastasis. Here, we review and discuss strategies targeting mutant p53, with focus on targeting the mutant p53 protein directly, and on the progress of identifying genes and pathways required in p53-mutant cells.

  9. Unfolding intermediates of the mutant His-107-Tyr of human ...

    Indian Academy of Sciences (India)

    The mutant His-107-Tyr of human carbonic anhydrase II (HCA II) is highly unstable and has long been linked to a misfolding disease known as carbonic anhydrase deficiency syndrome (CADS). High temperature unfolding trajectories of the mutant are obtained from classical molecular dynamics simulationsand analyzed in ...

  10. Assessment of Genetic diversity in mutant cowpea lines using ...

    African Journals Online (AJOL)

    FKOLADE

    2016-11-09

    Nov 9, 2016 ... for crop improvement, hence the need to broaden the genetic base of any crop. This study was done in order to further enhance this in cowpea. While assessing diversity and phylogenetic relationship with other mutants and their parents, each unique mutant was also characterized. Randomly amplified ...

  11. Differentially expressed genes in white egg 2 mutant of silkworm ...

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... egg 2 (w-2) has the same phenotypes as white egg 1 and white egg 3 mutants with white egg color, but its mechanism is more complicated than white egg 1 and white egg 3 mutants based on recent report (Tatematsu et al., 2011) which suggest that the silkworm w-2 locus existed multi-allelic mutations.

  12. Characterization of human glucocerebrosidase from different mutant alleles

    NARCIS (Netherlands)

    Ohashi, T.; Hong, C. M.; Weiler, S.; Tomich, J. M.; Aerts, J. M.; Tager, J. M.; Barranger, J. A.

    1991-01-01

    Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from

  13. Photosynthetic characterization of a rolled leaf mutant of rice ( Oryza ...

    African Journals Online (AJOL)

    A new rolling leaf rice mutant was identified which showed an apparently straighter longitudinal shape normal transverse rolling characters at all developing stages. The chlorophyll contents per fresh weight of this mutant leaves were lower than those of wild-type. The electron transfer rate (ETR) and photochemical ...

  14. Unfolding intermediates of the mutant His-107-Tyr of human ...

    Indian Academy of Sciences (India)

    Srabani Taraphder

    Abstract. The mutant His-107-Tyr of human carbonic anhydrase II (HCA II) is highly unstable and has long been linked to a misfolding disease known as carbonic anhydrase deficiency syndrome (CADS). High temperature unfolding trajectories of the mutant are obtained from classical molecular dynamics simulations.

  15. Decreased cariogenicity of a mutant of Streptococcus mutans

    NARCIS (Netherlands)

    Stoppelaar, J.D.; König, K.G.; Plasschaert, A.J.M.; Hoeven, J.S. van der

    A strain of Streptococcus mutans was treated with a mutagenic agent. This resulted in isolation of a mutant which, compared to the original strain, had lost the ability to form sticky deposits on hard surfaces in sucrose medium. Apart from colonial morphology, the mutant had not changed in any other

  16. Tumor-specific signaling to p53 is mimicked by Mdm2 inactivation in zebrafish: insights from mdm2 and mdm4 mutant zebrafish

    Science.gov (United States)

    Chua, J S; Liew, H P; Guo, L; Lane, D P

    2015-01-01

    In mice, the deletion of either Mdm2 or Mdm4 results in a p53-dependent embryonic lethality. We used zinc-finger nucleases to construct mutations in the mdm2 and mdm4 genes of zebrafish. Although the loss of mdm2 results in a p53-dependent early embryonic lethality, mdm4 mutant fish are viable and grow to adulthood. We also found that an in-frame five-amino acid deletion in mdm2 creates a novel hypomorphic allele. The lethal phenotype observed in the mdm2 mutant fish could be partially rescued by injecting mRNA encoding functional Mdm2, and this required the E3 ligase activity of the protein. Complete rescue was obtained by crossing the mdm2 mutant fish onto a p53M214K mutant background. Although p53 mutant fish on a wild-type mdm2 background were shown to accumulate high levels of p53 protein specifically in tumor tissues, we detected extensive staining of p53 in many normal tissues of the mdm2–p53M214K double-mutant fish. Our results are suggestive of the hypothesis that p53 protein accumulates during tumor formation as a result of tumor-specific inactivation of the Mdm2 pathway. PMID:25746004

  17. The application of an alanine-substituted mutant of the C-terminal fragment of Clostridium perfringens enterotoxin as a mucosal vaccine in mice.

    Science.gov (United States)

    Suzuki, Hidehiko; Kondoh, Masuo; Kakutani, Hideki; Yamane, Seiji; Uchida, Hiroshi; Hamakubo, Takao; Yagi, Kiyohito

    2012-01-01

    Efficient delivery of antigen to mucosal immune tissues is an essential part of mucosal vaccination. Claudin-4 is expressed on the epithelial cells that cover the mucosal immune tissues. We previously found that claudin-4-targeting is a promising strategy for mucosal vaccination by using a claudin-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Substitution of Asn and Ser at positions 309 and 313, respectively, with alanine increased the affinity of C-CPE for claudin-4. However, application of the C-CPE mutant as a mucosal vaccine has never been tried. Here, we investigated whether the C-CPE mutant could serve as a mucosal vaccine. We used ovalbumin (OVA) as a model antigen and fused the C-CPE mutant to it. The resultant fusion protein was bound to claudin-4. When mice were immunized with the C-CPE mutant-fused OVA, OVA-specific serum IgG and nasal IgA increased relative to levels in mice immunized with a C-CPE-fused antigen. Immunization with the C-CPE mutant-fused OVA activated Th1- and Th2-type responses and led to increased anti-tumor activity against OVA-expressing thymoma cells relative to that of mice immunized with the C-CPE-fused antigen. These findings suggest that the alanine-substituted C-CPE mutant shows promise as a claudin-targeted mucosal vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Mutants of Cre recombinase with improved accuracy

    Science.gov (United States)

    Eroshenko, Nikolai; Church, George M.

    2013-01-01

    Despite rapid advances in genome engineering technologies, inserting genes into precise locations in the human genome remains an outstanding problem. It has been suggested that site-specific recombinases can be adapted towards use as transgene delivery vectors. The specificity of recombinases can be altered either with directed evolution or via fusions to modular DNA-binding domains. Unfortunately, both wildtype and altered variants often have detectable activities at off-target sites. Here we use bacterial selections to identify mutations in the dimerization surface of Cre recombinase (R32V, R32M, and 303GVSdup) that improve the accuracy of recombination. The mutants are functional in bacteria, in human cells, and in vitro (except for 303GVSdup, which we did not purify), and have improved selectivity against both model off-target sites and the entire E. coli genome. We propose that destabilizing binding cooperativity may be a general strategy for improving the accuracy of dimeric DNA-binding proteins. PMID:24056590

  19. Gene expression profile analysis of Ligon lintless-1 (Li1) mutant reveals important genes and pathways in cotton leaf and fiber development.

    Science.gov (United States)

    Ding, Mingquan; Jiang, Yurong; Cao, Yuefen; Lin, Lifeng; He, Shae; Zhou, Wei; Rong, Junkang

    2014-02-10

    Ligon lintless-1 (Li1) is a monogenic dominant mutant of Gossypium hirsutum (upland cotton) with a phenotype of impaired vegetative growth and short lint fibers. Despite years of research involving genetic mapping and gene expression profile analysis of Li1 mutant ovule tissues, the gene remains uncloned and the underlying pathway of cotton fiber elongation is still unclear. In this study, we report the whole genome-level deep-sequencing analysis of leaf tissues of the Li1 mutant. Differentially expressed genes in leaf tissues of mutant versus wild-type (WT) plants are identified, and the underlying pathways and potential genes that control leaf and fiber development are inferred. The results show that transcription factors AS2, YABBY5, and KANDI-like are significantly differentially expressed in mutant tissues compared with WT ones. Interestingly, several fiber development-related genes are found in the downregulated gene list of the mutant leaf transcriptome. These genes include heat shock protein family, cytoskeleton arrangement, cell wall synthesis, energy, H2O2 metabolism-related genes, and WRKY transcription factors. This finding suggests that the genes are involved in leaf morphology determination and fiber elongation. The expression data are also compared with the previously published microarray data of Li1 ovule tissues. Comparative analysis of the ovule transcriptomes of Li1 and WT reveals that a number of pathways important for fiber elongation are enriched in the downregulated gene list at different fiber development stages (0, 6, 9, 12, 15, 18dpa). Differentially expressed genes identified in both leaf and fiber samples are aligned with cotton whole genome sequences and combined with the genetic fine mapping results to identify a list of candidate genes for Li1. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Resistance to thyroid hormone due to a novel thyroid hormone receptor mutant in a patient with hypothyroidism secondary to lingual thyroid and functional characterization of the mutant receptor.

    Science.gov (United States)

    Nakajima, Yasuyo; Yamada, Masanobu; Horiguchi, Kazuhiko; Satoh, Tetsurou; Hashimoto, Koshi; Tokuhiro, Etsuro; Onigata, Kazuhiko; Mori, Masatomo

    2010-08-01

    We describe a rare case of congenital hypothyroidism and an extremely high serum thyrotropin (TSH) level caused by a combination of resistance to thyroid hormone (RTH) and a lingual thyroid. As the RTH mutant, R316C, was new, the optimum dose of levothyroxine was unclear. To aid in assessment of the therapy, we characterized the mutant R316C thyroid hormone receptor (TR) and compared it with a common mutant, R316H, using in vitro studies. The patient was a newborn female having severe hypothyroidism with a free thyroxine level of 0.36 ng/dL and a serum TSH level of 177 microU/mL. A scintiscan showed ectopic lingual thyroid tissue without a normal thyroid gland. Supplementation with levothyroxine at a dose of >350 microg/day did not normalize the serum TSH level; however, the patient showed normal growth and intelligence at 14 years of age. Consistent with the results of a computer analysis, the binding of R316C to triiodothyronine (T3) was significantly decreased to 38% that of the wild type. Electrophoretic mobility shift assay demonstrated that like R316H, R316C did not form a homodimer, but formed a heterodimer with RXR. However, a glutathione-S-transferase pull-down assay showed reduced binding of R316C with NCoR in the absence of T3 and impaired release in the presence of T3. In addition, transient transfection experiments demonstrated that unlike R316H, R316C had severe impairment of transcriptional activity on genes both positively and negatively regulated by thyroid hormone. It also had a clear dominant negative effect on genes negatively, but not positively, regulated by thyroid hormone, including the TSH-releasing hormone and TSHbeta genes. This is the first reported case of a R316C TR mutation. The characteristics of the R316C mutant differed from those of the R316H mutant. Our findings suggest that R316C causes reduced association with and impaired release of NCoR, resulting in RTH predominantly at the pituitary level, and that slightly elevated serum

  1. Induction and characterization of Arabidopsis mutants by Ion beam

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Y. H.; Choi, J. D.; Park, J. Y.; Lee, J. R.; Sohn, H. S. [Gyeongbuk Institute for Bio Industry, Andong (Korea, Republic of)

    2008-03-15

    This study was conducted to search the proper conditions and times for irradiating proton beam to seeds generally used for induction of mutant. Arabidopsis as model plants has good characters that is a short generation time, producing a lot of seeds, sequenced genome, developed maker. This points were the best materials for plant breeding for this study. The data of inducing mutants of Arabidopsis is used to be applicate to crops have more longer generation that is the final goals of this study. The goals of this project were to inducing and characterizing arabidopsis mutants by the proton ion beam and {gamma}-ray. As well as, the purpose of this study was securing more than 10 lines of arabidopsis mutants in this project and also to know the changed DNA structure of the mutants using the basic data for applying to the more study

  2. Misfolded opsin mutants display elevated β-sheet structure.

    Science.gov (United States)

    Miller, Lisa M; Gragg, Megan; Kim, Tae Gyun; Park, Paul S-H

    2015-10-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself. Copyright © 2015 Federation of European Biochemical Societies. All rights reserved.

  3. Arabinose Kinase-Deficient Mutant of Arabidopsis thaliana 1

    Science.gov (United States)

    Dolezal, Olan; Cobbett, Christopher S.

    1991-01-01

    A mutant of Arabidopsis thaliana that is sensitive to exogenous l-arabinose has been isolated. Comparisons of growth of the wild type, mutant, and F1 and F2 progeny of crosses showed the arabinose-sensitive phenotype is semidominant and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed the mutation is linked to the eceriferum-2 locus on chromosome 4. In vivo incorporation of exogenous labeled l-arabinose into ethanol-insoluble polysaccharides was greatly reduced in the mutant with a concomitant accumulation of free labeled arabinose. Enzyme assays of crude plant extracts demonstrated a defect in arabinose kinase activity in the mutant. ImagesFigure 2Figure 3 PMID:16668327

  4. Developmental Biology: We Are All Walking Mutants.

    Science.gov (United States)

    Trainor, Paul A

    2016-01-01

    What is Developmental Biology? Developmental Biology is a discipline that evolved from the collective fields of embryology, morphology, and anatomy, which firmly established that structure underpins function. In its simplest terms, Developmental Biology has come to describe how a single cell becomes a completely formed organism. However, this definition of Developmental Biology is too narrow. Developmental Biology describes the properties of individual cells; their organization into tissues, organs, and organisms; their homeostasis, regeneration, aging, and ultimately death. Developmental Biology provides a context for cellular reprogramming, stem cell biology, regeneration, tissue engineering, evolutionary development and ecology, and involves the reiterated use of the same cellular mechanisms and signaling pathways throughout the lifespan of an organism. Using neural crest cells as an example, this review explores the contribution of Developmental Biology to our understanding of development, evolution, and disease. © 2016 Elsevier Inc. All rights reserved.

  5. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

    Science.gov (United States)

    García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

    2013-12-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga. Copyright © 2013 Elsevier GmbH. All rights reserved.

  6. Human liver cell trafficking mutants: characterization and whole exome sequencing.

    Directory of Open Access Journals (Sweden)

    Fei Yuan

    Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

  7. Lamin A/C mutants disturb sumo1 localization and sumoylation in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Émilie Boudreau

    Full Text Available A-type lamins A and C are nuclear intermediate filament proteins in which mutations have been implicated in multiple disease phenotypes commonly known as laminopathies. A few studies have implicated sumoylation in the regulation of A-type lamins. Sumoylation is a post-translational protein modification that regulates a wide range of cellular processes through the attachment of small ubiquitin-related modifier (sumo to various substrates. Here we showed that laminopathy mutants result in the mislocalization of sumo1 both in vitro (C2C12 cells overexpressing mutant lamins A and C and in vivo (primary myoblasts and myopathic muscle tissue from the Lmna(H222P/H222P mouse model. In C2C12 cells, we showed that the trapping of sumo1 in p.Asp192Gly, p.Gln353Lys, and p.Arg386Lys aggregates of lamin A/C correlated with an increased steady-state level of sumoylation. However, lamin A and C did not appear to be modified by sumo1. Our results suggest that mutant lamin A/C alters the dynamics of sumo1 and thus misregulation of sumoylation may be contributing to disease progression in laminopathies.

  8. PedonnanceofE3rly MatUring MutantS Derived from ''SuPa'~ Rice ...

    African Journals Online (AJOL)

    sig"ificant di.ffere-"~s between the mutants and their'parent for 'ail the maraders testeiiexcept 1 (j()(J grains weight and ~ipe weight The mutants .... reported in earlier rice improvement programmes

  9. 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

  10. Overexpression of RBBP6, alone or combined with mutant TP53, is predictive of poor prognosis in colon cancer.

    Directory of Open Access Journals (Sweden)

    Jian Chen

    Full Text Available Retinoblastoma binding protein 6 (RBBP6 plays an important role in chaperone-mediated ubiquitination and interacts with TP53 in carcinogenesis. However, the clinicopathologic significance of RBBP6 expression in colon cancer is unknown; in particular, the prognostic value of RBBP6 combined with TP53 expression has not been explored. Therefore, quantitative real-time PCR and western blot analyses were performed to detect RBBP6 expression in colon cancer tissues. RBBP6 and TP53 expression were assessed by immunohistochemistry in a tissue microarray format, in which the primary colon cancer tissue was paired with noncancerous tissue. Tissue specimens were obtained from 203 patients. We found that RBBP6 was overexpressed in colon tumorous tissues and was significantly associated with clinical stage, depth of tumor invasion, lymph node metastasis (LNM, distant metastasis, and histologic grade. Further studies revealed that a corresponding correlation between RBBP6 overexpression and mutant TP53 was evident in colon cancer (r = 0.450; P<0.001. RBBP6 expression was an independent prognostic factor for overall survival (OS and disease free survival (DFS. Interestingly, patients with tumors that had both RBBP6 overexpression and mutant TP53 protein accumulation relapsed and died within a significantly short period after surgery (P<0.001. Multivariate analysis showed that patients with LNM and patients with both RBBP6- and TP53-positive tumors had extremely poor OS (HR 6.75; 95% CI 2.63-17.35; P<0.001 and DFS (HR 8.08; 95% CI 2.80-23.30; P<0.001. These clinical findings indicate that the assessment of both RBBP6 and mutant TP53 expression will be helpful in predicting colon cancer prognosis.

  11. Genetic analysis of rice mutants responsible for narrow leaf phenotype and reduced vein number.

    Science.gov (United States)

    Kubo, Fumika Clara; Yasui, Yukiko; Kumamaru, Toshihiro; Sato, Yutaka; Hirano, Hiro-Yuki

    2017-03-17

    Leaves are a major site for photosynthesis and a key determinant of plant architecture. Rice produces thin and slender leaves, which consist of the leaf blade and leaf sheath separated by the lamina joint. Two types of vasculature, the large and small vascular bundles, run in parallel, together with a strong structure, the midrib. In this paper, we examined the function of four genes that regulate the width of the leaf blade and the vein number: NARROW LEAF1 (NAL1), NAL2, NAL3 and NAL7. We backcrossed original mutants of these genes with the standard wild-type rice, Taichung 65. We then compared the effect of each mutation on similar genetic backgrounds and examined genetic interactions of these genes. The nal1 single mutation and the nal2 nal3 double mutation showed a severe effect on leaf width, resulting in very narrow leaves. Although vein number was also reduced in the nal1 and nal2 nal3 mutants, the small vein number was more strongly reduced than the large vein number. In contrast, the nal7 mutation showed a milder effect on leaf width and vein number, and both the large and small veins were similarly affected. Thus, the genes responsible for narrow leaf phenotype seem to play distinct roles. The nal7 mutation showed additive effects on both leaf width and vein number, when combined with the nal1 single or the nal2 nal3 double mutation. In addition, observations of inner tissues revealed that cell differentiation was partially compromised in the nal2 nal3 nal7 mutant, consistent with the severe reduction in leaf width in this triple mutant.

  12. Bosutinib inhibits migration and invasion via ACK1 in KRAS mutant non-small cell lung cancer.

    Science.gov (United States)

    Tan, Daniel S W; Haaland, Benjamin; Gan, Jia Min; Tham, Su Chin; Sinha, Indrajit; Tan, Eng Huat; Lim, Kiat Hon; Takano, Angela; Krisna, Sai Sakktee; Thu, Minn Minn Myint; Liew, Hoe Peng; Ullrich, Axel; Lim, Wan-Teck; Chua, Boon Tin

    2014-01-24

    The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in "normal" tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches.

  13. Distribution of trace elements in normal and diseased mouse ileum and kidney tissues

    Energy Technology Data Exchange (ETDEWEB)

    Kirby, B.J.; Legge, G.J.F. (Micro Analytical Research Centre, School of Physics, Univ. of Melbourne, Parkville (Australia)); McArdle, H.; Danks, D.M. (Murdoch Inst., Royal Children' s Hospital, Parkville (Australia))

    1991-03-01

    A proton microprobe has been utilised to determine the distribution and relative concentration of Cu, Fe and Zn in 10 day old normal and brindled mouse small intestine and kidney tissues. High Cu levels were measured in the brindled mutant ileum and kidney tissues confirming previous tissue results. In the ileum, peripheral concentrations of both Fe and Cu in normal and mutant villi tips were observed. Ratios of X-ray yields from outer villus tip to inner villus tip irradiated areas were taken and compared for mutant and normal mice after normalising to the bremsstrahlung yield in each case. The ratio of outer to inner Fe concentrations in the epithelium were shown to be higher than normal in the diseased tissue. In the kidney, the high Cu concentration in the mutant tissue was found to be localised to within certain regions in the proximal tubule in the nephron. Accurate determination of the Cu distribution in the small intestine and kidney tissues of the mutant mouse will provide further information on where the defective reabsorption of Cu is occurring, and may contribute to a better understanding of the homologous human condition. (orig.).

  14. Pharmacological characterization of homobaclofen on wild type and mutant GABA(B)1b receptors coexpressed with the GABA(B)2 receptor

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Madsen, Bo E.; Krogsgaard-Larsen, P

    2001-01-01

    homogenate and in an assay of electrically induced contractions of guinea pig ileum. The results from the two tissues did, however, not correlate very well, and in order to further investigate these discrepancies, we have pharmacologically characterized these enantiomers on recombinant wild type and mutant...

  15. Mutation breedings in ornamental plants. Technique used for radiation induced mutant in begonia, chrysanthemum, aberia and winter daphne

    Energy Technology Data Exchange (ETDEWEB)

    Matsubara, Hisao

    1984-03-01

    Several methods of obtaining somatic mutant plants by el-ray irradiation on pieces of tissues as in vitro adventitious bud technique or small cutting methods with repeated pruning are described. The irradiation to the adventitious buds in the small pieces of organ cultured in vitro and to the small cuttings are employed. Culture beds of agar or of Japanese Kanuma soil were used in vitro culture. In these experiments, Japanese Kanuma soil bed in in vitro culture worked well for root development and transplant of the induced mutants. Combination with in vitro culture and repeated pruning technique were used for isolation and fixation of solid somatic mutant from small sectorial mutation induced by irradiation. This method was successful for begonia, chrysanthemum, aberia and winter daphne. These data indicates that most of the induced mutant plants were non-chimeric, while a few others were chimeric. Among the new varieties, ''Gin-Sei'', ''Ryoku-Ha'', ''Big-Cross'', ''Kaede-Iron'', ''Mei-Fu-Hana-Tsukubane-Utsugi'' and ''Daphne-el-3'' are non-chimeric, and ''Mini-Mini-Iron'' and ''Orange-Iron'' are chimeric. Moreover, these new varieties have remarkably differed in size and in color pattern from original variety. From the experimental results of somatic mutation, it is indicated that plant tissue culture have enormous potential in radiation breeding and in rapid propagation of the somatic mutant. (author).

  16. Whole Genome Sequencing-Based Mapping and Candidate Identification of Mutations from Fixed Zebrafish Tissue

    Directory of Open Access Journals (Sweden)

    Nicholas E. Sanchez

    2017-10-01

    Full Text Available As forward genetic screens in zebrafish become more common, the number of mutants that cannot be identified by gross morphology or through transgenic approaches, such as many nervous system defects, has also increased. Screening for these difficult-to-visualize phenotypes demands techniques such as whole-mount in situ hybridization (WISH or antibody staining, which require tissue fixation. To date, fixed tissue has not been amenable for generating libraries for whole genome sequencing (WGS. Here, we describe a method for using genomic DNA from fixed tissue and a bioinformatics suite for WGS-based mapping of zebrafish mutants. We tested our protocol using two known zebrafish mutant alleles, gpr126st49 and egr2bfh227, both of which cause myelin defects. As further proof of concept we mapped a novel mutation, stl64, identified in a zebrafish WISH screen for myelination defects. We linked stl64 to chromosome 1 and identified a candidate nonsense mutation in the F-box and WD repeat domain containing 7 (fbxw7 gene. Importantly, stl64 mutants phenocopy previously described fbxw7vu56 mutants, and knockdown of fbxw7 in wild-type animals produced similar defects, demonstrating that stl64 disrupts fbxw7. Together, these data show that our mapping protocol can map and identify causative lesions in mutant screens that require tissue fixation for phenotypic analysis.

  17. 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. PMID:26447695

  18. Mutant Resources for the Functional Analysis of the Rice Genome

    National Research Council Canada - National Science Library

    Nili Wang Tuan Long Wen Yao Lizhong Xiong Qifa Zhang Changyin Wu

    2013-01-01

    .... In order to systematically assign functions to all predicted genes in the rice genome, a large number of rice mutant lines, including those created by T-DNA insertion, Ds/dSpm tagging, Tos17 tagging...

  19. Hole poking and motor coordination in lurcher mutant mice.

    Science.gov (United States)

    Lalonde, R; Joyal, C C; Guastavino, J M; Botez, M I

    1993-07-01

    Lurcher mutant mice, a cerebellar mutant displaying ataxia and equilibrium deficits, had fewer hole pokes in a 16-hole matrix than normal mice. Lurcher mutants also took longer to reach a platform from a grid and to begin to climb a grid from the floor. However, the lurchers climbed as high as normal mice on the grid and their exploratory patterns of the holeboard were similar in many respects to normal mice, such as the ratio of center to peripheral hole exploration. In a wooden beam test, although lurchers did not differ from normal mice in terms of the amount of time spent on the beam or in the distance travelled, the mutants were found more often in unstable positions.

  20. Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii.

    Science.gov (United States)

    Sadana, J C; Shewale, J G; Deshpande, M V

    1979-10-01

    A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-beta-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-beta-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.

  1. Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii†

    Science.gov (United States)

    Sadana, J. C.; Shewale, J. G.; Deshpande, M. V.

    1979-01-01

    A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-β-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-β-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control. Images PMID:16345449

  2. Globulin gene expression in embryos of maize viviparous mutants

    Energy Technology Data Exchange (ETDEWEB)

    Kriz, A.R.; Wallace, M.S.; Paiva, R. (Univ. of Illinois, Urbana (USA))

    1990-02-01

    Expression of genes encoding the major Zea mays embryo globulins was examined in the maize precocious germination viviparous (vp) mutants. Comparison of globulin protein profiles of precociously germinating mutant embryos with those of normally germinating mature embryos revealed substantial differences with respect to the proteins encoded by the Glb1 gene. Analysis of Glb1 transcript levels in vp/vp embryos suggests that these mutants do not fully switch from a program of embryo maturation to one of germination. These preliminary studies indicate that the vp mutants provide an excellent system for the study of embryo maturation in maize. We also provide evidence for the positive regulation of Glb1 expression by the plant growth regulator abscisic acid.

  3. 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...

  4. 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

  5. Characterization of Pasteurella multocida mutants of low virulence.

    Science.gov (United States)

    Lee, M D; Glisson, J R; Wooley, R E; Brown, J

    1990-01-01

    Ten temperature-sensitive mutants of the Clemson University (CU) vaccine strain of Pasteurella multocida have been developed and were characterized by phenotypic attributes such as carbohydrate fermentation, antibiotic resistance, and membrane protein profiles. Some mutants were found to have lost the ability to utilize some substrates, notably xylose and gluconate, whereas others were able to ferment additional carbohydrates such as arabinose and rhamnose. CU was found to be resistant to sulfisoxazole, of intermediate resistance to bacitracin, and sensitive to rifampin; the sensitivity to these three antibiotics varied among the mutant strains, but 60% were resistant to rifampin. Membrane protein profiles demonstrated some changes in major bands, and there was variation in 50% of the mutants in proteins in the 31 kilodalton range. All strains were assayed for the presence of several virulence factors, and many were found to produce siderophore and to exhibit some degree of complement resistance.

