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Sample records for human mutant sod1

  1. Radio-sensitivity of the cells from amyotrophic lateral sclerosis model mice transfected with human mutant SOD1

    International Nuclear Information System (INIS)

    Wate, Reika; Ito, Hidefumi; Kusaka, Hirofumi; Takahashi, Sentaro; Kubota, Yoshihisa; Suetomi, Katsutoshi; Sato, Hiroshi; Okayasu, Ryuichi

    2005-01-01

    In order to clarify the possible involvement of oxidative damage induced by ionizing radiation in the onset and/or progression of familial amyotrophic lateral sclerosis (ALS), we studied radio-sensitivity in primary cells derived from ALS model mice expressing human mutant Cu/Zn superoxide dismutase (SOD1). The primary mouse cells expressed both mouse and the mutant human SOD1. The cell survival of the transgenic mice (with mutant SOD1), determined by counting cell numbers at a scheduled time after X-irradiation, is very similar to that of cells from wild type animals. The induction and repair of DNA damage in the transgenic cells, measured by single cell gel electrophoresis and pulsed field gel electrophoresis, are also similar to those of wild type cells. These results indicate that the human mutant SOD1 gene does not seem to contribute to the alteration of radio-sensitivity, at least in the fibroblastic cells used here. Although it is necessary to consider the difference in cell types between fibroblastic and neuronal cells, the present results may suggest that ionizing radiation is not primarily responsible for the onset of familial ALS with the SOD1 mutation, and that the excess risks are probably not a concern for radiation diagnosis and therapy in familial ALS patients. (author)

  2. A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants.

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    Luchinat, Enrico; Barbieri, Letizia; Banci, Lucia

    2017-12-12

    Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.

  3. S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects.

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    Antinone, Sarah E; Ghadge, Ghanashyam D; Ostrow, Lyle W; Roos, Raymond P; Green, William N

    2017-01-25

    Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord.

  4. ALS mutant SOD1 interacts with G3BP1 and affects stress granule dynamics.

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    Gal, Jozsef; Kuang, Lisha; Barnett, Kelly R; Zhu, Brian Z; Shissler, Susannah C; Korotkov, Konstantin V; Hayward, Lawrence J; Kasarskis, Edward J; Zhu, Haining

    2016-10-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approximately 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a critical role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathological cytoplasmic inclusions that co-localized with G3BP1-positive granules in spinal cord motor neurons. The co-localization was also observed in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are critical for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-positive stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metabolism alterations in ALS.

  5. Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

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    Kim, Ha Kun; Chung, Youn Wook; Chock, P Boon; Yim, Moon B

    2011-05-15

    Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H(2)O(2), mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. Published by Elsevier Inc.

  6. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

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    Ghadge, Ghanashyam D; Pavlovic, John D; Koduvayur, Sujatha P; Kay, Brian K; Roos, Raymond P

    2013-08-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Destabilizing protein polymorphisms in the genetic background direct phenotypic expression of mutant SOD1 toxicity.

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    Tali Gidalevitz

    2009-03-01

    Full Text Available Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles.

  8. Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

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    Roberts, Blaine R; Lim, Nastasia K H; McAllum, Erin J; Donnelly, Paul S; Hare, Dominic J; Doble, Philip A; Turner, Bradley J; Price, Katherine A; Lim, Sin Chun; Paterson, Brett M; Hickey, James L; Rhoads, Timothy W; Williams, Jared R; Kanninen, Katja M; Hung, Lin W; Liddell, Jeffrey R; Grubman, Alexandra; Monty, Jean-Francois; Llanos, Roxana M; Kramer, David R; Mercer, Julian F B; Bush, Ashley I; Masters, Colin L; Duce, James A; Li, Qiao-Xin; Beckman, Joseph S; Barnham, Kevin J; White, Anthony R; Crouch, Peter J

    2014-06-04

    Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1. Copyright © 2014 the authors 0270-6474/14/348021-11$15.00/0.

  9. An ALS-Associated Mutant SOD1 Rapidly Suppresses KCNT1 (Slack) Na+-Activated K+ Channels in Aplysia Neurons.

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    Zhang, Yalan; Ni, Weiming; Horwich, Arthur L; Kaczmarek, Leonard K

    2017-02-22

    Mutations that alter levels of Slack (KCNT1) Na + -activated K + current produce devastating effects on neuronal development and neuronal function. We now find that Slack currents are rapidly suppressed by oligomers of mutant human Cu/Zn superoxide dismutase 1 (SOD1), which are associated with motor neuron toxicity in an inherited form of amyotrophic lateral sclerosis (ALS). We recorded from bag cell neurons of Aplysia californica , a model system to study neuronal excitability. We found that injection of fluorescent wild-type SOD1 (wt SOD1YFP) or monomeric mutant G85R SOD1YFP had no effect on net ionic currents measured under voltage clamp. In contrast, outward potassium currents were significantly reduced by microinjection of mutant G85R SOD1YFP that had been preincubated at 37°C or of cross-linked dimers of G85R SOD1YFP. Reduction of potassium current was also seen with multimeric G85R SOD1YFP of ∼300 kDa or >300 kDa that had been cross-linked. In current clamp recordings, microinjection of cross-linked 300 kDa increased excitability by depolarizing the resting membrane potential, and decreasing the latency of action potentials triggered by depolarization. The effect of cross-linked 300 kDa on potassium current was reduced by removing Na + from the bath solution, or by knocking down levels of Slack using siRNA. It was also prevented by pharmacological inhibition of ASK1 (apoptosis signal-regulating kinase 1) or of c-Jun N-terminal kinase, but not by an inhibitor of p38 mitogen-activated protein kinase. These results suggest that soluble mutant SOD1 oligomers rapidly trigger a kinase pathway that regulates the activity of Na + -activated K + channels in neurons. SIGNIFICANCE STATEMENT Slack Na + -activated K + channels (KCNT1, K Na 1.1) regulate neuronal excitability but are also linked to cytoplasmic signaling pathways that control neuronal protein translation. Mutations that alter the amplitude of these currents have devastating effects on neuronal

  10. An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans.

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    Jiou Wang

    2009-01-01

    Full Text Available The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking.

  11. Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice.

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    Turner, Bradley J; Alfazema, Neza; Sheean, Rebecca K; Sleigh, James N; Davies, Kay E; Horne, Malcolm K; Talbot, Kevin

    2014-04-01

    Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1(G93A) mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1(G93A) mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1(G93A) mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. ALS-linked mutant SOD1 proteins promote Aβ aggregates in ALS through direct interaction with Aβ.

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    Jang, Ja-Young; Cho, Hyungmin; Park, Hye-Yoon; Rhim, Hyangshuk; Kang, Seongman

    2017-11-04

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons. Aggregation of ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) is a hallmark of a subset of familial ALS (fALS). Recently, intracellular amyloid-β (Aβ) is detected in motor neurons of both sporadic and familial ALS. We have previously shown that intracellular Aβ specifically interacts with G93A, an ALS-linked SOD1 mutant. However, little is known about the pathological and biological effect of this interaction in neurons. In this study, we have demonstrated that the Aβ-binding region is exposed on the SOD1 surface through the conformational changes due to misfolding of SOD1. Interestingly, we found that the intracellular aggregation of Aβ is enhanced through the direct interaction of Aβ with the Aβ-binding region exposed to misfolded SOD1. Ultimately, increased Aβ aggregation by this interaction promotes neuronal cell death. Consistent with this result, Aβ aggregates was three-fold higher in the brains of G93A transgenic mice than those of non Tg. Our study provides the first direct evidence that Aβ, an AD-linked factor, is associated to the pathogenesis of ALS and provides molecular clues to understand common aggregation mechanisms in the pathogenesis of neurodegenerative diseases. Furthermore, it will provide new insights into the development of therapeutic approaches for ALS. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components.

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    Şahin, Aslı; Held, Aaron; Bredvik, Kirsten; Major, Paxton; Achilli, Toni-Marie; Kerson, Abigail G; Wharton, Kristi; Stilwell, Geoff; Reenan, Robert

    2017-02-01

    Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies. Copyright © 2017 by the Genetics Society of America.

  14. Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis

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    Akshay Bhinge

    2017-04-01

    Full Text Available Summary: Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS, it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential. : In this article, Bhinge, Stanton, and colleagues use genome editing of patient-derived iPSCs to model ALS phenotypic defects in vitro. Transcriptomic analysis of disease MNs reveals activation of MAPK, AP1, WNT, cell-cycle, and p53 signaling in ALS MNs. Pharmacological screening uncovers activated ERK and JNK signaling as therapeutic targets in ALS. Keywords: ALS, SOD1, FUS, CRISPR-Cas9, p38, ERK, JNK, WNT, TP53, JUN

  15. Fisetin Exerts Antioxidant and Neuroprotective Effects in Multiple Mutant hSOD1 Models of Amyotrophic Lateral Sclerosis by Activating ERK.

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    Wang, T H; Wang, S Y; Wang, X D; Jiang, H Q; Yang, Y Q; Wang, Y; Cheng, J L; Zhang, C T; Liang, W W; Feng, H L

    2018-05-21

    Oxidative stress exhibits a central role in the course of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease commonly found to include a copper/zinc superoxide dismutase (SOD1) gene mutation. Fisetin, a natural antioxidant, has shown benefits in varied neurodegenerative diseases. The possible effect of fisetin in ALS has not been clarified as of yet. We investigated whether fisetin affected mutant hSOD1 ALS models. Three different hSOD1-related mutant models were used: Drosophila expressing mutant hSOD1 G85R , hSOD1 G93A NSC34 cells, and transgenic mice. Fisetin treatment provided neuroprotection as demonstrated by an improved survival rate, attenuated motor impairment, reduced ROS damage and regulated redox homeostasis compared with those in controls. Furthermore, fisetin increased the expression of phosphorylated ERK and upregulated antioxidant factors, which were reversed by MEK/ERK inhibition. Finally, fisetin reduced the levels of both mutant and wild-type hSOD1 in vivo and in vitro, as well as the levels of detergent-insoluble hSOD1 proteins. The results indicate that fisetin protects cells from ROS damage and improves the pathological behaviors caused by oxidative stress in disease models related to SOD1 gene mutations probably by activating ERK, thereby providing a potential treatment for ALS. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

  16. Overexpression of human SOD1 improves survival of mice susceptible to endotoxic shock

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    Charchaflieh J

    2012-07-01

    Full Text Available Jean Charchaflieh,1,2 Georges I Labaze,1 Pulsar Li,1 Holly Van Remmen,3 Haekyung Lee,1 Helen Stutz,1 Arlan Richardson,3 Asher Emanuel,1 Ming Zhang1,41Department of Anesthesiology, State University of New York (SUNY Downstate Medical Center, New York, NY, USA; 2Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USA; 3Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; 4Department of Cell Biology, State University of New York (SUNY Downstate Medical Center, New York, NY, USABackground: Protective effects of the antioxidant enzyme Cu-Zn superoxide dismutase (SOD1 against endotoxic shock have not been demonstrated in animal models. We used a murine model to investigate whether overexpression of SOD1 protects against endotoxic shock, and whether the genetic background of SOD1 affects its effective protective effects and susceptibility to endotoxic shock.Methods: Transgenic (tg mice overexpressing human SOD1 and control mice were divided into four groups based on their genetic background: (1 tg mice with mixed genetic background (tg-JAX; (2 wild-type (WT littermates of tg-JAX strain (WT-JAX; (3 tg mice with C57BL/6J background (tg-TX; (4 WT littermates of tg-TX strain (WT-TX. Activity of SOD1 in the intestine, heart, and liver of tg and control mice was confirmed using a polyacrylamide activity gel. Endotoxic shock was induced by intraperitoneal injection of lipopolysaccharide. Survival rates over 120 hours (mean, 95% confidence interval were analyzed using Kaplan–Meier survival curves.Results: Human SOD1 enzymatic activities were significantly higher in the intestine, heart, and liver of both tg strains (tg-JAX and tg-TX compared with their WT littermates (WT-JAX and WT-TX, respectively. Interestingly, the endogenous SOD1 activities in tg-JAX mice were decreased compared with their WT littermates (WT-JAX, but such aberrant changes were not

  17. Delayed Disease Onset and Extended Survival in the SOD1G93A Rat Model of Amyotrophic Lateral Sclerosis after Suppression of Mutant SOD1 in the Motor Cortex

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    Thomsen, Gretchen M.; Gowing, Genevieve; Latter, Jessica; Chen, Maximus; Vit, Jean-Philippe; Staggenborg, Kevin; Avalos, Pablo; Alkaslasi, Mor; Ferraiuolo, Laura; Likhite, Shibi; Kaspar, Brian K.

    2014-01-01

    Sporadic amyotrophic lateral sclerosis (ALS) is a fatal disease with unknown etiology, characterized by a progressive loss of motor neurons leading to paralysis and death typically within 3–5 years of onset. Recently, there has been remarkable progress in understanding inherited forms of ALS in which well defined mutations are known to cause the disease. Rodent models in which the superoxide dismutase-1 (SOD1) mutation is overexpressed recapitulate hallmark signs of ALS in patients. Early anatomical changes in mouse models of fALS are seen in the neuromuscular junctions (NMJs) and lower motor neurons, and selective reduction of toxic mutant SOD1 in the spinal cord and muscle of these models has beneficial effects. Therefore, much of ALS research has focused on spinal motor neuron and NMJ aspects of the disease. Here we show that, in the SOD1G93A rat model of ALS, spinal motor neuron loss occurs presymptomatically and before degeneration of ventral root axons and denervation of NMJs. Although overt cell death of corticospinal motor neurons does not occur until disease endpoint, we wanted to establish whether the upper motor neuron might still play a critical role in disease progression. Surprisingly, the knockdown of mutant SOD1 in only the motor cortex of presymptomatic SOD1G93A rats through targeted delivery of AAV9–SOD1–shRNA resulted in a significant delay of disease onset, expansion of lifespan, enhanced survival of spinal motor neurons, and maintenance of NMJs. This datum suggests an early dysfunction and thus an important role of the upper motor neuron in this animal model of ALS and perhaps patients with the disease. PMID:25411487

  18. Redox susceptibility of SOD1 mutants is associated with the differential response to CCS over-expression in vivo.

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    Son, Marjatta; Fu, Qiao; Puttaparthi, Krishna; Matthews, Christina M; Elliott, Jeffrey L

    2009-04-01

    Over-expression of CCS in G93A SOD1 mice accelerates neurological disease and enhances mitochondrial pathology. We studied the effect of CCS over-expression in transgenic mice expressing G37R, G86R or L126Z SOD1 mutations in order to understand factors which influence mitochondrial dysfunction. Over-expression of CCS markedly decreased survival and produced mitochondrial vacuolation in G37R SOD1 mice but not in G86R or L126Z SOD1 mice. Moreover, CCS/G37R SOD1 spinal cord showed specific reductions in mitochondrial complex IV subunits consistent with an isolated COX deficiency, while no such reductions were detected in CCS/G86R or CCS/L126Z SOD1 mice. CCS over-expression increased the ratio of reduced to oxidized SOD1 monomers in the spinal cords of G37R SOD1 as well as G93A SOD1 mice, but did not influence the redox state of G86R or L126Z SOD1 monomers. The effects of CCS on disease are SOD1 mutation dependent and correlate with SOD1 redox susceptibility.

  19. Mice overexpressing both non-mutated human SOD1 and mutated SOD1G93A genes: a competent experimental model for studying iron metabolism in amyotrophic lateral sclerosis

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    Anna eGajowiak

    2016-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease characterized by degeneration and loss of motor neurons in the spinal cord, brainstem and motor cortex. Up to 10% of ALS cases are inherited (familial, fALS and associated with mutations, frequently in the superoxide dismutase 1 (SOD1 gene. Rodent transgenic models of ALS are often used to elucidate a complex pathogenesis of this disease. Of importance, both ALS patients and animals carrying mutated human SOD1 gene show symptoms of oxidative stress and iron metabolism misregulation. The aim of our study was to characterize changes in iron metabolism in one of the most commonly used models of ALS – transgenic mice overexpressing human mutated SOD1G93A gene. We analyzed the expression of iron-related genes in asymptomatic, 2-month old and symptomatic, 4-month old SOD1G93A mice. In parallel, respective age-matched mice overexpressing human non-mutated SOD1 transgene and control mice were analyzed. We demonstrate that the overexpression of both SOD1 and SOD1G93A genes account for a substantial increase in SOD1 protein levels and activity in selected tissues and that not all the changes in iron metabolism genes expression are specific for the overexpression of the mutated form of SOD1.

  20. Enhanced tethered-flight duration and locomotor activity by overexpression of the human gene SOD1 in Drosophila motorneurons

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    Agavni Petrosyan

    2015-03-01

    Full Text Available Mutation of the human gene superoxide dismutase (hSOD1 is associated with the fatal neurodegenerative disease familial amyotrophic lateral sclerosis (Lou Gehrig’s disease. Selective overexpression of hSOD1 in Drosophila motorneurons increases lifespan to 140% of normal. The current study was designed to determine resistance to lifespan decline and failure of sensorimotor functions by overexpressing hSOD1 in Drosophila‘s motorneurons. First, we measured the ability to maintain continuous flight and wingbeat frequency (WBF as a function of age (5 to 50 days. Flies overexpressing hSOD1 under the D42-GAL4 activator were able to sustain flight significantly longer than controls, with the largest effect observed in the middle stages of life. The hSOD1-expressed line also had, on average, slower wingbeat frequencies in late, but not early life relative to age-matched controls. Second, we examined locomotor (exploratory walking behavior in late life when flies had lost the ability to fly (age ≥ 60 d. hSOD1-expressed flies showed significantly more robust walking activity relative to controls. Findings show patterns of functional decline dissimilar to those reported for other life-extended lines, and suggest that the hSOD1 gene not only delays death but enhances sensorimotor abilities critical to survival even in late life.

  1. Spinal cord homogenates from SOD1 familial amyotrophic lateral sclerosis induce SOD1 aggregation in living cells.

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    Edward Pokrishevsky

    Full Text Available Mutant Cu/Zn superoxide dismutase (SOD1 can confer its misfolding on wild-type SOD1 in living cells; the propagation of misfolding can also be transmitted between cells in vitro. Recent studies identified fluorescently-tagged SOD1G85R as a promiscuous substrate that is highly prone to aggregate by a variety of templates, in vitro and in vivo. Here, we utilized several SOD1-GFP reporter proteins with G37R, G85R, or G93A mutations in SOD1. We observed that human spinal cord homogenates prepared from SOD1 familial ALS (FALS can induce significantly more intracellular reporter protein aggregation than spinal cord homogenates from sporadic ALS, Alzheimer's disease, multiple system atrophy or healthy control individuals. We also determined that the induction of reporter protein aggregation by SOD1-FALS tissue homogenates can be attenuated by incubating the cells with the SOD1 misfolding-specific antibody 3H1, or the small molecule 5-fluorouridine. Our study further implicates SOD1 as the seeding particle responsible for the spread of SOD1-FALS neurodegeneration from its initial onset site(s, and demonstrates two potential therapeutic strategies for SOD1-mediated disease. This work also comprises a medium-throughput cell-based platform of screening potential therapeutics to attenuate propagated aggregation of SOD1.

  2. Peripheral motor axons of SOD1(G127X) mutant mice are susceptible to activity-dependent degeneration

    DEFF Research Database (Denmark)

    Alvarez Herrero, Susana; Calin, A; Graffmo, K S

    2013-01-01

    -onset, fast-progression SOD1(G127X) mouse model of amyotrophic lateral sclerosis to long-lasting, high-frequency repetitive activity. Tibial nerves were stimulated at ankle in 7 to 8-month-old SOD1(G127X) mice when they were clinically indistinguishable from wild-type (WT) mice. The evoked compound muscle......-concentrations. It is possible that in SOD1(G127X) there is inadequate energy-dependent Na(+)/K(+) pumping, which may lead to a lethal Na(+) overload....

  3. Structural and biophysical properties of metal-free pathogenic SOD1 mutants A4V and G93A

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    Galaleldeen, Ahmad; Strange, Richard W.; Whitson, Lisa J.; Antonyuk, Svetlana V.; Narayana, Narendra; Taylor, Alexander B.; Schuermann, Jonathan P.; Holloway, Stephen P.; Hasnain, S.Samar; Hart, P. John; (Texas-HSC); (Liverpool)

    2010-07-19

    Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the destruction of motor neurons in the spinal cord and brain. A subset of ALS cases are linked to dominant mutations in copper-zinc superoxide dismutase (SOD1). The pathogenic SOD1 variants A4V and G93A have been the foci of multiple studies aimed at understanding the molecular basis for SOD1-linked ALS. The A4V variant is responsible for the majority of familial ALS cases in North America, causing rapidly progressing paralysis once symptoms begin and the G93A SOD1 variant is overexpressed in often studied murine models of the disease. Here we report the three-dimensional structures of metal-free A4V and of metal-bound and metal-free G93A SOD1. In the metal-free structures, the metal-binding loop elements are observed to be severely disordered, suggesting that these variants may share mechanisms of aggregation proposed previously for other pathogenic SOD1 proteins.

  4. Absence of Nrf2 or its selective overexpression in neurons and muscle does not affect survival in ALS-linked mutant hSOD1 mouse models.

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    Marcelo R Vargas

    Full Text Available The nuclear factor erythroid 2-related factor 2 (Nrf2 governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1 cause familial forms of amyotrophic lateral sclerosis (ALS, a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1(G93A mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS.

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

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    Larmet Yves

    2011-04-01

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

  6. TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion.

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    Pokrishevsky, Edward; Grad, Leslie I; Cashman, Neil R

    2016-03-01

    Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TDP-43, FUS, C9ORF72, or can occur sporadically without recognized genetic mutations. Misfolded human wild-type (HuWt) SOD1 has been detected in both familial and sporadic ALS patients, despite mutations in SOD1 accounting for only 2% of total cases. We previously showed that accumulation of pathological TDP-43 or FUS coexist with misfolded HuWtSOD1 in patient motor neurons, and can trigger its misfolding in cultured cells. Here, we used immunocytochemistry and immunoprecipitation to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. In this system, intercellular spread of SOD1 misfolding is not accompanied by transmission of TDP-43 or FUS pathology. Our findings argue that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of propagated misfolding of SOD1.

  7. ZNStress: a high-throughput drug screening protocol for identification of compounds modulating neuronal stress in the transgenic mutant sod1G93R zebrafish model of amyotrophic lateral sclerosis.

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    McGown, Alexander; Shaw, Dame Pamela J; Ramesh, Tennore

    2016-07-26

    Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease with death on average within 2-3 years of symptom onset. Mutations in superoxide dismutase 1 (SOD1) have been identified to cause ALS. Riluzole, the only neuroprotective drug for ALS provides life extension of only 3 months on average. Thishighlights the need for compound screening in disease models to identify new neuroprotective therapies for this disease. Zebrafish is an emerging model system that is well suited for the study of diseasepathophysiology and also for high throughput (HT) drug screening. The mutant sod1 zebrafish model of ALS mimics the hallmark features of ALS. Using a fluorescence based readout of neuronal stress, we developed a high throughput (HT) screen to identify neuroprotective compounds. Here we show that the zebrafish screen is a robust system that can be used to rapidly screen thousands ofcompounds and also demonstrate that riluzole is capable of reducing neuronal stress in this model system. The screen shows optimal quality control, maintaining a high sensitivity and specificity withoutcompromising throughput. Most importantly, we demonstrate that many compounds previously failed in human clinical trials, showed no stress reducing activity in the zebrafish assay. We conclude that HT drug screening using a mutant sod1 zebrafish is a reliable model system which supplemented with secondary assays would be useful in identifying drugs with potential for neuroprotective efficacy in ALS.

  8. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

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    Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

    2016-05-13

    Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. © 2016 by The American Society for Biochemistry and Molecular

  9. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1)*

    Science.gov (United States)

    Harlan, Benjamin A.; Pehar, Mariana; Sharma, Deep R.; Beeson, Gyda; Beeson, Craig C.; Vargas, Marcelo R.

    2016-01-01

    Nicotinamide adenine dinucleotide (NAD+) participates in redox reactions and NAD+-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD+-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD+ as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1–7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD+ salvage pathway capable of resynthesizing NAD+ from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD+ levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD+ salvage pathway in astrocytes. Supplementation with the NAD+ precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD+ levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1–2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD+ content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD+ salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. PMID:27002158

  10. Quercetin Protects Primary Human Osteoblasts Exposed to Cigarette Smoke through Activation of the Antioxidative Enzymes HO-1 and SOD-1

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    Karl F. Braun

    2011-01-01

    Full Text Available Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS. The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO- 1 and superoxide-dismutase- (SOD- 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers.

  11. An inducer of VGF protects cells against ER stress-induced cell death and prolongs survival in the mutant SOD1 animal models of familial ALS.

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    Masamitsu Shimazawa

    2010-12-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is the most frequent adult-onset motor neuron disease, and recent evidence has suggested that endoplasmic reticulum (ER stress signaling is involved in the pathogenesis of ALS. Here we identified a small molecule, SUN N8075, which has a marked protective effect on ER stress-induced cell death, in an in vitro cell-based screening, and its protective mechanism was mediated by an induction of VGF nerve growth factor inducible (VGF: VGF knockdown with siRNA completely abolished the protective effect of SUN N8075 against ER-induced cell death, and overexpression of VGF inhibited ER-stress-induced cell death. VGF level was lower in the spinal cords of sporadic ALS patients than in the control patients. Furthermore, SUN N8075 slowed disease progression and prolonged survival in mutant SOD1 transgenic mouse and rat models of ALS, preventing the decrease of VGF expression in the spinal cords of ALS mice. These data suggest that VGF plays a critical role in motor neuron survival and may be a potential new therapeutic target for ALS, and SUN N8075 may become a potential therapeutic candidate for treatment of ALS.

  12. PGC-1 silencing compounds the perturbation of mitochondrial function caused by mutant SOD1 in skeletal muscle of ALS mouse model

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    Yan eQi

    2015-10-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a lethal neurodegenerative disease causing death of motor neurons. This study investigated the roles of energy metabolism in the pathogenesis of ALS in the SOD1(G93A transgenic mouse model. Control and SOD1(G93A mice were administered with shcontrol or shPGC-1α in combination with PBS or TZD for 8 weeks. Gene expression was analyzed by quantitative real-time PCR and western blot. ROS and fibrosis were assessed with a colorimetric kit and Sirius staining respectively. Inflammatory cytokines were measured using ELISA kits. The levels of tissue ROS and serum inflammatory cytokines were significantly higher in SOD1(G93A mice compared to control mice, and knocking down PGC-1α drastically increased cytokine levels in both control and SOD1(G93A mice. Muscle fibrosis was much severer in SOD1(G93A mice, and worsened by silencing PGC-1α and attenuate d by TZD. The expression levels of PGC-1α, SOD1, UCP2, and cytochrome C were substantially reduced by shPGC-1α and increased by TZD in muscle of both control and SOD1(G93A mice whereas the level of NF-B was significantly elevated in SOD1(G93A mice, which was further increased by PGC-1α silencing. These data indicated that disruption of energy homeostasis would exacerbate the pathological changes caused by SOD1 mutations to promote the pathogenesis of ALS.

  13. Structures of the G85R Variant of SOD1 in Familial Amyotrophic Lateral Sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Xiaohang; Antonyuk, Svetlana V.; Seetharaman, Sai V.; Whitson, Lisa J.; Taylor, Alexander B.; Holloway, Stephen P.; Strange, Richard W.; Doucette, Peter A.; Valentine, Joan Selverstone; Tiwari, Ashutosh; Hayward, Lawrence J.; Padua, Shelby; Cohlberg, Jeffrey A.; Hasnain, S. Samar; Hart, P. John (Texas-HSC); (Cal. State); (UMASS, MED); (UCLA); (Daresbury)

    2008-07-21

    Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.

  14. Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis

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    Savina Apolloni

    2014-09-01

    Full Text Available In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a ‘gene modifier’ in amyotrophic lateral sclerosis (ALS: the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG, a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising

  15. The Effects of Bee Venom Acupuncture on the Central Nervous System and Muscle in an Animal hSOD1G93A Mutant

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    MuDan Cai

    2015-03-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is caused by the degeneration of lower and upper motor neurons, leading to muscle paralysis and respiratory failure. However, there is no effective drug or therapy to treat ALS. Complementary and alternative medicine (CAM, including acupuncture, pharmacopuncture, herbal medicine, and massage is popular due to the significant limitations of conventional therapy. Bee venom acupuncture (BVA, also known as one of pharmacopunctures, has been used in Oriental medicine to treat inflammatory diseases. The purpose of this study is to investigate the effect of BVA on the central nervous system (CNS and muscle in symptomatic hSOD1G93A transgenic mice, an animal model of ALS. Our findings show that BVA at ST36 enhanced motor function and decreased motor neuron death in the spinal cord compared to that observed in hSOD1G93A transgenic mice injected intraperitoneally (i.p. with BV. Furthermore, BV treatment at ST36 eliminated signaling downstream of inflammatory proteins such as TLR4 in the spinal cords of symptomatic hSOD1G93A transgenic mice. However, i.p. treatment with BV reduced the levels of TNF-α and Bcl-2 expression in the muscle hSOD1G93A transgenic mice. Taken together, our findings suggest that BV pharmacopuncture into certain acupoints may act as a chemical stimulant to activate those acupoints and subsequently engage the endogenous immune modulatory system in the CNS in an animal model of ALS.

  16. Multiple intracerebroventricular injections of human umbilical cord mesenchymal stem cells delay motor neurons loss but not disease progression of SOD1G93A mice.

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    Sironi, Francesca; Vallarola, Antonio; Violatto, Martina Bruna; Talamini, Laura; Freschi, Mattia; De Gioia, Roberta; Capelli, Chiara; Agostini, Azzurra; Moscatelli, Davide; Tortarolo, Massimo; Bigini, Paolo; Introna, Martino; Bendotti, Caterina

    2017-12-01

    Stem cell therapy is considered a promising approach in the treatment of amyotrophic lateral sclerosis (ALS) and mesenchymal stem cells (MSCs) seem to be the most effective in ALS animal models. The umbilical cord (UC) is a source of highly proliferating fetal MSCs, more easily collectable than other MSCs. Recently we demonstrated that human (h) UC-MSCs, double labeled with fluorescent nanoparticles and Hoechst-33258 and transplanted intracerebroventricularly (ICV) into SOD1G93A transgenic mice, partially migrated into the spinal cord after a single injection. This prompted us to assess the effect of repeated ICV injections of hUC-MSCs on disease progression in SOD1G93A mice. Although no transplanted cells migrated to the spinal cord, a partial but significant protection of motor neurons (MNs) was found in the lumbar spinal cord of hUC-MSCs-treated SOD1G93A mice, accompanied by a shift from a pro-inflammatory (IL-6, IL-1β) to anti-inflammatory (IL-4, IL-10) and neuroprotective (IGF-1) environment in the lumbar spinal cord, probably linked to the activation of p-Akt survival pathway in both motor neurons and reactive astrocytes. However, this treatment neither prevented the muscle denervation nor delayed the disease progression of mice, emphasizing the growing evidence that protecting the motor neuron perikarya is not sufficient to delay the ALS progression. Copyright © 2017. Published by Elsevier B.V.

  17. An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis

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    Melissa S Rotunno

    2013-12-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s of sporadic ALS, which accounts for approximately 90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1 protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a toxic conformation that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS.

  18. FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis.

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    Kabashi, Edor; Bercier, Valérie; Lissouba, Alexandra; Liao, Meijiang; Brustein, Edna; Rouleau, Guy A; Drapeau, Pierre

    2011-08-01

    Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS). Recently, mutations in the Fused in sarcoma gene (FUS) were identified in familial (FALS) ALS cases and sporadic (SALS) patients. Similarly to TDP-43 (coded by TARDBP gene), FUS is an RNA binding protein. Using the zebrafish (Danio rerio), we examined the consequences of expressing human wild-type (WT) FUS and three ALS-related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS-related FUS mutations, R521H and R521C, unlike S57Δ, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C) and FUS (R521H) or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A). Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1.

  19. FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis.

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    Edor Kabashi

    2011-08-01

    Full Text Available Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS. Recently, mutations in the Fused in sarcoma gene (FUS were identified in familial (FALS ALS cases and sporadic (SALS patients. Similarly to TDP-43 (coded by TARDBP gene, FUS is an RNA binding protein. Using the zebrafish (Danio rerio, we examined the consequences of expressing human wild-type (WT FUS and three ALS-related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS-related FUS mutations, R521H and R521C, unlike S57Δ, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C and FUS (R521H or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A. Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1.

  20. The effect of amyotrophic lateral sclerosis-linked exogenous SOD1-G93A on electrophysiological properties and intracellular calcium in cultured rat astrocytes.

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    Milošević, Milena; Bataveljić, Danijela; Nikolić, Ljiljana; Bijelić, Dunja; Andjus, Pavle

    2016-01-01

    Over 150 mutations in the SOD1 gene that encodes Cu/Zn superoxide dismutase (SOD1) cause 20-25% of familial ALS, albeit without a known gain-of-function mechanism. ALS is also non-cell-autonomous, the interactions between motor neurons and their glial neighbours being implicated in disease progression. The aim here was to investigate the biophysical effects of the exogenous human mutant SOD1-G93A on rat astrocytes in culture. Primary cortical astrocyte cultures were treated with recombinant human apo- mSOD1-G93A vs. wild-type control (wtSOD1) and recorded by patch-clamp and calcium imaging. Results showed that exogenous mSOD1 as well as wtSOD1 induced a decrease of membrane resistance, the effect being persistent (up to 13 min) only for the mutant form. Similarly, whole-cell inward currents in astrocytes were augmented by both wt and mSOD1, but the effect was twice larger and only progressed continuously for the latter. Both forms of SOD1 also induced a rise in intracellular Ca(2+) activity, the effect being dependent on external Ca(2+) and again only persisted with mSOD1, becoming significantly different from wtSOD1 only at longer times (14 min). In conclusion, this study points to membrane permeability and Ca(2+) signalling as processes affected by SOD1-G93A that presents the humoral factor triggering the role of astrocytes in ALS pathophysiology.

  1. Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice.

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    Le Pecheur, Marie; Bourdon, Emmanuel; Paly, Evelyne; Farout, Luc; Friguet, Bertrand; London, Jacqueline

    2005-07-04

    Premature ageing, one of the characteristics of Down syndrome (DS), may involve oxidative stress and impairment of proteasome activity. Transgenic mice overexpressing the human copper/zinc superoxide dismutase (SOD1) gene are one of the first murine models for DS and it has been shown that SOD1 overexpression might be either deleterious or beneficial. Here, we show a reduction in proteasome activities in the cortex of SOD1 transgenic mice and an associated increase in the content of oxidized SOD1 protein. As we demonstrate that in vitro oxidized SOD can inhibit purified proteasome peptidase activities, modified SOD1 might be partially responsible for proteasome inhibition shown in SOD1 transgenic mice.

  2. Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

    Science.gov (United States)

    Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

    2008-05-02

    G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

  3. Comprehensive analysis to explain reduced or increased SOD1 enzymatic activity in ALS patients and their relatives.

    Science.gov (United States)

    Keskin, Isil; Birve, Anna; Berdynski, Mariusz; Hjertkvist, Karin; Rofougaran, Reza; Nilsson, Torbjörn K; Glass, Jonathan D; Marklund, Stefan L; Andersen, Peter M

    2017-08-01

    To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay. Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72 HRE . In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios. Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing.

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

    Science.gov (United States)

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

    2015-10-01

    Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1-G93A co-cultures. Deleting TNFR2 from SOD1-G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1-G93A/TNFR2-/- mice showed high phospho-TAR DNA-binding protein 43 (TDP-43) accumulation and low levels of acetyl-tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane-bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology. We show evidence of the involvement of neuronal and astroglial TNFR2 in the motor neuron degeneration in ALS. Both concur to cause motor neuron death in primary astrocyte/spinal neuron co-cultures. TNFR2 deletion partially protects motor neurons and sciatic nerves in SOD1-G93A mice but does not improve their symptoms and survival. However, TNFR2 could be a new target for multi-intervention therapies. © 2015 International Society for Neurochemistry.

  5. ZNStress: a high-throughput drug screening protocol for identification of compounds modulating neuronal stress in the transgenic mutant sod1G93R zebrafish model of amyotrophic lateral sclerosis

    OpenAIRE

    McGown, Alexander; Shaw, Dame Pamela J.; Ramesh, Tennore

    2016-01-01

    Background Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease with death on average within 2?3 years of symptom onset. Mutations in superoxide dismutase 1 (SOD1) have been identified to cause ALS. Riluzole, the only neuroprotective drug for ALS provides life extension of only 3 months on average. Thishighlights the need for compound screening in disease models to identify new neuroprotective therapies for this disease. Zebrafish is an emerging model system that is well ...

  6. Differential effects of phytotherapic preparations in the hSOD1 Drosophila melanogaster model of ALS

    Science.gov (United States)

    De Rose, Francescaelena; Marotta, Roberto; Talani, Giuseppe; Catelani, Tiziano; Solari, Paolo; Poddighe, Simone; Borghero, Giuseppe; Marrosu, Francesco; Sanna, Enrico; Kasture, Sanjay; Acquas, Elio; Liscia, Anna

    2017-01-01

    The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms. PMID:28102336

  7. Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology.

    Science.gov (United States)

    Son, Marjatta; Puttaparthi, Krishna; Kawamata, Hibiki; Rajendran, Bhagya; Boyer, Philip J; Manfredi, Giovanni; Elliott, Jeffrey L

    2007-04-03

    Cu, Zn superoxide dismutase (SOD1) has been detected within spinal cord mitochondria of mutant SOD1 transgenic mice, a model of familial ALS. The copper chaperone for SOD1 (CCS) provides SOD1 with copper, facilitates the conversion of immature apo-SOD1 to a mature holoform, and influences in yeast the cytosolic/mitochondrial partitioning of SOD1. To determine how CCS affects G93A-SOD1-induced disease, we generated transgenic mice overexpressing CCS and crossed them to G93A-SOD1 or wild-type SOD1 transgenic mice. Both CCS transgenic mice and CCS/wild-type-SOD1 dual transgenic mice are neurologically normal. In contrast, CCS/G93A-SOD1 dual transgenic mice develop accelerated neurological deficits, with a mean survival of 36 days, compared with 242 days for G93A-SOD1 mice. Immuno-EM and subcellular fractionation studies on the spinal cord show that G93A-SOD1 is enriched within mitochondria in the presence of CCS overexpression. Our results indicate that CCS overexpression in G93A-SOD1 mice produces severe mitochondrial pathology and accelerates disease course.

  8. In yeast redistribution of Sod1 to the mitochondrial intermembrane space provides protection against respiration derived oxidative stress.

    Science.gov (United States)

    Klöppel, Christine; Michels, Christine; Zimmer, Julia; Herrmann, Johannes M; Riemer, Jan

    2010-12-03

    The antioxidative enzyme copper-zinc superoxide dismutase (Sod1) is an important cellular defence system against reactive oxygen species (ROS). While the majority of this enzyme is localized to the cytosol, about 1% of the cellular Sod1 is present in the intermembrane space (IMS) of mitochondria. These amounts of mitochondrial Sod1 are increased for certain Sod1 mutants that are linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). To date, only little is known about the physiological function of mitochondrial Sod1. Here, we use the model system Saccharomyces cerevisiae to generate cells in which Sod1 is exclusively localized to the IMS. We find that IMS-localized Sod1 can functionally substitute wild type Sod1 and that it even exceeds the protective capacity of wild type Sod1 under conditions of mitochondrial ROS stress. Moreover, we demonstrate that upon expression in yeast cells the common ALS-linked mutant Sod1(G93A) becomes enriched in the mitochondrial fraction and provides an increased protection of cells from mitochondrial oxidative stress. Such an effect cannot be observed for the catalytically inactive mutant Sod1(G85R). Our observations suggest that the targeting of Sod1 to the mitochondrial IMS provides an increased protection against respiration-derived ROS. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Aberrant association of misfolded SOD1 with Na(+)/K(+)ATPase-α3 impairs its activity and contributes to motor neuron vulnerability in ALS

    NARCIS (Netherlands)

    Ruegsegger, Céline; Maharjan, Niran; Goswami, Anand; Filézac de L'Etang, Audrey; Weis, Joachim; Troost, Dirk; Heller, Manfred; Gut, Heinz; Saxena, Smita

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is an adult onset progressive motor neuron disease with no cure. Transgenic mice overexpressing familial ALS associated human mutant SOD1 are a commonly used model for examining disease mechanisms. Presently, it is well accepted that alterations in motor neuron

  10. Prognostic role of ‘prion-like propagation’ in SOD1-linked familial ALS: an alternative view

    Directory of Open Access Journals (Sweden)

    Keizo eSugaya

    2014-10-01

    Full Text Available ‘Prion-like propagation’ has recently been proposed for disease spread in Cu/Zn superoxide dismutase 1 (SOD1-linked familial amyotrophic lateral sclerosis (ALS. Pathological SOD1 conformers are presumed to propagate via cell-to-cell transmission. In this model, the risk-based kinetics of neuronal cell loss over time appears to be represented by a sigmoidal function that reflects the kinetics of intercellular transmission. Here, we describe an alternative view of prion-like propagation in SOD1-linked ALS−its relation to disease prognosis under the protective-aggregation hypothesis. Nucleation-dependent polymerization has been widely accepted as the molecular mechanism of prion propagation. If toxic species of misfolded SOD1, as soluble oligomers, are formed as on-pathway intermediates of nucleation-dependent polymerization, further fibril extension via sequential addition of monomeric mutant SOD1 would be protective against neurodegeneration. This is because the concentration of unfolded mutant SOD1 monomers, which serve as precursor of nucleation and toxic species of mutant SOD1, would decline in proportion to the extent of aggregation. The nucleation process requires that native conformers exist in an unfolded state that may result from escaping the cellular protein quality control machinery. However, prion-like propagation−SOD1 aggregated form self-propagates by imposing its altered conformation on normal SOD1−appears to antagonize the protective role of aggregate growth. The cross-seeding reaction with normal SOD1 would lead to a failure to reduce the concentration of unfolded mutant SOD1 monomers, resulting in continuous nucleation and subsequent generation of toxic species, and influence disease prognosis. In this alternative view, the kinetics of neuronal loss appears to be represented by an exponential function, with decreasing risk reflecting the protective role of aggregate and the potential for cross-seeding reactions between

  11. Misfolded SOD1 associated with motor neuron mitochondria alters mitochondrial shape and distribution prior to clinical onset.

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    Christine Vande Velde

    Full Text Available Mutations in superoxide dismutase (SOD1 are causative for inherited amyotrophic lateral sclerosis. A proportion of SOD1 mutant protein is misfolded onto the cytoplasmic face of mitochondria in one or more spinal cord cell types. By construction of mice in which mitochondrially targeted enhanced green fluorescent protein is selectively expressed in motor neurons, we demonstrate that axonal mitochondria of motor neurons are primary in vivo targets for misfolded SOD1. Mutant SOD1 alters axonal mitochondrial morphology and distribution, with dismutase active SOD1 causing mitochondrial clustering at the proximal side of Schmidt-Lanterman incisures within motor axons and dismutase inactive SOD1 producing aberrantly elongated axonal mitochondria beginning pre-symptomatically and increasing in severity as disease progresses. Somal mitochondria are altered by mutant SOD1, with loss of the characteristic cylindrical, networked morphology and its replacement by a less elongated, more spherical shape. These data indicate that mutant SOD1 binding to mitochondria disrupts normal mitochondrial distribution and size homeostasis as early pathogenic features of SOD1 mutant-mediated ALS.

  12. TFE-induced local unfolding and fibrillation of SOD1: bridging the experiment and simulation studies.

    Science.gov (United States)

    Kumar, Vijay; Prakash, Amresh; Pandey, Preeti; Lynn, Andrew M; Hassan, Md Imtaiyaz

    2018-05-18

    Misfolding and aggregation of Cu, Zn Superoxide dismutase (SOD1) is involved in the neurodegenerative disease, amyotrophic lateral sclerosis. Many studies have shown that metal-depleted, monomeric form of SOD1 displays substantial local unfolding dynamics and is the precursor for aggregation. Here, we have studied the structure and dynamics of different apo monomeric SOD1 variants associated with unfolding and aggregation in aqueous trifluoroethanol (TFE) through experiments and simulation. TFE induces partially unfolded β-sheet-rich extended conformations in these SOD1 variants, which subsequently develops aggregates with fibril-like characteristics. Fibrillation was achieved more easily in disulfide-reduced monomeric SOD1 when compared with wild-type and mutant monomeric SOD1. At higher concentrations of TFE, a native-like structure with the increase in α-helical content was observed. The molecular dynamics simulation results illustrate distinct structural dynamics for different regions of SOD1 variants and show uniform local unfolding of β-strands. The strands protected by the zinc-binding and electrostatic loops were found to unfold first in 20% (v/v) TFE, leading to a partial unfolding of β-strands 4, 5, and 6 which are prone to aggregation. Our results thus shed light on the role of local unfolding and conformational dynamics in SOD1 misfolding and aggregation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  13. Biological effects of CCS in the absence of SOD1 enzyme activation: implications for disease in a mouse model for ALS.