  6. 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.

  7. Uv- and Gamma-Radiation Sensitive Mutants of Arabidopsis Thaliana

    OpenAIRE

    Jiang, C Z; Yen, C. N.; Cronin, K; Mitchell, D.; Britt, A B

    1997-01-01

    Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and -independent pathways. The mechanism of the ``dark repair'' pathway is still unknown. To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light. Seven independently derived UV sensitive mutants were isolated from an EMS-mutagenized population. These fell in...

  8. [The behavioral development of the mutant "staggerer" mouse].

    Science.gov (United States)

    Guastavino, J M

    1978-01-01

    The behavioural study, in particular rearing environmental conditions, of the mutant mouse staggerer has shown that such animals may live more than 90 days. (he behavioural diagnosis of this mutation has been possible from the second week of life, using specific tests. A typical "bat posture" permits one to recognize the mutant from the normal Mouse. Locomotory and feeding behaviours also present late and various qualitatige particularities.

  9. Structure prediction of subtilisin BPN' mutants using molecular dynamics methods

    OpenAIRE

    Heiner, Andreas P.; Berendsen, Herman J.C.; van Gunsteren, Wilfred F.

    2017-01-01

    In this paper we describe the achievements and pitfalls encountered in doing structure predictions of protein mutants using molecular dynamics simulation techniques in which properties of atoms are slowly changed as a function of time. Basically the method consists of a thermodynamic integration (slow growth) calculation used for free energy determination, but aimed at structure prediction; this allows for a fast determination of the mutant structure. We compared the calculated structure of t...

  10. Fate of peptides in peptidase mutants of Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, E.R S; Mierau, I; Poolman, B.; Konings, W.N; Venema, G; Kok, J.

    The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepN, pepC, pepO, pepX and pepT were deleted, Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu-Gly-Gly, Gly-Phe-Leu, Leu-Gly-Pro,

  11. Salt tolerance, salt accumulation, and ionic homeostasis in an epidermal bladder-cell-less mutant of the common ice plant Mesembryanthemum crystallinum.

    Science.gov (United States)

    Agarie, Sakae; Shimoda, Toshifumi; Shimizu, Yumi; Baumann, Kathleen; Sunagawa, Haruki; Kondo, Ayumu; Ueno, Osamu; Nakahara, Teruhisa; Nose, Akihiro; Cushman, John C

    2007-01-01

    The aerial surfaces of the common or crystalline ice plant Mesembryanthemum crystallinum L., a halophytic, facultative crassulacean acid metabolism species, are covered with specialized trichome cells called epidermal bladder cells (EBCs). EBCs are thought to serve as a peripheral salinity and/or water storage organ to improve survival under high salinity or water deficit stress conditions. However, the exact contribution of EBCs to salt tolerance in the ice plant remains poorly understood. An M. crystallinum mutant lacking EBCs was isolated from plant collections mutagenized by fast neutron irradiation. Light and electron microscopy revealed that mutant plants lacked EBCs on all surfaces of leaves and stems. Dry weight gain of aerial parts of the mutant was almost half that of wild-type plants after 3 weeks of growth at 400 mM NaCl. The EBC mutant also showed reduced leaf succulence and leaf and stem water contents compared with wild-type plants. Aerial tissues of wild-type plants had approximately 1.5-fold higher Na(+) and Cl(-) content than the mutant grown under 400 mM NaCl for 2 weeks. Na(+) and Cl(-) partitioning into EBCs of wild-type plants resulted in lower concentrations of these ions in photosynthetically active leaf tissues than in leaves of the EBC-less mutant, particularly under conditions of high salt stress. Potassium, nitrate, and phosphate ion content decreased with incorporation of NaCl into tissues in both the wild type and the mutant, but the ratios of Na(+)/K(+) and Cl(-)/NO(3)(-)content were maintained only in the leaf and stem tissues of wild-type plants. The EBC mutant showed significant impairment in plant productivity under salt stress as evaluated by seed pod and seed number and average seed weight. These results clearly show that EBCs contribute to succulence by serving as a water storage reservoir and to salt tolerance by maintaining ion sequestration and homeostasis within photosynthetically active tissues of M. crystallinum.

  12. Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI and pseudoxanthoma elasticum (PXE

    Directory of Open Access Journals (Sweden)

    Alexander Apschner

    2014-07-01

    Full Text Available In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf, which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1, a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues – most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin. Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord

  13. Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)

    Science.gov (United States)

    Apschner, Alexander; Huitema, Leonie F. A.; Ponsioen, Bas; Peterson-Maduro, Josi; Schulte-Merker, Stefan

    2014-01-01

    In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues – most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype

  14. Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection.

    Science.gov (United States)

    Aalto, J; Pelkonen, S; Kalimo, H; Finne, J

    2001-10-01

    There is a molecular mimicry between the polysialic acid polysaccharide of bacterial pathogens causing sepsis and meningitis, and the carbohydrate units of the neural cell adhesion molecule NCAM. We investigated whether bacteriophage mutants with catalytically disabled endosialidase, which bind but do not cleave polysialic acid, could recognise and bind to bacterial and eukaryotic polysialic acid. In nitrocellulose dot blot assay the mutant bacteriophages, but not the wild-type phages, remained specifically bound to polysialic acid-containing bacteria including Escherichia coli K1 and K92, group B meningococci, Mannheimia (Pasteurella) haemolytica A2, and Moraxella nonliquefaciens. A minimum binding requirement was determined to be 10 sialyl residues in the polysialic acid chain. In Western blots the mutant phages specifically bound to the embryonic polysialylated form of NCAM, but not to the adult less sialylated form of the molecule. The mutant phages together with secondary anti-phage antibodies were subsequently successfully used in fluorescence microscopy of cultured cells and light microscopy of paraffin-embedded tissue sections as a probe for the eukaryotic polysialic acid. Thus, mutant bacteriophages of meningitis causing bacteria bind to and detect the molecularly mimicked polysialic acid of the neural cell adhesion molecule in host tissues.

  15. Hormone Profiling in Plant Tissues.

    Science.gov (United States)

    Müller, Maren; Munné-Bosch, Sergi

    2017-01-01

    Plant hormones are for a long time known to act as chemical messengers in the regulation of physiological processes during a plant's life cycle, from germination to senescence. Furthermore, plant hormones simultaneously coordinate physiological responses to biotic and abiotic stresses. To study the hormonal regulation of physiological processes, three main approaches have been used (1) exogenous application of hormones, (2) correlative studies through measurements of endogenous hormone levels, and (3) use of transgenic and/or mutant plants altered in hormone metabolism or signaling. A plant hormone profiling method is useful to unravel cross talk between hormones and help unravel the hormonal regulation of physiological processes in studies using any of the aforementioned approaches. However, hormone profiling is still particularly challenging due to their very low abundance in plant tissues. In this chapter, a sensitive, rapid, and accurate method to quantify all the five "classic" classes of plant hormones plus other plant growth regulators, such as jasmonates, salicylic acid, melatonin, and brassinosteroids is described. The method includes a fast and simple extraction procedure without time consuming steps as purification or derivatization, followed by optimized ultrahigh-performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UHPLC-MS/MS) analysis. This protocol facilitates the high-throughput analysis of hormone profiling and is applicable to different plant tissues.

  16. Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient.

    Science.gov (United States)

    Barateau, Alice; Vadrot, Nathalie; Agbulut, Onnik; Vicart, Patrick; Batonnet-Pichon, Sabrina; Buendia, Brigitte

    2017-04-24

    Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A.

  17. Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient

    Directory of Open Access Journals (Sweden)

    Alice Barateau

    2017-04-01

    Full Text Available Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A.

  18. Developmental patterns of 3-hydroxykynurenine accumulation in white and various other eye color mutants of Drosophila melanogaster.

    Science.gov (United States)

    Howells, A J; Summers, K M; Ryall, R L

    1977-12-01

    Several points of biochemical similarity between white and scarlet mutants suggest that both are defective in the transport of xanthommatin precursors. In both, accumulation of 3-hydroxykynurenine is negligible during larval life and occurs at only a slow rate during adult development. Larvae of both mutants also excrete 3H-3-hydroxykynurenine and 3H-kynurenine rapidly, which probably accounts for the normal levels of kynurenine during larval life. 3-Hydroxykynurenine levels are abnormal in all white mutants which were studied, although in two alleles which are strongly pigmented (w(sat) and w(col)) accumulation is enhanced rather than diminished. In w(a), larval accumulation is normal but accumulation during adult development is greatly diminished, suggesting that this mutation has a tissue-specific effect. Similar levels were found in zeste females. Of the 11 other eye color mutants tested, abnormal levels of 3-hydroxykynurenine were found in eight. In four of these (claret, light, lightoid, and pink), larval accumulation is negligible, suggesting that these have defects in the kynurenine transport system like scarlet and white. In three others, however (brown, karmoisin, and rosy), accumulation during larval life is enhanced. In cardinal accumulation is normal during larval life but is excessive during adult development. This evidence supports the suggestion that the cd mutation blocks the final step of xanthommatin synthesis.

  19. A Novel PTEN/Mutant p53/c-Myc/Bcl-XL Axis Mediates Context-Dependent Oncogenic Effects of PTEN with Implications for Cancer Prognosis and Therapy

    Directory of Open Access Journals (Sweden)

    Xiaoping Huang

    2013-08-01

    Full Text Available Phosphatase and tensin homolog located on chromosome 10 (PTEN is one of the most frequently mutated tumor suppressors in human cancer including in glioblastoma. Here, we show that PTEN exerts unconventional oncogenic effects in glioblastoma through a novel PTEN/mutant p53/c-Myc/Bcl-XL molecular and functional axis. Using a wide array of molecular, genetic, and functional approaches, we demonstrate that PTEN enhances a transcriptional complex containing gain-of-function mutant p53, CBP, and NFY in human glioblastoma cells and tumor tissues. The mutant p53/CBP/NFY complex transcriptionally activates the oncogenes c-Myc and Bcl-XL, leading to increased cell proliferation, survival, invasion, and clonogenicity. Disruption of the mutant p53/c-Myc/Bcl-XL axis or mutant p53/CBP/NFY complex reverses the transcriptional and oncogenic effects of PTEN and unmasks its tumor-suppressive function. Consistent with these data, we find that PTEN expression is associated with worse patient survival than PTEN loss in tumors harboring mutant p53 and that a small molecule modulator of p53 exerts greater antitumor effects in PTEN-expressing cancer cells. Altogether, our study describes a new signaling pathway that mediates context-dependent oncogenic/tumor-suppressive role of PTEN. The data also indicate that the combined mutational status of PTEN and p53 influences cancer prognosis and anticancer therapies that target PTEN and p53.

  20. The gravity persistent signal (gps) Mutants of Arabidopsis: Insights into Gravitropic Signal Transduction

    Science.gov (United States)

    Wyatt, S.

    The gravitropic response of Arabidopsis stems is rapid with a visible within 30 min and vertical reorientation within 2 h. However, horizontal gravistimulation for 3 h at 4°C does not cause curvature. When the stems are subsequently placed in the vertical position at RT, they bend in response to the previous, horizontal gravistimulation. These results indicate that the gravity perception step can occur at 4°C, but that part of the response is sensitive to cold. At 4°C, starch-containing amyloplasts in the endodermis of the inflorescence stems sedimented normally but auxin transport was abolished indicating that the cold treatment affected early events of the signal transduction pathway that occur after amyloplast sedimentation but prior to auxin transport. The gps mutants of Arabidopsis are a unique group of mutants that respond abnormally after gravistimulation at 4°C. gps1 shows no response to the cold gravistimulation, gps2 bends the wrong way as compared to wild type and gps3 over responds, bending past the anticipated curvature. The mutants were selected from a T-DNA tagged population. Cloning strategies based on the tag have been employed to identify the genes disrupted. GPS1 was cloned using TAIL PCR and is At3g20130, a cytochrome P450, CYP705A22, of unknown function. GPS1p::GFP fusions are being used to determine temporal and spatial expression of GPS1. The mutation in gps3 appears to disrupt a non-coding region downstream of At1g43950 No function has yet been determined for this region, but it appears that the mutation disrupts transcription of a transcription factor homologous to the DNA binding domain of an auxin response factor (ARF) 9-like protein. The identity of GPS2 is as yet unknown. The gps mutants represent potentially three independent aspects of signal transduction in the gravitropic response: perception or retention of the gravity signal (gps1), determination of the polarity of the response (gps2), and the tissue specificity of the

  1. An enhanced immune response of Mclk1⁺/⁻ mutant mice is associated with partial protection from fibrosis, cancer and the development of biomarkers of aging.

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    Dantong Wang

    Full Text Available The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1⁺/⁻ mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1⁺/⁻ mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1⁺/⁻ mutants, as well as during spontaneous tumorigenesis in Mclk1⁺/⁻Trp53⁺/⁻ double mutants. Tumour latency was increased by the Mclk1⁺/⁻ genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8⁺ T lymphocytes from Mclk1⁺/⁻ graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1⁺/⁻ mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process.

  2. Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure.

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    Wolf, Yochai; Boura-Halfon, Sigalit; Cortese, Nina; Haimon, Zhana; Sar Shalom, Hadas; Kuperman, Yael; Kalchenko, Vyacheslav; Brandis, Alexander; David, Eyal; Segal-Hayoun, Yifat; Chappell-Maor, Louise; Yaron, Avraham; Jung, Steffen

    2017-06-01

    Tissue macrophages provide immunological defense and contribute to the establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator Mecp2 in macrophages. Mice that lacked the gene encoding Mecp2, which is associated with Rett syndrome, in macrophages did not show signs of neurodevelopmental disorder but displayed spontaneous obesity, which was linked to impaired function of brown adipose tissue (BAT). Specifically, mutagenesis of a BAT-resident Cx3Cr1(+) macrophage subpopulation compromised homeostatic thermogenesis but not acute, cold-induced thermogenesis. Mechanistically, malfunction of BAT in pre-obese mice with mutant macrophages was associated with diminished sympathetic innervation and local titers of norepinephrine, which resulted in lower expression of thermogenic factors by adipocytes. Mutant macrophages overexpressed the signaling receptor and ligand PlexinA4, which might contribute to the phenotype by repulsion of sympathetic axons expressing the transmembrane semaphorin Sema6A. Collectively, we report a previously unappreciated homeostatic role for macrophages in the control of tissue innervation. Disruption of this circuit in BAT resulted in metabolic imbalance.

  3. Disturbed clockwork resetting in Sharp-1 and Sharp-2 single and double mutant mice.

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    Moritz J Rossner

    Full Text Available BACKGROUND: The circadian system provides the basis to anticipate and cope with daily recurrent challenges to maintain the organisms' homeostasis. De-synchronization of circadian feedback oscillators in humans causes 'jet lag', likely contributes to sleep-, psychiatric-, metabolic disorders and even cancer. However, the molecular mechanisms leading to the disintegration of tissue-specific clocks are complex and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Based on their circadian expression and cell culture experiments, the basic Helix-Loop-Helix (bHLH transcription factors SHARP-1(Dec2 and SHARP-2(Stra13/Dec1 were proposed as novel negative regulators of the molecular clock. To address their function in vivo, we generated Sharp-1 and Sharp-2 single and double mutant mice. Our experiments reveal critical roles for both factors in regulating period length, tissue-specific control of clock gene expression and entrainment to external cues. Light-pulse experiments and rapid delays of the light-dark cycle (experimental jet lag unravel complementary functions for SHARP-1 and SHARP-2 in controlling activity phase resetting kinetics. Moreover, we show that SHARP-1 and 2 can serve dual functions as repressors and co-activators of mammalian clock gene expression in a context-specific manner. This correlates with increased amplitudes of Per2 expression in the cortex and liver and a decrease in the suprachiasmatic nucleus (SCN of double mutant mice. CONCLUSIONS/SIGNIFICANCE: The existence of separate mechanisms regulating phase of entrainment, rhythm amplitude and period length has been postulated before. The differential effects of Sharp-deficiency on rhythmicity and behavioral re-entrainment, coupled to tissue-dependent regulatory functions, provide a new mechanistic basis to further understand the complex process of clock synchronizations.

  4. Disturbed clockwork resetting in Sharp-1 and Sharp-2 single and double mutant mice.

    Science.gov (United States)

    Rossner, Moritz J; Oster, Henrik; Wichert, Sven P; Reinecke, Lisa; Wehr, Michael C; Reinecke, Johannes; Eichele, Gregor; Taneja, Reshma; Nave, Klaus-Armin

    2008-07-23

    The circadian system provides the basis to anticipate and cope with daily recurrent challenges to maintain the organisms' homeostasis. De-synchronization of circadian feedback oscillators in humans causes 'jet lag', likely contributes to sleep-, psychiatric-, metabolic disorders and even cancer. However, the molecular mechanisms leading to the disintegration of tissue-specific clocks are complex and not well understood. Based on their circadian expression and cell culture experiments, the basic Helix-Loop-Helix (bHLH) transcription factors SHARP-1(Dec2) and SHARP-2(Stra13/Dec1) were proposed as novel negative regulators of the molecular clock. To address their function in vivo, we generated Sharp-1 and Sharp-2 single and double mutant mice. Our experiments reveal critical roles for both factors in regulating period length, tissue-specific control of clock gene expression and entrainment to external cues. Light-pulse experiments and rapid delays of the light-dark cycle (experimental jet lag) unravel complementary functions for SHARP-1 and SHARP-2 in controlling activity phase resetting kinetics. Moreover, we show that SHARP-1 and 2 can serve dual functions as repressors and co-activators of mammalian clock gene expression in a context-specific manner. This correlates with increased amplitudes of Per2 expression in the cortex and liver and a decrease in the suprachiasmatic nucleus (SCN) of double mutant mice. The existence of separate mechanisms regulating phase of entrainment, rhythm amplitude and period length has been postulated before. The differential effects of Sharp-deficiency on rhythmicity and behavioral re-entrainment, coupled to tissue-dependent regulatory functions, provide a new mechanistic basis to further understand the complex process of clock synchronizations.

  5. In vitro exploration of latent prothrombin mutants conveying antithrombin resistance.

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    Tamura, Shogo; Murata-Kawakami, Moe; Takagi, Yuki; Suzuki, Sachiko; Katsumi, Akira; Takagi, Akira; Kojima, Tetsuhito

    2017-09-20

    Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade. Copyright © 2017. Published by Elsevier Ltd.

  6. NRAS-mutant melanoma: current challenges and future prospect

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    Muñoz-Couselo E

    2017-08-01

    Full Text Available Eva Muñoz-Couselo,1,2 Ester Zamora Adelantado,1,2 Carolina Ortiz,1,2 Jesús Soberino García,3 José Perez-Garcia31Medical Oncology Department, Vall d’Hebron Hospital, Barcelona, Spain; 2Vall d’Hebron Institute of Oncology (VHIO, Barcelona, Spain; 3Baselga Institute of Oncology, Hospital Quirón, Barcelona, SpainAbstract: Melanoma is one of the most common cutaneous cancers worldwide. Activating mutations in RAS oncogenes are found in a third of all human cancers and NRAS mutations are found in 15%–20% of melanomas. The NRAS-mutant subset of melanoma is more aggressive and associated with poorer outcomes, compared to non-NRAS-mutant melanoma. Although immune checkpoint inhibitors and targeted therapies for BRAF-mutant melanoma are transforming the treatment of metastatic melanoma, the ideal treatment for NRAS-mutant melanoma remains unknown. Despite promising preclinical data, current therapies for NRAS-mutant melanoma remain limited, showing a modest increase in progression-free survival but without any benefit in overall survival. Combining MEK inhibitors with agents inhibiting cell cycling and the PI3K–AKT pathway appears to provide additional benefit; in particular, a strategy of MEK inhibition and CDK4/6 inhibition is likely to be a viable treatment option in the future. Patients whose tumors had NRAS mutations had better response to immunotherapy and better outcomes than patients whose tumors had other genetic subtypes, suggesting that immune therapies – especially immune checkpoint inhibitors – may be particularly effective as treatment options for NRAS-mutant melanoma. Improved understanding of NRAS-mutant melanoma will be essential to develop new treatment strategies for this subset of patients with melanoma.Keywords: metastatic melanoma, NRAS mutation, MEK inhibitor, immunotherapy, trametinib, binimetinib

  7. Defective glycinergic synaptic transmission in zebrafish motility mutants

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    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.

  8. Feeding on Leaves of the Glucosinolate Transporter Mutant gtr1gtr2 Reduces Fitness of Myzus persicae.

    Science.gov (United States)

    Madsen, Svend Roesen; Kunert, Grit; Reichelt, Michael; Gershenzon, Jonathan; Halkier, Barbara Ann

    2015-11-01

    As aphids are a pest on various crops worldwide, a better understanding of the interaction between aphids and plant host defenses is required. The green peach aphid (Myzus persicae) feeds on a variety of plant species, including the model plant Arabidopsis thaliana (Arabidopsis), in which glucosinolates function as a major part of the chemical defense. Several studies have shown that glucosinolates play a role in interactions between Arabidopsis and the green peach aphid. In this work, we used a recently identified Arabidopsis glucosinolate transporter mutant (gtr1gtr2 dKO), with altered glucosinolate content in the vasculature, to investigate the role of defense compound transport in aphid infestation. By monitoring aphid performance on caged leaves and analyzing glucosinolates in leaf tissue and phloem sap, as well as inside aphids, we examined if a change in spatial distribution of glucosinolates within a leaf influences aphid performance. Based on reduced glucosinolate content in the phloem sap of the transporter mutant, we hypothesized that aphids would perform better on gtr1gtr2 dKO leaves compared to WT. Unexpectedly, aphids performed poorly on gtr1gtr2 dKO leaves. Our data suggest that higher glucosinolate content in tissues surrounding the phloem of the double transporter mutant may play a role in reducing aphid performance on this genotype.

  9. Wheat streak mosaic virus Coat Protein Deletion Mutants Elicit More Severe Symptoms Than Wild-Type Virus in Multiple Cereal Hosts.

    Science.gov (United States)

    Tatineni, Satyanarayana; Elowsky, Christian; Graybosch, Robert A

    2017-12-01

    Previously, we reported that coat protein (CP) of Wheat streak mosaic virus (WSMV) (genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. In this study, we demonstrated that WSMV mutants with deletion of CP amino acids 58 to 84 but not of 36 to 57 induced severe chlorotic streaks and spots, followed by acute chlorosis in wheat, maize, barley, and rye compared with mild to moderate chlorotic streaks and mosaic symptoms by wild-type virus. Deletion of CP amino acids 58 to 84 from the WSMV genome accelerated cell-to-cell movement, with increased accumulation of genomic RNAs and CP, compared with the wild-type virus. Microscopic examination of wheat tissues infected by green fluorescent protein-tagged mutants revealed that infection by mutants lacking CP amino acids 58 to 84 caused degradation of chloroplasts, resulting in acute macroscopic chlorosis. The profile of CP-specific proteins was altered in wheat infected by mutants causing acute chlorosis, compared with mutants eliciting wild-type symptoms. All deletion mutants accumulated CP-specific major protein similarly to that in wild-type virus; however, mutants that elicit acute chlorosis failed to accumulate a 31-kDa minor protein compared with wild-type virus or mutants lacking amino acids 36 to 57. Taken together, these data suggest that deletion of CP amino acids 58 to 84 from the WSMV genome enhanced accumulation of CP and genomic RNA, altered CP-specific protein profiles, and caused severe symptom phenotypes in multiple cereal hosts.

  10. CBL exon 8/9 mutants activate the FLT3 pathway and cluster in core binding factor/11q deletion acute myeloid leukemia/myelodysplastic syndrome subtypes.

    Science.gov (United States)

    Reindl, Carola; Quentmeier, Hilmar; Petropoulos, Konstantin; Greif, Philipp A; Benthaus, Tobias; Argiropoulos, Bob; Mellert, Gudrun; Vempati, Sridhar; Duyster, Justus; Buske, Christian; Bohlander, Stefan K; Humphries, Keith R; Hiddemann, Wolfgang; Spiekermann, Karsten

    2009-04-01

    CBL is a negative regulator of activated receptor tyrosine kinases (RTK). In this study, we determined the frequency of CBL mutations in acute leukemias and evaluated the oncogenic potential of mutant CBL. The cDNA of 300 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL) patients and 82 human leukemic cell lines was screened for aberrations in the linker and RING finger domain of CBL. The oncogenic potential of identified mutants was evaluated in hematopoietic cells. We identified 3 of 279 AML/MDS patients expressing CBL exon 8/9 deletion mutants. Three of four cases at diagnosis expressed deleted transcripts missing exon 8 or exon 8/9. In remission samples a weak or no expression of mutant CBL was detected. No aberrations were found in normal hematopoietic tissues. One of 116 sequenced AML/MDS cases carried a R420G missense mutation. All AML/MDS patients with identified CBL mutants belonged to the core binding factor and 11q deletion AML subtypes. Functionally, CBL negatively regulated FMS-like tyrosine kinase 3 (FLT3) activity and interacted with human FLT3 via the autophosphorylation sites Y589 and Y599 and colocalized in vivo. Expression of CBLDeltaexon8 and CBLDeltaexon8+9 in FLT3-WT-Ba/F3 cells induced growth factor-independent proliferation associated with autophosphorylation of FLT3 and activated the downstream targets signal transducer and activator of transcription 5 (STAT5) and protein kinase B (AKT). FLT3 ligand-dependent hyperproliferation of CBL mutant cells could be abrogated by treatment with the FLT3 PTK inhibitor PKC412 (midostaurin). CBL exon8/9 mutants occur in genetically defined AML/MDS subtypes and transform hematopoietic cells by constitutively activating the FLT3 pathway. This phenotype resembles the one of mutated RTKs and suggests that CBL mutant AML patients might benefit from treatment with FLT3 PTK inhibitors.

  11. PIK3CA mutant tumors depend on oxoglutarate dehydrogenase

    Science.gov (United States)

    Ilic, Nina; Birsoy, Kıvanç; Aguirre, Andrew J.; Kory, Nora; Pacold, Michael E.; Singh, Shambhavi; Moody, Susan E.; DeAngelo, Joseph D.; Spardy, Nicole A.; Freinkman, Elizaveta; Weir, Barbara A.; Cowley, Glenn S.; Root, David E.; Asara, John M.; Vazquez, Francisca; Widlund, Hans R.; Sabatini, David M.; Hahn, William C.

    2017-01-01

    Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate–aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate–aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene. PMID:28396387

  12. Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants.

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    Sasaki, Yo; Margolin, Zachary; Borgo, Benjamin; Havranek, James J; Milbrandt, Jeffrey

    2015-07-10

    Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD(+) biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD(+) synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Development of Bacillus subtilis mutants to produce tryptophan in pigs.

    Science.gov (United States)

    Bjerre, Karin; Cantor, Mette D; Nørgaard, Jan V; Poulsen, Hanne D; Blaabjerg, Karoline; Canibe, Nuria; Jensen, Bent B; Stuer-Lauridsen, Birgitte; Nielsen, Bea; Derkx, Patrick M F

    2017-02-01

    To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution. A novel concept has been investigated-to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis by UV was combined with selection on Trp and purine analogues in an iterative process. Two mutants from different wild types were obtained, mutant 1 (M1) produced 1 mg Trp/l and mutant 2 (M2) 14 mg Trp/l. Genome sequence analysis revealed that M1 had three single nuclear polymorphisms (SNPs) and M2 had two SNPs compared to the wild type strains. In both mutants SNPs were found in genes regulating tryptophan synthesis. Reverse transcription PCR confirmed up-regulation of the tryptophan synthesis genes in both mutants, the expression was up to 3 times higher in M2 than in M1. Tryptophan-excreting B. subtilis strains were obtained with UV-mutagenesis and analogue selection and can be used in animal feed applications.