    Science.gov (United States)

    Proescher, Jody B; Son, Marjatta; Elliott, Jeffrey L; Culotta, Valeria C

    2008-06-15

    The CCS copper chaperone is critical for maturation of Cu, Zn-superoxide dismutase (SOD1) through insertion of the copper co-factor and oxidization of an intra-subunit disulfide. The disulfide helps stabilize the SOD1 polypeptide, which can be particularly important in cases of amyotrophic lateral sclerosis (ALS) linked to misfolding of mutant SOD1. Surprisingly, however, over-expressed CCS was recently shown to greatly accelerate disease in a G93A SOD1 mouse model for ALS. Herein we show that disease in these G93A/CCS mice correlates with incomplete oxidation of the SOD1 disulfide. In the brain and spinal cord, CCS over-expression failed to enhance oxidation of the G93A SOD1 disulfide and if anything, effected some accumulation of disulfide-reduced SOD1. This effect was mirrored in culture with a C244,246S mutant of CCS that has the capacity to interact with SOD1 but can neither insert copper nor oxidize the disulfide. In spite of disulfide effects, there was no evidence for increased SOD1 aggregation. If anything, CCS over-expression prevented SOD1 misfolding in culture as monitored by detergent insolubility. This protection against SOD1 misfolding does not require SOD1 enzyme activation as the same effect was obtained with the C244,246S allele of CCS. In the G93A SOD1 mouse, CCS over-expression was likewise associated with a lack of obvious SOD1 misfolding marked by detergent insolubility. CCS over-expression accelerates SOD1-linked disease without the hallmarks of misfolding and aggregation seen in other mutant SOD1 models. These studies are the first to indicate biological effects of CCS in the absence of SOD1 enzymatic activation.

  14. Import, maturation, and function of SOD1 and its copper chaperone CCS in the mitochondrial intermembrane space.

    Science.gov (United States)

    Kawamata, Hibiki; Manfredi, Giovanni

    2010-11-01

    Cu, Zn, superoxide dismutase (SOD1) is a ubiquitous enzyme localized in multiple cellular compartments, including mitochondria, where it concentrates in the intermembrane space (IMS). Similar to other small IMS proteins, the import and retention of SOD1 in the IMS is linked to its folding and maturation, involving the formation of critical intra- and intermolecular disulfide bonds. Therefore, the cysteine residues of SOD1 play a fundamental role in its IMS localization. IMS import of SOD1 involves its copper chaperone, CCS, whose mitochondrial distribution is regulated by the Mia40/Erv1 disulfide relay system in a redox-dependent manner: CCS promotes SOD1 maturation and retention in the IMS. The function of SOD1 in the IMS is still unknown, but it is plausible that it serves to remove superoxide released from the mitochondrial respiratory chain. Mutations in SOD1 cause familial amyotrophic lateral sclerosis (ALS), whose pathologic features include mitochondrial bioenergetic dysfunction. Mutant SOD1 localization in the IMS is not dictated by oxygen concentration and the Mia40/Erv1 system, but is primarily dependent on aberrant protein folding and aggregation. Mutant SOD1 localization and aggregation in the IMS might cause the mitochondrial abnormalities observed in familial ALS and could play a significant role in disease pathogenesis.

  15. Knocking down metabotropic glutamate receptor 1 improves survival and disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Milanese, Marco; Giribaldi, Francesco; Melone, Marcello; Bonifacino, Tiziana; Musante, Ilaria; Carminati, Enrico; Rossi, Pia I A; Vergani, Laura; Voci, Adriana; Conti, Fiorenzo; Puliti, Aldamaria; Bonanno, Giambattista

    2014-04-01

    Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1(G93A)), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1(G93A) background (SOD1(G93A)Grm1(crv4/+)), by crossing the SOD1(G93A) mutant mouse with the Grm1(crv4/+) mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1(G93A)Grm1(crv4/+) mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1(G93A) mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1(G93A)Grm1(crv4/+)compared to SOD1(G93A) mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact on mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  16. The effect of SOD1 mutation on cellular bioenergetic profile and viability in response to oxidative stress and influence of mutation-type.

    Directory of Open Access Journals (Sweden)

    Katie Richardson

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disorder characterized by the progressive degeneration of motor neurons. Substantial evidence implicates oxidative stress and mitochondrial dysfunction as early events in disease progression. Our aim was to ascertain whether mutation of the SOD1 protein increases metabolic functional susceptibility to oxidative stress. Here we used a motor neuron-like cell line (NSC34 stably transfected with various human mutant SOD1 transgenes (G93A, G37R, H48Q to investigate the impact of oxidative stress on cell viability and metabolic function within intact cells. NSC34 cells expressing mutant SOD1 showed a dose dependent reduction in cell viability when exposed to oxidative stress induced by hydrogen peroxide, with variation between mutations. The G93A transfectants showed greater cell death and LDH release compared to cells transfected with the other SOD1 mutations, and H48Q showed an accelerated decline at later time points. Differences in mitochondrial bioenergetics, including mitochondrial respiration, coupling efficiency and proton leak, were identified between the mutations, consistent with the differences observed in viability. NSC34 cells expressing G93A SOD1 displayed reduced coupled respiration and mitochondrial membrane potential compared to controls. Furthermore, the G93A mutation had significantly increased metabolic susceptibility to oxidative stress, with hydrogen peroxide increasing ROS production, reducing both cellular oxygen consumption and glycolytic flux in the cell. This study highlights bioenergetic defects within a cellular model of ALS and suggests that oxidative stress is not only detrimental to oxygen consumption but also glycolytic flux, which could lead to an energy deficit in the cell.

  17. Sod1 deficiency reduces incubation time in mouse models of prion disease.

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    Shaheen Akhtar

    Full Text Available Prion infections, causing neurodegenerative conditions such as Creutzfeldt-Jakob disease and kuru in humans, scrapie in sheep and BSE in cattle are characterised by prolonged and variable incubation periods that are faithfully reproduced in mouse models. Incubation time is partly determined by genetic factors including polymorphisms in the prion protein gene. Quantitative trait loci studies in mice and human genome-wide association studies have confirmed that multiple genes are involved. Candidate gene approaches have also been used and identified App, Il1-r1 and Sod1 as affecting incubation times. In this study we looked for an association between App, Il1-r1 and Sod1 representative SNPs and prion disease incubation time in the Northport heterogeneous stock of mice inoculated with the Chandler/RML prion strain. No association was seen with App, however, significant associations were seen with Il1-r1 (P = 0.02 and Sod1 (P<0.0001 suggesting that polymorphisms at these loci contribute to the natural variation observed in incubation time. Furthermore, following challenge with Chandler/RML, ME7 and MRC2 prion strains, Sod1 deficient mice showed highly significant reductions in incubation time of 20, 13 and 24%, respectively. No differences were detected in Sod1 expression or activity. Our data confirm the protective role of endogenous Sod1 in prion disease.

  18. ALS-associated mutation SOD1G93A leads to abnormal mitochondrial dynamics in osteocytes.

    Science.gov (United States)

    Wang, Huan; Yi, Jianxun; Li, Xuejun; Xiao, Yajuan; Dhakal, Kamal; Zhou, Jingsong

    2018-01-01

    mitochondrial fission, but suppresses the fusion activity. Our data provide the first evidence that mitochondria show abnormality in osteocytes derived from an ALS mouse model. The accumulation of mutant SOD1 G93A protein inside mitochondria directly causes dysfunction in mitochondrial dynamics in cultured MLO-Y4 osteocytes. In addition, the ALS mutation SOD1 G93A -mediated dysfunction in mitochondrial dynamics is associated with an enhanced apoptosis in osteocytes, which could be a potential mechanism underlying the bone loss during ALS progression. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Javier Sábado

    2014-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is an adult-onset progressive neurodegenerative disease affecting upper and lower motoneurons (MNs. Although the motor phenotype is a hallmark for ALS, there is increasing evidence that systems other than the efferent MN system can be involved. Mutations of superoxide dismutase 1 (SOD1 gene cause a proportion of familial forms of this disease. Misfolding and aggregation of mutant SOD1 exert neurotoxicity in a noncell autonomous manner, as evidenced in studies using transgenic mouse models. Here, we used the SOD1G93A mouse model for ALS to detect, by means of conformational-specific anti-SOD1 antibodies, whether misfolded SOD1-mediated neurotoxicity extended to neuronal types other than MNs. We report that large dorsal root ganglion (DRG proprioceptive neurons accumulate misfolded SOD1 and suffer a degenerative process involving the inflammatory recruitment of macrophagic cells. Degenerating sensory axons were also detected in association with activated microglial cells in the spinal cord dorsal horn of diseased animals. As large proprioceptive DRG neurons project monosynaptically to ventral horn MNs, we hypothesise that a prion-like mechanism may be responsible for the transsynaptic propagation of SOD1 misfolding from ventral horn MNs to DRG sensory neurons.

  20. Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity in E. coli via Mutation of Free Cysteine Residues

    Directory of Open Access Journals (Sweden)

    Kun Zhang

    2017-01-01

    Full Text Available Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1 can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields in E. coli. In this study, a wild-type hSOD1 (wtSOD1 and three mutant SOD1s (mhSOD1s, in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S increased the expression of soluble hSOD1 in E. coli whereas substitution of the internal Cys6 (mhSOD1/C6S decreased it. Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

  1. Resveratrol Derivative-Rich Melinjo Seed Extract Attenuates Skin Atrophy in Sod1-Deficient Mice

    Directory of Open Access Journals (Sweden)

    Kenji Watanabe

    2015-01-01

    Full Text Available The oxidative damages induced by a redox imbalance cause age-related changes in cells and tissues. Superoxide dismutase (SOD enzymes play a pivotal role in the antioxidant system and they also catalyze superoxide radicals. Since the loss of cytoplasmic SOD (SOD1 resulted in aging-like phenotypes in several types of murine tissue, SOD1 is essential for the maintenance of tissue homeostasis. Melinjo (Gnetum gnemon Linn seed extract (MSE contains trans-resveratrol (RSV and resveratrol derivatives, including gnetin C, gnemonoside A, and gnemonoside D. MSE intake also exerts no adverse events in human study. In the present studies, we investigated protective effects of MSE on age-related skin pathologies in mice. Orally MSE and RSV treatment reversed the skin thinning associated with increased oxidative damage in the Sod1−/− mice. Furthermore, MSE and RSV normalized gene expression of Col1a1 and p53 and upregulated gene expression of Sirt1 in skin tissues. In vitro experiments revealed that RSV significantly promoted the viability of Sod1−/− fibroblasts. These finding demonstrated that RSV in MSE stably suppressed an intrinsic superoxide generation in vivo and in vitro leading to protecting skin damages. RSV derivative-rich MSE may be a powerful food of treatment for age-related skin diseases caused by oxidative damages.

  2. Optimised and rapid pre-clinical screening in the SOD1(G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS.

    Directory of Open Access Journals (Sweden)

    Richard J Mead

    Full Text Available The human SOD1(G93A transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS. In that time, a great many insights into the toxicity of mutant SOD1 have been gained using this and other mutant SOD transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (3-4 months is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6 SOD1(G93A transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved.

  3. Optimised and rapid pre-clinical screening in the SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS).

    Science.gov (United States)

    Mead, Richard J; Bennett, Ellen J; Kennerley, Aneurin J; Sharp, Paul; Sunyach, Claire; Kasher, Paul; Berwick, Jason; Pettmann, Brigitte; Battaglia, Guiseppe; Azzouz, Mimoun; Grierson, Andrew; Shaw, Pamela J

    2011-01-01

    The human SOD1(G93A) transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS). In that time, a great many insights into the toxicity of mutant SOD1 have been gained using this and other mutant SOD transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (3-4 months) is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6) SOD1(G93A) transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved.

  4. Radioimmunoassay of serum SOD-1 in the elderly

    International Nuclear Information System (INIS)

    Ren Yu'an; Lin Baoyuan

    1995-01-01

    A RIA for serum SOD-1 was performed in 168 aged subjects including 47 aged healthy subjects and 121 aged patients as well as in 35 healthy young and adult cases serving as control. The measuring results are as follows: serum SOD-1 value of 47 aged healthy subjects are 279.42 +- 89.38 μg/l, 121 aged patients are 405.10 +- 181.29 μg/l, and 35 young and adult cases are 185.80 +- 56.44 μg/l. It shows the obvious difference between the aged group and control group. It also shows the obvious difference between the aged healthy subjects and aged patients. In addition, the clinical evaluation is also discussed

  5. Phenotype of transgenic mice carrying a very low copy number of the mutant human G93A superoxide dismutase-1 gene associated with amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Jeffrey S Deitch

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease of the motor neuron. While most cases of ALS are sporadic, 10% are familial (FALS with 20% of FALS caused by a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1. There is variability in sporadic ALS as well as FALS where even within the same family some siblings with the same mutation do not manifest disease. A transgenic (Tg mouse model of FALS containing 25 copies of the mutant human SOD1 gene demonstrates motor neuron pathology and progressive weakness similar to ALS patients, leading to death at approximately 130 days. The onset of symptoms and survival of these transgenic mice are directly related to the number of copies of the mutant gene. We report the phenotype of a very low expressing (VLE G93A SOD1 Tg carrying only 4 copies of the mutant G93ASOD1 gene. While weakness can start at 9 months, only 74% of mice 18 months or older demonstrate disease. The VLE mice show decreased motor neurons compared to wild-type mice as well as increased cytoplasmic translocation of TDP-43. In contrast to the standard G93A SOD1 Tg mouse which always develops motor weakness leading to death, not all VLE animals manifested clinical disease or shortened life span. In fact, approximately 20% of mice older than 24 months had no motor symptoms and only 18% of VLE mice older than 22 months reached end stage. Given the variable penetrance of clinical phenotype, prolonged survival, and protracted loss of motor neurons the VLE mouse provides a new tool that closely mimics human ALS. This tool will allow the study of pathologic events over time as well as the study of genetic and environmental modifiers that may not be causative, but can exacerbate or accelerate motor neuron disease.

  6. In-vivo effects of knocking-down metabotropic glutamate receptor 5 in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Bonifacino, Tiziana; Cattaneo, Luca; Gallia, Elena; Puliti, Aldamaria; Melone, Marcello; Provenzano, Francesca; Bossi, Simone; Musante, Ilaria; Usai, Cesare; Conti, Fiorenzo; Bonanno, Giambattista; Milanese, Marco

    2017-09-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1 G93A mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1 G93A mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1 G93A mice with reduced expression of mGluR5 (SOD1 G93A Grm5 -/+ ) by crossing the SOD1 G93A mutant mouse with the mGluR5 heterozigous Grm5 -/+ mouse. SOD1 G93A Grm5 -/+ mice showed prolonged survival probability and delayed pathology onset. These effects were associated to enhanced number of preserved MNs, decreased astrocyte and microglia activation, reduced cytosolic free Ca 2+ concentration, and regularization of abnormal Glu release in the spinal cord of SOD1 G93A Grm5 -/+ mice. Unexpectedly, only male SOD1 G93A Grm5 -/+ mice showed improved motor skills during disease progression vs. SOD1 G93A mice, while SOD1 G93A Grm5 -/+ females did not. These results demonstrate that a lower constitutive level of mGluR5 has a significant positive impact in mice with ALS and support the idea that blocking Group I mGluRs may represent a potentially effective pharmacological approach to the disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Canine degenerative myelopathy: biochemical characterization of superoxide dismutase 1 in the first naturally occurring non-human amyotrophic lateral sclerosis model.

    Science.gov (United States)

    Crisp, Matthew J; Beckett, Jeffrey; Coates, Joan R; Miller, Timothy M

    2013-10-01

    Mutations in canine superoxide dismutase 1 (SOD1) have recently been shown to cause canine degenerative myelopathy, a disabling neurodegenerative disorder affecting specific breeds of dogs characterized by progressive motor neuron loss and paralysis until death, or more common, euthanasia. This discovery makes canine degenerative myelopathy the first and only naturally occurring non-human model of amyotrophic lateral sclerosis (ALS), closely paralleling the clinical, pathological, and genetic presentation of its human counterpart, SOD1-mediated familial ALS. To further understand the biochemical role that canine SOD1 plays in this disease and how it may be similar to human SOD1, we characterized the only two SOD1 mutations described in affected dogs to date, E40K and T18S. We show that a detergent-insoluble species of mutant SOD1 is present in spinal cords of affected dogs that increases with disease progression. Our in vitro results indicate that both canine SOD1 mutants form enzymatically active dimers, arguing against a loss of function in affected homozygous animals. Further studies show that these mutants, like most human SOD1 mutants, have an increased propensity to form aggregates in cell culture, with 10-20% of cells possessing visible aggregates. Creation of the E40K mutation in human SOD1 recapitulates the normal enzymatic activity but not the aggregation propensity seen with the canine mutant. Our findings lend strong biochemical support to the toxic role of SOD1 in canine degenerative myelopathy and establish close parallels for the role mutant SOD1 plays in both canine and human disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Improving the Delivery of SOD1 Antisense Oligonucleotides to Motor Neurons Using Calcium Phosphate-Lipid Nanoparticles

    Directory of Open Access Journals (Sweden)

    Liyu Chen

    2017-08-01

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disease affecting the upper and lower motor neurons in the motor cortex and spinal cord. Abnormal accumulation of mutant superoxide dismutase I (SOD1 in motor neurons is a pathological hallmark of some forms of the disease. We have shown that the orderly progression of the disease may be explained by misfolded SOD1 cell-to-cell propagation, which is reliant upon its active endogenous synthesis. Reducing the levels of SOD1 is therefore a promising therapeutic approach. Antisense oligonucleotides (ASOs can efficiently silence proteins with gain-of-function mutations. However, naked ASOs have a short circulation half-life and are unable to cross the blood brain barrier (BBB warranting the use of a drug carrier for effective delivery. In this study, calcium phosphate lipid coated nanoparticles (CaP-lipid NPs were developed for delivery of SOD1 ASO to motor neurons. The most promising nanoparticle formulation (Ca/P ratio of 100:1, had a uniform spherical core–shell morphology with an average size of 30 nm, and surface charge (ζ-potential of −4.86 mV. The encapsulation efficiency of ASO was 48% and stability studies found the particle to be stable over a period of 20 days. In vitro experiments demonstrated that the negatively charged ASO-loaded CaP-lipid NPs could effectively deliver SOD1-targeted ASO into a mouse motor neuron-like cell line (NSC-34 through endocytosis and significantly down-regulated SOD1 expression in HEK293 cells. The CaP-lipid NPs exhibited a pH-dependant dissociation, suggesting that that the acidification of lysosomes is the likely mechanism responsible for facilitating intracellular ASO release. To demonstrate tissue specific delivery and localization of these NPs we performed in vivo microinjections into zebrafish. Successful delivery of these NPs was confirmed for the zebrafish brain, the blood stream, and the spinal cord. These results suggest that Ca

  9. Muscle Expression of SOD1G93A Triggers the Dismantlement of Neuromuscular Junction via PKC-Theta.

    Science.gov (United States)

    Dobrowolny, Gabriella; Martini, Martina; Scicchitano, Bianca Maria; Romanello, Vanina; Boncompagni, Simona; Nicoletti, Carmine; Pietrangelo, Laura; De Panfilis, Simone; Catizone, Angela; Bouchè, Marina; Sandri, Marco; Rudolf, Rüdiger; Protasi, Feliciano; Musarò, Antonio

    2018-04-20

    Neuromuscular junction (NMJ) represents the morphofunctional interface between muscle and nerve. Several chronic pathologies such as aging and neurodegenerative diseases, including muscular dystrophy and amyotrophic lateral sclerosis, display altered NMJ and functional denervation. However, the triggers and the molecular mechanisms underlying the dismantlement of NMJ remain unclear. Here we provide evidence that perturbation in redox signaling cascades, induced by muscle-specific accumulation of mutant SOD1 G93A in transgenic MLC/SOD1 G93A mice, is causally linked to morphological alterations of the neuromuscular presynaptic terminals, high turnover rate of acetylcholine receptor, and NMJ dismantlement. The analysis of potential molecular mechanisms that mediate the toxic activity of SOD1 G93A revealed a causal link between protein kinase Cθ (PKCθ) activation and NMJ disintegration. The study discloses the molecular mechanism that triggers functional denervation associated with the toxic activity of muscle SOD1 G93A expression and suggests the possibility of developing a new strategy to counteract age- and pathology-associated denervation based on pharmacological inhibition of PKCθ activity. Collectively, these data indicate that muscle-specific accumulation of oxidative damage can affect neuromuscular communication and induce NMJ dismantlement through a PKCθ-dependent mechanism. Antioxid. Redox Signal. 28, 1105-1119.

  10. Defining SOD1 ALS natural history to guide therapeutic clinical trial design.

    Science.gov (United States)

    Bali, Taha; Self, Wade; Liu, Jingxia; Siddique, Teepu; Wang, Leo H; Bird, Thomas D; Ratti, Elena; Atassi, Nazem; Boylan, Kevin B; Glass, Jonathan D; Maragakis, Nicholas J; Caress, James B; McCluskey, Leo F; Appel, Stanley H; Wymer, James P; Gibson, Summer; Zinman, Lorne; Mozaffar, Tahseen; Callaghan, Brian; McVey, April L; Jockel-Balsarotti, Jennifer; Allred, Peggy; Fisher, Elena R; Lopate, Glenn; Pestronk, Alan; Cudkowicz, Merit E; Miller, Timothy M

    2017-02-01

    Understanding the natural history of familial amyotrophic lateral sclerosis (ALS) caused by SOD1 mutations (ALS SOD1 ) will provide key information for optimising clinical trials in this patient population. To establish an updated natural history of ALS SOD1 . Retrospective cohort study from 15 medical centres in North America evaluated records from 175 patients with ALS with genetically confirmed SOD1 mutations, cared for after the year 2000. Age of onset, survival, ALS Functional Rating Scale (ALS-FRS) scores and respiratory function were analysed. Patients with the A4V (Ala-Val) SOD1 mutation (SOD1 A4V ), the largest mutation population in North America with an aggressive disease progression, were distinguished from other SOD1 mutation patients (SOD1 non-A4V ) for analysis. Mean age of disease onset was 49.7±12.3 years (mean±SD) for all SOD1 patients, with no statistical significance between SOD1 A4V and SOD1 non-A4V (p=0.72, Kruskal-Wallis). Total SOD1 patient median survival was 2.7 years. Mean disease duration for all SOD1 was 4.6±6.0 and 1.4±0.7 years for SOD1 A4V . SOD1 A4V survival probability (median survival 1.2 years) was significantly decreased compared with SOD1 non-A4V (median survival 6.8 years; p<0.0001, log-rank). A statistically significant increase in ALS-FRS decline in SOD1 A4V compared with SOD1 non-A4V participants (p=0.02) was observed, as well as a statistically significant increase in ALS-forced vital capacity decline in SOD1 A4V compared with SOD1 non-A4V (p=0.02). SOD1 A4V is an aggressive, but relatively homogeneous form of ALS. These SOD1-specific ALS natural history data will be important for the design and implementation of clinical trials in the ALS SOD1 patient population. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  11. Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis.

    Science.gov (United States)

    Parone, Philippe A; Da Cruz, Sandrine; Han, Joo Seok; McAlonis-Downes, Melissa; Vetto, Anne P; Lee, Sandra K; Tseng, Eva; Cleveland, Don W

    2013-03-13

    Mitochondria have been proposed as targets for toxicity in amyotrophic lateral sclerosis (ALS), a progressive, fatal adult-onset neurodegenerative disorder characterized by the selective loss of motor neurons. A decrease in the capacity of spinal cord mitochondria to buffer calcium (Ca(2+)) has been observed in mice expressing ALS-linked mutants of SOD1 that develop motor neuron disease with many of the key pathological hallmarks seen in ALS patients. In mice expressing three different ALS-causing SOD1 mutants, we now test the contribution of the loss of mitochondrial Ca(2+)-buffering capacity to disease mechanism(s) by eliminating ubiquitous expression of cyclophilin D, a critical regulator of Ca(2+)-mediated opening of the mitochondrial permeability transition pore that determines mitochondrial Ca(2+) content. A chronic increase in mitochondrial buffering of Ca(2+) in the absence of cyclophilin D was maintained throughout disease course and was associated with improved mitochondrial ATP synthesis, reduced mitochondrial swelling, and retention of normal morphology. This was accompanied by an attenuation of glial activation, reduction in levels of misfolded SOD1 aggregates in the spinal cord, and a significant suppression of motor neuron death throughout disease. Despite this, muscle denervation, motor axon degeneration, and disease progression and survival were unaffected, thereby eliminating mutant SOD1-mediated loss of mitochondrial Ca(2+) buffering capacity, altered mitochondrial morphology, motor neuron death, and misfolded SOD1 aggregates, as primary contributors to disease mechanism for fatal paralysis in these models of familial ALS.

  12. Genetic biomarkers for ALS disease in transgenic SOD1(G93A mice.

    Directory of Open Access Journals (Sweden)

    Ana C Calvo

    Full Text Available The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1(G93A mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturer's protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1(G93A mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10 could be considered potential genetic biomarkers of longevity in transgenic SOD1(G93A mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies.

  13. Oxidation of the tryptophan 32 residue of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity triggers the non-amyloid aggregation of the enzyme.

    Science.gov (United States)

    Coelho, Fernando R; Iqbal, Asif; Linares, Edlaine; Silva, Daniel F; Lima, Filipe S; Cuccovia, Iolanda M; Augusto, Ohara

    2014-10-31

    The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1(WT) and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp(32) residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp(32) residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1(WT) and hSOD1(G93A) mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp(32) residue in the process. The results showed that Trp(32) residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp(32) residue (bovine SOD1 and hSOD1(W32F) mutant). The results support a role for the oxidation products of the hSOD1-Trp(32) residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. ApoSOD1 lacking dismutase activity neuroprotects motor neurons exposed to beta-methylamino-L-alanine through the Ca2+/Akt/ERK1/2 prosurvival pathway

    Science.gov (United States)

    Petrozziello, Tiziana; Secondo, Agnese; Tedeschi, Valentina; Esposito, Alba; Sisalli, MariaJosè; Scorziello, Antonella; Di Renzo, Gianfranco; Annunziato, Lucio

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe human adult-onset neurodegenerative disease affecting lower and upper motor neurons. In >20% of cases, the familial form of ALS is caused by mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1). Interestingly, administration of wild-type SOD1 to SOD1G93A transgenic rats ameliorates motor symptoms through an unknown mechanism. Here we investigated whether the neuroprotective effects of SOD1 are due to the Ca2+-dependent activation of such prosurvival signaling pathway and not to its catalytic activity. To this aim, we also examined the mechanism of neuroprotective action of ApoSOD1, the metal-depleted state of SOD1 that lacks dismutase activity, in differentiated motor neuron-like NSC-34 cells and in primary motor neurons exposed to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA). Preincubation of ApoSOD1 and SOD1, but not of human recombinant SOD1G93A, prevented cell death in motor neurons exposed to L-BMAA. Moreover, ApoSOD1 elicited ERK1/2 and Akt phosphorylation in motor neurons through an early increase of intracellular Ca2+ concentration ([Ca2+]i). Accordingly, inhibition of ERK1/2 by siMEK1 and PD98059 counteracted ApoSOD1- and SOD1-induced neuroprotection. Similarly, transfection of the dominant-negative form of Akt in NSC-34 motor neurons and treatment with the selective PI3K inhibitor LY294002 prevented ApoSOD1- and SOD1-mediated neuroprotective effects in L-BMAA-treated motor neurons. Furthermore, ApoSOD1 and SOD1 prevented the expression of the two markers of L-BMAA-induced ER stress GRP78 and caspase-12. Collectively, our data indicate that ApoSOD1, which is devoid of any catalytic dismutase activity, exerts a neuroprotective effect through an early activation of Ca2+/Akt/ERK1/2 pro-survival pathway that, in turn, prevents ER stress in a neurotoxic model of ALS. PMID:28085149

  15. SOD1 Transcriptional and Posttranscriptional Regulation and Its Potential Implications in ALS

    Directory of Open Access Journals (Sweden)

    Pamela Milani

    2011-01-01

    Full Text Available Copper-zinc superoxide dismutase (SOD1 is a detoxifying enzyme localized in the cytosol, nucleus, peroxisomes, and mitochondria. The discovery that mutations in SOD1 gene cause a subset of familial amyotrophic lateral sclerosis (FALS has attracted great attention, and studies to date have been mainly focused on discovering mutations in the coding region and investigation at protein level. Considering that changes in SOD1 mRNA levels have been associated with sporadic ALS (SALS, a molecular understanding of the processes involved in the regulation of SOD1 gene expression could not only unravel novel regulatory pathways that may govern cellular phenotypes and changes in diseases but also might reveal therapeutic targets and treatments. This review seeks to provide an overview of SOD1 gene structure and of the processes through which SOD1 transcription is controlled. Furthermore, we emphasize the importance to focus future researches on investigating posttranscriptional mechanisms and their relevance to ALS.

  16. FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Deng, Han-Xiang; Zhai, Hong; Bigio, Eileen H; Yan, Jianhua; Fecto, Faisal; Ajroud, Kaouther; Mishra, Manjari; Ajroud-Driss, Senda; Heller, Scott; Sufit, Robert; Siddique, Nailah; Mugnaini, Enrico; Siddique, Teepu

    2010-06-01

    Amyotrophic lateral sclerosis (ALS) is a fatal disorder of motor neuron degeneration. Most cases of ALS are sporadic (SALS), but about 5 to 10% of ALS cases are familial (FALS). Recent studies have shown that mutations in FUS are causal in approximately 4 to 5% of FALS and some apparent SALS cases. The pathogenic mechanism of the mutant FUS-mediated ALS and potential roles of FUS in non-FUS ALS remain to be investigated. Immunostaining was performed on postmortem spinal cords from 78 ALS cases, including SALS (n = 52), ALS with dementia (ALS/dementia, n = 10), and FALS (n = 16). In addition, postmortem brains or spinal cords from 22 cases with or without frontotemporal lobar degeneration were also studied. In total, 100 cases were studied. FUS-immunoreactive inclusions were observed in spinal anterior horn neurons in all SALS and FALS cases, except for those with SOD1 mutations. The FUS-containing inclusions were also immunoreactive with antibodies to TDP43, p62, and ubiquitin. A fraction of tested FUS antibodies recognized FUS inclusions, and specific antigen retrieval protocol appeared to be important for detection of the skein-like FUS inclusions. Although mutations in FUS account for only a small fraction of FALS and SALS, our data suggest that FUS protein may be a common component of the cellular inclusions in non-SOD1 ALS and some other neurodegenerative conditions, implying a shared pathogenic pathway underlying SALS, non-SOD1 FALS, ALS/dementia, and related disorders. Our data also indicate that SOD1-linked ALS may have a pathogenic pathway distinct from SALS and other types of FALS.

  17. Imaging of glial cell morphology, SOD1 distribution and elemental composition in the brainstem and hippocampus of the ALS hSOD1G93A rat.

    Science.gov (United States)

    Stamenković, Stefan; Dučić, Tanja; Stamenković, Vera; Kranz, Alexander; Andjus, Pavle R

    2017-08-15

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor and cognitive domains of the CNS. Mutations in the Cu,Zn-superoxide dismutase (SOD1) cause 20% of familial ALS and provoke formation of intracellular aggregates and copper and zinc unbinding, leading to glial activation and neurodegeneration. Therefore, we investigated glial cell morphology, intracellular SOD1 distribution, and elemental composition in the brainstem and hippocampus of the hSOD1 G93A transgenic rat model of ALS. Immunostaining for astrocytes, microglia and SOD1 revealed glial proliferation and progressive tissue accumulation of SOD1 in both brain regions of ALS rats starting already at the presymptomatic stage. Glial cell morphology analysis in the brainstem of ALS rats revealed astrocyte activation occurring before disease symptoms onset, followed by activation of microglia. Hippocampal ALS astrocytes exhibited an identical reactive profile, while microglial morphology was unchanged. Additionally, ALS brainstem astrocytes demonstrated progressive SOD1 accumulation in the cell body and processes, while microglial SOD1 levels were reduced and its distribution limited to distal cell processes. In the hippocampus both glial cell types exhibited SOD1 accumulation in the cell body. X-ray fluorescence imaging revealed decreased P and increased Ca, Cl, K, Ni, Cu and Zn in the brainstem, and higher levels of Cl, Ni and Cu, but lower levels of Zn in the hippocampus of symptomatic ALS rats. These results bring new insights into the glial response during disease development and progression in motor as well as in non-motor CNS structures, and indicate disturbed tissue elemental homeostasis as a prominent hallmark of disease pathology. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Primary glia expressing the G93A-SOD1 mutation present a neuroinflammatory phenotype and provide a cellular system for studies of glial inflammation

    Directory of Open Access Journals (Sweden)

    Qi Min

    2006-01-01

    Full Text Available Abstract Detailed study of glial inflammation has been hindered by lack of cell culture systems that spontaneously demonstrate the "neuroinflammatory phenotype". Mice expressing a glycine → alanine substitution in cytosolic Cu, Zn-superoxide dismutase (G93A-SOD1 associated with familial amyotrophic lateral sclerosis (ALS demonstrate age-dependent neuroinflammation associated with broad-spectrum cytokine, eicosanoid and oxidant production. In order to more precisely study the cellular mechanisms underlying glial activation in the G93A-SOD1 mouse, primary astrocytes were cultured from 7 day mouse neonates. At this age, G93A-SOD1 mice demonstrated no in vivo hallmarks of neuroinflammation. Nonetheless astrocytes cultured from G93A-SOD1 (but not wild-type human SOD1-expressing transgenic mouse pups demonstrated a significant elevation in either the basal or the tumor necrosis alpha (TNFα-stimulated levels of proinflammatory eicosanoids prostaglandin E2 (PGE2 and leukotriene B4 (LTB4; inducible nitric oxide synthase (iNOS and •NO (indexed by nitrite release into the culture medium; and protein carbonyl products. Specific cytokine- and TNFα death-receptor-associated components were similarly upregulated in cultured G93A-SOD1 cells as assessed by multiprobe ribonuclease protection assays (RPAs for their mRNA transcripts. Thus, endogenous glial expression of G93A-SOD1 produces a metastable condition in which glia are more prone to enter an activated neuroinflammatory state associated with broad-spectrum increased production of paracrine-acting substances. These findings support a role for active glial involvement in ALS and may provide a useful cell culture tool for the study of glial inflammation.

  19. Stathmin 1/2-triggered microtubule loss mediates Golgi fragmentation in mutant SOD1 motor neurons

    NARCIS (Netherlands)

    Bellouze, Sarah; Baillat, Gilbert; Buttigieg, Dorothée; de la Grange, Pierre; Rabouille, Catherine; Haase, Georg

    2016-01-01

    BACKGROUND: Pathological Golgi fragmentation represents a constant pre-clinical feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) but its molecular mechanisms remain hitherto unclear. RESULTS: Here, we show that the severe Golgi fragmentation in transgenic

  20. Inhibitors of SOD1 Interaction as an Approach to Slow the Progressive Spread of ALS Symptoms

    Science.gov (United States)

    2016-07-01

    the progression of ALS caused by mutations in this protein . To accomplish this goal, we developed an assay that is based on the observation that the...force. In our assay , this force is the normal interaction that occurs when 2 individual SOD1 proteins come together to form a normal active enzyme...Using recombinant DNA, we create fusion proteins of SOD1 and each half of the luciferase enzyme. In the past year, we have characterized and optimized

  1. Genetic disruption of SOD1 gene causes glucose intolerance and impairs β-cell function.

    Science.gov (United States)

    Muscogiuri, Giovanna; Salmon, Adam B; Aguayo-Mazzucato, Cristina; Li, Mengyao; Balas, Bogdan; Guardado-Mendoza, Rodolfo; Giaccari, Andrea; Reddick, Robert L; Reyna, Sara M; Weir, Gordon; Defronzo, Ralph A; Van Remmen, Holly; Musi, Nicolas

    2013-12-01

    Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with intraperitoneal glucose tolerance tests. Peripheral and hepatic insulin sensitivity was quantitated with the euglycemic-hyperinsulinemic clamp. β-Cell function was determined with the hyperglycemic clamp and morphometric analysis of pancreatic islets. Genetic ablation of SOD1 caused glucose intolerance, which was associated with reduced in vivo β-cell insulin secretion and decreased β-cell volume. Peripheral and hepatic insulin sensitivity were not significantly altered in SOD1-null mice. High-fat diet caused glucose intolerance in WT mice but did not further worsen the glucose intolerance observed in standard chow-fed SOD1-null mice. Our findings suggest that oxidative stress per se does not play a major role in the pathogenesis of insulin resistance and demonstrate that oxidative stress caused by SOD1 ablation leads to glucose intolerance secondary to β-cell dysfunction.

  2. Unraveling ALS due to SOD1 mutation through the combination of brain and cervical cord MRI.

    Science.gov (United States)

    Agosta, Federica; Spinelli, Edoardo Gioele; Marjanovic, Ivan V; Stevic, Zorica; Pagani, Elisabetta; Valsasina, Paola; Salak-Djokic, Biljana; Jankovic, Milena; Lavrnic, Dragana; Kostic, Vladimir S; Filippi, Massimo

    2018-02-20

    To explore structural and functional changes of the brain and cervical cord in patients with amyotrophic lateral sclerosis (ALS) due to mutation in the superoxide dismutase ( SOD1 ) gene compared with sporadic ALS. Twenty patients with SOD1 ALS, 11 with sporadic ALS, and 33 healthy controls underwent clinical evaluation and brain MRI. Cortical thickness analysis, diffusion tensor MRI of the corticospinal tracts (CST) and corpus callosum, and resting-state functional connectivity were performed. Patients with ALS also underwent cervical cord MRI to evaluate cord cross-sectional area and magnetization transfer ratio (MTR). Patients with SOD1 ALS showed longer disease duration and slower rate of functional decline relative to those with sporadic ALS. No cortical thickness abnormalities were found in patients with ALS compared with controls. Fractional anisotropy showed that sporadic ALS patients had significant CST damage relative to both healthy controls ( p = 0.001-0.02) and SOD1-related ALS ( p = 0.05), although the latter showed alterations that were intermediate between controls and sporadic ALS. Functional hyperconnectivity of the motor cortex in the sensorimotor network was observed in patients with sporadic ALS relative to controls. Conversely, patients with SOD1 ALS showed lower cord cross-sectional area along the whole cervical cord relative to those with sporadic ALS ( p ALS showed cervical cord atrophy relative to those with sporadic ALS and a relative preservation of brain motor structural and functional networks. Neurodegeneration in SOD1 ALS is likely to occur primarily in the spinal cord. An objective and accurate estimate of spinal cord damage has potential in the future assessment of preventive SOD1 ALS therapies. © 2018 American Academy of Neurology.

  3. Acute intermittent hypoxia induced phrenic long-term facilitation despite increased SOD1 expression in a rat model of ALS.

    Science.gov (United States)

    Nichols, Nicole L; Satriotomo, Irawan; Harrigan, Daniel J; Mitchell, Gordon S

    2015-11-01

    Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron death. Since most ALS patients succumb to ventilatory failure from loss of respiratory motor neurons, any effective ALS treatment must preserve and/or restore breathing capacity. In rats over-expressing mutated super-oxide dismutase-1 (SOD1(G93A)), the capacity to increase phrenic motor output is decreased at disease end-stage, suggesting imminent ventilatory failure. Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity with potential to restore phrenic motor output in clinical disorders that compromise breathing. Since pLTF requires NADPH oxidase activity and reactive oxygen species (ROS) formation, it is blocked by NADPH oxidase inhibition and SOD mimetics in normal rats. Thus, we hypothesized that SOD1(G93A) (mutant; MT) rats do not express AIH-induced pLTF due to over-expression of active mutant superoxide dismutase-1. AIH-induced pLTF and hypoglossal (XII) LTF were assessed in young, pre-symptomatic and end-stage anesthetized MT rats and age-matched wild-type littermates. Contrary to predictions, pLTF and XII LTF were observed in MT rats at all ages; at end-stage, pLTF was actually enhanced. SOD1 levels were elevated in young and pre-symptomatic MT rats, yet superoxide accumulation in putative phrenic motor neurons (assessed with dihydroethidium) was unchanged; however, superoxide accumulation significantly decreased at end-stage. Thus, compensatory mechanisms appear to maintain ROS homoeostasis until late in disease progression, preserving AIH-induced respiratory plasticity. Following intrathecal injections of an NADPH oxidase inhibitor (apocynin; 600 μM; 12 μL), pLTF was abolished in pre-symptomatic, but not end-stage MT rats, demonstrating that pLTF is NADPH oxidase dependent in pre-symptomatic, but NADPH oxidase independent in end-stage MT rats. Mechanisms

  4. Determining the Effect of Catechins on SOD1 Conformation and Aggregation by Ion Mobility Mass Spectrometry Combined with Optical Spectroscopy

    Science.gov (United States)

    Zhao, Bing; Zhuang, Xiaoyu; Pi, Zifeng; Liu, Shu; Liu, Zhiqiang; Song, Fengrui

    2018-02-01

    The aggregation of Cu,Zn-superoxide dismutase (SOD1) plays an important role in the etiology of amyotrophic lateral sclerosis (ALS). For the disruption of ALS progression, discovering new drugs or compounds that can prevent SOD1 aggregation is important. In this study, ESI-MS was used to investigate the interaction of catechins and SOD1. The noncovalent complex of catechins that interact with SOD1 was found and retained in the gas phase under native ESI-MS condition. The conformation changes of SOD1 after binding with catechins were also explored via traveling wave ion mobility (IM) spectrometry. Epigallocatechin gallate (EGCG) can stabilize SOD1 conformation against unfolding in three catechins. To further evaluate the efficacy of EGCG, we monitored the fluorescence changes of dimer E2,E2,-SOD1(apo-SOD1, E:empty) with and without ligands under denaturation conditions, and found that EGCG can inhibit apo-SOD1 aggregation. In addition, the circular dichroism spectra of the samples showed that EGCG can decrease the β-sheet content of SOD1, which can produce aggregates. These results indicated that orthogonal separation dimension in the gas-phase IM coupled with ESI-MS (ESI-IM-MS) can potentially provide insight into the interaction between SOD1 and small molecules. The advantage is that it dramatically decreases the analysis time. Meantime, optical spectroscopy techniques can be used to confirm ESI-IM-MS results. [Figure not available: see fulltext.

  5. ATF3 expression precedes death of spinal motoneurons in amyotrophic lateral sclerosis-SOD1 transgenic mice and correlates with c-Jun phosphorylation, CHOP expression, somato-dendritic ubiquitination and Golgi fragmentation

    NARCIS (Netherlands)

    Vlug, Angela S; Teuling, Eva; Haasdijk, Elize D; French, Pim; Hoogenraad, Casper C; Jaarsma, Dick

    2005-01-01

    To obtain insight into the morphological and molecular correlates of motoneuron degeneration in amyotrophic lateral sclerosis (ALS) mice that express G93A mutant superoxide dismutase (SOD)1 (G93A mice), we have mapped and characterized 'sick' motoneurons labelled by the 'stress transcription

  6. Spatial constraints govern competition of mutant clones in human epidermis.

    Science.gov (United States)

    Lynch, M D; Lynch, C N S; Craythorne, E; Liakath-Ali, K; Mallipeddi, R; Barker, J N; Watt, F M

    2017-10-24

    Deep sequencing can detect somatic DNA mutations in tissues permitting inference of clonal relationships. This has been applied to human epidermis, where sun exposure leads to the accumulation of mutations and an increased risk of skin cancer. However, previous studies have yielded conflicting conclusions about the relative importance of positive selection and neutral drift in clonal evolution. Here, we sequenced larger areas of skin than previously, focusing on cancer-prone skin spanning five decades of life. The mutant clones identified were too large to be accounted for solely by neutral drift. Rather, using mathematical modelling and computational lattice-based simulations, we show that observed clone size distributions can be explained by a combination of neutral drift and stochastic nucleation of mutations at the boundary of expanding mutant clones that have a competitive advantage. These findings demonstrate that spatial context and cell competition cooperate to determine the fate of a mutant stem cell.

  7. Tempol moderately extends survival in a hSOD1(G93A ALS rat model by inhibiting neuronal cell loss, oxidative damage and levels of non-native hSOD1(G93A forms.