  14. Analysis of AtCry1 and Mutants

    Science.gov (United States)

    Burdick, Derek; Purvis, Adam; Ahmad, Margaret; Link, Justin J.; Engle, Dorothy

    Cryptochrome is an incredibly versatile protein that influences numerous biological processes such as plant growth, bird migration, and sleep cycles. Due to the versatility of this protein, understanding the mechanism would allow for advances in numerous fields such as crop growth, animal behavior, and sleep disorders. It is known that cryptochrome requires blue light to function, but the exact processes in the regulation of biological activity are still not fully understood. It is believed that the c-terminal domain of the protein undergoes a conformational change when exposed to blue light which allows for biological function. Three different non-functioning mutants were tested during this study to gain insight on the mechanism of cryptochrome. Absorbance spectra showed a difference between two of the mutants and the wild type with one mutant showing little difference. Immunoprecipitation experiments were also conducted to identify the different c-terminal responses of the mutants. By studying non functioning mutants of this protein, the mechanism of the protein can be further characterized. This two-month research experience in Paris allowed us to experience international and interdisciplinary collaborations in science and immerse in a different culture. The Borcer Fund for Student Research, Xavier University, Cincinnati, OH, and John Hauck Foundation.

  15. Normal aging modulates the neurotoxicity of mutant huntingtin.

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    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.

  16. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    Science.gov (United States)

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  17. Deregulation of the Egfr/Ras Signaling Pathway Induces Age-related Brain Degeneration in the Drosophila Mutant vap

    Science.gov (United States)

    Botella, José A.; Kretzschmar, Doris; Kiermayer, Claudia; Feldmann, Pascale; Hughes, David A.; Schneuwly, Stephan

    2003-01-01

    Ras signaling has been shown to play an important role in promoting cell survival in many different tissues. Here we show that upregulation of Ras activity in adult Drosophila neurons induces neuronal cell death, as evident from the phenotype of vacuolar peduncle (vap) mutants defective in the Drosophila RasGAP gene, which encodes a Ras GTPase-activating protein. These mutants show age-related brain degeneration that is dependent on activation of the EGF receptor signaling pathway in adult neurons, leading to autophagic cell death (cell death type 2). These results provide the first evidence for a requirement of Egf receptor activity in differentiated adult Drosophila neurons and show that a delicate balance of Ras activity is essential for the survival of adult neurons. PMID:12529440

  18. Wnt activation by wild type and mutant myocilin in cultured human trabecular meshwork cells.

    Directory of Open Access Journals (Sweden)

    Xiang Shen

    Full Text Available Myocilin is a gene linked to the most prevalent form of glaucoma, a major blinding disease. The trabecular meshwork (TM, a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The Pro³⁷⁰ to Leu (P370L mutation of myocilin is associated with severe glaucoma phenotypes and Gln³⁶⁸ stop (Q368X is the most common myocilin mutation reported. Myocilin, upon overexpression, has been shown to induce phenotypes that include a loss of actin stress fibers, an increase in the cAMP level and protein kinase A (PKA activity, as well as a reduction in the RhoA activity. We examined herein whether Wnt signaling pathway is involved in the myocilin phenotypes and whether P370L and Q368X mutants also display biological effects similar to those of the wild type myocilin.Wild type myocilin, when transfected into cultured human TM cells, induced a loss of actin stress fibers as judged by phalloidin staining. Such a loss was averted by treatment of secreted Frizzled-related protein 1 (sFRP1, an inhibitor of Wnt signaling. Consistent with the notion that Wnt pathway mediates the myocilin phenotype, Wnt activation was demonstrated by TOP/FOP-Flash reporter assays. Treatment of human TM cells of a Wnt activator, SB216763, as well as transfection of myocilin P370L and Q368X mutants all resulted in actin stress fiber loss, PKA activation and RhoA inactivation. The PKA elevation was obviated by the sFRP1 treatment, indicating that Wnt signaling was upstream that of PKA.The present study demonstrated that following forced expression of wild type myocilin, Wnt was activated, triggering in turn other myocilin-related alterations. P370L and Q368X mutations induced similar phenotypes, suggesting one possible mechanism how the mutants may lead to TM cell damage and pathology.

  19. Alopecia in a viable phospholipase C delta 1 and phospholipase C delta 3 double mutant.

    Directory of Open Access Journals (Sweden)

    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.

  20. The role of the lysyl binding site of tissue-type plasminogen activator in the interaction with a forming fibrin clot

    NARCIS (Netherlands)

    Bakker, A.H.F.; Weening-Verhoeff, E.J.D.; Verheijen, J.H.

    1995-01-01

    To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the

  1. Generation and Characterization of dickkopf3 Mutant Mice

    Science.gov (United States)

    del Barco Barrantes, Ivan; Montero-Pedrazuela, Ana; Guadaño-Ferraz, Ana; Obregon, Maria-Jesus; Martinez de Mena, Raquel; Gailus-Durner, Valérie; Fuchs, Helmut; Franz, Tobias J.; Kalaydjiev, Svetoslav; Klempt, Martina; Hölter, Sabine; Rathkolb, Birgit; Reinhard, Claudia; Morreale de Escobar, Gabriella; Bernal, Juan; Busch, Dirk H.; Wurst, Wolfgang; Wolf, Eckhard; Schulz, Holger; Shtrom, Svetlana; Greiner, Erich; Hrabé de Angelis, Martin; Westphal, Heiner; Niehrs, Christof

    2006-01-01

    dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity. PMID:16508007

  2. Some enzyme activities of acetate mutants of Yarrowia lypolytica

    Energy Technology Data Exchange (ETDEWEB)

    Robak, M.; Wojtatowicz, M.; Rymowicz, W. [Akademia Rolnicza, Wroclaw (Poland)

    1994-12-31

    Activity at the following enzymes: CS (oxaloacetate-lyase citrate), AH (citrate (isocitrate) hydrolyase), ICDH (threo-Ds-isocitrate: NADP oxidoreductase) and ICL (threo-Ds-isocitrate glyoxyglate-lyase) was measured at subsequent stages of citrate fermentation on glucose by wild type strain `Y, lipolytica A-101` and 2 acetate defective mutants, in order to recognize metabolic disorders in those mutants, which resulted in markedly improved homogeneity of citric acid production. Mutants did not show significant changes in activity of TCA cycle enzymes and ICL. Thus suggests that the control of citric:isocitric acid ratio is more difficult and it can also depend on transportation systems of both acids. (author). 19 refs, 3 figs, 2 tabs.

  3. The swimming activity of the staggerer mutant mouse.

    Science.gov (United States)

    Goodall, G; Guastavino, J M; Gheusi, G

    1986-09-01

    Four experiments investigated the swimming behaviour of staggerer mutant mice. The results partially confirmed previous reports that a mouse's swimming is unaffected by the staggerer mutation. In terms of speed and distance there are indeed no measurable differences between normal and staggerer mice, when first placed in the water. The stagger's resistance was however shown to be much lower than a normal's and the genetic difference was also associated with different styles of swimming. Furthermore, whereas the normal mouse's swimming behaviour evolves with increased time in the water, the staggerer's remains constant. The differences are interpreted on the basis of abnormal novelty reactions by the staggerer mutants. Thus, swimming appears to be a better tool for investigating the higher-level cognitive functions of this mutant than terrestrial locomotion. Copyright © 1986. Published by Elsevier B.V.

  4. 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. Copyright © 2010. Published by Elsevier B.V.

  5. Tissue bionics: examples in biomimetic tissue engineering.

    Science.gov (United States)

    Green, David W

    2008-09-01

    Many important lessons can be learnt from the study of biological form and the functional design of organisms as design criteria for the development of tissue engineering products. This merging of biomimetics and regenerative medicine is termed 'tissue bionics'. Clinically useful analogues can be generated by appropriating, modifying and mimicking structures from a diversity of natural biomatrices ranging from marine plankton shells to sea urchin spines. Methods in biomimetic materials chemistry can also be used to fabricate tissue engineering scaffolds with added functional utility that promise human tissues fit for the clinic.

  6. Tissue bionics: examples in biomimetic tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Green, David W [Bone and Joint Research Group, Developmental Origins of Health and Disease, General Hospital, University of Southampton, SO16 6YD (United Kingdom)], E-mail: Hindoostuart@googlemail.com

    2008-09-01

    Many important lessons can be learnt from the study of biological form and the functional design of organisms as design criteria for the development of tissue engineering products. This merging of biomimetics and regenerative medicine is termed 'tissue bionics'. Clinically useful analogues can be generated by appropriating, modifying and mimicking structures from a diversity of natural biomatrices ranging from marine plankton shells to sea urchin spines. Methods in biomimetic materials chemistry can also be used to fabricate tissue engineering scaffolds with added functional utility that promise human tissues fit for the clinic.

  7. Dissection of symbiosis and organ development by integrated transcriptome analysis of lotus japonicus mutant and wild-type plants.

    Directory of Open Access Journals (Sweden)

    Niels Høgslund

    Full Text Available Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this

  8. Dissection of Symbiosis and Organ Development by Integrated Transcriptome Analysis of Lotus japonicus Mutant and Wild-Type Plants

    Science.gov (United States)

    Høgslund, Niels; Radutoiu, Simona; Krusell, Lene; Voroshilova, Vera; Hannah, Matthew A.; Goffard, Nicolas; Sanchez, Diego H.; Lippold, Felix; Ott, Thomas; Sato, Shusei; Tabata, Satoshi; Liboriussen, Poul; Lohmann, Gitte V.; Schauser, Leif; Weiller, Georg F.; Udvardi, Michael K.; Stougaard, Jens

    2009-01-01

    Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful

  9. Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant

    Directory of Open Access Journals (Sweden)

    Edson Jiovany Ramírez-Nava

    2017-10-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD and G6PD Nefza (Leu323Pro, and the double mutant G6PD A− (Asn126Asp + Leu323Pro. The mutants showed lower residual activity (≤50% of WT G6PD and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.

  10. Genetic interactions among homologous recombination mutants in Candida albicans.

    Science.gov (United States)

    Bellido, Alberto; Andaluz, Encarnación; Gómez-Raja, Jonathan; Álvarez-Barrientos, Alberto; Larriba, Germán

    2015-01-01

    rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Qualidade de frutos de tomate da cv. Santa Clara, mutante de fruto amarelo e seus híbridos F1 Quality of tomato red fruit, cv. Santa Clara, its yellow mutant and respective F1 hybrid

    Directory of Open Access Journals (Sweden)

    Elizanilda R. do Rêgo

    1999-07-01

    Full Text Available A caracterização de alguns atributos físicos e químicos de frutos de tomate da cv. Santa Clara, um mutante de fruto amarelo, e o híbrido F1 obtido do cruzamento entre eles, foi feita com base nos teores de b-caroteno, licopeno, vitamina C, pH, matéria fresca, matéria seca, diâmetro do fruto, diâmetro do pericarpo, acidez e sólidos solúveis totais dos tecidos loculares e do pericarpo. Os frutos maduros amarelos apresentaram teores reduzidos de b-caroteno, licopeno e vitamina C, enquanto o híbrido apresentou teores semelhantes ao genótipo normal. Tanto o fruto mutante quanto o F1 apresentaram teor de pH menor que o do fruto normal. Não houve diferença significativa quanto a matéria fresca e seca totais, diâmetro do fruto e do pericarpo e acidez. Os teores de sólidos solúveis totais do pericarpo foram inferiores nos frutos do mutante amarelo e F1, em relação ao genótipo normal, enquanto os teores de sólidos solúveis totais da massa locular não diferiram, estatisticamente, entre os genótipos.The determination of some physical and chemical attributes of red fruits from tomato cv. Santa Clara, a yellow mutant and the F1 hybrid, were based on b-carotene, lycopene, vitamin C, pH, fresh and dry weight matter, pericarp and fruit diameter, titrateable acidity and total soluble solids. Ripe fruits of the yellow mutant showed reduced levels of b-carotene, lycopene and vitamin C, while fruits of the F1 hybrid showed levels of carotenoid pigments and vitamin C similar to the red fruit. Both mutant and F1 ripe fruits showed lower pH levels than the red parentals. Total fresh fruit and dry weight matter, pericarp and fruit diameter, and acidity were similar in the three genotypes studied. The total soluble-solids content of the pericarp was lower in the mutant and F1 fruits, whereas their concentration in the locule tissues was similar in all genotypes.

  12. Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Masayuki Hiraki

    2015-01-01

    Full Text Available KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR signaling may cause dysregulation of microRNA (miRNA and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS and v-akt murine thymoma viral oncogene homolog 1(AKT1, key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK and phosphoinositide 3-kinase (PI3K/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

  13. Analysis of the Maize dicer-like1 Mutant, fuzzy tassel, Implicates MicroRNAs in Anther Maturation and Dehiscence.

    Directory of Open Access Journals (Sweden)

    Sterling Field

    Full Text Available Sexual reproduction in plants requires development of haploid gametophytes from somatic tissues. Pollen is the male gametophyte and develops within the stamen; defects in the somatic tissues of the stamen and in the male gametophyte itself can result in male sterility. The maize fuzzy tassel (fzt mutant has a mutation in dicer-like1 (dcl1, which encodes a key enzyme required for microRNA (miRNA biogenesis. Many miRNAs are reduced in fzt, and fzt mutants exhibit a broad range of developmental defects, including male sterility. To gain further insight into the roles of miRNAs in maize stamen development, we conducted a detailed analysis of the male sterility defects in fzt mutants. Early development was normal in fzt mutant anthers, however fzt anthers arrested in late stages of anther maturation and did not dehisce. A minority of locules in fzt anthers also exhibited anther wall defects. At maturity, very little pollen in fzt anthers was viable or able to germinate. Normal pollen is tricellular at maturity; pollen from fzt anthers included a mixture of unicellular, bicellular, and tricellular pollen. Pollen from normal anthers is loaded with starch before dehiscence, however pollen from fzt anthers failed to accumulate starch. Our results indicate an absolute requirement for miRNAs in the final stages of anther and pollen maturation in maize. Anther wall defects also suggest that miRNAs have key roles earlier in anther development. We discuss candidate miRNAs and pathways that might underlie fzt anther defects, and also note that male sterility in fzt resembles water deficit-induced male sterility, highlighting a possible link between development and stress responses in plants.

  14. RFLP mapping of the barley homeotic mutant lax-a.

    Science.gov (United States)

    Laurie, D A; Pratchett, N; Allen, R L; Hantke, S S

    1996-07-01

    The lax-a homeotic mutant of barley has flowers in which lodicules are replaced by stamens (giving five stamens per flower). RFLP mapping of an F2 population from a Bonus lax-a (1) x H. spontaneum cross showed that the mutation was on the short arm of chromosome 7(5H), closely linked to the centromere. An additional F2 population was used to show that the lax-a mutation gave the five-stamen phenotype in all flowers of 6-rowed spikes and that hoods were elevated and reduced in size in lax-a/Hooded double-mutant plants.

  15. Mutant E. coli strain with increased succinic acid production

    Science.gov (United States)

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  16. Forward and reverse genetics: The LORE1 retrotransposon insertion mutants

    DEFF Research Database (Denmark)

    Fukai, Eigo; Malolepszy, Anna; Sandal, Niels Nørgaard

    2014-01-01

    The endogenous Lotus retrotransposon 1 (LORE1) transposes in the germ line of Lotus japonicus plants that carry an active element. This feature of LORE1 has been exploited for generation of a large non-transgenic insertion mutant population, where insertions have been annotated using next......-generation sequencing approaches. The LORE1 mutant lines are freely available and can be ordered online. Endogenous retrotransposons are also active in many other plant species. Based on the methods developed for LORE1 mutagenesis, it should be simple to establish similar systems in other species, once an appropriate...

  17. Mutant E. coli strain with increased succinic acid production

    Science.gov (United States)

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  18. Clear Plaque Mutants of Lactococcal Phage TP901-1

    DEFF Research Database (Denmark)

    Kot, Witold; Kilstrup, Mogens; Vogensen, Finn K.

    2016-01-01

    We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome...... protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1....

  19. A male sterile pepper (C. annuum L.) mutant.

    Science.gov (United States)

    Daskaloff, S

    1968-08-01

    1. After treatment of dry seeds of red pepperCapsicum annuum L. with X-rays a male-sterile mutant was discovered in the M2. 2. The male-sterile mutant segregates in a ratio of 3.28:1 (χ(2)=3.148, probability 0.07). 3. After an alternative cultivation of male-sterile plants and of a variety with good combining ability relatively good fruit-setting and seed production was obtained. 4. Grafting of male-sterile scions to normal stocks does not affect the male-sterile phenotype.

  20. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    Science.gov (United States)

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  1. American Association of Tissue Banks

    Science.gov (United States)

    ... Brightest in Tissue Banking FIND AATB ACCREDITED TISSUE BANK INSTITUTIONS SEARCH STANDARDS FOR TISSUE BANKING ACCESS CERTIFIED TISSUE BANK SPECIALISTS (CTBS) MANAGE QDEW Workshop | Baltimore, MD Save ...

  2. 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.

  3. In Vitro Root Development in Arabidopsis Thaliana Wild-Type and scr Mutants under Clinorotation

    Science.gov (United States)

    Kordyum, E. L.; Sarnatska, V. V.; Talalaiev, A. S.; Ovcharenko, Y. V.

    2008-06-01

    A task of our experiments was to study in vitro rhizogenesis in Arabidopsis thaliana wild type and scr mutants under slow horizontal clinorotation as a convenient model to clear up a question, whether root morphogenesis de novo will occur normally in simulated microgravity. Two methods for obtaining A. thaliana roots in vitro were used: 1) from the primary callus of leaf origin and 2) directly from leaf explants. Light and electron microscopy and RT-PCR were used for an analysis of the experimental materials. Graviperceptive cells differentiated in roots formed de novo from callus and leaf explants of wild type and scr mutants but did not function under clinorotation. Tissue and cell type patterning in a root proper as well as gene expression in all variants in the control and under clinorotation were similar that gives new evidence on normal morphogenesis in altered gravity. We proposed such model for performing the experiments on board the ISS to study morphogenesis in vitro, including differentiation of graviperceptive cells.

  4. Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington's Disease T Lymphocytes.

    Science.gov (United States)

    Miller, James R C; Träger, Ulrike; Andre, Ralph; Tabrizi, Sarah J

    2015-01-01

    Huntington's disease is a fatal neurodegenerative condition caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system is dysregulated in Huntington's disease and may contribute to its pathogenesis. However, it is not clear whether or to what extent the adaptive immune system is also involved. Here, we carry out the first comprehensive investigation of human ex vivo T lymphocytes in Huntington's disease, focusing on the frequency of a range of T lymphocyte subsets, as well as analysis of proliferation, cytokine production and gene transcription. In contrast to the innate immune system, the intrinsic phenotype of T lymphocytes does not appear to be affected by the presence of mutant huntingtin, with Huntington's disease T lymphocytes exhibiting no significant functional differences compared to control cells. The transcriptional profile of T lymphocytes also does not appear to be significantly affected, suggesting that peripheral immune dysfunction in Huntington's disease is likely to be mediated primarily by the innate rather than the adaptive immune system. This study increases our understanding of the effects of Huntington's disease on peripheral tissues, while further demonstrating the differential effects of the mutant protein on different but related cell types. Finally, this study suggests that the potential use of novel therapeutics aimed at modulating the Huntington's disease innate immune system should not be extended to include the adaptive immune system.

  5. Loss of Cell Adhesion Increases Tumorigenic Potential of Polarity Deficient Scribble Mutant Cells.

    Directory of Open Access Journals (Sweden)

    Indrayani Waghmare

    Full Text Available Epithelial polarity genes are important for maintaining tissue architecture, and regulating growth. The Drosophila neoplastic tumor suppressor gene scribble (scrib belongs to the basolateral polarity complex. Loss of scrib results in disruption of its growth regulatory functions, and downregulation or mislocalization of Scrib is correlated to tumor growth. Somatic scribble mutant cells (scrib- surrounded by wild-type cells undergo apoptosis, which can be prevented by introduction of secondary mutations that provide a growth advantage. Using genetic tools in Drosophila, we analyzed the phenotypic effects of loss of scrib in different growth promoting backgrounds. We investigated if a central mechanism that regulates cell adhesion governs the growth and invasive potential of scrib mutant cells. Here we show that increased proliferation, and survival abilities of scrib- cells in different genetic backgrounds affect their differentiation, and intercellular adhesion. Further, loss of scrib is sufficient to cause reduced cell survival, activation of the JNK pathway and a mild reduction of cell adhesion. Our data show that for scrib cells to induce aggressive tumor growth characterized by loss of differentiation, cell adhesion, increased proliferation and invasion, cooperative interactions that derail signaling pathways play an essential role in the mechanisms leading to tumorigenesis. Thus, our study provides new insights on the effects of loss of scrib and the modification of these effects via cooperative interactions that enhance the overall tumorigenic potential of scrib deficient cells.

  6. Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling.

    Science.gov (United States)

    Herz, Hans-Martin; Morgan, Marc; Gao, Xin; Jackson, Jessica; Rickels, Ryan; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Eissenberg, Joel C; Shilatifard, Ali

    2014-08-29

    Histone H3 lysine(27)-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. We established a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing derepression of PRC2 target genes and developmental perturbations. Similarly, an H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M-containing nucleosomes, and its misregulation in Drosophila results in changes of H3K9 methylation levels and heterochromatic silencing defects. We have established histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin signaling pathways. Copyright © 2014, American Association for the Advancement of Science.

  7. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    Energy Technology Data Exchange (ETDEWEB)

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-01

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  8. LOXL2 catalytically inactive mutants mediate epithelial-to-mesenchymal transition

    Directory of Open Access Journals (Sweden)

    Eva P. Cuevas

    2014-01-01

    Lysyl-oxidase-like 2 (LOXL2 is a member of the lysyl oxidase family that catalyzes the cross-linking of collagens or elastins in the extracellular matrix, thus regulating the tensile strength of tissues. However, many reports have suggested different intracellular roles for LOXL2, including the ability to regulate gene transcription and tumor progression. We previously reported that LOXL2 mediates epithelial-to-mesenchymal transition (EMT by Snail1-dependent and independent mechanisms, related to E-cadherin silencing and downregulation of epidermal differentiation and cell polarity components, respectively. Whether or not the catalytic activity of LOXL2 is required to induce/sustain EMT is actually unknown. Here we show that LOXL2 catalytic inactive mutants collaborate with Snail1 in E-cadherin gene repression to trigger EMT and, in addition, promote FAK/Src pathway activation to support EMT. These findings reveal a non-conventional role of LOXL2 on regulating epithelial cell plasticity.

  9. Mutant connexin 50 (S276F) inhibits channel and hemichannel ...

    Indian Academy of Sciences (India)

    The mutant and wild-type Cx50 were expressed in equal levels and could efficiently localize to the plasma membrane without transportation and assembly ... Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Donghu Road 169#, Wuhan, Hubei 430071, People's Republic of China; Hubei Cancer ...

  10. Evaluation of high yielding mutants of Hordeumvulgare cultivar Izgrev

    Directory of Open Access Journals (Sweden)

    B. Dyulgerova

    2017-06-01

    Full Text Available Abstract. Seeds of Hordeum vulgare L. cultivar Izgrev were treated with different concentrations of sodium azide to induce genetic variability for the selection of genotypes with improved traits. After passing through different stages of selection, 18 promising mutants were selected for further studies. Eighteen mutants and their parent and national standard cultivar Veslets were evaluated in Complete Block Design with four replications. The research was conducted in 2013 – 2014 and 2014 – 2015 growing seasons in the experimental field of the Institute of Agriculture Karnobat, Southeastern Bulgaria. The characters studied included days to heading, plant height, lodging, peduncle length, spike length, awn length, spikelet number per spike, grain number per spike, grain weight per spike, 1000 grains weight and grain yield. Wide variation among mutant lines was observed for different traits. Mutant lines M4/16 and M 3/14 produced significantly greater grain yield than the parent and standard cultivar. Positive changes in lodging tolerance, grain number per spike, grain weight per spike, 1000 grains weightwere also observed. This study showed positive effects in the use of mutation in inducing improvement for grain yield and some yield related traits.

  11. Locating a modifier gene of Ovum mutant through crosses between ...

    Indian Academy of Sciences (India)

    RESEARCH ARTICLE Volume 95 Issue 2 June 2016 pp 297-302 ... mouse; DDK syndrome; ovum mutant; modifier gene; quantitative trait loci ... Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/+) females and males of other ...

  12. Characterization of a novel curled-cotyledons mutant in soybean ...

    African Journals Online (AJOL)

    ARL

    mutant, the embryo sac becomes smaller and bulbous, and ultrastructure of developing cotyledons exhibits ... has higher protein and oil content, and has altered seed ultrastructure. The purpose of this experiment is to evaluate the features of soybean curled-cotyledons ..... mitochondria of soybean seedling cotyledons.

  13. Enhanced longevity in tau mutant Syrian hamsters, Mesocricetus auratus

    NARCIS (Netherlands)

    Oklejewicz, Malgorzata; Daan, Serge

    The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span

  14. Molecular analysis of sex chromosome-linked mutants in the ...

    Indian Academy of Sciences (India)

    2010-09-06

    Sep 6, 2010 ... In B. mori, polyploid individuals with different sex chromo- some constitutions can be ... RNA was extracted from the ovary, testis, and fat body of a fifth-instar larva, and from the an- tennae of 10 moths. The primer set .... For example, the body of sk. (stick, 4–25.8) mutant larvae is firm to the touch, while that.

  15. Characterization of resistant tomato mutants to bacterial canker ...

    African Journals Online (AJOL)

    Yomi

    2012-04-19

    Apr 19, 2012 ... A small scale ethylmethanesulfonate (EMS) mutation was used to obtain resistant mutant plants to bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis isolate 2 (Cmm2). Susceptible EBR3 tomato line (200) seeds were mutagenised with the chemical EMS. Of the ...

  16. Growth properties of Cellulomonas flavigena mutants affected in cellulose utilization.

    Science.gov (United States)

    Béguin, P; Eisen, H

    1978-01-01

    The role of cellobiose metabolism in cellulose utilization by Cellulomonas flavigena was investigated by studying mutants unable to grow on cellobiose or cellulose. The results show that the ability to utilize cellulose is strictly dependent on the ability to utilize cellobiose. PMID:415038

  17. 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...

  18. Molecular analysis of mutants of the Neurospora adenylosuccinate ...

    Indian Academy of Sciences (India)

    2012-08-07

    Aug 7, 2012 ... Abstract. 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 ...

  19. Performance of early maturing mutants derived from 'supa' rice ...

    African Journals Online (AJOL)

    Days to 50% flowering and 1000 grain weight exerted negative direct effect on yield. Changes in grain quality were also observed emphasizing the importance of conducting cooking and taste panel tests. Keywords: Early maturity, grain guality, rice mutants, Oryza saliva, path coefficient. Tanzania J. Agri. Sc. (2001) Vol 4, ...

  20. Isolation and partial characterization of carotenoid mutants of ...

    African Journals Online (AJOL)

    Global Journal of Pure and Applied Sciences ... Three major pigments isolated by high performance liquid chromatography (HPLC) were characterized by their absorption maxima, partition ratios in light ... High performance liquid chromatography was used to compare pigments of the wild-type with those of the mutants.

  1. Characterization of mutant cowpea [ Vigna unguiculata (L) Walp ...

    African Journals Online (AJOL)

    Phylogenetic relationship and polymorphism was detected in 10 cowpea lines comprising of leaf, flower and stem mutants, their putative parents and an exotic accession using 10 random ... Genetic distance ranged from 0.05 to 0.30 based on AFLP markers, while it ranged between 0.13 and 0.44 for RAPD markers. Cluster ...

  2. Clustering common bean mutants based on heterotic groupings ...

    African Journals Online (AJOL)

    The objective of this study was to cluster bean mutants from a bean mutation breeding programme, based on heterotic groupings. This was achieved by genotyping 16 bean genotypes, using 21 Simple Sequence Repeats (SSR) bean markers. From the results, three different clusters A, B and C, were obtained suggesting ...

  3. Insulator dysfunction and oncogene activation in IDH mutant gliomas.

    Science.gov (United States)

    Flavahan, William A; Drier, Yotam; Liau, Brian B; Gillespie, Shawn M; Venteicher, Andrew S; Stemmer-Rachamimov, Anat O; Suvà, Mario L; Bernstein, Bradley E

    2016-01-07

    Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

  4. IGFBP2 expression predicts IDH-mutant glioma patient survival.

    Science.gov (United States)

    Huang, Lin Eric; Cohen, Adam L; Colman, Howard; Jensen, Randy L; Fults, Daniel W; Couldwell, William T

    2017-01-03

    Mutations of the isocitrate dehydrogenase (IDH) 1 and 2 genes occur in ~80% of lower-grade (WHO grade II and grade III) gliomas. Mutant IDH produces (R)-2-hydroxyglutarate, which induces DNA hypermethylation and presumably drives tumorigenesis. Interestingly, IDH mutations are associated with improved survival in glioma patients, but the underlying mechanism for the difference in survival remains unclear. Through comparative analyses of 286 cases of IDH-wildtype and IDH-mutant lower-grade glioma from a TCGA data set, we report that IDH-mutant gliomas have increased expression of tumor-suppressor genes (NF1, PTEN, and PIK3R1) and decreased expression of oncogenes(AKT2, ARAF, ERBB2, FGFR3, and PDGFRB) and glioma progression genes (FOXM1, IGFBP2, and WWTR1) compared with IDH-wildtype gliomas. Furthermore, each of these genes is prognostic in overall gliomas; however, within the IDH-mutant group, none remains prognostic except IGFBP2 (encodinginsulin-like growth factor binding protein 2). Through validation in an independent cohort, we show that patients with low IGFBP2 expressiondisplay a clear advantage in overall and disease-free survival, whereas those with high IGFBP2 expressionhave worse median survival than IDH-wildtype patients. These observations hold true across different histological and molecular subtypes of lower-grade glioma. We propose therefore that an unexpected biological consequence of IDH mutations in glioma is to ameliorate patient survival by promoting tumor-suppressor signaling while inhibiting that of oncogenes, particularly IGFBP2.

  5. 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.

  6. Development and evaluation of drought resistant mutant germ ...

    African Journals Online (AJOL)

    terms of relative water content, free proline concentration and yield. The yield ... are suitable for boiling and canning. .... definition the permanent wilting point is the soil water content at which plants do not recover turgor overnight, but will recover if water is applied. Selected mutant and control plants were planted in pots in a.

  7. Complementation of sweet corn mutants: a method for grouping ...

    Indian Academy of Sciences (India)

    accumulate sugars at the expense of starch and have low total carbohydrate at the mature kernel stage (Boyer and. Shannon 1984). At 18–21 days after pollination (harvest stage of sweet corn), these mutants have four to eight times higher total sugar than the normal corn (Holder et al. 1974). Due to comparative high sugar ...

  8. Characterization of resistant tomato mutants to bacterial canker ...

    African Journals Online (AJOL)

    A small scale ethylmethanesulfonate (EMS) mutation was used to obtain resistant mutant plants to bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis isolate 2 (Cmm2). Susceptible EBR3 tomato line (200) seeds were mutagenised with the chemical EMS. Of the constructed M2 population, ...

  9. A dwarf wheat mutant is associated with increased drought ...

    African Journals Online (AJOL)

    Owner

    'Green revolution' genes encode mutant gibberellin response modulators. Nature 400 (6741):. 256-61. Zhang et al. 1057. Peng J, Carol P, Richards DE, King KE, Cowling RJ, Murphy GP,. Harberd NP (1997). The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses Genes Dev.

  10. Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy.

    Directory of Open Access Journals (Sweden)

    Xin Dai

    Full Text Available KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors.

  11. Modelling the evolution and spread of HIV immune escape mutants.

    Science.gov (United States)

    Fryer, Helen R; Frater, John; Duda, Anna; Roberts, Mick G; Phillips, Rodney E; McLean, Angela R

    2010-11-18

    During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level.

  12. Enhanced sporulation and toxin production by a mutant derivative of ...

    African Journals Online (AJOL)

    fatima

    based medium. Maximum spore and crystal proteins were produced at 40°C with corn steep liquor as nitrogen source and hydrol as a carbon source. The best mutant MUV7 supported .... containing 10 L of culture medium as described earlier (Ghribi et al.,. 2004). ..... Saccharomyces cerevisiae ITV strain (Ortiz-Muniz et al.,.

  13. nitrosoguanidine-induced cadmium resistant mutants of Aspergillus ...

    Indian Academy of Sciences (India)

    Unknown

    of S. cerevisiae (Inouhe et al 1989) were used for under- standing the molecular genetics of cadmium toxicity and ... The pH of the medium was adjusted to 6⋅4 before autoclaving. Cadmium-resistant mutants after isolation ..... Saccharomyces cerevisiae; Biochem. Biophys. Acta 993 51–55. Kimura M, Otaki N and Imano M ...

  14. Development of a mutant strain of Bacillus subtilis showing ...

    African Journals Online (AJOL)

    Through fermentation experiments, it was confirmed that the mutant strain, TH-49, was not capable of using acetoin accumulated in broth as its energy sources for growth after glucose was consumed. This phenomenon was inconsistent with that the majorities of bacteria accumulate acetoin as stored energy sources and ...

  15. Transcriptional Analysis of serk1 and serk3 coreceptor mutants

    NARCIS (Netherlands)

    Esse, van Wilma; Hove, ten Colette A.; Guzzonato, Francesco; Esse, van Peter; Boekschoten, Mark; Ridder, Lars; Vervoort, Jacques; Vries, de Sacco C.

    2016-01-01

    Somatic embryogenesis receptor kinases (SERKs) are ligand-binding coreceptors that are able to combine with different ligandperceiving receptors such as BRASSINOSTEROID INSENSITIVE1 (BRI1) and FLAGELLIN-SENSITIVE2. Phenotypical analysis of serk single mutants is not straightforward because

  16. Screening of allyl alcohol resistant mutant of Rhizopus oryzae and ...

    African Journals Online (AJOL)

    Ethanol is a main by-product in the fermentation broth of Rhizopus oryzae during the production of high-optical purity L-lactic acid. By screening the lower activity of alcohol dehydrogenase (ADH) mutant, thus decreasing the flux of pyruvic acid to ethanol may be a virtual method for increasing the conversion rate of glucose ...

  17. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. A dwarf wheat mutant is associated with increased drought ...

    African Journals Online (AJOL)

    ... was significantly higher than Jingdong 6. Most of the s-dwarf seedlings survived in recovering experiement after water loss. The stalk of s-dwarf seedling also showed reduced gravitropism. This is the first report about a new dwarf wheat mutant associated with increased drought resistance and altered stalk gravitropism.

  19. Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila

    Indian Academy of Sciences (India)

    2014-07-20

    Jul 20, 2014 ... from the mutation. As a result, Pdf01 mutant flies locomote with precise rhythmicity generated by one ultradian oscilla- tor. In future studies, we would like to dissect this system using genetic tools available in Drosophila, for example by silencing a particular set of Pdf-positive neurons. Circadian rhythms are ...

  20. Siim Nestor soovitab : Mutant Disco. Azymuth. Klubis Hollywood / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2003-01-01

    Mutant Disco klubis Prive 4. juulil. Brasiilia jazz-trio Azmuth klubis BonBon 5. juulil. Pidustuste sarja Hip Hop Cafe sünnipäeva tähistamisest klubis Hollywood 4. juulil, üritusest Ibiza Night 5. juulil

  1. Photophysics and optical switching in green fluorescent protein mutants

    NARCIS (Netherlands)

    Creemers, T.M.H.; Lock, A.J.; Subramaniam, V.; Jovin, T.M.; Völker, S.

    2000-01-01

    We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo- interconverted among three conformations and are, therefore, not photostable 'one-color' systems as previously believed. From our experiments we have further derived

  2. Genetic characterization of glossy-leafed mutant broccoli lines

    Science.gov (United States)

    Glossy mutants of Brassica oleracea L. have reduced or altered epicuticular wax on the surface of their leaves as compared to wild-type plants, conveying a shiny green appearance. Mutations conferring glossiness are common and have been found in most B. oleracea crop varieties, including cauliflower...

  3. Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1 mutants.

    Science.gov (United States)

    Labonne, Jonathan D J; Dorweiler, Jane E; McGinnis, Karen M

    2013-04-01

    Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1-1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1-1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression.

  4. Mutant p53 as a target for cancer treatment.

    Science.gov (United States)

    Duffy, Michael J; Synnott, Naoise C; Crown, John

    2017-09-01

    TP53 (p53) is the single most frequently altered gene in human cancers, with mutations being present in approximately 50% of all invasive tumours. However, in some of the most difficult-to-treat cancers such as high-grade serous ovarian cancers, triple-negative breast cancers, oesophageal cancers, small-cell lung cancers and squamous cell lung cancers, p53 is mutated in at least 80% of samples. Clearly, therefore, mutant p53 protein is an important candidate target against which new anticancer treatments could be developed. Although traditionally regarded as undruggable, several compounds such as p53 reactivation and induction of massive apoptosis-1 (PRIMA-1), a methylated derivative and structural analogue of PRIMA-1, i.e. APR-246, 2-sulfonylpyrimidines such as PK11007, pyrazoles such as PK7088, zinc metallochaperone-1 (ZMC1), a third generation thiosemicarbazone developed by Critical Outcome Techonologies Inc. (COTI-2) as well as specific peptides have recently been reported to reactive mutant p53 protein by converting it to a form exhibiting wild-type properties. Consistent with the reactivation of mutant p53, these compounds have been shown to exhibit anticancer activity in preclinical models expressing mutant p53. To date, two of these compounds, i.e. APR-246 and COTI-2 have progressed to clinical trials. A phase I/IIa clinical trial with APR-246 reported no major adverse effect. Currently, APR-246 is undergoing a phase Ib/II trial in patients with advanced serous ovarian cancer, while COTI-2 is being evaluated in a phase I trial in patients with advanced gynaecological cancers. It remains to be shown however, whether any mutant p53 reactivating compound has efficacy for the treatment of human cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.

    Science.gov (United States)

    Chachoua, Ilyas; Pecquet, Christian; El-Khoury, Mira; Nivarthi, Harini; Albu, Roxana-Irina; Marty, Caroline; Gryshkova, Vitalina; Defour, Jean-Philippe; Vertenoeil, Gaëlle; Ngo, Anna; Koay, Ann; Raslova, Hana; Courtoy, Pierre J; Choong, Meng Ling; Plo, Isabelle; Vainchenker, William; Kralovics, Robert; Constantinescu, Stefan N

    2016-03-10

    Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling. © 2016 by The American Society of Hematology.

  6. Metabolic reprogramming in mutant IDH1 glioma cells.

    Directory of Open Access Journals (Sweden)

    Jose L Izquierdo-Garcia

    Full Text Available Mutations in isocitrate dehydrogenase (IDH 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS-detectable changes in the cellular metabolome.Two genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by 1H-MRS. Principal Component Analysis was used to analyze the MRS data.Principal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts.The IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.

  7. Elevation of Urinary 2-Hydroxyglutarate in IDH-Mutant Glioma.

    Science.gov (United States)

    Fathi, Amir T; Nahed, Brian V; Wander, Seth A; Iafrate, A John; Borger, Darrell R; Hu, Ranliang; Thabet, Ashraf; Cahill, Daniel P; Perry, Ashley M; Joseph, Christelle P; Muzikansky, Alona; Chi, Andrew S

    2016-02-01

    Recurrent mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes, which are frequent in gliomas, result in marked accumulation of the metabolic by-product 2-hydroxyglutarate (2-HG) within tumors. In other malignancies, such as acute myeloid leukemia, presence of IDH mutation is associated with elevated 2-HG levels in serum or urine compartments. Circulating 2-HG in patients with glial malignancies has not been thoroughly investigated. In this study, we analyzed 2-HG levels in the serum and urine of a large set of patients with IDH-mutant and IDH-wild-type glioma, and the cerebrospinal fluid (CSF) from a subset of this cohort. We found that 2-HG was elevated in the urine of patients with IDH-mutant versus IDH-wild-type glioma, although no significant differences in 2-HG levels were observed in the serum or the small set of CSF samples obtained. Among patients with IDH-mutant glioma, 2-HG levels did not differ based on the histopathologic grade, genetic subtype (TP53 mutant or 1p/19q codeleted), presence of a canonical (IDH1 R132H) or noncanonical (any other IDH variant) mutation, or treatment type. Our finding suggests that urinary 2-HG is increased among patients with IDH-mutant gliomas, and may represent a future surrogate, noninvasive biomarker to aid in diagnosis, prognosis, and management. Patients with glioma who harbor mutations in isocitrate dehydrogenase genes showed selective elevation of the oncometabolite 2-hydroxyglutarate in the urine. Similar elevations were not identified in the serum or cerebrospinal fluid. 2-Hydroxyglutarate may serve as a useful, noninvasive biomarker to stratify patients newly diagnosed with glioma with regard to prognosis and management. ©AlphaMed Press.

  8. Arabidopsis mutant bik1 exhibits strong resistance to Plasmodiophora brassicae

    Directory of Open Access Journals (Sweden)

    Tao Chen

    2016-09-01

    Full Text Available Botrytis-induced kinase1 (BIK1, a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1 and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2 and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40-50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2 and npr1-1 (non-expresser of PR genes that regulate systemic acquired resistance (SAR mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms enhance the resistance to clubroot disease.

  9. Multiplexed lasing in tissues

    Science.gov (United States)

    Chen, Yu-Cheng; Chen, Qiushu; Fan, Xudong

    2017-02-01

    Biolasers are an emerging technology for next generation biochemical detection and clinical applications. Progress has recently been made to achieve lasing from biomolecules and single living cells. Tissues, which consist of cells embedded in extracellular matrix, mimic more closely the actual complex biological environment in a living body and therefore are of more practical significance. Here, we developed a highly versatile tissue laser platform, in which tissues stained with fluorophores are sandwiched in a high-Q Fabry-Pérot microcavity. Distinct lasing emissions from muscle and adipose tissues stained respectively with fluorescein isothiocyanate (FITC) and boron-dipyrromethene (BODIPY), and hybrid muscle/adipose tissue with dual-staining were achieved with a threshold of only 10 μJ/mm2. Additionally, we investigated how tissue structure/geometry, tissue thickness, and staining dye concentration affect the tissue laser. It is further found that, despite large fluorescence spectral overlap between FITC and BODIPY in tissues, their lasing emissions could be clearly distinguished and controlled due to their narrow lasing bands and different lasing thresholds, thus enabling highly multiplexed detection. Our tissue laser platform can be broadly applicable to various types of tissues/diseases. It provides a new tool for a wide range of biological and biomedical applications, such as diagnostics/screening of tissues and identification/monitoring of biological transformations in tissue engineering.

  10. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery

    Science.gov (United States)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized...

  11. Human GLTP and mutant forms of ACD11 suppress cell death in the Arabidopsis acd11 mutant

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; McKinney, Lea V; Pike, Helen

    2008-01-01

    The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11...

  12. Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant.

    Science.gov (United States)

    Silber, J; Bazin, C; Le Menn, A

    1989-09-01

    Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vgB/+ flies are reared on these substances (vgB is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies. Our data suggest that the vg+ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.

  13. Efficient transduction of LEDGF/p75 mutant cells by complementary gain-of-function HIV-1 integrase mutant viruses

    Directory of Open Access Journals (Sweden)

    Hao Wang

    2014-01-01

    Full Text Available Controlling the specificity of retroviral DNA integration could improve the safety of gene therapy vectors, and fusions of heterologous chromatin binding modules to the integrase (IN–binding domain from the lentiviral integration host cofactor lens epithelium–derived growth factor (LEDGF/p75 are a promising retargeting strategy. We previously proposed the utility of IN mutant lentiviral vectors that are selectively activated by complementary LEDGF/p75 variants, and our initial modifications in human immunodeficiency virus type 1 IN and LEDGF/p75 supported about 13% of wild-type vector transduction activity. Here we describe the selection and characterization of the K42E gain-of-function mutation in IN, which greatly improves the efficiency of this system. Both K42E and initial reverse-charge mutations in IN negatively affected reverse transcription and integration, yet when combined together boosted viral transduction efficiency to ∼75% of the wild-type vector in a manner dependent on a complementary LEDGF/p75 variant. Although the K42E mutation conferred functional gains to IN mutant viral reverse transcription and integration, only the integration boost depended on the engineered LEDGF/p75 mutant. We conclude that the specificity of lentiviral retargeting strategies based on heterologous LEDGF/p75 fusion proteins will benefit from our optimized system that utilizes the unique complementation properties of reverse-charge IN mutant viral and LEDGF/p75 host proteins.

  14. Laser/tissue interaction.

    Science.gov (United States)

    Dederich, D N

    1991-01-01

    When laser light impinges on tissue, it can reflect, scatter, be absorbed, or transmit to the surrounding tissue. Absorption controls to a great degree the extent to which reflection, scattering and transmission occur, and wavelength is the primary determinant of absorption. The CO2 laser is consistently absorbed by most materials and tissues and the Nd-YAG laser wavelength is preferentially absorbed in pigmented tissues. The factors which determine the initial tissue effect include the laser wavelength, laser power, laser waveform, tissue optical properties, and tissue thermal properties. There are almost an infinite number of combinations of these factors possible, many of which would result in unacceptable damage to the tissues. This underscores the need to thoroughly test any particular combination of these factors on the conceptual, in-vitro, and in-vivo level before a treatment is offered.

  15. Plant tissue culture techniques

    Directory of Open Access Journals (Sweden)

    Rolf Dieter Illg

    1991-01-01

    Full Text Available Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus or organized tissues or organs put in culture, under controlled sterile conditions.

  16. FRD tissue archive

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The fishery genetics tissue collection has over 80,000 tissues stored in 95% ethanol representing fishes and invertebrates collected globally but with a focus on the...

  17. Drosophila non-muscle myosin II motor activity determines the rate of tissue folding

    Science.gov (United States)

    Vasquez, Claudia G; Heissler, Sarah M; Billington, Neil; Sellers, James R; Martin, Adam C

    2016-01-01

    Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the Drosophila regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing Drosophila embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing Drosophila embryos. DOI: http://dx.doi.org/10.7554/eLife.20828.001 PMID:28035903

  18. Adipose tissue fibrosis

    OpenAIRE

    Buechler, Christa; Krautbauer, Sabrina; Eisinger, Kristina

    2015-01-01

    The increasing prevalence of obesity causes a major interest in white adipose tissue biology. Adipose tissue cells are surrounded by extracellular matrix proteins whose composition and remodeling is of crucial importance for cell function. The expansion of adipose tissue in obesity is linked to an inappropriate supply with oxygen and hypoxia development. Subsequent activation of hypoxia inducible factor 1 (HIF-1) inhibits preadipocyte differentiation and initiates adipose tissue fibrosis. The...

  19. Bioreactors for tissue engineering.

    Science.gov (United States)

    Chen, Huang-Chi; Hu, Yu-Chen

    2006-09-01

    Bioreactors are essential in tissue engineering, not only because they provide an in vitro environment mimicking in vivo conditions for the growth of tissue substitutes, but also because they enable systematic studies of the responses of living tissues to various mechanical and biochemical cues. The basic principles of bioreactor design are reviewed, the bioreactors commonly used for the tissue engineering of cartilage, bone and cardiovascular systems are assessed in terms of their performance and usefulness. Several novel bioreactor types are also reviewed.

  20. HdhQ111 Mice Exhibit Tissue Specific Metabolite Profiles that Include Striatal Lipid Accumulation.

    Directory of Open Access Journals (Sweden)

    Jeffrey B Carroll

    Full Text Available The HTT CAG expansion mutation causes Huntington's Disease and is associated with a wide range of cellular consequences, including altered metabolism. The mutant allele is expressed widely, in all tissues, but the striatum and cortex are especially vulnerable to its effects. To more fully understand this tissue-specificity, early in the disease process, we asked whether the metabolic impact of the mutant CAG expanded allele in heterozygous B6.HdhQ111/+ mice would be common across tissues, or whether tissues would have tissue-specific responses and whether such changes may be affected by diet. Specifically, we cross-sectionally examined steady state metabolite concentrations from a range of tissues (plasma, brown adipose tissue, cerebellum, striatum, liver, white adipose tissue, using an established liquid chromatography-mass spectrometry pipeline, from cohorts of 8 month old mutant and wild-type littermate mice that were fed one of two different high-fat diets. The differential response to diet highlighted a proportion of metabolites in all tissues, ranging from 3% (7/219 in the striatum to 12% (25/212 in white adipose tissue. By contrast, the mutant CAG-expanded allele primarily affected brain metabolites, with 14% (30/219 of metabolites significantly altered, compared to wild-type, in striatum and 11% (25/224 in the cerebellum. In general, diet and the CAG-expanded allele both elicited metabolite changes that were predominantly tissue-specific and non-overlapping, with evidence for mutation-by-diet interaction in peripheral tissues most affected by diet. Machine-learning approaches highlighted the accumulation of diverse lipid species as the most genotype-predictive metabolite changes in the striatum. Validation experiments in cell culture demonstrated that lipid accumulation was also a defining feature of mutant HdhQ111 striatal progenitor cells. Thus, metabolite-level responses to the CAG expansion mutation in vivo were tissue specific and

  1. Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

    Science.gov (United States)

    Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

    2016-01-10

    To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph). Copyright © 2015 Elsevier B.V. All rights reserved.

  2. 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.

  3. Overproduction of delta-endotoxins by sporeless Bacillus thuringiensis mutants obtained by nitrous acid mutagenesis.

    Science.gov (United States)

    Ben Khedher, Saoussen; Zouari, Nabil; Messaddeq, Nadia; Schultz, Patrick; Jaoua, Samir

    2011-01-01

    Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.

  4. Vascularization Tissue Engineering

    NARCIS (Netherlands)

    Rouwkema, Jeroen; Rivron, N.C.; van Blitterswijk, Clemens

    2008-01-01

    Tissue engineering has been an active field of research for several decades now. However, the amount of clinical applications in the field of tissue engineering is still limited. One of the current limitations of tissue engineering is its inability to provide sufficient blood supply in the initial

  5. Tertiary lymphoid tissue

    Science.gov (United States)

    Di Caro, Giuseppe; Marchesi, Federica

    2014-01-01

    Tumor-infiltrating lymphocytes influence colorectal cancer progression. We have recently documented that tertiary lymphoid tissue in the colorectal cancer microenvironment orchestrates lymphocyte infiltration and that tertiary lymphoid tissue and lymphocytes cooperate in a coordinated antitumor immune response to improve patient outcome. Thus, tertiary lymphoid tissue represents a potential target in the design of tailored immune-based therapeutic approaches. PMID:25083321

  6. Development of tissue bank

    Directory of Open Access Journals (Sweden)

    R P Narayan

    2012-01-01

    Full Text Available The history of tissue banking is as old as the use of skin grafting for resurfacing of burn wounds. Beneficial effects of tissue grafts led to wide spread use of auto and allograft for management of varied clinical conditions like skin wounds, bone defects following trauma or tumor ablation. Availability of adequate amount of tissues at the time of requirement was the biggest challenge that forced clinicians to find out techniques to preserve the living tissue for prolonged period of time for later use and thus the foundation of tissue banking was started in early twentieth century. Harvesting, processing, storage and transportation of human tissues for clinical use is the major activity of tissue banks. Low temperature storage of processed tissue is the best preservation technique at present. Tissue banking organization is a very complex system and needs high technical expertise and skilled personnel for proper functioning in a dedicated facility. A small lapse/deviation from the established protocol leads to loss of precious tissues and or harm to recipients as well as the risk of transmission of deadly diseases and tumors. Strict tissue transplant acts and stringent regulations help to streamline the whole process of tissue banking safe for recipients and to community as whole.

  7. Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.

    Science.gov (United States)

    Zhang, Honghao; Kamiya, Nobuhiro; Tsuji, Takehito; Takeda, Haruko; Scott, Greg; Rajderkar, Sudha; Ray, Manas K; Mochida, Yoshiyuki; Allen, Benjamin; Lefebvre, Veronique; Hung, Irene H; Ornitz, David M; Kunieda, Tetsuo; Mishina, Yuji

    2016-12-01

    Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

  8. Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.

    Directory of Open Access Journals (Sweden)

    Honghao Zhang

    2016-12-01

    Full Text Available Ellis-van Creveld (EvC syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN. While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

  9. A sorghum (Sorghum bicolor mutant with altered carbon isotope ratio.

    Directory of Open Access Journals (Sweden)

    Govinda Rizal

    Full Text Available Recent efforts to engineer C4 photosynthetic traits into C3 plants such as rice demand an understanding of the genetic elements that enable C4 plants to outperform C3 plants. As a part of the C4 Rice Consortium's efforts to identify genes needed to support C4 photosynthesis, EMS mutagenized sorghum populations were generated and screened to identify genes that cause a loss of C4 function. Stable carbon isotope ratio (δ13C of leaf dry matter has been used to distinguishspecies with C3 and C4 photosynthetic pathways. Here, we report the identification of a sorghum (Sorghum bicolor mutant with a low δ13C characteristic. A mutant (named Mut33 with a pale phenotype and stunted growth was identified from an EMS treated sorghum M2 population. The stable carbon isotope analysis of the mutants showed a decrease of 13C uptake capacity. The noise of random mutation was reduced by crossing the mutant and its wildtype (WT. The back-cross (BC1F1 progenies were like the WT parent in terms of 13C values and plant phenotypes. All the BC1F2 plants with low δ13C died before they produced their 6th leaf. Gas exchange measurements of the low δ13C sorghum mutants showed a higher CO2 compensation point (25.24 μmol CO2.mol-1air and the maximum rate of photosynthesis was less than 5μmol.m-2.s-1. To identify the genetic determinant of this trait, four DNA pools were isolated; two each from normal and low δ13C BC1F2 mutant plants. These were sequenced using an Illumina platform. Comparison of allele frequency of the single nucleotide polymorphisms (SNPs between the pools with contrasting phenotype showed that a locus in Chromosome 10 between 57,941,104 and 59,985,708 bps had an allele frequency of 1. There were 211 mutations and 37 genes in the locus, out of which mutations in 9 genes showed non-synonymous changes. This finding is expected to contribute to future research on the identification of the causal factor differentiating C4 from C3 species that can be used

  10. Autophagy drives epidermal deterioration in a Drosophila model of tissue aging.

    Science.gov (United States)

    Scherfer, Christoph; Han, Violet C; Wang, Yan; Anderson, Aimee E; Galko, Michael J

    2013-04-01

    Organismal lifespan has been the primary readout in aging research. However, how longevity genes control tissue-specific aging remains an open question. To examine the crosstalk between longevity programs and specific tissues during aging, biomarkers of organ-specific aging are urgently needed. Since the earliest signs of aging occur in the skin, we sought to examine skin aging in a genetically tractable model. Here we introduce a Drosophila model of skin aging. The epidermis undergoes a dramatic morphological deterioration with age that includes membrane and nuclear loss. These changes were decelerated in a long-lived mutant and accelerated in a short-lived mutant. An increase in autophagy markers correlated with epidermal aging. Finally, the epidermis of Atg7 mutants retained younger characteristics, suggesting that autophagy is a critical driver of epidermal aging. This is surprising given that autophagy is generally viewed as protective during aging. Since Atg7 mutants are short-lived, the deceleration of epidermal aging in this mutant suggests that in the epidermis healthspan can be uncoupled from longevity. Because the aging readout we introduce here has an early onset and is easily visualized, genetic dissection using our model should identify other novel mechanisms by which lifespan genes feed into tissue-specific aging.