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    Edlaine Linares

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease characterized by the progressive dysfunction and death of motor neurons by mechanisms that remain unclear. Evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. Cyclic nitroxides are an alternative worth exploring because they are multifunctional antioxidants that display low toxicity in vivo. Here, we examine the effects of the cyclic nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl on ALS onset and progression in transgenic female rats over-expressing the mutant hSOD1(G93A . Starting at 7 weeks of age, a high dose of tempol (155 mg/day/rat in the rat´s drinking water had marginal effects on the disease onset but decelerated disease progression and extended survival by 9 days. In addition, tempol protected spinal cord tissues as monitored by the number of neuronal cells, and the reducing capability and levels of carbonylated proteins and non-native hSOD1 forms in spinal cord homogenates. Intraperitoneal tempol (26 mg/rat, 3 times/week extended survival by 17 days. This group of rats, however, diverted to a decelerated disease progression. Therefore, it was inconclusive whether the higher protective effect of the lower i.p. dose was due to higher tempol bioavailability, decelerated disease development or both. Collectively, the results show that tempol moderately extends the survival of ALS rats while protecting their cellular and molecular structures against damage. Thus, the results provide proof that cyclic nitroxides are alternatives worth to be further tested in animal models of ALS.

  8. Oxidant production and SOD1 protein expression in single skeletal myofibers from Down syndrome mice

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    Patrick M. Cowley

    2017-10-01

    Full Text Available Down syndrome (DS is a genetic condition caused by the triplication of chromosome 21. Persons with DS exhibit pronounced muscle weakness, which also occurs in the Ts65Dn mouse model of DS. Oxidative stress is thought to be an underlying factor in the development of DS-related pathologies including muscle dysfunction. High-levels of oxidative stress have been attributed to triplication and elevated expression of superoxide dismutase 1 (SOD1; a gene located on chromosome 21. The elevated expression of SOD1 is postulated to increase production of hydrogen peroxide and cause oxidative injury and cell death. However, it is unknown whether SOD1 protein expression is associated with greater oxidant production in skeletal muscle from Ts65Dn mice. Thus, our objective was to assess levels of SOD1 expression and oxidant production in skeletal myofibers from the flexor digitorum brevis obtained from Ts65Dn and control mice. Measurements of oxidant production were obtained from myofibers loaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA in the basal state and following 15 min of stimulated unloaded contraction. Ts65Dn myofibers exhibited a significant decrease in basal DCF emissions (p 0.05. Myofibers from Ts65Dn mice tended to be smaller and myonuclear domain was lower (p < 0.05. In summary, myofibers from Ts65Dn mice exhibited decreased basal DCF emissions that were coupled with elevated protein expression of SOD1. Stimulated contraction in isolated myofibers did not affect DCF emissions in either group. These findings suggest the skeletal muscle dysfunction in the adult Ts65Dn mouse is not associated with skeletal muscle oxidative stress.

  9. Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant.

    Science.gov (United States)

    Bruijn, L I; Beal, M F; Becher, M W; Schulz, J B; Wong, P C; Price, D L; Cleveland, D W

    1997-07-08

    Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515-518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

  10. A Cystine-Rich Whey Supplement (Immunocal® Delays Disease Onset and Prevents Spinal Cord Glutathione Depletion in the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

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    Erika K. Ross

    2014-12-01

    Full Text Available Depletion of the endogenous antioxidant, glutathione (GSH, underlies progression of the devastating neurodegenerative disease, amyotrophic lateral sclerosis (ALS. Thus, strategies aimed at elevating GSH may yield new therapeutics for ALS. Here, we investigated the effects of a unique non-denatured whey protein supplement, Immunocal®, in the transgenic Gly position 93 to Ala (G93A mutant hSOD1 (hSOD1G93A mouse model of ALS. Immunocal® is rich in the GSH precursor, cystine, and is therefore capable of bolstering GSH content. Transgenic hSOD1G93A mice receiving Immunocal® displayed a significant delay in disease onset compared to untreated hSOD1G93A controls. Additionally, Immunocal® treatment significantly decreased the rate of decline in grip strength and prevented disease-associated reductions in whole blood and spinal cord tissue GSH levels in end-stage hSOD1G93A mice. However, Immunocal® did not extend survival, likely due to its inability to preserve the mitochondrial GSH pool in spinal cord. Combination treatment with Immunocal® and the anti-glutamatergic compound, riluzole, delayed disease onset and extended survival in hSOD1G93A mice. These findings demonstrate that sustaining tissue GSH with Immunocal® only modestly delays disease onset and slows the loss of skeletal muscle strength in hSOD1G93A mice. Moreover, the inability of Immunocal® to rescue mitochondrial GSH in spinal cord provides a possible mechanism for its lack of effect on survival and is a limiting factor in the potential utility of this supplement as a therapeutic for ALS.

  11. Degenerative myelopathy in German Shepherd Dog: comparison of two molecular assays for the identification of the SOD1:c.118G>A mutation.

    Science.gov (United States)

    Capucchio, Maria Teresa; Spalenza, Veronica; Biasibetti, Elena; Bottero, Maria Teresa; Rasero, Roberto; Dalmasso, Alessandra; Sacchi, Paola

    2014-02-01

    Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.

  12. Yield of two mutant lines of soybean for human consumption

    International Nuclear Information System (INIS)

    Salmeron E, J.; Mastache L, A. A.; Diaz V, G. E.; Valencia E, F.; Ranfla C, R.; Melendez P, M.; Cervantes S, T.; De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.

    2009-01-01

    The present work has the objective of to evaluate the yield and the agronomic behavior of 2 mutant lines of soybean for human consumption, obtained by means of a process of recurrent irradiation of soybean seed ISAAEG-BM 2 with gammas of Co 60 and selection in the generation R 4 M 18 . For the variable yield significant statistical differences were not observed, but considering the rest of the evaluated agronomic characteristics the mutant lines L 6 and Bombona they were excellent with values of 3,934.6 and 3,806.8 Kg ha- 1 to 15% of grain humidity, they also possess excellent genetic characteristics result of the irradiations and selections of these new genetic materials. (Author)

  13. Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

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    Zippelius Annette

    2009-06-01

    Full Text Available Abstract Background Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS. Results To gain insights into the molecular mechanisms of SOD1G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT and G93A (SH-SY5YG93A expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation. Conclusion In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations

  14. SOD1 aggregation in ALS mice shows simplistic test tube behavior.

    Science.gov (United States)

    Lang, Lisa; Zetterström, Per; Brännström, Thomas; Marklund, Stefan L; Danielsson, Jens; Oliveberg, Mikael

    2015-08-11

    A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.

  15. Pyrimethamine significantly lowers cerebrospinal fluid Cu/Zn superoxide dismutase in amyotrophic lateral sclerosis patients with SOD1 mutations.

    Science.gov (United States)

    Lange, Dale J; Shahbazi, Mona; Silani, Vincenzo; Ludolph, Albert C; Weishaupt, Jochen H; Ajroud-Driss, Senda; Fields, Kara G; Remanan, Rahul; Appel, Stanley H; Morelli, Claudia; Doretti, Alberto; Maderna, Luca; Messina, Stefano; Weiland, Ulrike; Marklund, Stefan L; Andersen, Peter M

    2017-06-01

    Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1-transgenic animal models. Pyrimethamine produces dose-dependent SOD1 reduction in cell culture systems. A previous phase 1 trial showed pyrimethamine lowers SOD1 levels in leukocytes in patients with SOD1 mutations. This study investigated whether pyrimethamine lowered SOD1 levels in the cerebrospinal fluid (CSF) in patients carrying SOD1 mutations linked to familial amyotrophic lateral sclerosis (fALS/SOD1). A multicenter (5 sites), open-label, 9-month-duration, dose-ranging study was undertaken to determine the safety and efficacy of pyrimethamine to lower SOD1 levels in the CSF in fALS/SOD1. All participants underwent 3 lumbar punctures, blood draw, clinical assessment of strength, motor function, quality of life, and adverse effect assessments. SOD1 levels were measured in erythrocytes and CSF. Pyrimethamine was measured in plasma and CSF. Appel ALS score, ALS Functional Rating Scale-Revised, and McGill Quality of Life Single-Item Scale were measured at screening, visit 6, and visit 9. We enrolled 32 patients; 24 completed 6 visits (18 weeks), and 21 completed all study visits. A linear mixed effects model showed a significant reduction in CSF SOD1 at visit 6 (p < 0.001) with a mean reduction of 13.5% (95% confidence interval [CI] = 8.4-18.5) and at visit 9 (p < 0.001) with a mean reduction of 10.5% (95% CI = 5.2-15.8). Pyrimethamine is safe and well tolerated in ALS. Pyrimethamine is capable of producing a significant reduction in total CSF SOD1 protein content in patients with ALS caused by different SOD1 mutations. Further long-term studies are warranted to assess clinical efficacy. Ann Neurol 2017;81:837-848. © 2017 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

  16. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-01-01

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

  17. APP/SOD1 overexpressing mice present reduced neuropathic pain sensitivity.

    Science.gov (United States)

    Kotulska, Katarzyna; Larysz-Brysz, Magdalena; LePecheur, Marie; Marcol, Wiesław; Olakowska, Edyta; Lewin-Kowalik, Joanna; London, Jacqueline

    2011-07-15

    There are controversies regarding pain expression in mentally disabled people, including Down syndrome patients. The aim of this study was to examine neuropathic pain-related behavior and peripheral nerve regeneration in mouse model of Down syndrome. Sciatic nerves of double transgenic mice, overexpressing both amyloid precursor protein (APP) and Cu/Zn superoxide dismutase (SOD1) genes, and FVB/N wild type mice were transected and immediately resutured. Evaluation of autotomy and functional recovery was carried out during 4-week follow-up. We found markedly less severe autotomy in transgenic animals, although the onset of autotomy was significantly delayed in control mice. Interestingly, neuroma formation at the injury site was significantly more prominent in transgenic animals. Sciatic function index outcome was better in transgenic mice than in wild-type group. Histological evaluation revealed no statistically significant differences in the number of GAP-43-positive growth cones and macrophages in the distal stump of the transected nerve between groups. However, in transgenic animals, the regenerating axons were arranged more chaotically. The number of Schwann cells in the distal stump of the transected nerves was significantly lower in transgenic mice. The number of surviving motoneurons was markedly decreased in transgenic group. We measured also the atrophy of denervated muscles and found it decreased in APP/SOD1 overexpressing mice. Taken together, in this model of Down syndrome, we observed increased neuroma formation and decreased autotomy after peripheral nerve injury. Our findings suggest that APP/SOD1 overexpressing mice are less sensitive for neuropathic pain associated with neuroma. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu, Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

    Science.gov (United States)

    Ibrahim, Osama M. A.; Dogru, Murat; Matsumoto, Yukihiro; Igarashi, Ayako; Kojima, Takashi; Wakamatsu, Tais Hitomi; Inaba, Takaaki; Shimizu, Takahiko; Shimazaki, Jun; Tsubota, Kazuo

    2014-01-01

    Purpose The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 −/−) mouse. Methods Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 −/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed. Results Corneal vital staining scores in the Sod1 −/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 −/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 −/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 −/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 −/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 −/− mice. Conclusions Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 −/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations. PMID:25036096

  19. A Model of Oxidative Stress Management: Moderation of Carbohydrate Metabolizing Enzymes in SOD1-Null Drosophila melanogaster

    Science.gov (United States)

    Bernard, Kristine E.; Parkes, Tony L.; Merritt, Thomas J. S.

    2011-01-01

    The response to oxidative stress involves numerous genes and mutations in these genes often manifest in pleiotropic ways that presumably reflect perturbations in ROS-mediated physiology. The Drosophila melanogaster SOD1-null allele (cSODn108) is proposed to result in oxidative stress by preventing superoxide breakdown. In SOD1-null flies, oxidative stress management is thought to be reliant on the glutathione-dependent antioxidants that utilize NADPH to cycle between reduced and oxidized form. Previous studies suggest that SOD1-null Drosophila rely on lipid catabolism for energy rather than carbohydrate metabolism. We tested these connections by comparing the activity of carbohydrate metabolizing enzymes, lipid and triglyceride concentration, and steady state NADPH:NADP+ in SOD1-null and control transgenic rescue flies. We find a negative shift in the activity of carbohydrate metabolizing enzymes in SOD1-nulls and the NADP+-reducing enzymes were found to have significantly lower activity than the other enzymes assayed. Little evidence for the catabolism of lipids as preferential energy source was found, as the concentration of lipids and triglycerides were not significantly lower in SOD1-nulls compared with controls. Using a starvation assay to impact lipids and triglycerides, we found that lipids were indeed depleted in both genotypes when under starvation stress, suggesting that oxidative damage was not preventing the catabolism of lipids in SOD1-null flies. Remarkably, SOD1-nulls were also found to be relatively resistant to starvation. Age profiles of enzyme activity, triglyceride and lipid concentration indicates that the trends observed are consistent over the average lifespan of the SOD1-nulls. Based on our results, we propose a model of physiological response in which organisms under oxidative stress limit the production of ROS through the down-regulation of carbohydrate metabolism in order to moderate the products exiting the electron transport chain. PMID

  20. Mechanisms of Enhanced Phrenic Long-Term Facilitation in SOD1G93A Rats

    Science.gov (United States)

    Satriotomo, Irawan; Grebe, Ashley M.

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease, causing muscle paralysis and death from respiratory failure. Effective means to preserve/restore ventilation are necessary to increase the quality and duration of life in ALS patients. At disease end-stage in a rat ALS model (SOD1G93A), acute intermittent hypoxia (AIH) restores phrenic nerve activity to normal levels via enhanced phrenic long-term facilitation (pLTF). Mechanisms enhancing pLTF in end-stage SOD1G93A rats are not known. Moderate AIH-induced pLTF is normally elicited via cellular mechanisms that require the following: Gq-protein-coupled 5-HT2 receptor activation, new BDNF synthesis, and MEK/ERK signaling (the Q pathway). In contrast, severe AIH elicits pLTF via a distinct mechanism that requires the following: Gs-protein-coupled adenosine 2A receptor activation, new TrkB synthesis, and PI3K/Akt signaling (the S pathway). In end-stage male SOD1G93A rats and wild-type littermates, we investigated relative Q versus S pathway contributions to enhanced pLTF via intrathecal (C4) delivery of small interfering RNAs targeting BDNF or TrkB mRNA, and MEK/ERK (U0126) or PI3 kinase/Akt (PI828) inhibitors. In anesthetized, paralyzed and ventilated rats, moderate AIH-induced pLTF was abolished by siBDNF and UO126, but not siTrkB or PI828, demonstrating that enhanced pLTF occurs via the Q pathway. Although phrenic motor neuron numbers were decreased in end-stage SOD1G93A rats (∼30% survival; p phrenic motor neurons (p phrenic motor plasticity results from amplification of normal cellular mechanisms versus addition/substitution of alternative mechanisms. Greater understanding of mechanisms underlying phrenic motor plasticity in ALS may guide development of new therapies to preserve and/or restore breathing in ALS patients. PMID:28500219

  1. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    Science.gov (United States)

    Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

    2015-01-01

    We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

  2. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    Directory of Open Access Journals (Sweden)

    Silvina Epsztejn-Litman

    Full Text Available We report on the derivation of a diploid 46(XX human embryonic stem cell (HESC line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19, monitoring the expression of two parentally imprinted genes (SNRPN and H19 and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

  3. Ligand and proton exchange dynamics in recombinant human myoglobin mutants.

    Science.gov (United States)

    Lambright, D G; Balasubramanian, S; Boxer, S G

    1989-05-05

    Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively

  4. NH exchange in point mutants of human ubiquitin.

    Science.gov (United States)

    Jahr, Nicole; Fiedler, Erik; Günther, Robert; Hofmann, Hans-Jörg; Berger, Stefan

    2011-08-01

    Several point mutants of human ubiquitin (Ub(T9V), Ub(F45W), Ub(F45G), and Ub(A46S)) were prepared by recombinant techniques. The NH exchange rate constants were measured by the NMR diffusion and the MEXICO methods and compared with those in the wild type to examine the influence of structural changes and to improve the understanding of this important reaction in studies of protein folding and denaturation. The observed changes follow qualitatively the polarity and steric alterations caused by the introduced amino acids. Attempts to reproduce quantitatively the observed changes by modeling studies and molecular dynamics simulations were not satisfactory. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Reactive oxygen species on bone mineral density and mechanics in Cu,Zn superoxide dismutase (Sod1) knockout mice

    International Nuclear Information System (INIS)

    Smietana, Michael J.; Arruda, Ellen M.; Faulkner, John A.; Brooks, Susan V.; Larkin, Lisa M.

    2010-01-01

    Research highlights: → Reactive oxygen species (ROS) are considered to be a factor in the onset of a number of age-associated conditions, including loss of BMD. → Cu,Zn-superoxide dismutase (Sod1) deficient mice have increased ROS, reduced bone mineral density, decreased bending stiffness, and decreased strength compared to WT controls. → Increased ROS caused by the deficiency of Sod1, may be responsible for the changes in BMD and bone mechanics and therefore represent an appropriate model for studying mechanisms of age-associated bone loss. -- Abstract: Reactive oxygen species (ROS) play a role in a number of degenerative conditions including osteoporosis. Mice deficient in Cu,Zn-superoxide dismutase (Sod1) (Sod1 -/- mice) have elevated oxidative stress and decreased muscle mass and strength compared to wild-type mice (WT) and appear to have an accelerated muscular aging phenotype. Thus, Sod1 -/- mice may be a good model for evaluating the effects of free radical generation on diseases associated with aging. In this experiment, we tested the hypothesis that the structural integrity of bone as measured by bending stiffness (EI; N/mm 2 ) and strength (MPa) is diminished in Sod1 -/- compared to WT mice. Femurs were obtained from male and female WT and Sod1 -/- mice at 8 months of age and three-point bending tests were used to determine bending stiffness and strength. Bones were also analyzed for bone mineral density (BMD; mg/cc) using micro-computed tomography. Femurs were approximately equal in length across all groups, and there were no significant differences in BMD or EI with respect to gender in either genotype. Although male and female mice demonstrated similar properties within each genotype, Sod1 -/- mice exhibited lower BMD and EI of femurs from both males and females compared with gender matched WT mice. Strength of femurs was also lower in Sod1 -/- mice compared to WT as well as between genders. These data indicate that increased oxidative stress

  6. 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...

  7. Mechanisms of Enhanced Phrenic Long-Term Facilitation in SOD1G93A Rats.

    Science.gov (United States)

    Nichols, Nicole L; Satriotomo, Irawan; Allen, Latoya L; Grebe, Ashley M; Mitchell, Gordon S

    2017-06-14

    Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease, causing muscle paralysis and death from respiratory failure. Effective means to preserve/restore ventilation are necessary to increase the quality and duration of life in ALS patients. At disease end-stage in a rat ALS model ( SOD1 G93A ), acute intermittent hypoxia (AIH) restores phrenic nerve activity to normal levels via enhanced phrenic long-term facilitation (pLTF). Mechanisms enhancing pLTF in end-stage SOD1 G93A rats are not known. Moderate AIH-induced pLTF is normally elicited via cellular mechanisms that require the following: G q -protein-coupled 5-HT 2 receptor activation, new BDNF synthesis, and MEK/ERK signaling (the Q pathway). In contrast, severe AIH elicits pLTF via a distinct mechanism that requires the following: G s -protein-coupled adenosine 2A receptor activation, new TrkB synthesis, and PI3K/Akt signaling (the S pathway). In end-stage male S OD1 G93A rats and wild-type littermates, we investigated relative Q versus S pathway contributions to enhanced pLTF via intrathecal (C4) delivery of small interfering RNAs targeting BDNF or TrkB mRNA, and MEK/ERK (U0126) or PI3 kinase/Akt (PI828) inhibitors. In anesthetized, paralyzed and ventilated rats, moderate AIH-induced pLTF was abolished by siBDNF and UO126, but not siTrkB or PI828, demonstrating that enhanced pLTF occurs via the Q pathway. Although phrenic motor neuron numbers were decreased in end-stage SOD1 G93A rats (∼30% survival; p phrenic motor neurons ( p phrenic motor plasticity results from amplification of normal cellular mechanisms versus addition/substitution of alternative mechanisms. Greater understanding of mechanisms underlying phrenic motor plasticity in ALS may guide development of new therapies to preserve and/or restore breathing in ALS patients. Copyright © 2017 the authors 0270-6474/17/375834-12$15.00/0.

  8. 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.

  9. Advanced age-related denervation and fiber-type grouping in skeletal muscle of SOD1 knockout mice.

    Science.gov (United States)

    Kostrominova, Tatiana Y

    2010-11-30

    In this study skeletal muscles from 1.5- and 10-month-old Cu/Zn superoxide dismutase (SOD1) homozygous knockout (JLSod1(-/-)) mice obtained from The Jackson Laboratory (C57Bl6/129SvEv background) were compared with muscles from age- and sex-matched heterozygous (JLSod1(+/-)) littermates. The results of this study were compared with previously published data on two different strains of Sod1(-/-) mice: one from Dr. Epstein's laboratory (ELSod1(-/-); C57Bl6 background) and the other from Cephalon, Inc. (CSod1(-/-); 129/CD-1 background). Grouping of succinate dehydrogenase-positive fibers characterized muscles of Sod1(-/-) mice from all three strains. The 10-month-old Sod1(-/-)C and JL mice displayed pronounced denervation of the gastrocnemius muscle, whereas the ELSod1(-/-) mice displayed a small degree of denervation at this age, but developed accelerated age-related denervation later on. Denervation markers were up-regulated in skeletal muscle of 10-month-old JLSod1(-/-) mice. This study is the first to show that metallothionein mRNA and protein expression was up-regulated in the skeletal muscle of 10-month-old JLSod1(-/-) mice and was mostly localized to the small atrophic muscle fibers. In conclusion, all three strains of Sod1(-/-) mice develop accelerated age-related muscle denervation, but the genetic background has significant influence on the progress of denervation. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. Differential survival among sSOD-1* genotypes in Chinook Salmon

    Science.gov (United States)

    Hayes, Michael C.; Reisenbichler, Reginald R.; Rubin, Stephen P.; Wetzel, Lisa A.; Marshall , Anne R.

    2011-01-01

    Differential survival and growth were tested in Chinook salmon Oncorhynchus tshawytscha expressing two common alleles, *–100 and *–260, at the superoxide dismutase locus (sSOD-1*). These tests were necessary to support separate studies in which the two alleles were used as genetic marks under the assumption of mark neutrality. Heterozygous adults were used to produce progeny with –100/–100, –100/–260, and –260/–260 genotypes that were reared in two natural streams and two hatcheries in the states of Washington and Oregon. The latter also were evaluated as returning adults. In general, the genotype ratios of juveniles reared at hatcheries were consistent with high survival and little or no differential survival in the hatchery. Adult returns at one hatchery were significantly different from the expected proportions, and the survival of the –260/–260 genotype was 0.56–0.89 times that of the –100/–100 genotype over four year-classes. Adult returns at a second hatchery (one year-class) were similar but not statistically significant: survival of the –260/–260genotype relative to the –100/–100 genotype was 0.76. The performance of the heterozygote group was intermediate at both hatcheries. Significant differences in growth were rarely observed among hatchery fish (one year-class of juveniles and one age-class of adult males) but were consistent with greater performance for the –100/–100 genotype. Results from two groups of juveniles reared in streams (one year-class from each stream) suggested few differences in growth, but the observed genotype ratios were significantly different from the expected ratios in one stream. Those differences were consistent with the adult data; survival for the –260/–260 genotype was 76% of that of the –100/–100 genotype. These results, which indicate nonneutrality among sSOD-1* genotypes, caused us to modify our related studies and suggest caution in the interpretation of results and analyses in

  11. Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Savina Apolloni

    2017-11-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a late-onset motor neuron disease where activated glia release pro-inflammatory cytokines that trigger a vicious cycle of neurodegeneration in the absence of resolution of inflammation. Given the well-established role of histamine as a neuron-to-glia alarm signal implicated in brain disorders, the aim of this study was to investigate the expression and regulation of the histaminergic pathway in microglial activation in ALS mouse model and in humans. By examining the contribution of the histaminergic system to ALS, we found that particularly via H1 and H4 receptors, histamine promoted an anti-inflammatory profile in microglia from SOD1-G93A mice by modulating their activation state. A decrease in NF-κB and NADPH oxidase 2 with an increase in arginase 1 and P2Y12 receptor was induced by histamine only in the ALS inflammatory environment, but not in the healthy microglia, together with an increase in IL-6, IL-10, CD163, and CD206 phenotypic markers in SOD1-G93A cells. Moreover, histaminergic H1, H2, H3, and H4 receptors, and histamine metabolizing enzymes histidine decarboxylase, histamine N-methyltransferase, and diamine oxidase were found deregulated in spinal cord, cortex, and hypothalamus of SOD1-G93A mice during disease progression. Finally, by performing a meta-analysis study, we found a modulated expression of histamine-related genes in cortex and spinal cord from sporadic ALS patients. Our findings disclose that histamine acts as anti-inflammatory agent in ALS microglia and suggest a dysregulation of the histaminergic signaling in ALS.

  12. Molecular and biochemical analyses of spontaneous and X-ray-induced mutants in human lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Liber, H L; Call, K M; Little, J B

    1987-05-01

    The authors have isolated a series of 14 spontaneously arising and 28 X-ray-induced mutants at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in human lymphoblastoid cells. Among the spontaneous mutants, 5/14 (36%) had detectable alterations in their restriction fragment pattern after hybridization with a human cDNA probe for hgprt. Of the 10 remaining mutants, 4 had partial HGPRT enzyme activity, which suggested that they contained point mutations. Among the 28 mutants induced by 150 rad of X-rays, 15 (54%) had deletions of part or all of the hgprt gene. Of the remaining 13 (18% overall) 5 had partial HGPRT enzyme activity, which suggested that they contained point mutations. These data imply that in this human cell system, X-rays induce both point mutants which have residual enzyme activity as well as mutations involving relatively large deletions of DNA. 48 reference, 1 figure, 4 tables.

  13. CCS and SOD1 mRNA are reduced after copper supplementation in peripheral mononuclear cells of individuals with high serum ceruloplasmin concentration.

    Science.gov (United States)

    Suazo, Miriam; Olivares, Felipe; Mendez, Marco A; Pulgar, Rodrigo; Prohaska, Joseph R; Arredondo, Miguel; Pizarro, Fernando; Olivares, Manuel; Araya, Magdalena; González, Mauricio

    2008-04-01

    The limits of copper homeostatic regulation in humans are not known, making it difficult to define the milder effects of early copper excess. Furthermore, a robust assay to facilitate the detection of early stages of copper excess is needed. To address these issues, we assessed changes in relative mRNA abundance of methallothionein 2A (MT2A), prion (PrP), amyloid precursor-like protein 2 (APLP2), Cu/Zn superoxide dismutase (SOD1) and its copper chaperone (CCS) in peripheral mononuclear cells (PMNCs) from healthy adults representing the 5% highest and lowest extremes in the distribution curve of serum ceruloplasmin (Cp) concentrations of 800 individuals. The intracellular Cu content was also determined. PMNCs were isolated from individuals before and after exposure to a single daily dose of 10 mg Cu (as CuSO(4)) for 2 months. Results showed that although there were fluctuations in serum Cp values of the samples assessed before copper exposure, no significant differences were observed in cell copper content or in the relative abundance of MT2A, PrP and APLP2 transcripts in PMNCs. Also, these values were not modified after copper supplementation. However, CCS and SOD1 mRNA levels were reduced in PMNCs after copper supplementation in the individuals with the high Cp values, suggesting that they should be further explored as biomarkers of moderate copper overload in humans.

  14. Functional analysis of mutant human carnitine acylcarnitine translocases in yeast

    NARCIS (Netherlands)

    IJlst, L.; van Roermund, C. W.; Iacobazzi, V.; Oostheim, W.; Ruiter, J. P.; Williams, J. C.; Palmieri, F.; Wanders, R. J.

    2001-01-01

    Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine translocase (CACT). We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency. CACT activities in fibroblasts of both

  15. Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Kronenberg, A; Little, J B

    1989-04-01

    In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, the authors have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells iraadiated with either fast neutrons or accelerated argon ions. Individual muant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loses and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbAl locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes. (author). 51 refs.; 2 figs.; 6 tabs.

  16. Induction of diphtheria toxin-resistant mutants in human cells by ultraviolet light

    International Nuclear Information System (INIS)

    Rocchi, P.; Ferreri, A.M.; Capucci, A.; Prodi, G.

    1981-01-01

    Stable spontaneous mutants resistant to the protein synthesis inhibitor diphtheria toxin (DT) have been selected in human cell line EUE at a very low frequency (less than 8 x 10(-6)). U.v.-induced mutation has been quantitatively measured: treatment of cells with u.v. light increased the frequencies of diphtheria toxin resistant (DTr) mutants up to 1000-fold. The maximum recovery of DTr mutants was observed after a short expression period, for all u.v. doses tested, and was followed by a decrease in mutation frequency on subsequent passages

  17. Induction of diphtheria toxin-resistant mutants in human cells by ultraviolet light

    International Nuclear Information System (INIS)

    Rocchi, P.; Ferreri, A.M.; Capucci, A.; Prodi, G.

    1981-01-01

    Stable spontaneous mutants resistant to the protein synthesis inhibitor diphtheria toxin (DT) have been selected in human cell line EUE at a very low frequency ( -6 ). U.v.-induced mutation has been quantitatively measured: treatment of cells with u.v. light increased the frequencies of diphtheria toxin resistant (DTsup(r)) mutants up to 1000-fold. The maximum recovery of DTsup(r) mutants was observed after a short expression period, for all u.v. doses tested, and was followed by a decrease in mutation frequency on subsequent passages. (author)

  18. Analysis of Serum Cytokines and Single-Nucleotide Polymorphisms of SOD1, SOD2, and CAT in Erysipelas Patients

    Directory of Open Access Journals (Sweden)

    Charles C. Emene

    2017-01-01

    Full Text Available Increased free radical production had been documented in group A (β-hemolytic streptococcus infection cases. Comparing 71 erysipelas patients to 55 age-matched healthy individuals, we sought for CAT, SOD1, and SOD2 single polymorphism mutation (SNPs interactions with erysipelas’ predisposition and serum cytokine levels in the acute and recovery phases of erysipelas infection. Whereas female patients had a higher predisposition to erysipelas, male patients were prone to having a facial localization of the infection. The presence of SOD1 G7958, SOD2 T2734, and CAT C262 alleles was linked to erysipelas’ predisposition. T and C alleles of SOD2 T2734C individually were linked to patients with bullous and erythematous erysipelas, respectively. G and A alleles of SOD1 G7958A individually were associated with lower limbs and higher body part localizations of the infection, respectively. Serum levels of IL-1β, CCL11, IL-2Rα, CXCL9, TRAIL, PDGF-BB, and CCL4 were associated with symptoms accompanying the infection, while IL-6, IL-9, IL-10, IL-13, IL-15, IL-17, G-CSF, and VEGF were associated with predisposition and recurrence of erysipelas. While variations of IL-1β, IL-7, IL-8, IL-17, CCL5, and HGF were associated with the SOD2 T2734C SNP, variations of PDFG-BB and CCL2 were associated with the CAT C262T SNP.

  19. Measuring Neuromuscular Junction Functionality in the SOD1(G93A) Animal Model of Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Rizzuto, Emanuele; Pisu, Simona; Musarò, Antonio; Del Prete, Zaccaria

    2015-09-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to motor neuron degeneration, alteration in neuromuscular junctions (NMJs), muscle atrophy, and paralysis. To investigate the NMJ functionality in ALS we tested, in vitro, two innervated muscle types excised from SOD1(G93A) transgenic mice at the end-stage of the disease: the Soleus, a postural muscle almost completely paralyzed at that stage, and the diaphragm, which, on the contrary, is functional until death. To this aim we employed an experimental protocol that combined two types of electrical stimulation: the direct stimulation and the stimulation through the nerve. The technique we applied allowed us to determine the relevance of NMJ functionality separately from muscle contractile properties in SOD1(G93A) animal model. Functional measurements revealed that the muscle contractility of transgenic diaphragms is almost unaltered in comparison to control muscles, while transgenic Soleus muscles were severely compromised. In contrast, when stimulated via the nerve, both transgenic muscle types showed a strong decrease of the contraction force, a slowing down of the kinetic parameters, as well as alterations in the neurotransmission failure parameter. All together, these results confirm a severely impaired functionality in the SOD1(G93A) neuromuscular junctions.

  20. Wildtype motoneurons, ALS-Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling.

    Science.gov (United States)

    Madji Hounoum, Blandine; Mavel, Sylvie; Coque, Emmanuelle; Patin, Franck; Vourc'h, Patrick; Marouillat, Sylviane; Nadal-Desbarats, Lydie; Emond, Patrick; Corcia, Philippe; Andres, Christian R; Raoul, Cédric; Blasco, Hélène

    2017-04-01

    The selective degeneration of motoneuron that typifies amyotrophic lateral sclerosis (ALS) implicates non-cell-autonomous effects of astrocytes. However, mechanisms underlying astrocyte-mediated neurotoxicity remain largely unknown. According to the determinant role of astrocyte metabolism in supporting neuronal function, we propose to explore the metabolic status of astrocytes exposed to ALS-associated conditions. We found a significant metabolic dysregulation including purine, pyrimidine, lysine, and glycerophospholipid metabolism pathways in astrocytes expressing an ALS-causing mutated superoxide dismutase-1 (SOD1) when co-cultured with motoneurons. SOD1 astrocytes exposed to glutamate revealed a significant modification of the astrocyte metabolic fingerprint. More importantly, we observed that SOD1 mutation and glutamate impact the cellular shuttling of lactate between astrocytes and motoneurons with a decreased in extra- and intra-cellular lactate levels in astrocytes. Based on the emergent strategy of metabolomics, this work provides novel insight for understanding metabolic dysfunction of astrocytes in ALS conditions and opens the perspective of therapeutics targets through focusing on these metabolic pathways. GLIA 2017 GLIA 2017;65:592-605. © 2017 Wiley Periodicals, Inc.

  1. A 50 bp deletion in the SOD1 promoter lowers enzyme expression but is not associated with ALS in Sweden.

    Science.gov (United States)

    Ingre, Caroline; Wuolikainen, Anna; Marklund, Stefan L; Birve, Anna; Press, Rayomand; Andersen, Peter M

    2016-01-01

    Mutations in the superoxide dismutase (SOD1) gene have been linked to amyotrophic lateral sclerosis (ALS). A 50 base pair (bp) deletion of SOD1 has been suggested to reduce transcription and to be associated with later disease onset in ALS. This study was aimed to reveal if the 50 bp deletion influenced SOD1 enzymatic activity, occurrence and phenotype of the disease in a Swedish ALS/control cohort. Blood samples from 512 Swedish ALS patients and 354 Swedish controls without coding SOD1 mutations were analysed for the 50 bp deletion allele. The enzymatic activity of SOD1 in erythrocytes was analysed and genotype-phenotype correlations were assessed. Results demonstrated that the genotype frequencies of the 50 bp deletion were all found to be in Hardy-Weinberg equilibrium. No significant differences were found for age of onset, disease duration or site of onset. SOD1 enzymatic activity showed a statistically significant decreasing trend in the control group, in which the allele was associated with a 5% reduction in SOD1 activity. The results suggest that the 50 bp deletion has a moderate reducing effect on SOD1 synthesis. No modulating effects, however, were found on ALS onset, phenotype and survival in the Swedish population.

  2. Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

    Directory of Open Access Journals (Sweden)

    Jinglan Liu

    2009-05-01

    Full Text Available Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS. Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

  3. Postactivation depression of the Ia EPSP in motoneurons is reduced in both the G127X SOD1 model of amyotrophic lateral sclerosis and in aged mice

    DEFF Research Database (Denmark)

    Hedegaard, Anne; Lehnhoff, Janna; Moldovan, Mihai

    2015-01-01

    Post Activation Depression (PActD) of Ia afferent EPSPs in spinal motoneurons results in a long lasting depression of the stretch reflex. PActD is of clinical interest as it has been shown to be reduced in a number of spastic disorders. Using in vivo intracellular recordings of Ia EPSPs in adult...... mice, we demonstrate that PActD in adult (100-220 days) C57BL/6J mice is both qualitatively and quantitatively similar to that which has been observed in larger animals with respect to both magnitude (approximately 20% depression of EPSPs at 0.5 ms after a stimuli train) and time course (returning...... Sclerosis (ALS). Using the G127X SOD1 mutant mouse, an ALS model with a prolonged asymptomatic phase and fulminant symptom onset, we observed that PActD is significantly reduced at both pre-symptomatic (16% depression) and symptomatic (17.3% depression) time points compared to aged-matched controls (22...

  4. Comparison of dendritic calcium transients in juvenile wild type and SOD1G93A mouse lumbar motoneurons

    Directory of Open Access Journals (Sweden)

    Katharina Ann Quinlan

    2015-04-01

    Full Text Available Previous studies of spinal motoneurons in the SOD1 mouse model of amyotrophic lateral sclerosis have shown alterations long before disease onset, including increased dendritic branching, increased persistent Na+ and Ca2+ currents, and impaired axonal transport. In this study dendritic Ca2+ entry was investigated using 2 photon excitation fluorescence microscopy and whole-cell patch-clamp of juvenile (P4-11 motoneurons. Neurons were filled with both Ca2+ Green-1 and Texas Red dextrans, and line scans performed throughout. Steps were taken to account for different sources of variability, including 1 dye filling and laser penetration, 2 dendritic anatomy, and 3 the time elapsed from the start of recording. First, Ca2+ Green-1 fluorescence was normalized by Texas Red; next, neurons were reconstructed so anatomy could be evaluated; finally, time was recorded. Customized software detected the largest Ca2+ transients (area under the curve from each line scan and matched it with parameters above. Overall, larger dendritic diameter and shorter path distance from the soma were significant predictors of larger transients, while time was not significant up to 2 hours (data thereafter was dropped. However, Ca2+ transients showed additional variability. Controlling for previous factors, significant variation was found between Ca2+ signals from different processes of the same neuron in 3/7 neurons. This could reflect differential expression of Ca2+ channels, local neuromodulation or other variations. Finally, Ca2+ transients in SOD1G93A motoneurons were significantly smaller than in non-transgenic motoneurons. In conclusion, motoneuron processes show highly variable Ca2+ transients, but these transients are smaller overall SOD1G93A motoneurons.

  5. Seeking homeostasis: Temporal trends in respiration, oxidation, and calcium in SOD1 G93A Amyotrophic Lateral Sclerosis mice

    Directory of Open Access Journals (Sweden)

    Cameron W Irvin

    2015-07-01

    Full Text Available Impairments in mitochondria, oxidative regulation, and calcium homeostasis have been well documented in numerous amyotrophic lateral sclerosis (ALS experimental models, especially in the superoxide dismutase 1 glycine 93 to alanine (SOD1 G93A transgenic mouse. However, the timing of these deficiencies has been debatable. In a systematic review of 45 articles, we examine experimental measurements of cellular respiration, mitochondrial mechanisms, oxidative markers, and calcium regulation. We evaluate the quantitative magnitude and statistical temporal trend of these aggregated assessments in high transgene copy SOD1 G93A mice compared to wild type mice. Analysis of overall trends reveals cellular respiration, intracellular ATP, and corresponding mitochondrial elements (Cox, cytochrome c, complex I, enzyme activity are depressed for the entire lifespan of the SOD1 G93A mouse. Oxidant markers (H2O2, 8OH2’dG, MDA are initially similar to wild type but are double that of wild type by the time of symptom onset despite early post-natal elevation of protective heat shock proteins. All aspects of calcium regulation show early disturbances, although a notable and likely compensatory convergence to near wild type levels appears to occur between 40-80 days (pre-onset, followed by a post-onset elevation in intracellular calcium. The identified temporal trends and compensatory fluctuations provide evidence that the cause of ALS may lay within failed homeostatic regulation, itself, rather than any one particular perturbing event or cellular mechanism. We discuss the vulnerabilities of motoneurons to regulatory instability and possible hypotheses regarding failed regulation and its potential treatment in ALS.

  6. Neuroprotective Effect of Bexarotene in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

    Science.gov (United States)

    Riancho, Javier; Ruiz-Soto, María; Berciano, María T.; Berciano, José; Lafarga, Miguel

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive weakness and muscle atrophy related to the loss of upper and lower motor neurons (MNs) without a curative treatment. There is experimental evidence suggesting that retinoids may be involved in ALS pathogenesis. Bexarotene (Bxt) is a retinoid-X receptor agonist used in the treatment of cutaneous lymphoma with a favorable safety profile whose effects have been recently investigated in other neurodegenerative diseases. In this study, we analyze the potential therapeutic effect of Bxt in the SOD1G93A mouse model of ALS. Mice were treated with Bxt or vehicle five times per week from day 60 onward. Survival, weight, and neuromuscular function studies together with histological and biochemical analyses were performed. Bxt significantly delayed motor function deterioration, ameliorated the loss of body weight, and extended mice survival up to 30% of the symptomatic period. Histological analyses of the lumbosacral spinal cord revealed that Bxt markedly delayed the early motor-neuron degeneration occurring at presymptomatic stages in ALS-transgenic mice. Bxt treatment contributed to preserve the MN homeostasis in the SOD1G93A mice. Particularly, it reduced the neuronal loss and the chromatolytic response, induced nucleolar hypertrophy, decreased the formation of ubiquitylated inclusions, and modulated the lysosomal response. As an agonist of the retinoic-X receptor (RXR) pathway, Bxt notably increased the nuclear expression of the RXRα throughout transcriptionally active euchromatin domains. Bxt also contributed to protect the MN environment by reducing reactive astrogliosis and preserving perisomatic synapsis. Overall, these neuroprotective effects suggest that treatment with Bxt could be useful in ALS, particularly in those cases related to SOD1 mutations. PMID:26190974

  7. A botanical containing freeze dried açai pulp promotes healthy aging and reduces oxidative damage in sod1 knockdown flies

    OpenAIRE

    Laslo, Mara; Sun, Xiaoping; Hsiao, Cheng-Te; Wu, Wells W.; Shen, Rong-Fong; Zou, Sige

    2012-01-01

    Superoxide dismutase 1 (SOD1), a critical enzyme against oxidative stress, is implicated in aging and degenerative diseases. We previously showed that a nutraceutical containing freeze-dried açai pulp promotes survival of flies fed a high-fat diet or sod1 knockdown flies fed a standard diet. Here, we investigated the effect of açai supplementation initiated at the early or late young adulthood on lifespan, physiological function, and oxidative damage in sod1 knockdown flies. We found that Aça...

  8. Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

    DEFF Research Database (Denmark)

    Roszko, Kelly L; Bi, Ruiye; Gorvin, Caroline M

    2017-01-01

    in patients with autosomal-dominant hypocalcemia type 2 (ADH2), an inherited disorder of hypocalcemia, low parathyroid hormone (PTH), and hyperphosphatemia. We have generated knockin mice harboring the point mutation GNA11 c.C178T (p.Arg60Cys) identified in ADH2 patients. The mutant mice faithfully replicated...... human ADH2. They also exhibited low bone mineral density and increased skin pigmentation. Treatment with NPS 2143, a negative allosteric modulator of the calcium-sensing receptor (CASR), increased PTH and calcium concentrations in WT and mutant mice, suggesting that the gain-of-function effect of GNA11...

  9. orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET.

    Science.gov (United States)

    DeGennaro, Matthew; McBride, Carolyn S; Seeholzer, Laura; Nakagawa, Takao; Dennis, Emily J; Goldman, Chloe; Jasinskiene, Nijole; James, Anthony A; Vosshall, Leslie B

    2013-06-27

    Female mosquitoes of some species are generalists and will blood-feed on a variety of vertebrate hosts, whereas others display marked host preference. Anopheles gambiae and Aedes aegypti have evolved a strong preference for humans, making them dangerously efficient vectors of malaria and Dengue haemorrhagic fever. Specific host odours probably drive this strong preference because other attractive cues, including body heat and exhaled carbon dioxide (CO2), are common to all warm-blooded hosts. Insects sense odours via several chemosensory receptor families, including the odorant receptors (ORs), membrane proteins that form heteromeric odour-gated ion channels comprising a variable ligand-selective subunit and an obligate co-receptor called Orco (ref. 6). Here we use zinc-finger nucleases to generate targeted mutations in the orco gene of A. aegypti to examine the contribution of Orco and the odorant receptor pathway to mosquito host selection and sensitivity to the insect repellent DEET (N,N-diethyl-meta-toluamide). orco mutant olfactory sensory neurons have greatly reduced spontaneous activity and lack odour-evoked responses. Behaviourally, orco mutant mosquitoes have severely reduced attraction to honey, an odour cue related to floral nectar, and do not respond to human scent in the absence of CO2. However, in the presence of CO2, female orco mutant mosquitoes retain strong attraction to both human and animal hosts, but no longer strongly prefer humans. orco mutant females are attracted to human hosts even in the presence of DEET, but are repelled upon contact, indicating that olfactory- and contact-mediated effects of DEET are mechanistically distinct. We conclude that the odorant receptor pathway is crucial for an anthropophilic vector mosquito to discriminate human from non-human hosts and to be effectively repelled by volatile DEET.