  11. Adipose tissue fibrosis.

    Science.gov (United States)

    Buechler, Christa; Krautbauer, Sabrina; Eisinger, Kristina

    2015-05-15

    The increasing prevalence of obesity causes a major interest in white adipose tissue biology. Adipose tissue cells are surrounded by extracellular matrix proteins whose composition and remodeling is of crucial importance for cell function. The expansion of adipose tissue in obesity is linked to an inappropriate supply with oxygen and hypoxia development. Subsequent activation of hypoxia inducible factor 1 (HIF-1) inhibits preadipocyte differentiation and initiates adipose tissue fibrosis. Thereby adipose tissue growth is limited and excess triglycerides are stored in ectopic tissues. Stressed adipocytes and hypoxia contribute to immune cell immigration and activation which further aggravates adipose tissue fibrosis. There is substantial evidence that adipose tissue fibrosis is linked to metabolic dysfunction, both in rodent models and in the clinical setting. Peroxisome proliferator activated receptor gamma agonists and adiponectin both reduce adipose tissue fibrosis, inflammation and insulin resistance. Current knowledge suggests that antifibrotic drugs, increasing adipose tissue oxygen supply or HIF-1 antagonists will improve adipose tissue function and thereby ameliorate metabolic diseases.

  12. Cell and Tissue Engineering

    CERN Document Server

    2012-01-01

    “Cell and Tissue Engineering” introduces the principles and new approaches in cell and tissue engineering. It includes both the fundamentals and the current trends in cell and tissue engineering, in a way useful both to a novice and an expert in the field. The book is composed of 13 chapters all of which are written by the leading experts. It is organized to gradually assemble an insight in cell and tissue function starting form a molecular nano-level, extending to a cellular micro-level and finishing at the tissue macro-level. In specific, biological, physiological, biophysical, biochemical, medical, and engineering aspects are covered from the standpoint of the development of functional substitutes of biological tissues for potential clinical use. Topics in the area of cell engineering include cell membrane biophysics, structure and function of the cytoskeleton, cell-extracellular matrix interactions, and mechanotransduction. In the area of tissue engineering the focus is on the in vitro cultivation of ...

  13. Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila.

    Science.gov (United States)

    Seki, Yuuichi; Tanimura, Teiichi

    2014-09-01

    A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. We used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, the Pigment-dispersing factor mutant (Pdf 01) demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. Our results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours.

  14. Dynamic void distribution in myoglobin and five mutants

    Science.gov (United States)

    Jiang, Yingying; Kirmizialtin, Serdal; Sanchez, Isaac C.

    2014-02-01

    Globular proteins contain cavities/voids that play specific roles in controlling protein function. Elongated cavities provide migration channels for the transport of ions and small molecules to the active center of a protein or enzyme. Using Monte Carlo and Molecular Dynamics on fully atomistic protein/water models, a new computational methodology is introduced that takes into account the protein's dynamic structure and maps all the cavities in and on the surface. To demonstrate its utility, the methodology is applied to study cavity structure in myoglobin and five of its mutants. Computed cavity and channel size distributions reveal significant differences relative to the wild type myoglobin. Computer visualization of the channels leading to the heme center indicates restricted ligand access for the mutants consistent with the existing interpretations. The new methodology provides a quantitative measure of cavity structure and distributions and can become a valuable tool for the structural characterization of proteins.

  15. Characterizing visible and invisible cell wall mutant phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Carpita, Nicholas C.; McCann, Maureen C.

    2015-04-06

    About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with ‘invisible’ phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.

  16. Butyric acid tolerance of rice mutant M4 families

    Directory of Open Access Journals (Sweden)

    Mauricio Marini Kopp

    2007-01-01

    Full Text Available Hydromorphic soils have a low drainage capacity and are used mainly for the cultivation of irrigated rice.This condition favors the development of anaerobic microorganisms that produce phytotoxic substances. The objective of thisstudy was to evaluate the response of rice mutants to the phytotoxicity caused by butyric acid under anaerobic conditions. Theexperiment consisted of four treatments arranged in a randomized block design. Plants of 40 families were grown in ahydroponic system and the measured variables were root length and length of aerial part (LAP, number of roots (NR androot dry matter (RDM and aerial part dry matter (DMAP. The analysis of variance was performed, the relative performancecalculated and linear regressions were fitted. Only the treatment effect for NR and effect of interaction for LAP were notsignificant. Root length was most affected by the acid and the regressions expressed positive as well as negative effects for acidtolerance in the mutant families.

  17. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...... in which apoptosis can be studied using the novel, temperature sensitive mutant, cdc77. The cdc77 cells are defective in a G1 process, and die show the characteristc signs of apoptosis: condensation of the chromatin, degradation of the inner nuclear membrane, dilation of the space between the nuclear...

  18. Development and evaluation of drought resistant mutant germ ...

    African Journals Online (AJOL)

    The aim of this project was to select cowpea plants with improved levels of drought resistance without alteration to the colour of the testa or the growth form. Seed from M2 to M5 generations (M = mutant) were used in the study. The M2 to M4 seeds were planted and evaluated in wooden boxes in the greenhouse and in the ...

  19. Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes.

    Directory of Open Access Journals (Sweden)

    Mohammad Haeri

    Full Text Available Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and Rho(P23H-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS, accumulating in the OS to a concentration of ∼0.1% compared to endogenous opsin. Rho(P23H-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with Rho(P23H-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The Rho(P23H-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing Rho(P23H, suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.

  20. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine

  1. Wheat ABA-insensitive mutants result in reduced grain dormancy

    Science.gov (United States)

    Schramm, Elizabeth C.; Nelson, Sven K.

    2014-01-01

    This paper describes the isolation of wheat mutants in the hard red spring Scarlet resulting in reduced sensitivity to the plant hormone abscisic acid (ABA) during seed germination. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature seeds. Wheat sensitivity to ABA gradually decreases with dry after-ripening. Scarlet grain normally fails to germinate when fully dormant, shows ABA sensitive germination when partially after-ripened, and becomes ABA insensitive when after-ripened for 8–12 months. Scarlet ABA-insensitive (ScABI) mutants were isolated based on the ability to germinate on 5 µM ABA after only 3 weeks of after-ripening, a condition under which Scarlet would fail to germinate. Six independent seed-specific mutants were recovered. ScABI 1, ScABI2, ScABI3 and ScABI4 are able to germinate more efficiently than Scarlet at up to 25 µM ABA. The two strongest ABA insensitive lines, ScABI3 and ScABI4, both proved to be partly dominant suggesting that they result from gain-of-function mutations. The ScABI1, ScABI2, ScABI3, ScABI4, and ScABI5 mutants after-ripen more rapidly than Scarlet. Thus, ABA insensi-tivity is associated with decreased grain dormancy in Scarlet wheat. This suggests that ABA sensitivity is an important factor controlling grain dormancy in wheat, a trait that impacts seedling emergence and pre-harvest sprouting resistance. PMID:25431501

  2. Xylitol production by a Pichia stipitis D-xylulokinase mutant

    Science.gov (United States)

    Yong-Su Jin; Jose Cruz; Thomas W. Jeffries

    2005-01-01

    Xylitol production by Pichia stipitis FPL-YS30, a xyl3-Ä1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-Ä1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g...

  3. Strain-Dependent Anterior Segment Dysgenesis and Progression to Glaucoma in Col4a1 Mutant Mice

    Science.gov (United States)

    Mao, Mao; Smith, Richard S.; Alavi, Marcel V.; Marchant, Jeffrey K.; Cosma, Mihai; Libby, Richard T.; John, Simon W. M.; Gould, Douglas B.

    2015-01-01

    Purpose Mutations in the gene encoding collagen type IV alpha 1 (COL4A1) cause multisystem disorders including anterior segment dysgenesis (ASD) and optic nerve hypoplasia. The penetrance and severity of individual phenotypes depends on genetic context. Here, we tested the effects of a Col4a1 mutation in two different genetic backgrounds to compare how genetic context influences ocular dysgenesis, IOP, and progression to glaucoma. Methods Col4a1 mutant mice maintained on a C57BL/6J background were crossed to either 129S6/SvEvTac or CAST/EiJ and the F1 progeny were analyzed by slit-lamp biomicroscopy and optical coherence tomography. We also measured IOPs and compared tissue sections of eyes and optic nerves. Results. We found that the CAST/EiJ inbred strain has a relatively uniform and profound suppression on the effects of Col4a1 mutation and that mutant CASTB6F1 mice were generally only very mildly affected. In contrast, mutant 129B6F1 mice had more variable and severe ASD and IOP dysregulation that were associated with glaucomatous signs including lost or damaged retinal ganglion cell axons and excavation of the optic nerve head. Conclusions. Ocular defects in Col4a1 mutant mice model ASD and glaucoma that are observed in a subset of patients with COL4A1 mutations. We demonstrate that different inbred strains of mice give graded severities of ASD and we detected elevated IOP and glaucomatous damage in 129B6F1, but not CASTB6F1 mice that carried a Col4a1 mutation. These data demonstrate that genetic context differences are one factor that may contribute to the variable penetrance and severity of ASD and glaucoma in patients with COL4A1 mutations. PMID:26567795

  4. dhm1, an Arabidopsis mutant with increased sensitivity to alkamides shows tumorous shoot development and enhanced lateral root formation.

    Science.gov (United States)

    Pelagio-Flores, Ramón; Ortiz-Castro, Randy; López-Bucio, José

    2013-04-01

    The control of cell division by growth regulators is critical to proper shoot and root development. Alkamides belong to a class of small lipid amides involved in plant morphogenetic processes, from which N-isobutyl decanamide is one of the most active compounds identified. This work describes the isolation and characterization of an N-isobutyl decanamide-hypersensitive (dhm1) mutant of Arabidopsis (Arabidopsis thaliana). dhm1 seedlings grown in vitro develop disorganized tumorous tissue in petioles, leaves and stems. N-isobutyl decanamide treatment exacerbates the dhm1 phenotype resulting in widespread production of callus-like structures in the mutant. Together with these morphological alterations in shoot, dhm1 seedlings sustained increased lateral root formation and greater sensitivity to alkamides in the inhibition of primary root growth. The mutants also show reduced etiolation when grown in darkness. When grown in soil, adult dhm1 plants were characterized by reduced plant size, and decreased fertility. Genetic analysis indicated that the mutant phenotype segregates as a single recessive Mendelian trait. Developmental alterations in dhm1 were related to an enhanced expression of the cell division marker CycB1-uidA both in the shoot and root system, which correlated with altered expression of auxin and cytokinin responsive gene markers. Pharmacological inhibition of auxin transport decreased LR formation in WT and dhm1 seedlings in a similar manner, indicating that auxin transport is involved in the dhm1 root phenotype. These data show an important role of alkamide signaling in cell proliferation and plant architecture remodeling likely acting through the DHM1 protein.

  5. PRC2-mediated repression of SMARCA2 predicts EZH2 inhibitor activity in SWI/SNF mutant tumors.

    Science.gov (United States)

    Januario, Thomas; Ye, Xiaofen; Bainer, Russell; Alicke, Bruno; Smith, Tunde; Haley, Benjamin; Modrusan, Zora; Gould, Stephen; Yauch, Robert L

    2017-10-30

    Subunits of the SWI/SNF chromatin remodeling complex are frequently mutated in human cancers leading to epigenetic dependencies that are therapeutically targetable. The dependency on the polycomb repressive complex (PRC2) and EZH2 represents one such vulnerability in tumors with mutations in the SWI/SNF complex subunit, SNF5; however, whether this vulnerability extends to other SWI/SNF subunit mutations is not well understood. Here we show that a subset of cancers harboring mutations in the SWI/SNF ATPase, SMARCA4, is sensitive to EZH2 inhibition. EZH2 inhibition results in a heterogenous phenotypic response characterized by senescence and/or apoptosis in different models, and also leads to tumor growth inhibition in vivo. Lower expression of the SMARCA2 paralog was associated with cellular sensitivity to EZH2 inhibition in SMARCA4 mutant cancer models, independent of tissue derivation. SMARCA2 is suppressed by PRC2 in sensitive models, and induced SMARCA2 expression can compensate for SMARCA4 and antagonize PRC2 targets. The induction of SMARCA2 in response to EZH2 inhibition is required for apoptosis, but not for growth arrest, through a mechanism involving the derepression of the lysomal protease cathepsin B. Expression of SMARCA2 also delineates EZH2 inhibitor sensitivity for other SWI/SNF complex subunit mutant tumors, including SNF5 and ARID1A mutant cancers. Our data support monitoring SMARCA2 expression as a predictive biomarker for EZH2-targeted therapies in the context of SWI/SNF mutant cancers. Published under the PNAS license.

  6. The wheat mutant DELLA-encoding gene (Rht-B1c) affects plant photosynthetic responses to cadmium stress.

    Science.gov (United States)

    Dobrikova, Anelia G; Yotsova, Ekaterina K; Börner, Andreas; Landjeva, Svetlana P; Apostolova, Emilia L

    2017-05-01

    Тhe sensitivity to cadmium (Cd) stress of two near-isogenic wheat lines with differences at the Rht-B1 locus, Rht-B1a (tall wild type, encoding DELLA proteins) and Rht-B1c (dwarf mutant, encoding modified DELLA proteins), was investigated. The effects of 100 μM CdCl2 on plant growth, pigment content and functional activity of the photosynthetic apparatus of wheat seedlings grown on a nutrient solution were evaluated through a combination of PAM chlorophyll fluorescence, oxygen evolution, oxidation-reduction kinetics of P700 and 77 K fluorescence. The results showed that the wheat mutant (Rht-B1c) was more tolerant to Cd stress compared to the wild type (Rht-B1a), as evidenced by the lower reductions in plant growth and pigment content, lower inhibition of photosystem I (PSI) and photosystem II (PSII) photochemistry and of the oxygen evolution measured with Clark-type and Joliot-type electrodes. Furthermore, the enhanced Cd tolerance was accompanied by increased Cd accumulation within mutant plant tissues. The molecular mechanisms through which the Rht-B1c mutation improves plant tolerance to Cd stress involve structural alterations in the mutant photosynthetic membranes leading to better protection of the Mn cluster of oxygen-evolving complex and increased capacity for PSI cyclic electron transport, protecting photochemical activity of the photosynthetic apparatus under stress. This study suggests a role for the Rht-B1c-encoded DELLA proteins in protective mechanisms and tolerance of the photosynthetic apparatus in wheat plants exposed to heavy metals stress. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Lipid metabolic perturbation is an early-onset phenotype in adult spinster mutants: a Drosophila model for lysosomal storage disorders.

    Science.gov (United States)

    Hebbar, Sarita; Khandelwal, Avinash; Jayashree, R; Hindle, Samantha J; Chiang, Yin Ning; Yew, Joanne Y; Sweeney, Sean T; Schwudke, Dominik

    2017-12-15

    Intracellular accumulation of lipids and swollen dysfunctional lysosomes are linked to several neurodegenerative diseases, including lysosomal storage disorders (LSD). Detailed characterization of lipid metabolic changes in relation to the onset and progression of neurodegeneration is currently missing. We systematically analyzed lipid perturbations in spinster (spin) mutants, a Drosophila model of LSD-like neurodegeneration. Our results highlight an imbalance in brain ceramide and sphingosine in the early stages of neurodegeneration, preceding the accumulation of endomembranous structures, manifestation of altered behavior, and buildup of lipofuscin. Manipulating levels of ceramidase and altering these lipids in spin mutants allowed us to conclude that ceramide homeostasis is the driving force in disease progression and is integral to spin function in the adult nervous system. We identified 29 novel physical interaction partners of Spin and focused on the lipid carrier protein, Lipophorin (Lpp). A subset of Lpp and Spin colocalize in the brain and within organs specialized for lipid metabolism (fat bodies and oenocytes). Reduced Lpp protein was observed in spin mutant tissues. Finally, increased levels of lipid metabolites produced by oenocytes in spin mutants allude to a functional interaction between Spin and Lpp, underscoring the systemic nature of lipid perturbation in LSD. © 2017 Hebbar et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. Syndactyly in a novel Fras1(rdf) mutant results from interruption of signals for interdigital apoptosis.

    Science.gov (United States)

    Hines, Elizabeth A; Verheyden, Jamie M; Lashua, Amber J; Larson, Sarah C; Branchfield, Kelsey; Domyan, Eric T; Gao, Juan; Harvey, Julie F; Herriges, John C; Hu, Linghan; Mcculley, David J; Throckmorton, Kurt; Yokoyama, Shigetoshi; Ikeda, Akihiro; Xu, Guoliang; Sun, Xin

    2016-04-01

    Fras1 encodes an extracellular matrix protein that is critical for the establishment of the epidermal basement membrane during gestation. In humans, mutations in FRAS1 cause Fraser Syndrome (FS), a pleiotropic condition with many clinical presentations such as limb, eye, kidney, and craniofacial deformations. Many of these defects are mimicked by loss of Fras1 in mice, and are preceded by the formation of epidermal blisters in utero. In this study, we identified a novel ENU-derived rounded foot (rdf) mouse mutant with highly penetrant hindlimb soft-tissue syndactyly, among other structural defects. Mapping and sequencing revealed that rdf is a novel loss-of-function nonsense allele of Fras1 (Fras1(rdf)). Focusing on the limb, we found that the Fras1(rdf) syndactyly phenotype originates from loss of interdigital cell death (ICD). Despite normal expression of bone morphogenetic protein (BMP) ligands and their receptors, the BMP downstream target gene Msx2, which is also necessary and sufficient to promote ICD, was down-regulated in the interdigital regions of Fras1(rdf) hindlimb buds. The close correlation between limb bud epidermal blistering, decreased Msx2 expression, and reduced ICD in the Fras1(rdf) hindlimb buds suggests that epithelium detachment from the mesenchyme may create a physical gap that interrupts the transmission of BMP, among other signals, resulting in soft tissue syndactyly. © 2016 Wiley Periodicals, Inc.

  9. Temperature Sensitivity of Neural Tube Defects in Zoep Mutants.

    Science.gov (United States)

    Ma, Phyo; Swartz, Morgan R; Kindt, Lexy M; Kangas, Ashley M; Liang, Jennifer Ostrom

    2015-12-01

    Neural tube defects (NTD) occur when the flat neural plate epithelium fails to fold into the neural tube, the precursor to the brain and spinal cord. Squint (Sqt/Ndr1), a Nodal ligand, and One-eyed pinhead (Oep), a component of the Nodal receptor, are required for anterior neural tube closure in zebrafish. The NTD in sqt and Zoep mutants are incompletely penetrant. The penetrance of several defects in sqt mutants increases upon heat or cold shock. In this project, undergraduate students tested whether temperature influences the Zoep open neural tube phenotype. Single pairs of adults were spawned at 28.5°C, the normal temperature for zebrafish, and one half of the resulting embryos were moved to 34°C at different developmental time points. Analysis of variance indicated temperature and clutch/genetic background significantly contributed to the penetrance of the open neural tube phenotype. Heat shock affected the embryos only at or before the midblastula stage. Many factors, including temperature changes in the mother, nutrition, and genetic background, contribute to NTD in humans. Thus, sqt and Zoep mutants may serve as valuable models for studying the interactions between genetics and the environment during neurulation.

  10. Genes and Alcohol Consumption: Studies with Mutant Mice

    Science.gov (United States)

    Mayfield, Jody; Arends, Michael A.; Harris, R. Adron; Blednov, Yuri A.

    2017-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. PMID:27055617

  11. Drosophila melanogaster White Mutant w1118 Undergo Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    María José Ferreiro

    2018-01-01

    Full Text Available Key scientific discoveries have resulted from genetic studies of Drosophila melanogaster, using a multitude of transgenic fly strains, the majority of which are constructed in a genetic background containing mutations in the white gene. Here we report that white mutant flies from w1118 strain undergo retinal degeneration. We observed also that w1118 mutants have progressive loss of climbing ability, shortened life span, as well as impaired resistance to various forms of stress. Retinal degeneration was abolished by transgenic expression of mini-white+ in the white null background w1118. We conclude that beyond the classical eye-color phenotype, mutations in Drosophila white gene could impair several biological functions affecting parameters like mobility, life span and stress tolerance. Consequently, we suggest caution and attentiveness during the interpretation of old experiments employing white mutant flies and when planning new ones, especially within the research field of neurodegeneration and neuroprotection. We also encourage that the use of w1118 strain as a wild-type control should be avoided.

  12. Characterization of tryptophan synthase alpha subunit mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Radwanski, E R; Barczak, A J; Last, R L

    1996-12-13

    Three mutations in the Arabidopsis thaliana gene encoding the alpha subunit of tryptophan synthase were isolated by selection for resistance to 5-methylanthranilate or 5-fluoroindole, toxic analogs of tryptophan pathway intermediates. Plants homozygous for trp3-1 and trp3-2 are light-conditional tryptophan auxotrophs, while trp3-100 is a more leaky mutant. Genetic complementation crosses demonstrated that the three mutations are allelic to each other, and define a new complementation group. All three mutants have decreased steady-state levels of tryptophan synthase alpha protein, and the trp3-100 polypeptide exhibits altered electrophoretic mobility. All three mutations were shown to be in the TSA1 (tryptophan synthase alpha subunit) structural gene by several criteria. Firstly, the trp3-1 mutation is linked to TSA1 on the bottom of chromosome 3. Secondly, the trp3-1 mutation was complemented when transformed with the wild-type TSA1 gene. Finally, DNA sequence analysis of the TSA1 gene revealed a single transition mutation in each trp3 mutant.

  13. Allele-selective suppression of mutant genes in polyglutamine diseases.

    Science.gov (United States)

    Liu, Chia-Rung; Cheng, Tzu-Hao

    2015-01-01

    Polyglutamine (polyQ) diseases are heritable dominant neurological disorders, caused by abnormal CAG tri-nucleotide expansion in the coding sequence of affected genes. Extension of CAG repeats results in the production of aberrant gene products that are deleterious to neurons, such as transcripts with a CAG stem-loop secondary structure, and proteins containing a long stretch of polyQ residues. Thus, determining methods for the prevention or elimination of these mutant gene products from neuronal cells and translating this knowledge to clinical application are currently important goals in the fields of neurology and neurogenetics. Recently, several studies have revealed intriguing findings related to the allele-selective regulation of CAG-expanded genes, and have proposed novel designs to selectively diminish the mutant polyQ proteins. In this review, we focus on the genes, genetically engineered proteins, and oligonucleotides that show potential to modulate the expression of mutant genes. We also discuss their respective molecular functions at the levels of transcription, translation, and post-translation.

  14. Coloboma hyperactive mutant exhibits delayed neurobehavioral developmental milestones.

    Science.gov (United States)

    Heyser, C J; Wilson, M C; Gold, L H

    1995-11-21

    The coloboma mutation (Cm) is a neutron-irradiation induced gene deletion located on the distal portion of mouse chromosome 2. This deletion region includes a gene encoding the synaptic vesicle docking fusion protein, synaptosomal-associated protein of 25 kDa (SNAP-25). The resulting mutation is semi-dominant with heterozygote mice exhibiting a triad of phenotypic abnormalities that comprise profound spontaneous hyperactivity, head bobbing and a prominent eye dysmorphology. Because the expression pattern of two SNAP-25 isoforms begins to change during the first postnatal week, neurobehavioral developmental milestones were examined in order to determine if the expression of the coloboma behavioral phenotype could be detected during this period of postnatal development. The early classification of coloboma mutant offspring may help to further describe the penetrance of this mutation as well as the contribution of developmental changes to the adult behavioral phenotype. The coloboma mutation resulted in delays in some tests of complex motor skills including righting reflex and bar holding. In addition, coloboma mutants were characterized by body weight differences (first appearance day 7) and hyperreactivity to touch (day 11) and head bobbing (day 14). These data demonstrate disruptions in the time course of attaining developmental milestones in coloboma mutants and provide further evidence supporting the hypotheses that alterations in Snap gene expression are associated with functional behavioral consequences in developing offspring.

  15. Molecular Imaging Of Metabolic Reprogramming In Mutant IDH Cells

    Directory of Open Access Journals (Sweden)

    Pavithra eViswanath

    2016-03-01

    Full Text Available Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG. Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG. In turn, 2-HG, which has been termed an oncometabolite, inhibits key α-KG- dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprogramming that extends beyond 2-HG production, and this reprogramming often differs from what has been previously reported in other cancer types. In this review we will discuss in detail what is known to date about the metabolic reprogramming of mutant IDH cells and how this reprogramming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  16. Molecular Imaging of Metabolic Reprograming in Mutant IDH Cells.

    Science.gov (United States)

    Viswanath, Pavithra; Chaumeil, Myriam M; Ronen, Sabrina M

    2016-01-01

    Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG). In turn, 2-HG, which has been termed an "oncometabolite," inhibits key α-KG-dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprograming that extends beyond 2-HG production, and this reprograming often differs from what has been previously reported in other cancer types. In this review, we will discuss in detail what is known to date about the metabolic reprograming of mutant IDH cells, and how this reprograming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells, and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  17. Functional Analysis of Jasmonates in Rice through Mutant Approaches

    Directory of Open Access Journals (Sweden)

    Rohit Dhakarey

    2016-03-01

    Full Text Available Jasmonic acid, one of the major plant hormones, is, unlike other hormones, a lipid-derived compound that is synthesized from the fatty acid linolenic acid. It has been studied intensively in many plant species including Arabidopsis thaliana, in which most of the enzymes participating in its biosynthesis were characterized. In the past 15 years, mutants and transgenic plants affected in the jasmonate pathway became available in rice and facilitate studies on the functions of this hormone in an important crop. Those functions are partially conserved compared to other plant species, and include roles in fertility, response to mechanical wounding and defense against herbivores. However, new and surprising functions have also been uncovered by mutant approaches, such as a close link between light perception and the jasmonate pathway. This was not only useful to show a phenomenon that is unique to rice but also helped to establish this role in plant species where such links are less obvious. This review aims to provide an overview of currently available rice mutants and transgenic plants in the jasmonate pathway and highlights some selected roles of jasmonate in this species, such as photomorphogenesis, and abiotic and biotic stress.

  18. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

    Science.gov (United States)

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-02-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Genes and Alcohol Consumption: Studies with Mutant Mice.