  10. Differential Motor Neuron Impairment and Axonal Regeneration in Sporadic and Familiar Amyotrophic Lateral Sclerosis with SOD-1 Mutations: Lessons from Neurophysiology

    OpenAIRE

    Bocci, Tommaso; Pecori, Chiara; Giorli, Elisa; Briscese, Lucia; Tognazzi, Silvia; Caleo, Matteo; Sartucci, Ferdinando

    2011-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a degenerative disorder of the motor system. About 10% of cases are familial and 20% of these families have point mutations in the Cu/Zn superoxide dismutase 1 (SOD-1) gene. SOD-1 catalyses the superoxide radical (O−2) into hydrogen peroxide and molecular oxygen. The clinical neurophysiology in ALS plays a fundamental role in differential diagnosis between the familial and sporadic forms and in the assessment of its severity and progression. Sixty ALS pa...

  11. Cell Transformation by PTP1B Truncated Mutants Found in Human Colon and Thyroid Tumors.

    Science.gov (United States)

    Mei, Wenhan; Wang, Kemin; Huang, Jian; Zheng, Xinmin

    2016-01-01

    Expression of wild-type protein tyrosine phosphatase (PTP) 1B may act either as a tumor suppressor by dysregulation of protein tyrosine kinases or a tumor promoter through Src dephosphorylation at Y527 in human breast cancer cells. To explore whether mutated PTP1B is involved in human carcinogenesis, we have sequenced PTP1B cDNAs from human tumors and found splice mutations in ~20% of colon and thyroid tumors. The PTP1BΔE6 mutant expressed in these two tumor types and another PTP1BΔE5 mutant expressed in colon tumor were studied in more detail. Although PTP1BΔE6 revealed no phosphatase activity compared with wild-type PTP1B and the PTP1BΔE5 mutant, its expression induced oncogenic transformation of rat fibroblasts without Src activation, indicating that it involved signaling pathways independent of Src. The transformed cells were tumourigenic in nude mice, suggesting that the PTP1BΔE6 affected other molecule(s) in the human tumors. These observations may provide a novel therapeutic target for colon and thyroid cancer.

  12. EGFR inhibitor erlotinib delays disease progression but does not extend survival in the SOD1 mouse model of ALS.

    Directory of Open Access Journals (Sweden)

    Claire E Le Pichon

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease that causes progressive paralysis due to motor neuron death. Several lines of published evidence suggested that inhibition of epidermal growth factor receptor (EGFR signaling might protect neurons from degeneration. To test this hypothesis in vivo, we treated the SOD1 transgenic mouse model of ALS with erlotinib, an EGFR inhibitor clinically approved for oncology indications. Although erlotinib failed to extend ALS mouse survival it did provide a modest but significant delay in the onset of multiple behavioral measures of disease progression. However, given the lack of protection of motor neuron synapses and the lack of survival extension, the small benefits observed after erlotinib treatment appear purely symptomatic, with no modification of disease course.

  13. Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

    DEFF Research Database (Denmark)

    Ermakova, Inessa; Boldyreff, Brigitte; Issinger, Olaf-Georg

    2003-01-01

    structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two...

  14. Differential motor neuron impairment and axonal regeneration in sporadic and familiar amyotrophic lateral sclerosis with SOD-1 mutations: lessons from neurophysiology.

    Science.gov (United States)

    Bocci, Tommaso; Pecori, Chiara; Giorli, Elisa; Briscese, Lucia; Tognazzi, Silvia; Caleo, Matteo; Sartucci, Ferdinando

    2011-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a degenerative disorder of the motor system. About 10% of cases are familial and 20% of these families have point mutations in the Cu/Zn superoxide dismutase 1 (SOD-1) gene. SOD-1 catalyses the superoxide radical (O(-2)) into hydrogen peroxide and molecular oxygen. The clinical neurophysiology in ALS plays a fundamental role in differential diagnosis between the familial and sporadic forms and in the assessment of its severity and progression. Sixty ALS patients (34 males; 26 females) were enrolled in the study and examined basally (T0) and every 4 months (T1, T2, and T3). Fifteen of these patients are SOD-1 symptomatic mutation carriers (nine males, six females). We used Macro-EMG and Motor Unit Number Estimation (MUNE) in order to evaluate the neuronal loss and the re-innervation process at the onset of disease and during follow-up period. SOD-1 mutation carriers have a higher number of motor units at the moment of diagnosis when compared with the sporadic form, despite a more dramatic drop in later stages. Moreover, in familiar SOD-1 ALS there is not a specific time interval in which the axonal regeneration can balance the neuronal damage. Taken together, these results strengthen the idea of a different pathogenetic mechanism at the base of sALS and fALS.

  15. Differential Motor Neuron Impairment and Axonal Regeneration in Sporadic and Familiar Amyotrophic Lateral Sclerosis with SOD-1 Mutations: Lessons from Neurophysiology

    Directory of Open Access Journals (Sweden)

    Tommaso Bocci

    2011-12-01

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a degenerative disorder of the motor system. About 10% of cases are familial and 20% of these families have point mutations in the Cu/Zn superoxide dismutase 1 (SOD-1 gene. SOD-1 catalyses the superoxide radical (O−2 into hydrogen peroxide and molecular oxygen. The clinical neurophysiology in ALS plays a fundamental role in differential diagnosis between the familial and sporadic forms and in the assessment of its severity and progression. Sixty ALS patients (34 males; 26 females were enrolled in the study and examined basally (T0 and every 4 months (T1, T2, and T3. Fifteen of these patients are SOD-1 symptomatic mutation carriers (nine males, six females. We used Macro-EMG and Motor Unit Number Estimation (MUNE in order to evaluate the neuronal loss and the re-innervation process at the onset of disease and during follow-up period. Results and Discussion: SOD-1 mutation carriers have a higher number of motor units at the moment of diagnosis when compared with the sporadic form, despite a more dramatic drop in later stages. Moreover, in familiar SOD-1 ALS there is not a specific time interval in which the axonal regeneration can balance the neuronal damage. Taken together, these results strengthen the idea of a different pathogenetic mechanism at the base of sALS and fALS.

  16. Influence of genetic variations in the SOD1 gene on the development of ascites and spontaneous bacterial peritonitis in decompensated liver cirrhosis

    DEFF Research Database (Denmark)

    Schwab, Sebastian; Lehmann, Jennifer; Lutz, Philipp

    2017-01-01

    BACKGROUND: The balance between generation and elimination of reactive oxygen species by superoxide dismutase (SOD) is crucially involved in the pathophysiology of liver cirrhosis. Reactive oxygen species damage cells and induce inflammation/fibrosis, but also play a critical role in immune defense...... in carriers of rs1041740. In this cohort, rs1041740 was not associated with survival. CONCLUSION: These data suggest a complex role of SOD1 in different processes leading to complications of liver cirrhosis. rs1041740 might be associated with the development of ascites and possibly plays a role in SBP once...... from pathogens. As both processes are involved in the development of liver cirrhosis and its complications, genetic variation of the SOD1 gene was investigated. PATIENTS AND METHODS: Two SOD1 single nucleotide polymorphisms (rs1041740 and rs3844942) were analyzed in 49 cirrhotic patients undergoing...

  17. Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin

    International Nuclear Information System (INIS)

    Chen Wenshu; Lee Yijang; Yu Yichu; Hsaio Chinghui

    2010-01-01

    Purpose: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Methods and Materials: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of γ-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Results: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G 2 /M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. Conclusions: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

  18. Progranulin is neurotrophic in vivo and protects against a mutant TDP-43 induced axonopathy.

    Directory of Open Access Journals (Sweden)

    Angela S Laird

    Full Text Available Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43 is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS and frontotemporal lobe dementia (FTLD. These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration.

  19. Gene expression changes in spinal motoneurons of the SOD1G93A transgenic model for ALS after treatment with G-CSF

    Science.gov (United States)

    Henriques, Alexandre; Kastner, Stefan; Chatzikonstantinou, Eva; Pitzer, Claudia; Plaas, Christian; Kirsch, Friederike; Wafzig, Oliver; Krüger, Carola; Spoelgen, Robert; Gonzalez De Aguilar, Jose-Luis; Gretz, Norbert; Schneider, Armin

    2015-01-01

    Background: Amyotrophic lateral sclerosis (ALS) is an incurable fatal motoneuron disease with a lifetime risk of approximately 1:400. It is characterized by progressive weakness, muscle wasting, and death ensuing 3–5 years after diagnosis. Granulocyte-colony stimulating factor (G-CSF) is a drug candidate for ALS, with evidence for efficacy from animal studies and interesting data from pilot clinical trials. To gain insight into the disease mechanisms and mode of action of G-CSF, we performed gene expression profiling on isolated lumbar motoneurons from SOD1G93A mice, the most frequently studied animal model for ALS, with and without G-CSF treatment. Results: Motoneurons from SOD1G93A mice present a distinct gene expression profile in comparison to controls already at an early disease stage (11 weeks of age), when treatment was initiated. The degree of deregulation increases at a time where motor symptoms are obvious (15 weeks of age). Upon G-CSF treatment, transcriptomic deregulations of SOD1G93A motoneurons were notably restored. Discriminant analysis revealed that SOD1 mice treated with G-CSF has a transcriptom close to presymptomatic SOD1 mice or wild type mice. Some interesting genes modulated by G-CSF treatment relate to neuromuscular function such as CCR4-NOT or Prss12. Conclusions: Our data suggest that G-CSF is able to re-adjust gene expression in symptomatic SOD1G93A motoneurons. This provides further arguments for G-CSF as a promising drug candidate for ALS. PMID:25653590

  20. Gene expression changes in spinal motoneurons of the SOD1G93A transgenic model for ALS after treatment with G-CSF.

    Directory of Open Access Journals (Sweden)

    Alexandre eHenriques

    2015-01-01

    Full Text Available ABSTRACTBackgroundAmyotrophic lateral sclerosis (ALS is an incurable fatal motoneuron disease with a lifetime risk of approximately 1:400. It is characterized by progressive weakness, muscle wasting, and death ensuing 3-5 years after diagnosis. Granulocyte-colony stimulating factor (G-CSF is a drug candidate for ALS, with evidence for efficacy from animal studies and interesting data from pilot clinical trials. To gain insight into the disease mechanisms and mode of action of G-CSF, we performed gene expression profiling on isolated lumbar motoneurons from SOD1G93A mice, the most frequently studied animal model for ALS, with and without G-CSF treatment. ResultsMotoneurons from SOD1G93A mice present a distinct gene expression profile in comparison to controls already at an early disease stage (11 weeks of age, when treatment was initiated. The degree of deregulation increases at a time where motor symptoms are obvious (15 weeks of age. Upon G-CSF treatment, transcriptomic deregulations of SOD1G93A motoneurons were notably restored. Discriminant analysis revealed that SOD1 mice treated with G-CSF has a transcriptom close to presymptomatic SOD1 mice or wild type mice. Some interesting genes modulated by G-CSF treatment relate to neuromuscular function such as CCR4-NOT or Prss12.ConclusionsOur data suggest that G-CSF is able to re-adjust gene expression in symptomatic SOD1G93A motoneurons. This provides further arguments for G-CSF as a promising drug candidate for ALS.

  1. Gene expression changes in spinal motoneurons of the SOD1(G93A) transgenic model for ALS after treatment with G-CSF.

    Science.gov (United States)

    Henriques, Alexandre; Kastner, Stefan; Chatzikonstantinou, Eva; Pitzer, Claudia; Plaas, Christian; Kirsch, Friederike; Wafzig, Oliver; Krüger, Carola; Spoelgen, Robert; Gonzalez De Aguilar, Jose-Luis; Gretz, Norbert; Schneider, Armin

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is an incurable fatal motoneuron disease with a lifetime risk of approximately 1:400. It is characterized by progressive weakness, muscle wasting, and death ensuing 3-5 years after diagnosis. Granulocyte-colony stimulating factor (G-CSF) is a drug candidate for ALS, with evidence for efficacy from animal studies and interesting data from pilot clinical trials. To gain insight into the disease mechanisms and mode of action of G-CSF, we performed gene expression profiling on isolated lumbar motoneurons from SOD1(G93A) mice, the most frequently studied animal model for ALS, with and without G-CSF treatment. Motoneurons from SOD1(G93A) mice present a distinct gene expression profile in comparison to controls already at an early disease stage (11 weeks of age), when treatment was initiated. The degree of deregulation increases at a time where motor symptoms are obvious (15 weeks of age). Upon G-CSF treatment, transcriptomic deregulations of SOD1(G93A) motoneurons were notably restored. Discriminant analysis revealed that SOD1 mice treated with G-CSF has a transcriptom close to presymptomatic SOD1 mice or wild type mice. Some interesting genes modulated by G-CSF treatment relate to neuromuscular function such as CCR4-NOT or Prss12. Our data suggest that G-CSF is able to re-adjust gene expression in symptomatic SOD1(G93A) motoneurons. This provides further arguments for G-CSF as a promising drug candidate for ALS.

  2. Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells

    International Nuclear Information System (INIS)

    Labadie, Karine; Pelletier, Isabelle; Saulnier, Aure; Martin, Javier; Colbere-Garapin, Florence

    2004-01-01

    Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal Caco-2 cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost 2 years [J. Virol. 74 (2000) 3001], were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days postvaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 (S3) strain. In contrast, three VDPVs excreted 136, 442, and 637 days postvaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cell surface

  3. Presymptomatic Treatment with Acetylcholinesterase Antisense Oligonucleotides Prolongs Survival in ALS (G93A-SOD1 Mice

    Directory of Open Access Journals (Sweden)

    Gotkine Marc

    2013-01-01

    Full Text Available Objective. Previous research suggests that acetylcholinesterase (AChE may be involved in ALS pathogenesis. AChE enzyme inhibitors can upregulate AChE transcription which in certain contexts can have deleterious (noncatalytic effects, making them theoretically harmful in ALS, whilst AChE antisense-oligonucleotides (mEN101, which downregulate AChE may be beneficial. Our aim was to investigate whether downregulation of AChE using mEN101 is beneficial in an ALS mouse model. Methods. ALS (G93A-SOD1 mice received saline, mEN101, inverse-EN101, or neostigmine. Treatments were administered from 5 weeks. Disease-onset and survival were recorded. Additional mice were sacrificed for pathological analysis at 15 weeks of age. In a follow-up experiment treatment was started at the symptomatic stage at a higher dose. Results. mEN101 given at the presymptomatic (but not symptomatic stage prolonged survival and attenuated motor-neuron loss in ALS mice. In contrast, neostigmine exacerbated the clinical parameters. Conclusions. These results suggest that AChE may be involved in ALS pathogenesis. The accelerated disease course with neostigmine suggests that any beneficial effects of mEN101 occur through a non-catalytic rather than cholinergic mechanism.

  4. Analysis of human HPRT- deletion mutants by the microarray-CGH (comparative genomic hybridization)

    International Nuclear Information System (INIS)

    Kodaira, M.; Sasaki, K.; Tagawa, H.; Omine, H.; Kushiro, J.; Takahashi, N.; Katayama, H.

    2003-01-01

    We are trying to evaluate genetic effects of radiation on human using mutation frequency as an indicator. For the efficient detection of mutations, it is important to understand the mechanism and the characteristics of radiation-induced mutations. We have started the analysis of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutants induced by X-ray in order to clarify the deletion size and the mutation-distribution. We analyzed 39 human X-ray induced HPRT-deletion mutants by using the microarray-CGH. The array for this analysis contains 57 BAC clones covering as much as possible of the 4Mb of the 5' side and 10Mb of the 3' side of the HPRT gene based on the NCBI genome database. DNA from parent strain and each HPRT-mutant strain are labeled with Cy5 and Cy3 respectively, and were mixed and hybridized on the array. Fluorescent intensity ratio of the obtained spots was analyzed using software we developed to identify clones corresponding to the deletion region. The deletion in these strains ranged up to 3.5 Mb on the 5' side and 6 Mb on the 3' side of the HPRT gene. Deletions in 13 strains ended around BAC clones located at about 3 Mb on the 5' side. On the 3' side, deletions extended up to the specific clones located at 1.5 Mb in 11 strains. The mutations seem to be complex on the 3' end of deletion; some accompanied duplications with deletions and others could not be explained by one mutation event. We need to confirm these results, taking into account the experimental reproducibility and the accuracy of the published genetic map. The results of the research using the microarray-CGH help us to search the regions where deletions are easily induced and to identify the factors affecting the range of deletions

  5. Yield of two mutant lines of soybean for human consumption;Rendimiento de dos lineas mutantes de soya para consumo humano

    Energy Technology Data Exchange (ETDEWEB)

    Salmeron E, J.; Mastache L, A. A.; Diaz V, G. E.; Valencia E, F.; Ranfla C, R.; Melendez P, M. [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 Postgraduados, 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., E-mail: csaegro@prodigy.net.m [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, Ocoyoacac 52750, Estado de Mexico (Mexico)

    2009-07-01

    The present work has the objective of to evaluate the yield and the agronomic behavior of 2 mutant lines of soybean for human consumption, obtained by means of a process of recurrent irradiation of soybean seed ISAAEG-BM{sub 2} with gammas of Co{sup 60} and selection in the generation R{sub 4}M{sub 18}. For the variable yield significant statistical differences were not observed, but considering the rest of the evaluated agronomic characteristics the mutant lines L{sub 6} and Bombona they were excellent with values of 3,934.6 and 3,806.8 Kg ha-{sup 1} to 15% of grain humidity, they also possess excellent genetic characteristics result of the irradiations and selections of these new genetic materials. (Author)

  6. The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

    International Nuclear Information System (INIS)

    Secondo, Agnese; De Mizio, Mariarosaria; Zirpoli, Laura; Santillo, Mariarosaria; Mondola, Paolo

    2008-01-01

    The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calcium concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation. These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor

  7. Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS.

    Directory of Open Access Journals (Sweden)

    Svitlana Garbuzova-Davis

    2007-11-01

    Full Text Available The blood-brain barrier (BBB, blood-spinal cord barrier (BSCB, and blood-cerebrospinal fluid barrier (BCSFB control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.Evans Blue (EB dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease.

  8. Glycerol restores the p53 function in human lingual cancer cells bearing mutant p53

    International Nuclear Information System (INIS)

    Ota, Ichiro; Yane, Katsunari; Yuki, Kazue; Kanata, Hirokazu; Hosoi, Hiroshi; Miyahara, Hiroshi

    2001-01-01

    Mutations in p53, tumor suppressor gene, have recently been shown to have an impact on the clinical course of several human tumors, including head and neck cancers. The genetic status of the p53 gene has been focused on as the most important candidate among various cancer-related genes for prognosis-predictive assays of cancer therapy. We examined the restoration of radiation- or cisplatin (CDDP)-induced p53-dependent apoptosis in human lingual cancer cells. The results suggest that glycerol is effective in inducing a conformational change of p53 and restoring normal function of mutant p53, leading to enhanced radiosensitivity or chemosensitivity through the induction of apoptosis. We have also represented the same results in vivo as in vitro. Thus, this novel tool for enhancement of radiosensitivity or chemosensitivity in cancer cells bearing m p53 may be applicable for p53-targeted cancer therapy. (author)

  9. Correction of β-thalassemia mutant by base editor in human embryos

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    Puping Liang

    2017-09-01

    Full Text Available Abstract β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

  10. Molecular analysis of human argininosuccinate lyase: Mutant characterization and alternative splicing of the coding region

    International Nuclear Information System (INIS)

    Walker, D.C.; McCloskey, D.A.; Simard, L.R.; McInnes, R.R.

    1990-01-01

    Argininosuccinic acid lyase (ASAL) deficiency is a clinically heterogeneous autosomal recessive urea cycle disorder. The authors previously established by complementation analysis that 29 ASAL-deficient patients have heterogeneous mutations in a single gene. To prove that the ASAL structural gene is the affected locus, they sequenced polymerase chain reaction-amplified ASAL cDNA of a representative mutant from the single complementation group. Fibroblast strain 944 from a late-onset patient who was the product of a consanguineous mating, had only a single base-pair change in the coding region, a C-283→ T transition at a CpG dinucleotide in exon 3. This substitution converts Arg-95 to Cys (R95C), occurs in a stretch of 13 residues that is identical in yeast and human ASAL, and was present in both of the patient's alleles but not in 14 other mutant or 10 normal alleles. They observed that amplified cDNA from mutant 944 and normal cells (liver, keratinocytes, lymphoblasts, and fibroblasts) contained, in addition to the expected 5' 513-base-pair band, a prominent 318-base-pair ASAL band formed by the splicing of exon 2 from the transcript. The short transcript maintains the ASAL reading frame but removes Lys-51, a residue that may be essential for catalysis, since it binds the argininosuccinate substrate. They conclude (i) that the identification of the R95C mutation in strain 944 demonstrates that virtually all ASAL deficiency results from defects in the ASAL structural gene and (ii) that minor alternative splicing of the coding region occurs at the ASAL locus

  11. Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.

    Science.gov (United States)

    Xia, Zhen-Wei; Zhou, Wen-Pu; Cui, Wen-Jun; Zhang, Xue-Hong; Shen, Qing-Xiang; Li, Yun-Zhu; Yu, Shan-Chang

    2004-08-15

    To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities. Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1. Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

  12. Tryptophan 32 potentiates aggregation and cytotoxicity of a copper/zinc superoxide dismutase mutant associated with familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Taylor, David M; Gibbs, Bernard F; Kabashi, Edor; Minotti, Sandra; Durham, Heather D; Agar, Jeffrey N

    2007-06-01

    One familial form of the neurodegenerative disease, amyotrophic lateral sclerosis, is caused by gain-of-function mutations in the gene encoding copper/zinc superoxide dismutase (SOD-1). This study provides in vivo evidence that normally occurring oxidative modification to SOD-1 promotes aggregation and toxicity of mutant proteins. The oxidation of Trp-32 was identified as a normal modification being present in both wild-type enzyme and SOD-1 with the disease-causing mutation, G93A, isolated from erythrocytes. Mutating Trp-32 to a residue with a slower rate of oxidative modification, phenylalanine, decreased both the cytotoxicity of mutant SOD-1 and its propensity to form cytoplasmic inclusions in motor neurons of dissociated mouse spinal cord cultures.

  13. Mutation induction in γ-irradiated primary human bronchial epithelial cells and molecular analysis of the HPRT- mutants

    International Nuclear Information System (INIS)

    Suzuki, Keiji; Hei, Tom K.