    Science.gov (United States)

    Mayfield, J; Arends, M A; Harris, R A; Blednov, Y A

    2016-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. © 2016 Elsevier Inc. All rights reserved.

  20. Virulence of Burkholderia mallei quorum-sensing mutants.

    Science.gov (United States)

    Majerczyk, Charlotte; Kinman, Loren; Han, Tony; Bunt, Richard; Greenberg, E Peter

    2013-05-01

    Many Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved.

  1. Recombination Phenotypes of Escherichia coli greA Mutants

    Directory of Open Access Journals (Sweden)

    Poteete Anthony R

    2011-03-01

    Full Text Available Abstract Background The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination. Results Escherichia coli mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A greA mutant and a greA deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination. Conclusion These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.

  2. Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

    Science.gov (United States)

    Gardiol, A; Arias, A; Cerveñansky, C; Gaggero, C; Martínez-Drets, G

    1980-10-01

    A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.

  3. Pattern formation mechanisms in motility mutants of Myxococcus xanthus

    CERN Document Server

    Starruss, Joern; Jakovljevic, Vladimir; Sogaard-Andersen, Lotte; Deutsch, Andreas; Baer, Markus

    2016-01-01

    Formation of spatial patterns of cells is a recurring theme in biology and often depends on regulated cell motility. Motility of M. xanthus depends on two motility machineries: the S-engine and A-engine. Moving M. xanthus cells can organize into spreading colonies or spore-filled fruiting bodies depending on their nutritional status. To understand these two pattern formation processes and the contributions by the two motility machineries, as well as cell reversal, we analyze spatial self-organization in 3 strains: i) a mutant that moves unidirectionally without reversing by the A-motility system only, ii) a unidirectional mutant that is also equipped with the S-motility system, and iii) the wild-type that, in addition to the two motility systems, reverses its direction of movement. The mutant moving by the A-engine illustrates that collective motion in the form of large moving clusters can arise in gliding bacteria due to steric interactions of the rod-shaped cells, without the need of invoking any biochemica...

  4. Incomplete nonsense-mediated decay of mutant lamin A/C mRNA provokes dilated cardiomyopathy and ventricular tachycardia.

    Science.gov (United States)

    Geiger, Stephanie K; Bär, Harald; Ehlermann, Philipp; Wälde, Sarah; Rutschow, Désirée; Zeller, Raphael; Ivandic, Boris T; Zentgraf, Hanswalter; Katus, Hugo A; Herrmann, Harald; Weichenhan, Dieter

    2008-03-01

    We have identified a family in which several members died of sudden cardiac death or suffer from dilated cardiomyopathy (DCM) and rhythm disturbances. Mutation screening revealed co-segregation of a novel nonsense mutation (pR321X) in the lamin A gene, LMNA, with the disease. Lamin A, and its smaller splice form lamin C are nuclear intermediate filament proteins forming a major part of the lamina, which is underlying the inner nuclear membrane. They are involved in the organization of heterochromatin and both in DNA replication and transcription. Recently, an increasing number of missense mutations in LMNA have been discovered to cause various types of rare diseases. Here, we investigated the causal role of the new nonsense mutation for the disease. Quantification of wild type and mutant lamin A mRNA from explanted myocardial tissue and cultured fibroblasts revealed an up to 30-fold reduction in the relative amount of the mutant transcript indicating that its synthesis was massively down-regulated by nonsense-mediated mRNA decay (NMD). Correspondingly, we did not detect the mutant truncated lamin A by Western blot analysis in extracts of patient fibroblasts and cardiac muscle tissue. Both wild type lamin A and C were present, however, in normal quantities. The immunohistochemical analyses of patient tissues revealed a normal distribution of lamin A/C and of major inner nuclear membrane proteins such as emerin and the lamin B receptor. Moreover, both chromatin distribution and nuclear shape were normal. However, over-expression of truncated lamin A in HeLa cells by transient transfection caused major disturbances of lamin A organization within both the nucleoplasm and the cytoplasm. In addition, after treatment of patient fibroblasts with the proteasome inhibitor epoxomicin, mutant truncated lamin A was detected in relatively high levels by Western blotting demonstrating that it is synthesized in these cells. Therefore, we conclude that NMD is not sufficient to

  5. Inhibition of Rhizobium etli Polysaccharide Mutants by Phaseolus vulgaris Root Compounds

    OpenAIRE

    Eisenschenk, Linda; Diebold, Ronald; Perez-Lesher, Jeanett; Peterson, Andrew C.; Kent Peters, N.; Noel, K. Dale

    1994-01-01

    Crude bean root extracts of Phaseolus vulgaris were tested for inhibition of the growth of several polysaccharide mutants of Rhizobium etli biovar phaseoli CE3. Mutants deficient only in exopolysaccharide and some mutants deficient only in the O-antigen of the lipopolysaccharide were no more sensitive than the wild-type strain to the extracts, whereas mutants defective in both lipopolysaccharide and exopolysaccharide were much more sensitive. The inhibitory activity was found at much higher l...

  6. Bacterial adherence to eucaryotic cells: isolation of lymphocyte-binding mutants.

    Science.gov (United States)

    Mayer, E P; Teodorescu, M

    1980-01-01

    A procedure for obtaining bacterial mutants that bind to eucaryotic cells is described. This procedure takes advantage of the ability of the mutants to obtain a required nutrient from the eucaryotic cells. We used this procedure to isolate mutants of Escherichia coli that bind to mouse lymphocytes. We show that the mutants identify some immunoglobulin-bearing lymphocytes and some non-immunoglobulin-bearing lymphocytes. PMID:6995342

  7. Bacterial adherence to eucaryotic cells: isolation of lymphocyte-binding mutants.

    OpenAIRE

    Mayer, E P; Teodorescu, M

    1980-01-01

    A procedure for obtaining bacterial mutants that bind to eucaryotic cells is described. This procedure takes advantage of the ability of the mutants to obtain a required nutrient from the eucaryotic cells. We used this procedure to isolate mutants of Escherichia coli that bind to mouse lymphocytes. We show that the mutants identify some immunoglobulin-bearing lymphocytes and some non-immunoglobulin-bearing lymphocytes.

  8. Selection of 5-fluorocytosine-resistant mutants from an Aspergillus niger citric acid-producing strain

    Directory of Open Access Journals (Sweden)

    Conte Ana Paula de F.

    2003-01-01

    Full Text Available Mutants of Aspergillus niger N402, induced by UV mutagenesis, were selected and tested for resistance or sensitivity to 5-fluorocytosine. Some mutants showed increased citric acid production, which did not correlate with the intracellular amount of protein or ammonium ion. The resistance to 5-fluorocytosine proved to be a rational approach for isolation of new mutants with improved production of citric acid. The best mutant (FR13 accumulated double the amount of citric acid produced by the parental strain.

  9. Regeneration of periodontal tissues: guided tissue regeneration.

    Science.gov (United States)

    Villar, Cristina C; Cochran, David L

    2010-01-01

    The concept that only fibroblasts from the periodontal ligament or undifferentiated mesenchymal cells have the potential to re-create the original periodontal attachment has been long recognized. Based on this concept, guided tissue regeneration has been applied with variable success to regenerate periodontal defects. Quantitative analysis of clinical outcomes after guided tissue regeneration suggests that this therapy is a successful and predictable procedure to treat narrow intrabony defects and class II mandibular furcations, but offers limited benefits in the treatment of other types of periodontal defects.

  10. Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer.

    Science.gov (United States)

    Silva, Jerson L; Cino, Elio A; Soares, Iaci N; Ferreira, Vitor F; A P de Oliveira, Guilherme

    2018-01-16

    Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer's, Parkinson's, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53-MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP

  11. Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA.

    Science.gov (United States)

    Remon, J; Caramella, C; Jovelet, C; Lacroix, L; Lawson, A; Smalley, S; Howarth, K; Gale, D; Green, E; Plagnol, V; Rosenfeld, N; Planchard, D; Bluthgen, M V; Gazzah, A; Pannet, C; Nicotra, C; Auclin, E; Soria, J C; Besse, B

    2017-04-01

    Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by the T790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib when T790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advanced EGFR-mutant NSCLC patients. ctDNA T790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48 EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. Progressing T790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage of T790M positive in ctDNA. The ctDNA T790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4-NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue.

  12. A fasciclin-like arabinogalactan-protein (FLA mutant of Arabidopsis thaliana, fla1, shows defects in shoot regeneration.

    Directory of Open Access Journals (Sweden)

    Kim L Johnson

    Full Text Available BACKGROUND: The fasciclin-like arabinogalactan-proteins (FLAs are an enigmatic class of 21 members within the larger family of arabinogalactan-proteins (AGPs in Arabidopsis thaliana. Located at the cell surface, in the cell wall/plasma membrane, they are implicated in many developmental roles yet their function remains largely undefined. Fasciclin (FAS domains are putative cell-adhesion domains found in extracellular matrix proteins of organisms from all kingdoms, but the juxtaposition of FAS domains with highly glycosylated AGP domains is unique to plants. Recent studies have started to elucidate the role of FLAs in Arabidopsis development. FLAs containing a single FAS domain are important for the integrity and elasticity of the plant cell wall matrix (FLA11 and FLA12 and FLA3 is involved in microspore development. FLA4/SOS5 with two FAS domains and two AGP domains has a role in maintaining proper cell expansion under salt stressed conditions. The role of other FLAs remains to be uncovered. METHOD/PRINCIPAL FINDINGS: Here we describe the characterisation of a T-DNA insertion mutant in the FLA1 gene (At5g55730. Under standard growth conditions fla1-1 mutants have no obvious phenotype. Based on gene expression studies, a putative role for FLA1 in callus induction was investigated and revealed that fla1-1 has a reduced ability to regenerate shoots in an in vitro shoot-induction assay. Analysis of FLA1p:GUS reporter lines show that FLA1 is expressed in several tissues including stomata, trichomes, the vasculature of leaves, the primary root tip and in lateral roots near the junction of the primary root. CONCLUSION: The results of the developmental expression of FLA1 and characterisation of the fla1 mutant support a role for FLA1 in the early events of lateral root development and shoot development in tissue culture, prior to cell-type specification.

  13. A fasciclin-like arabinogalactan-protein (FLA) mutant of Arabidopsis thaliana, fla1, shows defects in shoot regeneration.

    Science.gov (United States)

    Johnson, Kim L; Kibble, Natalie A J; Bacic, Antony; Schultz, Carolyn J

    2011-01-01

    The fasciclin-like arabinogalactan-proteins (FLAs) are an enigmatic class of 21 members within the larger family of arabinogalactan-proteins (AGPs) in Arabidopsis thaliana. Located at the cell surface, in the cell wall/plasma membrane, they are implicated in many developmental roles yet their function remains largely undefined. Fasciclin (FAS) domains are putative cell-adhesion domains found in extracellular matrix proteins of organisms from all kingdoms, but the juxtaposition of FAS domains with highly glycosylated AGP domains is unique to plants. Recent studies have started to elucidate the role of FLAs in Arabidopsis development. FLAs containing a single FAS domain are important for the integrity and elasticity of the plant cell wall matrix (FLA11 and FLA12) and FLA3 is involved in microspore development. FLA4/SOS5 with two FAS domains and two AGP domains has a role in maintaining proper cell expansion under salt stressed conditions. The role of other FLAs remains to be uncovered. Here we describe the characterisation of a T-DNA insertion mutant in the FLA1 gene (At5g55730). Under standard growth conditions fla1-1 mutants have no obvious phenotype. Based on gene expression studies, a putative role for FLA1 in callus induction was investigated and revealed that fla1-1 has a reduced ability to regenerate shoots in an in vitro shoot-induction assay. Analysis of FLA1p:GUS reporter lines show that FLA1 is expressed in several tissues including stomata, trichomes, the vasculature of leaves, the primary root tip and in lateral roots near the junction of the primary root. The results of the developmental expression of FLA1 and characterisation of the fla1 mutant support a role for FLA1 in the early events of lateral root development and shoot development in tissue culture, prior to cell-type specification.

  14. Gγ1 + Gγ2 ≠ Gβ: Heterotrimeric G Protein Gγ-Deficient Mutants Do Not Recapitulate All Phenotypes of Gβ-Deficient Mutants1[C][W][OA

    Science.gov (United States)

    Trusov, Yuri; Zhang, Wei; Assmann, Sarah M.; Botella, José Ramón

    2008-01-01

    Heterotrimeric G proteins are signaling molecules ubiquitous among all eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains one Gα (GPA1), one Gβ (AGB1), and two Gγ subunit (AGG1 and AGG2) genes. The Gβ requirement of a functional Gγ subunit for active signaling predicts that a mutant lacking both AGG1 and AGG2 proteins should phenotypically resemble mutants lacking AGB1 in all respects. We previously reported that Gβ- and Gγ-deficient mutants coincide during plant pathogen interaction, lateral root development, gravitropic response, and some aspects of seed germination. Here, we report a number of phenotypic discrepancies between Gβ- and Gγ-deficient mutants, including the double mutant lacking both Gγ subunits. While Gβ-deficient mutants are hypersensitive to abscisic acid inhibition of seed germination and are hyposensitive to abscisic acid inhibition of stomatal opening and guard cell inward K+ currents, none of the available Gγ-deficient mutants shows any deviation from the wild type in these responses, nor do they show the hypocotyl elongation and hook development defects that are characteristic of Gβ-deficient mutants. In addition, striking discrepancies were observed in the aerial organs of Gβ- versus Gγ-deficient mutants. In fact, none of the distinctive traits observed in Gβ-deficient mutants (such as reduced size of cotyledons, leaves, flowers, and siliques) is present in any of the Gγ single and double mutants. Despite the considerable amount of phenotypic overlap between Gβ- and Gγ-deficient mutants, confirming the tight relationship between Gβ and Gγ subunits in plants, considering the significant differences reported here, we hypothesize the existence of new and as yet unknown elements in the heterotrimeric G protein signaling complex. PMID:18441222

  15. Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation.

    Science.gov (United States)

    Liu, Jia; Saito, Kan; Maruya, Yuriko; Nakamura, Takashi; Yamada, Aya; Fukumoto, Emiko; Ishikawa, Momoko; Iwamoto, Tsutomu; Miyazaki, Kanako; Yoshizaki, Keigo; Ge, Lihong; Fukumoto, Satoshi

    2016-03-31

    Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling.

  16. Inverse polymerase chain reaction for rapid gene isolation in Arabidopsis thaliana insertion mutants

    NARCIS (Netherlands)

    Vanderhaeghen, R.; Scheres, B.J.G.; Montagu, M. van; Lijsebetten, M. van

    1992-01-01

    Recently, many mutants have been isolated in the model plant Arabidopsis thaliana by the insertion of the Agrobacterium tumefaciens T-DNA into the plant genome. Instead of applying Southern analysis on these insertion mutants and to avoid the construction of mutant- derived genomic libraries,

  17. Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli

    DEFF Research Database (Denmark)

    Brown, S; Brickman, E R; Beckwith, J

    1981-01-01

    We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon inser...

  18. Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3

    NARCIS (Netherlands)

    van Vugt-Lussenburg, B.M.A.; Damsten, M.C.; Maasdijk, D.M.; Vermeulen, N.P.E.; Commandeur, J.N.M.

    2006-01-01

    Recently, we described a triple mutant of the bacterial cytochrome P450 BM3 as the first mutant with affinity for drug-like compounds. In this paper, we show that this mutant, but not wild-type BM3, is able to metabolise testosterone and several drug-like molecules such as amodiaquine,

  19. Identification of a novel Lymantria dispar nucleopolyhedrovirus mutant that exhibits abnormal polyhedron formation and virion occlusion

    Science.gov (United States)

    James M. Slavicek; Melissa J. Mercer; Dana Pohlman; Mary Ellen Kelly; David S. Bischoff

    1998-01-01

    In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. Ld

  20. Differential analysis in Proteome of Space Induced Rice and Soybean Mutants

    Science.gov (United States)

    Wang, W.; Lu, B.; Gu, D.; Han, S.; Gao, Y.; Sun, Y.

    To investigate the change trends of proteome induced in space environment we chose 3 Rice mutants 2 Soybean mutants and the seeds which were selected as high yields high tillering rice blast resistance soybean insect pest resistance and wider leaf shape individually after abroad Recoverable Satellite JB-1 for 15 days in 1996 and their corresponding controls Two-dimensional gel electrophoresis 2-D with Coomassie Brilliant Blue staining and PDQuest TM software analysis found that In 6 rice samples 329 pm 35 protein spots were detected in controls whereas 298 pm 37 protein spots detected in mutants representing a 9 decrease 69 pm 27 protein spots were lost in mutants while 37 pm 14 protein spots appeared additionally showing 11 protein spots were lost in mutants 58 protein spots were significantly regulated in mutants with 16 pm 7 up- and 42 pm 18 down-regulated which occupied 5 and 14 of the total average mutants spots separately In 3 soybean leaf samples 263 pm 12 protein spots were detected in controls whereas 255 pm 20 protein spots detected in mutants representing a 3 decrease 49 pm 10 protein spots were lost in mutants while 36 pm 16 protein spots appeared additionally showing 5 protein spots lost in mutants 51 protein spots were significantly regulated in mutants with 25 pm 7 up- and 26 pm 15 down-regulated which occupied 9 8 and 10 2 of the total average mutants spots separately In 3 soybean seed samples 208 pm 41 protein spots were

  1. Isolation, characterization, and expression analyses of tryptophan aminotransferase genes in a maize dek18 mutant

    Science.gov (United States)

    The dek18 mutant of maize has decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology in maize seed development. Seeds of the dek18 mutants are smaller compared to wild type seeds and the vegetative d...

  2. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Bengtsson, Camilla F.; Olsen, Maja E.; Brandt, Luise Ørsted

    2011-01-01

    Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient...... genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little...... systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear...

  3. Grain product of 34 soya mutant lines;Rendimiento de grano de 34 lineas mutantes de soya

    Energy Technology Data Exchange (ETDEWEB)

    Salmeron E, J.; Mastache L, A. A.; Valencia E, F.; Diaz V, G. E. [Colegio Superior Agropecuario del Estado de Guerrero, Vicente Guerrero No. 81, Col. Centro, 40000 Iguala, Guerrero (Mexico); Cervantes S, T. [Instituto de Recursos Geneticos y Productividad, Colegio de Posgraduados, Carretera Mexico-Texcoco Km. 36.5, Montecillo, 56230 Texcoco, Estado de Mexico (Mexico); De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.; Gatica T, M. A. [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2009-07-01

    This work was development with the objective of obtaining information of the agronomic behavior of 34 soya mutant lines (R{sub 4}M{sub 18}) for human consumption and this way to select the 2 better lines. The genetic materials were obtained starting from the variety ISAAEG-B M2 by means of the application of recurrent radiation with Co{sup 60} gammas, to a dose of 350 Gray for the first two generations and both later to 200 Gray and selection during 17 cycles, being obtained the 34 better lines mutants with agronomic characteristic wanted and good flavor. The obtained results were that the mutant lines L{sub 25} and L{sub 32} produced the major quantity in branches/plant number with 7.5 and 7.25, pods/plant number with 171.25 and 167, grains/plant number with 350.89 and 333.07 and grain product (ton/ha) to 15% of humidity 5.15 and 4.68 ton/ha, respectively. (Author)

  4. Tissue engineering in dentistry.

    Science.gov (United States)

    Abou Neel, Ensanya Ali; Chrzanowski, Wojciech; Salih, Vehid M; Kim, Hae-Won; Knowles, Jonathan C

    2014-08-01

    of this review is to inform practitioners with the most updated information on tissue engineering and its potential applications in dentistry. The authors used "PUBMED" to find relevant literature written in English and published from the beginning of tissue engineering until today. A combination of keywords was used as the search terms e.g., "tissue engineering", "approaches", "strategies" "dentistry", "dental stem cells", "dentino-pulp complex", "guided tissue regeneration", "whole tooth", "TMJ", "condyle", "salivary glands", and "oral mucosa". Abstracts and full text articles were used to identify causes of craniofacial tissue loss, different approaches for craniofacial reconstructions, how the tissue engineering emerges, different strategies of tissue engineering, biomaterials employed for this purpose, the major attempts to engineer different dental structures, finally challenges and future of tissue engineering in dentistry. Only those articles that dealt with the tissue engineering in dentistry were selected. There have been a recent surge in guided tissue engineering methods to manage periodontal diseases beyond the traditional approaches. However, the predictable reconstruction of the innate organisation and function of whole teeth as well as their periodontal structures remains challenging. Despite some limited progress and minor successes, there remain distinct and important challenges in the development of reproducible and clinically safe approaches for oral tissue repair and regeneration. Clearly, there is a convincing body of evidence which confirms the need for this type of treatment, and public health data worldwide indicates a more than adequate patient resource. The future of these therapies involving more biological approaches and the use of dental tissue stem cells is promising and advancing. Also there may be a significant interest of their application and wider potential to treat disorders beyond the craniofacial region. Considering the

  5. Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.

    Directory of Open Access Journals (Sweden)

    Victoria A Lawson

    Full Text Available BACKGROUND: The accumulation of protease resistant conformers of the prion protein (PrP(res is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. METHODOLOGY/PRINCIPAL FINDING: In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP(res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS from the PrP(C substrate was found to specifically prevent PrP(res formation seeded by mouse derived PrP(Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP(res formation, while having no effect on PrP(res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. CONCLUSIONS/SIGNIFICANCE: Cofactor requirements for PrP(res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

  6. Characterisation of prostate cancer lesions in heterozygous Men1 mutant mice

    Directory of Open Access Journals (Sweden)

    Tong Wei-Ming

    2010-07-01

    Full Text Available Abstract Background Mutations of the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1 syndrome. Our group and others have shown that Men1 disruption in mice recapitulates MEN1 pathology. Intriguingly, rare lesions in hormone-dependent tissues, such as prostate and mammary glands, were also observed in the Men1 mutant mice. Methods To study the occurrence of prostate lesions, we followed a male mouse cohort of 47 Men1+/- mice and 23 age-matched control littermates, starting at 18 months of age, and analysed the prostate glands from the cohort. Results Six Men1+/- mice (12.8% developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin encoded by the Men1 gene was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions. Using immunostaining for the androgen receptor and p63, a basal epithelial cell marker, we demonstrated that the menin-negative prostate cancer cells did not display p63 expression and that the androgen receptor was expressed but more heterogeneous in these lesions. Furthermore, our data showed that the expression of the cyclin-dependent kinase inhibitor CDKN1B (p27, a Men1 target gene known to be inactivated during prostate cell tumorigenesis, was notably decreased in the prostate cancers that developed in the mutant mice. Conclusion Our work suggests the possible involvement of Men1 inactivation in the tumorigenesis of the prostate gland.

  7. The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant.

    Science.gov (United States)

    Tiruneh, Bayu Sisay; Kim, Byung-Hoon; Gallie, Daniel R; Roy, Bijoyita; von Arnim, Albrecht G

    2013-12-30

    Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1. The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs. These data represent the first genome

  8. New mutants and their chromosome assignment in the fly Megaselia scalaris.

    Science.gov (United States)

    Traut, W; Traut, G; Mertl, H G; Egelhaaf, A

    1994-01-01

    We describe six new mutants, spontaneous or EMS-induced, and a previously isolated radiation-induced mutant of Megaselia scalaris Loew (Diptera, Phoridae). Five are eye-color mutants, one affects the segmentation of the abdomen, and one disturbs the regular form and arrangement of ommatidia in the compound eye. A complementation test exposed two pairs of alleles among the five eye-color mutants, leaving three different loci that affect eye pigmentation. Two of them influence the amount of xanthommatin synthesized, and one blocks the ommochrome pathway at the kynurenine step. We established chromosome assignment of the mutants by crossbreeding them with Y chromosome-autosome translocation strains.

  9. Xanthine Dehydrogenase (XDH) cross-reacting material in mutants of Drosophila melanogaster deficient in XDH activity.

    Science.gov (United States)

    Browder, L W; Tucker, L; Wilkes, J

    1982-02-01

    Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin and cin mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.

  10. Identification of An Arsenic Tolerant Double Mutant With a Thiol-Mediated Component And Increased Arsenic Tolerance in PhyA Mutants

    Energy Technology Data Exchange (ETDEWEB)

    Sung, D.Y.; Lee, D.; Harris, H.; Raab, A.; Feldmann, J.; Meharg, A.; Kumabe, B.; Komives, E.A.; Schroeder, J.I.; /SLAC, SSRL /Sydney U. /Aberdeen U. /UC, San Diego

    2007-04-06

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

  11. Adipose tissue macrophages

    NARCIS (Netherlands)

    Boutens, Lily; Stienstra, Rinke

    2016-01-01

    Inflammation originating from the adipose tissue is considered to be one of the main driving forces for the development of insulin resistance and type 2 diabetes in obese individuals. Although a plethora of different immune cells shapes adipose tissue inflammation, this review is specifically

  12. Undifferentiated Connective Tissue Disease

    Science.gov (United States)

    ... Examples of connective tissue diseases include lupus , scleroderma , rheumatoid arthritis , Sjögren's syndrome , myositis and vasculitis . There are many people who have features of connective tissue disease, however, they do not fulfill the diagnostic criteria established for any one disease. In such ...

  13. Engineering Vascularized Adipose Tissue

    NARCIS (Netherlands)

    F. Verseijden (Femke)

    2011-01-01

    textabstractA large portion of the plastic and reconstructive surgical procedures performed each year is aimed at repairing soft tissue defects, which result for example from traumatic injury or tumor resections. Large soft tissue defects, lead to a change in function and ‘normal’ body contour,

  14. Tissue engineered aortic valve

    OpenAIRE

    Dohmen, P M

    2012-01-01

    Several prostheses are available to replace degenerative diseased aortic valves with unique advantages and disadvantages. Bioprotheses show excellent hemodynamic behavior and low risk of thromboembolic complications, but are limited by tissue deterioration. Mechanical heart valves have extended durability, but permanent anticoagulation is mandatory. Tissue engineering created a new generation heart valve, which overcome limitations of biological and mechanical heart valves due to remodelling,...

  15. Resveratrol Antagonizes Antimicrobial Lethality and Stimulates Recovery of Bacterial Mutants.

    Directory of Open Access Journals (Sweden)

    Yuanli Liu

    Full Text Available Reactive oxygen species (ROS; superoxide, peroxide, and hydroxyl radical are thought to contribute to the rapid bactericidal activity of diverse antimicrobial agents. The possibility has been raised that consumption of antioxidants in food may interfere with the lethal action of antimicrobials. Whether nutritional supplements containing antioxidant activity are also likely to interfere with antimicrobial lethality is unknown. To examine this possibility, resveratrol, a popular antioxidant dietary supplement, was added to cultures of Escherichia coli and Staphylococcus aureus that were then treated with antimicrobial and assayed for bacterial survival and the recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Resveratrol, at concentrations likely to be present during human consumption, caused a 2- to 3-fold reduction in killing during a 2-hr treatment with moxifloxacin or kanamycin. At higher, but still subinhibitory concentrations, resveratrol reduced antimicrobial lethality by more than 3 orders of magnitude. Resveratrol also reduced the increase in reactive oxygen species (ROS characteristic of treatment with quinolone (oxolinic acid. These data support the general idea that the lethal activity of some antimicrobials involves ROS. Surprisingly, subinhibitory concentrations of resveratrol promoted (2- to 6-fold the recovery of rifampicin-resistant mutants arising from the action of ciprofloxacin, kanamycin, or daptomycin. This result is consistent with resveratrol reducing ROS to sublethal levels that are still mutagenic, while the absence of resveratrol allows ROS levels to high enough to kill mutagenized cells. Suppression of antimicrobial lethality and promotion of mutant recovery by resveratrol suggests that the antioxidant may contribute to the emergence of resistance to several antimicrobials, especially if new derivatives and/or formulations of resveratrol markedly increase bioavailability.

  16. Analysis of Escherichia coli mutants with a linear respiratory chain.

    Directory of Open Access Journals (Sweden)

    Sonja Steinsiek

    Full Text Available The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important.

  17. The antiandrogenic effect of finasteride against a mutant androgen receptor

    Science.gov (United States)

    Chhipa, Rishi Raj; Zhang, Haitao; Ip, Clement

    2011-01-01

    Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild-type AR. When PC-3 cells, which are AR-null, were transfected with either the wild-type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy. PMID:21386657

  18. A sialidase mutant displaying trans-sialidase activity.

    Science.gov (United States)

    Paris, Gastón; Ratier, Laura; Amaya, María Fernanda; Nguyen, Tong; Alzari, Pedro M; Frasch, Alberto Carlos C

    2005-01-28

    Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to beta-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T.rangeli sialidase (TrSA) and T.cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studying the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA(5mut)) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA(5mut) revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.

  19. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Rodermel, Steven

    2015-11-16

    The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

  20. Mutant prevention concentrations of daptomycin for Enterococcus faecium clinical isolates.

    Science.gov (United States)

    Sinel, Clara; Jaussaud, Clara; Auzou, Michel; Giard, Jean-Christophe; Cattoir, Vincent

    2016-10-01

    Owing to the emergence of vancomycin-resistant Enterococcus faecium, treatment of enterococcal infections has become challenging. Although spontaneous in vitro resistance frequencies are low, the emergence of resistance is increasingly reported during daptomycin therapy. The mutant selection window (MSW), comprised between the minimum inhibitory concentration (MIC) and the mutant prevention concentration (MPC), corresponds to the concentration range within which resistant mutants may be selected. Since no data are available for enterococci, the aim of this study was to determine MPCs and MSWs for 12 representative E. faecium clinical isolates. MICs and MPCs were determined by broth microdilution and agar dilution methods, respectively. A basic MSW-derived pharmacodynamic analysis was also performed using mean maximum plasma concentration (Cmax) values obtained with dosages from 4 to 12 mg/kg. MICs and MPCs of daptomycin ranged from 0.5 to 4 mg/L and from 2 to 32 mg/L, respectively, with no correlation between them. The wideness of MSWs ranged from 2× to 32× MIC. Mean plasma Cmax values of daptomycin were calculated from 55 to 174.5 mg/L when using a dosage from 4 to 12 mg/kg. All Cmax values were above the MPCs whatever the dosage. Taking into account the protein binding of daptomycin (ca. 90%), the unbound fraction Cmax was just within the MSW in 67-92% of strains at recommended dosages (4-6 mg/kg) and was above the MPC for the majority of strains only with the highest dosage (12 mg/kg). This study shows that free daptomycin Cmax values usually fell into MSWs when using lower dosages (<10 mg/kg). Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  1. clustering common bean mutants based on heterotic groupings ...

    African Journals Online (AJOL)

    ACSS

    diversité génétique entre les haricots mutants, ces groupes (A, B et C) peuvent être considérés comme des groupements hétérotiques. Selon le trait phénotypique considéré, le croisement de deux des génotypes appartenant à des groupes différents peut générer de la vigueur hybride. Par ailleurs, pour créer une variabilité ...

  2. Using PATIMDB to create bacterial transposon insertion mutant libraries.

    Science.gov (United States)

    Urbach, Jonathan M; Wei, Tao; Liberati, Nicole; Grenfell-Lee, Daniel; Villanueva, Jacinto; Wu, Gang; Ausubel, Frederick M

    2009-04-01

    PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software.

  3. Environmental features determining successful rearing in the mutant mouse staggerer.

    Science.gov (United States)

    Guastavino, J M

    1984-02-01

    The mutant mouse staggerer is unable to rear her pups to weaning unless special precautions are taken. The following environmental conditions were found to contribute to compensate for the maternal behavioral deficits of the staggerer: (1) the foster pups used in a constraining box to stimulate the lactating staggerer mother are 4 days old. (2) The mother is enforced to stay in close physical contact with these pups for at least 12 hours immediately after delivery. (3) The staggerer pups are transferred to a normal lactating mother to suckle her for the first 12 hours of life.

  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...

  5. Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants

    Energy Technology Data Exchange (ETDEWEB)

    Phanchaisri, Boonrak [Science and Technology Research Institute, Chiang Mai University, Chiang Mai 50200 (Thailand); Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Samsang, Nuananong [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, Liang Deng; Singkarat, Somsorn [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Anuntalabhochai, Somboon, E-mail: soanu.1@gmail.com [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2012-06-01

    The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50-60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively.

  6. Disease causing mutants of TDP-43 nucleic acid binding domains are resistant to aggregation and have increased stability and half-life.

    Science.gov (United States)

    Austin, James A; Wright, Gareth S A; Watanabe, Seiji; Grossmann, J Günter; Antonyuk, Svetlana V; Yamanaka, Koji; Hasnain, S Samar

    2014-03-18

    Over the last two decades many secrets of the age-related human neural proteinopathies have been revealed. A common feature of these diseases is abnormal, and possibly pathogenic, aggregation of specific proteins in the effected tissue often resulting from inherent or decreased structural stability. An archetype example of this is superoxide dismutase-1, the first genetic factor to be linked with amyotrophic lateral sclerosis (ALS). Mutant or posttranslationally modified TAR DNA binding protein-32 (TDP-43) is also strongly associated with ALS and an increasingly large number of other neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). Cytoplasmic mislocalization and elevated half-life is a characteristic of mutant TDP-43. Furthermore, patient age at the onset of disease symptoms shows a good inverse correlation with mutant TDP-43 half-life. Here we show that ALS and FTLD-associated TDP-43 mutations in the central nucleic acid binding domains lead to elevated half-life and this is commensurate with increased thermal stability and inhibition of aggregation. It is achieved without impact on secondary, tertiary, or quaternary structure. We propose that tighter structural cohesion contributes to reduced protein turnover, increasingly abnormal proteostasis and, ultimately, faster onset of disease symptoms. These results contrast our perception of neurodegenerative diseases as misfolded proteinopathies and delineate a novel path from the molecular characteristics of mutant TDP-43 to aberrant cellular effects and patient phenotype.

  7. Sorghum bmr6 mutant analysis demonstrates that a shared MYB1 transcription factor binding site in the promoter links the expression of genes in related pathways.

    Science.gov (United States)

    Li, Jieqin; Wang, Lihua; Zhan, Qiuwen; Liu, Yanlong; Fu, Bisheng; Wang, Chunming

    2013-11-01

    Sorghum is not only an important cereal crop but also a biofuel crop. The sorghum brown midrib mutant 6 (bmr6) has a reduced lignin content in the cell walls and vascular tissues, which could potentially be advantageous for cellulosic biofuel production. Meanwhile, both dry matter yield and plant height were decreased in the bmr6 mutant. To identify genes affected in the mutant, differential gene expression analysis was performed for bmr6 and the wild type. As a result, a total of 1,052 differentially expressed genes were detected between the two samples, of which 166 genes were downregulated and 886 genes were upregulated. Five hundred seventy-nine of the 1,052 differentially expressed genes could be assigned to 154 documented pathways. These pathways mainly included primary and secondary metabolism. Therefore, mutation of the bmr6 gene, which impaired the biosynthesis of lignin, ultimately affected the expression of these genes associated with the growth and development of sorghum. Except for the bmr6 gene, 11 key enzyme genes of monolignols biosynthesis were upregulated. Promoter analysis identified that these genes have common MYB sites. It revealed that a feedback mechanism existed in the pathway and a MYB1 transcription factor (Sb02g031190) could associate with the upregulation of these genes in sorghum. In this study, we investigated gene expressions at a global level in sorghum bmr6 mutant and provided valuable insights into the mechanisms of lignin biosynthesis.

  8. Impaired colonization and infection of tomato roots by the Deltafrp1 mutant of Fusarium oxysporum correlates with reduced CWDE gene expression.

    Science.gov (United States)

    Jonkers, Wilfried; Rodrigues, Christopher D Andrade; Rep, Martijn

    2009-05-01

    The vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici efficiently invades roots and colonizes vascular tissues of its host tomato. For these processes, the F-box protein Frp1 is required. The Fusarium oxysporum Deltafrp1 mutant was characterized in detail to uncover the cause of its colonization defect. Using growth assays, we could attribute poor root colonization to reduced assimilation of organic acids, amino acids (except proline), or polysaccharides, singly or in combination. External root colonization by the Deltafrp1 mutant is restored by the addition of 0.1% glucose or proline but infection still does not occur. This is due to the inability of the Deltafrp1 mutant to penetrate the roots, as demonstrated by the lack of expression of SIX1 in the Deltafrp1 strain, which is a gene exclusively expressed inside roots, and loss of cell wall-degrading enzyme (CWDE) gene expression. Many of the metabolic defects of the Deltafrp1 strain can be attributed to reduced expression of the ICL1 (isocitrate lyase) gene. Strikingly, an Deltaicl1 mutant is still fully pathogenic and capable of external root colonization. We conclude that the inability of the Deltafrp1 strain to colonize and invade roots is not primarily due to metabolic defects but can be attributed to reduced expression of several CWDE genes.

  9. Multi-scale biomechanical remodeling in aging and genetic mutant murine mitral valve leaflets: insights into Marfan syndrome.

    Science.gov (United States)

    Gould, Russell A; Sinha, Ravi; Aziz, Hamza; Rouf, Rosanne; Dietz, Harry C; Judge, Daniel P; Butcher, Jonathan

    2012-01-01

    Mitral valve degeneration is a key component of the pathophysiology of Marfan syndrome. The biomechanical consequences of aging and genetic mutation in mitral valves are poorly understood because of limited tools to study this in mouse models. Our aim was to determine the global biomechanical and local cell-matrix deformation relationships in the aging and Marfan related Fbn1 mutated murine mitral valve. To conduct this investigation, a novel stretching apparatus and gripping method was implemented to directly quantify both global tissue biomechanics and local cellular deformation and matrix fiber realignment in murine mitral valves. Excised mitral valve leaflets from wild-type and Fbn1 mutant mice from 2 weeks to 10 months in age were tested in circumferential orientation under continuous laser optical imaging. Mouse mitral valves stiffen with age, correlating with increases in collagen fraction and matrix fiber alignment. Fbn1 mutation resulted in significantly more compliant valves (modulus 1.34 ± 0.12 vs. 2.51 ± 0.31 MPa, respectively, Paging. In comparison, Fbn1 mutated valves have decoupled cellular deformation and fiber alignment with tissue stretch. Taken together, quantitative understanding of multi-scale murine planar tissue biomechanics is essential for establishing consequences of aging and genetic mutations. Decoupling of local cell-matrix deformation kinematics with global tissue stretch may be an important mechanism of normal and pathological biomechanical remodeling in valves.

  10. miRNA-197 and miRNA-184 are associated with brain metastasis in EGFR-mutant lung cancers.

    Science.gov (United States)

    Remon, J; Alvarez-Berdugo, D; Majem, M; Moran, T; Reguart, N; Lianes, P

    2016-02-01

    The prognostic value of EGFR mutation in lung cancer patients with brain metastases is uncertain and therapeutic efficacy with EGFR TKI is limited. Looking for biomarkers closely related with early tumor changes and brain metastases in non-small cell lung cancer is warranted. MicroRNAs (miRNAs) are frequently deregulated in lung cancer. The objective of this study was to investigate whether some miRNAs are related with brain metastasis risk in EGFR-mutant non-small cell lung cancer patients. miRNA quantification was retrospectively performed in formalin-fixed, extracranial paraffin-embedded adenocarcinoma tumor tissue available from 17 human samples of advanced non-small cell lung cancer patients. Samples were classified as brain metastasis group (5 EGFR-mutant patients with initial BM, EGFRm-BM+; and 6 EGFR wild-type patients with initial BM) and the control group (6 EGFR-mutant NSCLC patients without BM). The RNA obtained was preamplified and retro-transcribed, and the miRNA was quantified with the TaqMan OpenArray Human MiRNA Panel in the QuantStudio™ 12 K Flex Real-Time PCR system. miRNA-197 and miRNA-184 showed a significant higher expression in EGFRm-BM+ group than in the control group (p = 0.017 and p = 0.01, for miRNA-197 and miRNA-184, respectively), with a trend toward overexpression in BM group compared with the control group (p = 0.08 and p = 0.065, for miRNA-197 and miRNA-184, respectively), without differences in expression in BM group according to EGFR mutational status (EGFR wild type vs. EGFR-mutant: p = 0.175 and p = 0.117, for miRNA-197, miRNA-184 respectively). miRNA-197 and miRNA-184 are overexpressed in EGFR-mutant patients with BM and they might be a new biomarker for stratifying the risk of BM in this subpopulation.

  11. Características fisiológicas de microtomateiros fitocromo-mutantes Physiological characteristics of micro-tomato (Lycopersicon esculentum P. Miller) phytochrome-mutants

    National Research Council Canada - National Science Library

    Hyrandir Cabral de Melo; Evaristo Mauro de Castro; Ângela Maria Soares; Cynthia de Oliveira; Sílvio Júnio Ramos

    2009-01-01

    ... (Lycopersicon esculentum P. Miller cv. Micro-Tom) fitocromo-mutantes. A cultivar Micro-Tom e os mutantes aurea (deficiente na biossíntese do cromóforo dos fitocromos), atroviolacea (atv) e high pigment1 (hp1...

  12. 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 ...

  13. DENTAL PULP TISSUE ENGINEERING

    Science.gov (United States)

    Demarco, FF; Conde, MCM; Cavalcanti, B; Casagrande, L; Sakai, V; Nör, JE

    2013-01-01

    Dental pulp is a highly specialized mesenchymal tissue, which have a restrict regeneration capacity due to anatomical arrangement and post-mitotic nature of odontoblastic cells. Entire pulp amputation followed by pulp-space disinfection and filling with an artificial material cause loss of a significant amount of dentin leaving as life-lasting sequelae a non-vital and weakened tooth. However, regenerative endodontics is an emerging field of modern tissue engineering that demonstrated promising results using stem cells associated with scaffolds and responsive molecules. Thereby, this article will review the most recent endeavors to regenerate pulp tissue based on tissue engineering principles and providing insightful information to readers about the different aspects enrolled in tissue engineering. Here, we speculate that the search for the ideal combination of cells, scaffolds, and morphogenic factors for dental pulp tissue engineering may be extended over future years and result in significant advances in other areas of dental and craniofacial research. The finds collected in our review showed that we are now at a stage in which engineering a complex tissue, such as the dental pulp, is no longer an unachievable and the next decade will certainly be an exciting time for dental and craniofacial research. PMID:21519641

  14. Fitness of Salmonella mutants resistant to antimicrobial peptides.

    Science.gov (United States)

    Lofton, Hava; Anwar, Naeem; Rhen, Mikael; Andersson, Dan I

    2015-02-01

    To examine the effects of mutations in the waaY, phoP and pmrB genes, which confer resistance to antimicrobial peptides (AMPs), on fitness of Salmonella Typhimurium. Survival during low pH, oxidative stress, stationary-phase incubation, exposure to serum and bile and growth in mice and laboratory media were determined by time-kills, disc inhibition assays, competition experiments and optical density measurements. Individual mutations in the waaY gene (involved in LPS core biosynthesis) and in the phoP and pmrB genes (part of two different two-component regulatory systems, phoPQ and pmrAB) conferred no or minor effects on bacterial survival during stressful in vitro conditions or in mice. In contrast, a waaY-phoP-pmrB triple mutant was compromised under most assay conditions. Results from this study show that AMP resistance can be cost-free, as assessed by several assays that attempt to mimic the conditions a bacterium might encounter within a host. Our findings imply that future therapeutic use of AMPs could select for fit mutants with cross-resistance to human defence peptides and that potential resistance development in response to therapeutic use of AMPs needs to be carefully monitored. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes.

    Science.gov (United States)

    Lyozin, George T; Kosaka, Yasuhiro; Bhattacharje, Gourab; Yost, H Joseph; Brunelli, Luca

    2017-04-03

    Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i.e., selectable markers, this approach is prone to false positives and is labor-intensive because it requires the subsequent removal of the selectable marker. We created Founder Principle-driven Enrichment (FPE), a method based on the population genetics "founder principle," to directly isolate rare mutant BACs, without any selectable marker, from liquid cultures via the polymerase chain reaction (PCR). Here, we provide a detailed description of FPE, including protocols for BAC recombineering and PCR screening. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  16. Nonselective enrichment for yeast adenine mutants by flow cytometry

    Science.gov (United States)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  17. Treatment of MDR1 Mutant Dogs with Macrocyclic Lactones

    Science.gov (United States)

    Geyer, Joachim; Janko, Christina

    2012-01-01

    P-glycoprotein, encoded by the multidrug resistance gene MDR1, is an ATP-driven drug efflux pump which is highly expressed at the blood-brain barrier of vertebrates. Drug efflux of macrocyclic lactones by P-glycoprotein is highly relevant for the therapeutic safety of macrocyclic lactones, as thereby GABA-gated chloride channels, which are confined to the central nervous system in vertebrates, are protected from high drug concentrations that otherwise would induce neurological toxicity. A 4-bp deletion mutation exists in the MDR1 gene of many dog breeds such as the Collie and the Australian Shepherd, which results in the expression of a non-functional P-glycoprotein and is associated with multiple drug sensitivity. Accordingly, dogs with homozygous MDR1 mutation are in general prone to neurotoxicity by macrocyclic lactones due to their increased brain penetration. Nevertheless, treatment of these dogs with macrocyclic lactones does not inevitably result in neurological symptoms, since, the safety of treatment highly depends on the treatment indication, dosage, route of application, and the individual compound used as outlined in this review. Whereas all available macrocyclic lactones can safely be administered to MDR1 mutant dogs at doses usually used for heartworm prevention, these dogs will experience neurological toxicity following a high dose regimen which is common for mange treatment in dogs. Here, we review and discuss the neurotoxicological potential of different macrocyclic lactones as well as their treatment options in MDR1 mutant dogs. PMID:22039792

  18. Neonatal vestibular stimulation and mating in cerebellar mutants.

    Science.gov (United States)

    Guastavino, J M; Larsson, K; Allain, C; Jaisson, P

    1993-05-01

    Two cerebellar mutants, staggerer and reeler, and their congenic nonmutants were used in this experiment. Experimental animals were subjected to intense rotational stimulation on a tilted plane during the first 3 weeks of life, while controls were left nonstimulated. The capacity for mating, as evidenced by vaginal plugs or the occurrence of pregnancy, was assayed during two periods: between 36 and 89 days of age (Experiment A) and between 90 and 120 days of age (Experiment B). During Experiment A the mutants as well as the normals were caged inter se with partners of the opposite sex. During Experiment B the animals were caged with intact, sexually experienced partners. The animals were examined daily for evidence of mating. During Experiment A, only 3 of the 89 couples participating in this study showed evidence of mating. During Experiment B, the number of males of both strains which had mated increased significantly. The staggerer females showed a relatively high level of mating activity, whether stimulated or not. The reeler females, in contrast, rarely mated, although early stimulation significantly increased the level of sexual efficiency. The majority of the normal males and females mated, whether stimulated or not. It was concluded that massive motor-sensory stimulation in infancy, improving gait and body balance in staggerer and reeler mice, may also improve mating efficiency.

  19. Family feud in chemosensitvity: p73 and mutant p53.

    Science.gov (United States)

    Irwin, Meredith S

    2004-03-01

    The importance of p53 in chemotherapy-induced apoptosis of cancer cells is well established. p53 plays a critical role in the cellular response to DNA damage by regulating genes involved in cell cycle progression, apoptosis, and genomic stability. As a result, p53 tumor status is a critical determinant of both responses to anti-cancer treatment and clinical prognosis. Interestingly, tumors expressing certain mutant forms of p53 ("gain of function") are particularly resistant to chemotherapy, even when compared to cells that lack any detectable p53. Until recently, the explanation for this enhanced chemoresistance was not clear. Recent studies have shown that the p53 homologues, p73 and p63, are also activated by chemotherapies, leading to tumor cell death. Now the discovery that mutant p53 interacts with p73, and that regulation of this interaction by a p53 polymorphism can modulate chemosensitvity provide a new model for how p53-family interactions can influence the response of tumors to anti-cancer therapies. Since p53 mutations are found in more than 50% of human tumors, strategies aimed at manipulating these interactions may prove useful in enhancing the chemotherapy response, and perhaps, overcoming chemoresistance.

  20. Structural dataset for the PPARγ V290M mutant

    Directory of Open Access Journals (Sweden)

    Ana C. Puhl

    2016-06-01

    Full Text Available Loss-of-function mutation V290M in the ligand-binding domain of the peroxisome proliferator activated receptor γ (PPARγ is associated with a ligand resistance syndrome (PLRS, characterized by partial lipodystrophy and severe insulin resistance. In this data article we discuss an X-ray diffraction dataset that yielded the structure of PPARγ LBD V290M mutant refined at 2.3 Å resolution, that allowed building of 3D model of the receptor mutant with high confidence and revealed continuous well-defined electron density for the partial agonist diclofenac bound to hydrophobic pocket of the PPARγ. These structural data provide significant insights into molecular basis of PLRS caused by V290M mutation and are correlated with the receptor disability of rosiglitazone binding and increased affinity for corepressors. Furthermore, our structural evidence helps to explain clinical observations which point out to a failure to restore receptor function by the treatment with a full agonist of PPARγ, rosiglitazone.

  1. Identification and Characterization of Spontaneous Auxotrophic Mutants in Fusarium langsethiae

    Directory of Open Access Journals (Sweden)

    Olga Gavrilova

    2017-03-01

    Full Text Available Analysis of 49 strains of Fusarium langsethiae originating from northern Europe (Russia, Finland, Sweden, UK, Norway, and Latvia revealed the presence of spontaneous auxotrophic mutants that reflect natural intraspecific diversity. Our investigations detected that 49.0% of F. langsethiae strains were auxotrophic mutants for biotin, and 8.2% of the strains required thiamine as a growth factor. They failed to grow on vitamin-free media. For both prototrophic and auxotrophic strains, no growth defect was observed in rich organic media. Without essential vitamins, a significant reduction in the growth of the auxotrophic strains results in a decrease of the formation of T-2 toxin and diacetoxyscirpenol. In addition, all analysed F. langsethiae strains were distinguished into two subgroups based on PCR product sizes. According to our results, 26 and 23 strains of F. langsethiae belong to subgroups I and II respectively. We determined that the deletion in the intergenic spacer (IGS region of the rDNA of F. langsethiae belonging to subgroup II is linked with temperature sensitivity and causes a decrease in strain growth at 30 °C. Four thiamine auxotrophic strains were found in subgroup I, while 21 biotin auxotrophic strains were detected in subgroups II. To the best of our knowledge, the spontaneous mutations in F. langsethiae observed in the present work have not been previously reported.

  2. Radiometric prescreen for antitumor activity with Saccharomyces cerevisiae mutant strain.

    Science.gov (United States)

    Speedie, M K; Fique, D V; Blomster, R N

    1980-07-01

    After modification, a technique for radiometrically measuring bacterial growth has been applied to a mutant strain of Saccharomyces cerevisiae. The assay is based on inhibition of 14CO2 release from [14C]glucose, which provides an extremely sensitive measure of cellular respiratory activity and growth. The criterion for antitumor activity is the differential inhibition of wild-type and mutant (distorted cell membrane) strains of the yeast. The system was optimized for medium, time of incubation, temperature, and size of inoculum. Known antitumor agents, including bleomycin, actinomycin D, adriamycin, and ellipticine were tested in the system, and differential inhibition was observed. Vincristine showed no inhibitory effects at the concentrations tried. The sensitivity for 20% inhibition ranged from 0.8 micrograms of adriamycin per ml to 0.14 mg of ellipticine per ml. Antifungal agents such as amphotericin B exhibited no differential inhibition. Antibacterial agents were inactive. This method may provide a rapid, sensitive, in vitro quantitative assay for antitumor agents which could be applied to a variety of assay needs and which can be run with facilities and equipment available in most laboratories.

  3. Analysis of Stomatal Patterning in Selected Mutants of MAPK Pathways

    KAUST Repository

    Felemban, Abrar

    2016-05-01

    Stomata are cellular valves in plants that play an essential role in the regulation of gas exchange and are distributed in the epidermis of aerial organs. In Arabidopsis thaliana, stomatal production and development are coordinated by the mitogen-activated protein kinase (MAPK) signalling pathway, which modulates a variety of other processes, including cell proliferation, regulation of cytokinesis, programed cell death, and response to abiotic and biotic stress. The environment also plays a role in stomatal development, by influencing the frequency at which stomata develop in leaves. This thesis presents an analysis of stomatal development in Arabidopsis mutants in two MAPK pathways: MEKK1-MKK1/MKK2-MPK4, and MAP3K17/18-MKK3. Obtained results demonstrate the effect of stress conditions on stomatal development and specify the involvement of analysed MAPK in stomatal patterning. First, both analysed pathways modulate stomatal patterning in Arabidopsis cotyledons. Second, plant growth-promoting bacteria tested enhance stomatal density and affect guard cell morphology. Third, the sucrose or mannitol treatment increases defects in stomatal patterning. Finally, salt stress or high temperature can suppress stomatal defects in mutants of the MEKK1-MKK1/MKK2-MPK4 pathway.

  4. Computational identification of adaptive mutants using the VERT system

    Directory of Open Access Journals (Sweden)

    Winkler James

    2012-04-01

    Full Text Available Background Evolutionary dynamics of microbial organisms can now be visualized using the Visualizing Evolution in Real Time (VERT system, in which several isogenic strains expressing different fluorescent proteins compete during adaptive evolution and are tracked using fluorescent cell sorting to construct a population history over time. Mutations conferring enhanced growth rates can be detected by observing changes in the fluorescent population proportions. Results Using data obtained from several VERT experiments, we construct a hidden Markov-derived model to detect these adaptive events in VERT experiments without external intervention beyond initial training. Analysis of annotated data revealed that the model achieves consensus with human annotation for 85-93% of the data points when detecting adaptive events. A method to determine the optimal time point to isolate adaptive mutants is also introduced. Conclusions The developed model offers a new way to monitor adaptive evolution experiments without the need for external intervention, thereby simplifying adaptive evolution efforts relying on population tracking. Future efforts to construct a fully automated system to isolate adaptive mutants may find the algorithm a useful tool.

  5. Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase[C][W][OPEN

    Science.gov (United States)

    Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

    2014-01-01

    The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants. PMID:24357602

  6. Analyses of tomato fruit brightness mutants uncover both cutin-deficient and cutin-abundant mutants and a new hypomorphic allele of GDSL lipase.

    Science.gov (United States)

    Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

    2014-02-01

    The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

  7. Rapporteur report: other tissues.

    Science.gov (United States)

    Sienkiewicz, Z

    2003-01-01

    This report covers the session devoted to 'other tissues'. It considers the effects of internal electric fields such as those induced by exposure to weak, extremely low frequency (ELF) electromagnetic fields, on cardiac physiology, neuroendocrine (pineal) function and on the processes of tissue repair and embryonic development. Summaries are provided for each of the papers presented, and the major aspects of the plenary session are discussed. Overall, these tissues and processes were not considered to be sensitive to the direct effects of weak ELF fields, although indirect effects may occur via field induced changes to the central nervous system.

  8. Rapporteur report: Other tissues

    Energy Technology Data Exchange (ETDEWEB)

    Sienkiewicz, Z

    2003-07-01

    This report covers the session devoted to other tissues. It considers the effects of internal electric fields such as those induced by exposure to weak, extremely low frequency (ELF) electromagnetic fields, on cardiac physiology, neuroendocrine (pineal) function and on the processes of tissue repair and embryonic development. Summaries are provided for each of the papers presented, and the major aspects of the plenary session are discussed. Overall, these tissues and processes were not considered to be sensitive to the direct effects of weak ELF fields, although indirect effects may occur via field induced changes to the central nervous system. (author)

  9. Altered lignification in mur1-1 a mutant deficient in GDP-L-fucose synthesis with reduced RG-II cross linking.

    Directory of Open Access Journals (Sweden)

    Aline Voxeur

    Full Text Available In the plant cell wall, boron links two pectic domain rhamnogalacturonan II (RG-II chains together to form a dimer and thus contributes to the reinforcement of cell adhesion. We studied the mur1-1 mutant of Arabidopsis thaliana which has lost the ability to form GDP-fucose in the shoots and show that the extent of RG-II cross-linking is reduced in the lignified stem of this mutant. Surprisingly, MUR1 mutation induced an enrichment of resistant interunit bonds in lignin and triggered the overexpression of many genes involved in lignified tissue formation and in jasmonic acid signaling. The defect in GDP-fucose synthesis induced a loss of cell adhesion at the interface between stele and cortex, as well as between interfascicular fibers. This led to the formation of regenerative xylem, where tissue detachment occurred, and underlined a loss of resistance to mechanical forces. Similar observations were also made on bor1-3 mutant stems which are altered in boron xylem loading, leading us to suggest that diminished RG-II dimerization is responsible for regenerative xylem formation.

  10. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

    Directory of Open Access Journals (Sweden)

    Juliana Oliveira Lima

    Full Text Available Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120 h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

  11. Skeletal muscle tissue engineering

    National Research Council Canada - National Science Library

    Bach, A. D; Beier, J. P; Stern‐Staeter, J; Horch, R. E

    2004-01-01

    The reconstruction of skeletal muscle tissue either lost by traumatic injury or tumor ablation or functional damage due to myopathies is hampered by the lack of availability of functional substitution...

  12. Breast reconstruction - natural tissue

    Science.gov (United States)

    ... muscle flap; TRAM; Latissimus muscle flap with a breast implant; DIEP flap; DIEAP flap; Gluteal free flap; Transverse upper gracilis flap; TUG; Mastectomy - breast reconstruction with natural tissue; Breast cancer - breast reconstruction ...

  13. Synovial tissue research

    DEFF Research Database (Denmark)

    Orr, Carl; Sousa, Elsa; Boyle, David L

    2017-01-01

    The synovium is the major target tissue of inflammatory arthritides such as rheumatoid arthritis. The study of synovial tissue has advanced considerably throughout the past few decades from arthroplasty and blind needle biopsy to the use of arthroscopic and ultrasonographic technologies that enable...... easier visualization and improve the reliability of synovial biopsies. Rapid progress has been made in using synovial tissue to study disease pathogenesis, to stratify patients, to discover biomarkers and novel targets, and to validate therapies, and this progress has been facilitated by increasingly...... diverse and sophisticated analytical and technological approaches. In this Review, we describe these approaches, and summarize how their use in synovial tissue research has improved our understanding of rheumatoid arthritis and identified candidate biomarkers that could be used in disease diagnosis...

  14. Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system.

    Science.gov (United States)

    Arai, Sayuri; Hayashihara, Kayoko; Kanamoto, Yuki; Shimizu, Kazunori; Hirokawa, Yasutaka; Hanai, Taizo; Murakami, Akio; Honda, Hiroyuki

    2017-08-01

    Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2 , low productivity, and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported. In this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30 g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72 h incubation under light with or without 10 g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. Biotechnol. Bioeng. 2017;114: 1771-1778. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Tissue Culture in Microgravity

    Science.gov (United States)

    Pellis, Neal R.; Duray, Paul H.; Hatfill, Steven J.

    1997-01-01

    Attempts to simulate normal tissue micro-environments in vitro have been thwarted by the complexity and plasticity of the extracellular matrix, which is important in regulating cytoskeletal and nuclear matrix proteins. Gravity is one of the problems, tending to separate components that should be kept together. For space shuttle experiments, NASA engineers devised a double-walled rotating bioreactor, which is proving to be a useful tissue culture device on earth as well as in space.

  16. Bypassing damaged nervous tissue

    CERN Document Server

    Shneider, M N

    2016-01-01

    We show the principal ability of bypassing damaged demyelinated portions of nervous tissue, thereby restoring its normal function for the passage of action potentials. We carry out a theoretical analysis on the basis of the synchronization mechanism of action potential propagation along a bundle of neurons, proposed recently in [1]. And we discuss the feasibility of implement a bypass to restore damaged nervous tissue and creating an artificial neuron network.

  17. Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Anne Grünewald

    Full Text Available BACKGROUND: Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD. The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7, as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and

  18. Live Cell Imaging of Endogenous RNAs Using Pumilio Homology Domain Mutants: Principles and Applications.

    Science.gov (United States)

    Yoshimura, Hideaki

    2018-01-16

    Recently, dynamic changes in the location of RNA in space and time in living cells have become a target of interest in biology because of their essential roles in controlling physiological phenomena. To visualize RNA, methods for the fluorescent labeling of RNA in living cells have been developed. For RNA labeling, oligonucleotide-based RNA probes have mainly been used because of their high selectivity for target RNAs. By contrast, protein-based RNA probes have not been used widely because of their lack of design flexibility, although they have various potential advantages compared with nucleotide-based probes, such as controllability of intracellular localization, high detectability, and ease of introduction into cells and transgenic organisms in a cell type and tissue specific manner by genetic engineering techniques. This Perspective focuses on a possible approach to the development of protein-based RNA probes using Pumilio homology domain (PUM-HD) mutants. The PUM-HD is a domain of an RNA binding protein that allows custom-made modifications to recognize a given eight-base RNA sequence. PUM-HD-based RNA probes have been applied to visualize various RNAs in living cells. Here, the techniques and RNA imaging results obtained using the PUM-HD are introduced.

  19. Biochemical analysis of plant protection afforded by a nonpathogenic endophytic mutant of Colletotrichum magna

    Science.gov (United States)

    Redman, R.S.; Freeman, S.; Clifton, D.R.; Morrel, J.; Brown, G.; Rodriguez, R.J.

    1999-01-01

    A nonpathogenic mutant of Colletotrichum magna (path-1) was previously shown to protect watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) seedlings from anthracnose disease elicited by wild-type C. magna. Disease protection was observed in stems of path-1-colonized cucurbits but not in cotyledons, indicating that path-1 conferred tissue-specific and/or localized protection. Plant biochemical indicators of a localized and systemic (peroxidase, phenylalanine ammonia-lyase, lignin, and salicylic acid) 'plant-defense' response were investigated in anthracnose-resistant and -susceptible cultivars of cucurbit seedlings exposed to four treatments: (1) water (control), (2) path-1 conidia, (3) wild-type conidia, and (4) challenge conditions (inoculation into path-1 conidia for 48 h and then exposure to wild-type conidia). Collectively, these analyses indicated that disease protection in path-1 colonized plants was correlated with the ability of these plants to mount a defense response more rapidly and to equal or greater levels than plants exposed to wild-type C. magna alone. Watermelon plants colonized with path-1 were also protected against disease caused by Colletotrichum orbiculare and Fusarium oxysporum. A model based on the kinetics of plant-defense activation is presented to explain the mechanism of path-1-conferred disease protection.

  20. Biochemical analysis of plant protection afforded by a nonpathogenic endophytic mutant of Colletotrichum magna

    Energy Technology Data Exchange (ETDEWEB)

    Redman, R.S.; Rodriguez, R.J. (Geological Survey, Seattle, WA (United States) Univ. of Washington, Seattle, WA (United States). Dept. of Botany); Clifton, D.R.; Morrel, J.; Brown, G. (Geological Survey, Seattle, WA (United States)); Freeman, S. (Volcani Center, Bet Dagan (Israel). Dept. of Plant Pathology)

    1999-02-01

    A nonpathogenic mutant of Colletotrichum magna (path-1) was previously shown to protect watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) seedlings from anthracnose disease elicited by wild-type C. magna. Disease protection was observed in stems of path-1-colonized cucurbits but not in cotyledons, indicating that path-1 conferred tissue-specific and/or localized protection. Plant biochemical indicators of a localized and systemic (peroxidase, phenylalanine ammonia-lyase, lignin, and salicylic acid) plant-defense response were investigated in anthracnose-resistant and-susceptible cultivars of cucurbit seedlings exposed to four treatments: (1) water (control), (2) path-1 conidia, (3) wild-type conidia, and (4) challenge conditions (inoculation into path-1 conidia for 48 h and then exposure to wild-type conidia). Collectively, these analyses indicated that disease protection in path-1-colonized plants was correlated with the ability of these plants to mount a defense response more rapidly and to equal or greater levels than plants exposed to wild-type C. magna alone. Watermelon plants colonized with path-1 were also protected against disease caused by Colletotrichum orbiculare and Fusarium oxysporum. A model based on the kinetics of plant-defense activation is presented to explain the mechanism of path-1-conferred disease protection.

  1. Hexachlorophene Is a Potent KCNQ1/KCNE1 Potassium Channel Activator Which Rescues LQTs Mutants

    Science.gov (United States)

    Zheng, Yueming; Zhu, Xuejing; Zhou, Pingzheng; Lan, Xi; Xu, Haiyan; Li, Min; Gao, Zhaobing

    2012-01-01

    The voltage-gated KCNQ1 potassium channel is expressed in cardiac tissues, and coassembly of KCNQ1 with an auxiliary KCNE1 subunit mediates a slowly activating current that accelerates the repolarization of action potential in cardiomyocytes. Mutations of KCNQ1 genes that result in reduction or loss of channel activity cause prolongation of repolarization during action potential, thereby causing long QT syndrome (LQTs). Small molecule activators of KCNQ1/KCNE1 are useful both for understanding the mechanism of the complex activity and for developing therapeutics for LQTs. In this study we report that hexachlorophene (HCP), the active component of the topical anti-infective prescription drug pHisoHex, is a KCNQ1/KCNE1 activator. HCP potently increases the current amplitude of KCNQ1/KCNE1 expressed by stabilizing the channel in an open state with an EC50 of 4.61±1.29 μM. Further studies in cardiomyocytes showed that HCP significantly shortens the action potential duration at 1 μM. In addition, HCP is capable of rescuing the loss of function of the LQTs mutants caused by either impaired activation gating or phosphatidylinositol-4,5-bisphosphate (PIP2) binding affinity. Our results indicate HCP is a novel KCNQ1/KCNE1 activator and may be a useful tool compound for the development of LQTs therapeutics. PMID:23251633

  2. Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Hassan Abdel-Latif A. Mohamed

    2006-06-01

    Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

  3. Mutant pso8-1 of Saccharomyces cerevisiae, sensitive to photoactivated psoralens, UV radiation, and chemical mutagens, contains a rad6 missense mutant allele.

    Science.gov (United States)

    Rolla, H; Grey, M; Schmidt, C L; Niegemann, E; Brendel, M; Henriques, J A P

    2002-07-01

    A novel mutant isolate of Saccharomyces cerevisiae, sensitive to photoactivated mono- and bi-functional psoralens, to UV at 254 nm (UVC), and to nitrosoguanidine, was found to complement the photoactivated psoralen-sensitivity phenotype conferred by the pso1- pso7 mutations and was therefore named pso8-1. A constructed pso8-1 rad4-4 double mutant was super-sensitive to UVC, thus indicating a synergistic interaction of the two mutant alleles. Molecular cloning via complementation of the pso8 mutant's sensitivity phenotype and genetic studies revealed that pso8 is allelic to RAD6. While a pso8-1 mutant had low mutagen-induced mutability, homoallelic diploids showed nearly wild-type sporulation. Sequence analysis of the mutant allele showed pso8-1 to contain a novel, hitherto undescribed T-->C transition in nucleotide position 191, leading to a substitution by leucine of a highly conserved proline at position 64, Rad6-[P64L], which may have severe consequences for the tertiary structure (and hence binding to Rad18p) of the mutant protein.

  4. Integrative genome analysis of somatic p53 mutant osteosarcomas identifies Ets2-dependent regulation of small nucleolar RNAs by mutant p53 protein.

    Science.gov (United States)

    Pourebrahim, Rasoul; Zhang, Yun; Liu, Bin; Gao, Ruli; Xiong, Shunbin; Lin, Patrick P; McArthur, Mark J; Ostrowski, Michael C; Lozano, Guillermina

    2017-09-15

    TP53 is the most frequently mutated gene in human cancer. Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that contribute to metastasis, but the mechanisms mediating these functions remain poorly defined in vivo. To elucidate how mutant p53 GOF drives metastasis, we developed a traceable somatic osteosarcoma mouse model that is initiated with either a single p53 mutation (p53R172H) or p53 loss in osteoblasts. Our study confirmed that p53 mutant mice developed osteosarcomas with increased metastasis as compared with p53-null mice. Comprehensive transcriptome RNA sequencing (RNA-seq) analysis of 16 tumors identified a cluster of small nucleolar RNAs (snoRNAs) that are highly up-regulated in p53 mutant tumors. Regulatory element analysis of these deregulated snoRNA genes identified strong enrichment of a common Ets2 transcription factor-binding site. Homozygous deletion of Ets2 in p53 mutant mice resulted in strong down-regulation of snoRNAs and reversed the prometastatic phenotype of mutant p53 but had no effect on osteosarcoma development, which remained 100% penetrant. In summary, our studies identify Ets2 inhibition as a potential therapeutic vulnerability in p53 mutant osteosarcomas. © 2017 Pourebrahim et al.; Published by Cold Spring Harbor Laboratory Press.

  5. Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia).

    Science.gov (United States)

    Jain, D K; Bordeleau, L M

    1990-12-01

    Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif (r) showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.

  6. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

    Science.gov (United States)

    Howden, R; Goldsbrough, P B; Andersen, C R; Cobbett, C S

    1995-04-01

    An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants.

  7. Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus

    Energy Technology Data Exchange (ETDEWEB)

    Pierson, B.K.; Keith, L.M.; Leovy, J.G.

    1984-07-01

    Mutants deficient in the production of bateriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus, Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions. 11 references, 4 figures, 3 tables.

  8. Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, P. Manish [Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008 (India); Brannigan, James A., E-mail: jab@ysbl.york.ac.uk [York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW (United Kingdom); Prabhune, Asmita; Pundle, Archana [Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008 (India); Turkenburg, Johan P.; Dodson, G. Guy [York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW (United Kingdom); Suresh, C. G., E-mail: jab@ysbl.york.ac.uk [Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008 (India)

    2005-01-01

    The production, crystallization and characterization of three inactive mutants of penicillin V acylase from B. sphaericus in their respective precursor and processed forms are reported. The space groups are different for the native enzyme and the mutants. The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme.

  9. Novel polyketide metabolites from Streptomyces rimosus mutant strain R1059.

    Science.gov (United States)

    Deseo, Myrna A; Hunter, lain S; Waterman, Peter G

    2005-12-01

    Three novel polyketide metabolites were isolated from laboratory-scale fermentation of the Streptomyces rimosus mutant strain R1059. Structural elucidation of the compounds was based on NMR experiments. The compounds were characterized as naphthalene derivatives: (rel)-4beta,8-dihydroxy-3alpha-hydroxymethyl-4alpha-methyl-1,2,3,4-tetrahydronaphthalene1-one (1), 4xi8-dihydroxy-3-hydroxymethyl-4xi-methyl-1,4-dihydronaphthalene-1-one (2) and (rel)-4beta,8-dihydroxy-3alpha-O-[alpha-glucopyranosyl]hydroxymethyl-4alpha-methyl-1,2,3,4-tetrahydronaphthalene-1-one (3). The compounds isolated appear to be derived via a shorter polyketide backbone than oxytetracycline (4), the normal end-product made by the parent of this strain. Compound 3 was the glucoside of 1 and must be formed as a post-PKS reaction by the activation of a glycosyl transferase, which has not been reported in this species before.

  10. Induction of Male sterile mutants in vegetable crops

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Nobuhiko (Hokkaido National Agricultural Experiment Station, Sapporo (Japan))

    1982-03-01

    The cultivars of vegetable crops in Japan are almost all F/sub 1/ hybrid lines. These hybrid cultivars are superior in yield, quality and uniformity by heterosis, and play an important role in the protection of breeder's rights. Utilization of male sterile mutants has such advantages as the reduction of cost for F/sub 1/ production by saving labor, production of better seeds, that is, pollination without emasculation and avoidance of contamination caused by self pollination. Male sterility must be used for some species in which seed production is difficult because of tiny flowers and meager seed production by artificial crossing such as carrot and onion, and those in which pollination by bag or emasculation is expensive such as tomato, and sweet pepper. However, for vegetable crop breeeding, the induction and use of genetic male sterility are more difficult than for other crops, considering the economy and efficiency of research because the type of cultivars needed changes rapidly.

  11. Characterization Of Laccase T-DNA Mutants In Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Andersen, Jeppe R; Asp, Torben; Mansfield, Shawn

    Laccases (P-diphenol:O2 oxidoreductase; EC 1.10.3.2), also termed laccase-like multicopper oxidases, are blue copper-containing oxidases which comprise multigene families in plants. In the Arabidopsis thaliana genome, 17 laccase genes (LAC1 to LAC17) have been annotated. To identify laccases...... involved in cell wall biosynthesis in Arabidopsis primary stems we have developed homozygous T-DNA mutants for 14 individual laccases. Six laccases are highly expressed in the wild type primary stem, four of which (LAC2, LAC4, LAC12, and LAC17) show correlated gene expression with one to several genes (e...... different and distinct biochemical pathways and that laccases might be involved in polymerization of both polysaccharides and monolignols in the Arabidopsis cell wall....

  12. Longevity mutants do not establish any "new science" of ageing.

    Science.gov (United States)

    Holliday, Robin; Rattan, Suresh I S

    2010-08-01

    The biological reasons for ageing are now well known, so it is no longer an unsolved problem in biology. Furthermore, there is only one science of ageing, which is continually advancing. The significance and importance of the mutations that lengthen the lifespan of invertebrates can be assessed only in relationship to previous well-established studies of ageing. The mutant strains of model organisms that increase longevity have altered nutrient signalling pathways similar to the effects of dietary restriction, and so it is likely that there is a shift in the trade-off between reproduction and maintenance of the soma. To believe that the isolation and characterisation of a few invertebrate mutations (as well as those in yeast) will "galvanise" the field and provide new insights into human ageing is an extreme point of view which does not recognize the huge progress in ageing research that has been made in the last 50 years or so.

  13. Validating regulatory predictions from diverse bacteria with mutant fitness data.

    Directory of Open Access Journals (Sweden)

    Shiori Sagawa

    Full Text Available Although transcriptional regulation is fundamental to understanding bacterial physiology, the targets of most bacterial transcription factors are not known. Comparative genomics has been used to identify likely targets of some of these transcription factors, but these predictions typically lack experimental support. Here, we used mutant fitness data, which measures the importance of each gene for a bacterium's growth across many conditions, to test regulatory predictions from RegPrecise, a curated collection of comparative genomics predictions. Because characterized transcription factors often have correlated fitness with one of their targets (either positively or negatively, correlated fitness patterns provide support for the comparative genomics predictions. At a false discovery rate of 3%, we identified significant cofitness for at least one target of 158 TFs in 107 ortholog groups and from 24 bacteria. Thus, high-throughput genetics can be used to identify a high-confidence subset of the sequence-based regulatory predictions.

  14. E151 (sym15), a pleiotropic mutant of pea (Pisum sativum L.), displays low nodule number, enhanced mycorrhizae, delayed lateral root emergence, and high root cytokinin levels.

    Science.gov (United States)

    Jones, James M C; Clairmont, Lindsey; Macdonald, Emily S; Weiner, Catherine A; Emery, R J Neil; Guinel, Frédérique C

    2015-07-01

    In legumes, the formation of rhizobial and mycorrhizal root symbioses is a highly regulated process which requires close communication between plant and microorganism. Plant mutants that have difficulties establishing symbioses are valuable tools for unravelling the mechanisms by which these symbioses are formed and regulated. Here E151, a mutant of Pisum sativum cv. Sparkle, was examined to characterize its root growth and symbiotic defects. The symbioses in terms of colonization intensity, functionality of micro-symbionts, and organ dominance were compared between the mutant and wild type. The endogenous cytokinin (CK) and abscisic acid (ABA) levels and the effect of the exogenous application of these two hormones were determined. E151 was found to be a low and delayed nodulator, exhibiting defects in both the epidermal and cortical programmes though a few mature and functional nodules develop. Mycorrhizal colonization of E151 was intensified, although the fungal functionality was impaired. Furthermore, E151 displayed an altered lateral root (LR) phenotype compared with that of the wild type whereby LR emergence is initially delayed but eventually overcome. No differences in ABA levels were found between the mutant and the wild type, but non-inoculated E151 exhibited significantly high CK levels. It is hypothesized that CK plays an essential role in differentially mediating the entry of the two micro-symbionts into the cortex; whereas it would inhibit the entry of the rhizobia in that tissue, it would promote that of the fungus. E151 is a developmental mutant which may prove to be a useful tool in further understanding the role of hormones in the regulation of beneficial root symbioses. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  15. Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls

    Directory of Open Access Journals (Sweden)

    Andreia Michelle Smith-Moritz

    2015-08-01

    Full Text Available The CELLULOSE SYNTHASE-LIKE F6 (CslF6 gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG, a cell wall polysaccharide that is hypothesized to be a tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of three day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell was of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

  16. Boc modifies the holoprosencephaly spectrum of Cdo mutant mice

    Directory of Open Access Journals (Sweden)

    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.

  17. HCN channels in the heart: lessons from mouse mutants.

    Science.gov (United States)

    Herrmann, S; Hofmann, F; Stieber, J; Ludwig, A

    2012-05-01

    Hyperpolarization-activated cation channels generate the I(f) current in the heart. In the sino-atrial node (SAN), I(f) is thought to play an essential role in setting the heart rate and mediating its autonomic control. This review focuses on the role of I(f) in pacemaking and non-pacemaking cardiomyocytes and the resulting therapeutic implications. HCN4 represents the principal isoform underlying sino-atrial I(f) , but other isoforms may also be of importance. To examine the functional role of cardiac channels, several mouse mutants, most of them targeting HCN4, have been generated by different groups. Unexpectedly, these lines display greatly different and as yet unexplained phenotypes. We provide an overview about these HCN mutants and suggest an interpretation of the functional significance of I(f) in the SAN in light of these studies. HCN channels are also present in ventricular myocytes, and an up-regulation of I(f) in the hypertrophic and failing heart may contribute to arrhythmogenesis. Inhibition of I(f) by HCN channel blockers is a novel approach in the treatment of cardiac disorders, and ivabradine is approved for treatment of stable angina pectoris. Remarkably, a recent clinical trial assessing this substance in heart failure showed a significantly improved outcome. The mechanism underlying this beneficial effect is not yet clear and might lie beyond heart rate slowing. Thus, the growing knowledge about cardiac HCN channels will undoubtedly promote the development of the promising class of HCN channel blockers. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  18. Mutant HSP70 Reverses Autoimmune Depigmentation in Vitiligo

    Science.gov (United States)

    Mosenson, Jeffrey A.; Zloza, Andrew; Nieland, John D.; Garrett-Mayer, Elizabeth; Eby, Jonathan M.; Huelsmann, Erica J.; Kumar, Previn; Denman, Cecele J.; Lacek, Andrew T.; Kohlhapp, Frederick J.; Alamiri, Ahmad; Hughes, Tasha; Bines, Steven D.; Kaufman, Howard L.; Overbeck, Andreas; Mehrotra, Shikhar; Hernandez, Claudia; Nishimura, Michael I.; Guevara-Patino, Jose A.; Le Poole, I. Caroline

    2013-01-01

    Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell–mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo. PMID:23447019

  19. Glycine receptor mouse mutants: model systems for human hyperekplexia.

    Science.gov (United States)

    Schaefer, Natascha; Langlhofer, Georg; Kluck, Christoph J; Villmann, Carmen

    2013-11-01

    Human hyperekplexia is a neuromotor disorder caused by disturbances in inhibitory glycine-mediated neurotransmission. Mutations in genes encoding for glycine receptor subunits or associated proteins, such as GLRA1, GLRB, GPHN and ARHGEF9, have been detected in patients suffering from hyperekplexia. Classical symptoms are exaggerated startle attacks upon unexpected acoustic or tactile stimuli, massive tremor, loss of postural control during startle and apnoea. Usually patients are treated with clonazepam, this helps to dampen the severe symptoms most probably by up-regulating GABAergic responses. However, the mechanism is not completely understood. Similar neuromotor phenotypes have been observed in mouse models that carry glycine receptor mutations. These mouse models serve as excellent tools for analysing the underlying pathomechanisms. Yet, studies in mutant mice looking for postsynaptic compensation of glycinergic dysfunction via an up-regulation in GABAA receptor numbers have failed, as expression levels were similar to those in wild-type mice. However, presynaptic adaptation mechanisms with an unusual switch from mixed GABA/glycinergic to GABAergic presynaptic terminals have been observed. Whether this presynaptic adaptation explains the improvement in symptoms or other compensation mechanisms exist is still under investigation. With the help of spontaneous glycine receptor mouse mutants, knock-in and knock-out studies, it is possible to associate behavioural changes with pharmacological differences in glycinergic inhibition. This review focuses on the structural and functional characteristics of the various mouse models used to elucidate the underlying signal transduction pathways and adaptation processes and describes a novel route that uses gene-therapeutic modulation of mutated receptors to overcome loss of function mutations. © 2013 The British Pharmacological Society.

  20. A mutant gene that increases gibberellin production in Brassica

    Energy Technology Data Exchange (ETDEWEB)

    Rood, S.B. (Univ. of Lethbridge, Alberta (Canada)); Williams, P.H. (Univ. of Wisconsin, Madison (USA)); Pearce, D.; Pharis, R.P. (Univ. of Calgary, Alberta (Canada)); Murofushi, Noboru (Univ. of Tokyo (Japan)); Mander, L.N. (Australian National Univ., Canberra (Australia))

    1990-07-01

    A single gene mutant (elongated internode (ein/ein)) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A{sub 3} (GA{sub 3}) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA{sub 1} and GA{sub 3} were estimated by gas chromatography-selected ion monitoring using ({sup 2}H)GA{sub 1} as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA{sub 20} and GA{sub 1}, and the rate of GA{sub 19} metabolism were simultaneously analyzed. Levels of GA{sub 1} and GA{sub 20} were 4.6- and 12.9-fold higher, respectively, and conversions to GA{sub 20} and GA{sub 1} were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA{sub 1} biosynthesis in ein, the conversion of ({sup 3}H)GA{sub 20} to ({sup 3}H) GA{sub 1} was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA{sub 1} biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A{sub 1} and A{sub 3}.