    1996-01-01

    We have examined various radiobiological parameters using commercially-available primary normal human bronchial epithelial (NHBE) cells, which can be subcultured more than 20 population doublings, and have established the mutation system in order to characterize the molecular changes in γ-irradiated primary cells. The survival curve, obtained after irradiation of cells with 137 Cs γ-rays, indicates that the D 0 , D q , and n values are 1.34 Gy, 1.12 Gy, and 2.3, respectively. The induction of HPRT - mutation was dose-dependent and the mutant fraction increased in a non-linear fashion. Since the doubling number of NHBE cells is limited, DNA was extracted directly from the single mutant colonies and alteration in the HPRT gene locus was analyzed using multiplex PCR technique. Among spontaneous mutants, the proportion with total and partial deletions of the gene was 10.0% (2/20) and 60.0% (12/20), respectively, while 30.0% (6/20) did not have any detectable changes in the nine exons examined. On the other hand, the fraction of total deletion increased by more than 2-fold among mutants induced by γ-rays in that 26.3% (10/38) of them showed the total gene deletions. Twenty-five out of 38 γ-induced mutants (65.8%) had partial deletions and 3 mutants (7.9%) had no detectable alteration. The present results showed that γ-irradiation efficiently induced HPRT gene mutation in primary human epithelial cells and that most of the induced mutants suffered larger deletions compared to that observed in spontaneous mutants. This system provides a useful tool for determination of mutagenicity and understanding the molecular mechanisms of environmental carcinogens in primary human bronchial cells

  14. Kinetic and structural evidences on human prolidase pathological mutants suggest strategies for enzyme functional rescue.

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    Roberta Besio

    Full Text Available Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients' fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.

  15. Autoradiographic detection of diphtheria toxin resistant mutants in human diploid fibroblasts

    International Nuclear Information System (INIS)

    Gupta, R.S.; Singh, B.

    1985-01-01

    An autoradiographic procedure for the detection of diphtheria toxin (DT) resistant (Dip/sub R/) mutants in human diploid fibroblast (HDF) cells has been developed. The assay is based on the observation that when HDFs from confluent cultures are seeded in medium containing 0.01 flocculating units/ml or higher concentration of DT, protein synthesis in sensitive cells is severely inhibited by 4-6 hr. If at this or later time, a radiolabeled protein precursor (eg, 3 H-leucine) is added to the culture, it is almost exclusively incorporated into the resistant cells, which are then readily identified by autoradiography. These studies provide strong evidence that the labeled cells identified by autoradiography are bona fide Dip/sub R/ mutants. The detection of Dip/sub R/ cells by autoradiography is apparently not affected by the presence of the sensitive cells in the mixtures. The spontaneous frequency of Dip/sub R/ cells in HDFs has been found to be in the range of 1-5 x 10 -6 , and this increases in a dose dependent manner upon treatment with the mutagen ethyl methanesulfonate. These results indicate that the autoradiographic assay could be used for quantitative mutagenesis. Since the autoradiographic assay does not depend on cell division, it may prove useful in estimating the incidence of pre-existing mutations in cell populations that either do not divide or have very limited growth potential (eg, lymphocytes, muscle cells, neurons, senescent fibroblasts, etc.)

  16. Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation.

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    Michael P Hefferan

    Full Text Available Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1 causes an inherited form of Amyotrophic Lateral Sclerosis (ALS. Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs into the lumbar spinal cord of SOD1(G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1(G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1(G93A animals.Presymptomatic SOD1(G93A rats (60-65 days old received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1(G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1(G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1(G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50-65% loss of large caliber descending motor axons.These data demonstrate that in order to achieve a more

  17. Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A.

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    Diana J M van den Wollenberg

    Full Text Available Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

  18. Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

    Science.gov (United States)

    Quinn, D; Orci, L; Ravazzola, M; Moore, H P

    1991-06-01

    Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway.

  19. Structural characterization of V57D and V57P mutants of human cystatin C, an amyloidogenic protein

    Energy Technology Data Exchange (ETDEWEB)

    Orlikowska, Marta; Szymańska, Aneta [University of Gdansk, Sobieskiego 18/19, 80-952 Gdansk (Poland); Borek, Dominika; Otwinowski, Zbyszek [University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8816 (United States); Skowron, Piotr; Jankowska, Elżbieta, E-mail: elaj@chem.univ.gda.pl [University of Gdansk, Sobieskiego 18/19, 80-952 Gdansk (Poland)

    2013-04-01

    Val57 point mutants of human cystatin C, which were designed to assess the influence of changes in the properties of the L1 loop on the dimerization propensity, were structurally characterized. Wild-type human cystatin C (hCC wt) is a low-molecular-mass protein (120 amino-acid residues, 13 343 Da) that is found in all nucleated cells. Physiologically, it functions as a potent regulator of cysteine protease activity. While the biologically active hCC wt is a monomeric protein, all crystallization efforts to date have resulted in a three-dimensional domain-swapped dimeric structure. In the recently published structure of a mutated hCC, the monomeric fold was preserved by a stabilization of the conformationally constrained loop L1 caused by a single amino-acid substitution: Val57Asn. Additional hCC mutants were obtained in order to elucidate the relationship between the stability of the L1 loop and the propensity of human cystatin C to dimerize. In one mutant Val57 was substituted by an aspartic acid residue, which is favoured in β-turns, and in the second mutant proline, a residue known for broadening turns, was substituted for the same Val57. Here, 2.26 and 3.0 Å resolution crystal structures of the V57D andV57P mutants of hCC are reported and their dimeric architecture is discussed in terms of the stabilization and destabilization effects of the introduced mutations.

  20. A Novel Iron Chelator-Radical Scavenger Ameliorates Motor Dysfunction and Improves Life Span and Mitochondrial Biogenesis in SOD1G93A ALS Mice.

    Science.gov (United States)

    Golko-Perez, Sagit; Amit, Tamar; Bar-Am, Orit; Youdim, Moussa B H; Weinreb, Orly

    2017-02-01

    The aim of the present study was to evaluate the therapeutic effect of the novel neuroprotective multitarget brain permeable monoamine oxidase inhibitor/iron chelating-radical scavenging drug, VAR10303 (VAR), co-administered with high-calorie/energy-supplemented diet (ced) in SOD1 G93A transgenic amyotrophic lateral sclerosis (ALS) mice. Administration of VAR-ced was initiated after the appearance of disease symptoms (at day 88), as this regimen is comparable with the earliest time at which drug therapy could start in ALS patients. Using this rescue protocol, we demonstrated in the current study that VAR-ced treatment provided several beneficial effects in SOD1 G93A mice, including improvement in motor performance, elevation of survival time, and attenuation of iron accumulation and motoneuron loss in the spinal cord. Moreover, VAR-ced treatment attenuated neuromuscular junction denervation and exerted a significant preservation of myofibril regular morphology, associated with a reduction in the expression levels of genes related to denervation and atrophy in the gastrocnemius (GNS) muscle in SOD1 G93A mice. These effects were accompanied by upregulation of mitochondrial DNA and elevated activities of complexes I and II in the GNS muscle. We have also demonstrated that VAR-ced treatment upregulated the mitochondrial biogenesis master regulator, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) and increased PGC-1α-targeted metabolic genes and proteins, such as, PPARγ, UCP1/3, NRF1/2, Tfam, and ERRα in GNS muscle. These results provide evidence of therapeutic potential of VAR-ced in SOD1 G93A mice with underlying molecular mechanisms, further supporting the importance role of multitarget iron chelators in ALS treatment.

  1. Bee Venom Acupuncture Augments Anti-Inflammation in the Peripheral Organs of hSOD1G93A Transgenic Mice.

    Science.gov (United States)

    Lee, Sun-Hwa; Choi, Sun-Mi; Yang, Eun Jin

    2015-07-29

    Amyotrophic lateral sclerosis (ALS) includes progressively degenerated motor neurons in the brainstem, motor cortex, and spinal cord. Recent reports demonstrate the dysfunction of multiple organs, including the lungs, spleen, and liver, in ALS animals and patients. Bee venom acupuncture (BVA) has been used for treating inflammatory diseases in Oriental Medicine. In a previous study, we demonstrated that BV prevented motor neuron death and increased anti-inflammation in the spinal cord of symptomatic hSOD1G93A transgenic mice. In this study, we examined whether BVA's effects depend on acupuncture point (ST36) in the organs, including the liver, spleen and kidney, of hSOD1G93A transgenic mice. We found that BV treatment at ST36 reduces inflammation in the liver, spleen, and kidney compared with saline-treatment at ST36 and BV injected intraperitoneally in symptomatic hSOD1G93A transgenic mice. Those findings suggest that BV treatment combined with acupuncture stimulation is more effective at reducing inflammation and increasing immune responses compared with only BV treatment, at least in an ALS animal model.

  2. A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS.

    Science.gov (United States)

    Hatzipetros, Theo; Kidd, Joshua D; Moreno, Andy J; Thompson, Kenneth; Gill, Alan; Vieira, Fernando G

    2015-10-06

    The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

  3. Administration of 4-(α-L-Rhamnosyloxy-benzyl Isothiocyanate Delays Disease Phenotype in SOD1G93A Rats: A Transgenic Model of Amyotrophic Lateral Sclerosis

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    Maria Galuppo

    2015-01-01

    Full Text Available 4-(α-L-Rhamnosyloxy-benzyl glucosinolate (glucomoringin, GMG is a compound found in Moringa oleifera seeds. Myrosinase-catalyzed hydrolysis at neutral pH of GMG releases the biologically active compound 4-(α-L-rhamnosyloxy-benzyl isothiocyanate (GMG-ITC. The present study was designed to test the potential therapeutic effectiveness of GMG-ITC to counteract the amyotrophic lateral sclerosis (ALS using SOD1tg rats, which physiologically develops SOD1G93A at about 16 weeks of life, and can be considered a genetic model of disease. Rats were treated once a day with GMG (10 mg/Kg bioactivated with myrosinase (20 µL/rat via intraperitoneal (i.p. injection for two weeks before disease onset and the treatment was prolonged for further two weeks before the sacrifice. Immune-inflammatory markers as well as apoptotic pathway were investigated to establish whether GMG-ITC could represent a new promising tool in clinical practice to prevent ALS. Achieved data display clear differences in molecular and biological profiles between treated and untreated SOD1tg rats leading to guessing that GMG-ITC can interfere with the pathophysiological mechanisms at the basis of ALS development. Therefore, GMG-ITC produced from myrosinase-catalyzed hydrolysis of pure GMG could be a candidate for further studies aimed to assess its possible use in clinical practice for the prevention or to slow down this disease.

  4. Rescue of a pathogenic mutant human glucagon receptor by pharmacological chaperones.

    Science.gov (United States)

    Yu, Run; Chen, Chun-Rong; Liu, Xiaohong; Kodra, János T

    2012-10-01

    We have previously demonstrated that a homozygous inactivating P86S mutation of the glucagon receptor (GCGR) causes a novel human disease of hyperglucagonemia, pancreatic α-cell hyperplasia, and pancreatic neuroendocrine tumors (Mahvash disease). The mechanisms for the decreased activity of the P86S mutant (P86S) are abnormal receptor localization to the endoplasmic reticulum (ER) and defective interaction with glucagon. To search for targeted therapies for Mahvash disease, we examined whether P86S can be trafficked to the plasma membrane by pharmacological chaperones and whether novel glucagon analogs restore effective receptor interaction. We used enhanced green fluorescent protein-tagged P86S stably expressed in HEK 293 cells to allow fluorescence imaging and western blotting and molecular modeling to design novel glucagon analogs in which alanine 19 was replaced with serine or asparagine. Incubation at 27 °C largely restored normal plasma membrane localization and normal processing of P86S but osmotic chaperones had no effects. The ER stressors thapsigargin and curcumin partially rescued P86S. The lipophilic GCGR antagonist L-168,049 also partially rescued P86S, so did Cpd 13 and 15 to a smaller degree. The rescued P86S led to more glucagon-stimulated cAMP production and was internalized by glucagon. Compared with the native glucagon, the novel glucagon analogs failed to stimulate more cAMP production by P86S. We conclude that the mutant GCGR is partially rescued by several pharmacological chaperones and our data provide proof-of-principle evidence that Mahvash disease can be potentially treated with pharmacological chaperones. The novel glucagon analogs, however, failed to interact with P86S more effectively.

  5. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Bin Huang

    Full Text Available Huntingtin (Htt is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q and mutant (46Q and 128Q Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

  6. A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.

    Science.gov (United States)

    Wang, Yuru; Havel, Jocelyn; Beal, Peter A

    2015-11-20

    Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.

  7. Elevated mRNA-levels of distinct mitochondrial and plasma membrane Ca2+ transporters in individual hypoglossal motor neurons of endstage SOD1 transgenic mice.

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    Tobias eMühling

    2014-11-01

    Full Text Available Disturbances in Ca2+ homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS, a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca2+ homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs from a common ALS mouse model, endstage superoxide dismutase SOD1G93A transgenic mice, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca2+ uptake capacity and elevated Ca2+ extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca2+ transporters in individual, choline-acetyltransferase (ChAT positive hMNs from wildtype (WT and endstage SOD1G93A mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1G93A mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca2+ transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca2+ transporters MCU/MICU1, Letm1 and UCP2 in remaining hMNs from endstage SOD1G93A mice. These higher expression-levels of mitochondrial Ca2+ transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na2+/Ca2+exchanger NCX1 was also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a novel neuroprotective strategy for ALS.

  8. The effects of 3% diquafosol sodium eye drop application on meibomian gland and ocular surface alterations in the Cu, Zn-superoxide dismutase-1 (Sod1) knockout mice.

    Science.gov (United States)

    Ikeda, Keisuke; Simsek, Cem; Kojima, Takashi; Higa, Kazunari; Kawashima, Motoko; Dogru, Murat; Shimizu, Takahiko; Tsubota, Kazuo; Shimazaki, Jun

    2018-04-01

    The purpose of the study is to investigate the effect of 3% diquafosol sodium eye drops on meibomian gland and ocular surface alterations in the superoxide dismutase-1 (Sod1 -/- ) mice in comparison to the wild-type mouse. Three percent diquafosol sodium eye drop was instilled to 20 eyes of 10 50-week-old male Sod1 -/- mice and 22 eyes of 11 C57BL/6 strain 50-week-old wild-type (WT) male mice six times a day for 2 weeks. Aqueous tear secretion quantity was measured with phenol red-impregnated cotton threads without anesthesia. Tear film stability and corneal epithelial damage were assessed by fluorescein and lissamine green staining. We also performed oil red O (ORO) lipid staining to evaluate the lipid changes in the meibomian glands. Meibomian gland specimens underwent hematoxylin and eosin staining to examine histopathological changes and meibomian gland acinar unit density after sacrifice. Immunohistochemistry staining was performed using cytokeratin 4, cytokeratin 13, and transglutaminase-1 antibodies. Quantitative real-time polymerase chain reaction for cytokeratin 4, cytokeratin 13, and transglutaminase-1 mRNA expression was also performed. The aqueous tear quantity, the mean tear film breakup time, and the number of lipid droplets significantly improved in the Sod1 -/- mice with treatment. The mean meibomian acinar unit density did not change in the Sod1 -/- mice and WT mice after treatment. Application of 3% diquafosol sodium eye drop significantly decreased the corneal fluorescein and lissamine green staining scores in the Sod1 -/- mice after 2 weeks. We showed a notable increase in cytokeratin 4, cytokeratin 13 immunohistochemistry staining, and cytokeratin 4, cytokeratin 13 mRNA expressions with a marked decrease in immunohistochemistry staining and significant decline in mRNA expression of transglutaminase-1 after 3% diquafosol sodium treatment. Topical application of 3% diquafosol sodium eye drop improved the number of lipid droplets, tear stability

  9. Restoration of tumor suppressor miR-34 inhibits human p53-mutant gastric cancer tumorspheres

    International Nuclear Information System (INIS)

    Ji, Qing; Hao, Xinbao; Meng, Yang; Zhang, Min; DeSano, Jeffrey; Fan, Daiming; Xu, Liang

    2008-01-01

    MicroRNAs (miRNAs), some of which function as oncogenes or tumor suppressor genes, are involved in carcinogenesis via regulating cell proliferation and/or cell death. MicroRNA miR-34 was recently found to be a direct target of p53, functioning downstream of the p53 pathway as a tumor suppressor. miR-34 targets Notch, HMGA2, and Bcl-2, genes involved in the self-renewal and survival of cancer stem cells. The role of miR-34 in gastric cancer has not been reported previously. In this study, we examined the effects of miR-34 restoration on p53-mutant human gastric cancer cells and potential target gene expression. Human gastric cancer cells were transfected with miR-34 mimics or infected with the lentiviral miR-34-MIF expression system, and validated by miR-34 reporter assay using Bcl-2 3'UTR reporter. Potential target gene expression was assessed by Western blot for proteins, and by quantitative real-time RT-PCR for mRNAs. The effects of miR-34 restoration were assessed by cell growth assay, cell cycle analysis, caspase-3 activation, and cytotoxicity assay, as well as by tumorsphere formation and growth. Human gastric cancer Kato III cells with miR-34 restoration reduced the expression of target genes Bcl-2, Notch, and HMGA2. Bcl-2 3'UTR reporter assay showed that the transfected miR-34s were functional and confirmed that Bcl-2 is a direct target of miR-34. Restoration of miR-34 chemosensitized Kato III cells with a high level of Bcl-2, but not MKN-45 cells with a low level of Bcl-2. miR-34 impaired cell growth, accumulated the cells in G1 phase, increased caspase-3 activation, and, more significantly, inhibited tumorsphere formation and growth. Our results demonstrate that in p53-deficient human gastric cancer cells, restoration of functional miR-34 inhibits cell growth and induces chemosensitization and apoptosis, indicating that miR-34 may restore p53 function. Restoration of miR-34 inhibits tumorsphere formation and growth, which is reported to be

  10. Comparison of HPRT mutant frequency in human peripheral blood lymphocytes of smokers and non-smokers

    International Nuclear Information System (INIS)

    Vivek Kumar, P.R.; Mary Mohankumar, N.; Chatterjee, Indranil; Jeevanram, R.K.

    2003-01-01

    The mutant frequency of hypoxanthine guanine phospho ribosyl transferase (HPRT) has been studied in peripheral blood lymphocytes of six non-smokers and six smokers. The mutant frequency was studied by following a Uniform Operating Protocol (UOP). The mean lymphocyte cloning efficiency of non-smokers and smokers was about 31 %. The mean mutant frequency obtained in smokers showed a marginal increase compared to that of non-smokers, but they were not significantly different (P= 0.1416). This paper discusses the methodology adopted and the results obtained with the preliminary finding. (author)

  11. Chemical Rescue of Enzymes: Proton Transfer in Mutants of Human Carbonic Anhydrase II

    Science.gov (United States)

    Maupin, C. Mark; Castillo, Norberto; Taraphder, Srabani; Tu, Chingkuang; McKenna, Robert; Silverman, David N.; Voth, Gregory A.

    2011-01-01

    In human carbonic anhydrase II (HCA II) the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II give a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site specific mutations intended to disrupt 4MI binding have demonstrated these sites to be non-productive. In the present work MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway PMID:21452838

  12. Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Cirullo, R.E.; Dana, S.; Wasmuth, J.J.

    1983-01-01

    A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure

  13. Non-invasive assessment of animal exercise stress: real-time PCR of GLUT4, COX2, SOD1 and HSP70 in avalanche military dog saliva.

    Science.gov (United States)

    Diverio, S; Guelfi, G; Barbato, O; Di Mari, W; Egidi, M G; Santoro, M M

    2015-01-01

    Exercise has been shown to increase mRNA expression of a growing number of genes. The aim of this study was to assess if mRNA expression of the metabolism- and oxidative stress-related genes GLUT4 (glucose transporter 4), COX2 (cyclooxygenase 2), SOD1 (superoxide dismutase 1) and HSP70 (heat shock protein 70) in saliva changes following acute exercise stress in dogs. For this purpose, 12 avalanche dogs of the Italian Military Force Guardia di Finanza were monitored during simulation of a search for a buried person in an artificial avalanche area. Rectal temperature (RT) and saliva samples were collected the day before the trial (T0), immediately after the descent from a helicopter at the onset of a simulated avalanche search and rescue operation (T1), after the discovery of the buried person (T2) and 2 h later (T3). Expressions of GLUT4, SOD1, COX2 and HSP70 were measured by real-time PCR. The simulated avalanche search and rescue operation was shown to exert a significant effect on RT, as well as on the expression of all metabolism- and oxidative stress-related genes investigated, which peaked at T2. The observed expression patterns indicate an acute exercise stress-induced upregulation, as confirmed by the reductions in expression at T3. Moreover, our findings indicate that saliva is useful for assessing metabolism- and oxidative stress-related genes without the need for restraint, which could affect working dog performance.

  14. Comparison of the crystal structure and function to wild-type and His25Ala mutant human heme oxygenase-1.

    Science.gov (United States)

    Zhou, Wen-Pu; Zhong, Wen-Wei; Zhang, Xue-Hong; Ding, Jian-Ping; Zhang, Zi-Li; Xia, Zhen-Wei

    2009-03-01

    Human heme oxygenase-1 (hHO-1) is a rate-limiting enzyme in heme metabolism. It regulates serum bilirubin level. Site-directed mutagenesis studies indicate that the proximal residue histidine 25 (His25) plays a key role in hHO-1 activity. A highly purified hHO-1 His25Ala mutant was generated and crystallized with a new expression system. The crystal structure of the mutant was determined by X-ray diffraction technology and molecular replacement at the resolution of 2.8 A, and the model of hHO-1 His25Ala mutant was refined. The final crystallographic and free R factors were 0.245 and 0.283, respectively. The standard bond length deviation was 0.007 A, and the standard bond angle deviation was 1.3 degrees . The mutation of His25 to Ala led to an empty pocket underneath the ferric ion in the heme, leading to loss of binding iron ligand. Although this did not cause an overall structural change, the enzymatic activity of the mutant hHO-1 was reduced by 90%. By supplementing imidazole, the HO-1 activity was restored approximately 90% to its normal level. These data suggest that Ala25 remains unchanged in the structure compared to His25, but the important catalytic function of hHO-1 is lost. Thus, it appears that His25 is a crucial residue for proper hHO-1 catalysis.

  15. Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

    Science.gov (United States)

    Nakayama, Takuya; Fisher, Marilyn; Nakajima, Keisuke; Odeleye, Akinleye O; Zimmerman, Keith B; Fish, Margaret B; Yaoita, Yoshio; Chojnowski, Jena L; Lauderdale, James D; Netland, Peter A; Grainger, Robert M

    2015-12-15

    Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Functional phenotypic rescue of Caenorhabditis elegans neuroligin-deficient mutants by the human and rat NLGN1 genes.

    Directory of Open Access Journals (Sweden)

    Fernando Calahorro

    Full Text Available Neuroligins are cell adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin encoding genes lead to autism spectrum disorder and/or mental retardation. Caenorhabditis elegans mutants deficient in nlg-1, an orthologue of human neuroligin genes, have defects in different behaviors. Here we show that the expression of human NLGN1 or rat Nlgn1 cDNAs in C. elegans nlg-1 mutants rescues the fructose osmotic strength avoidance and gentle touch response phenotypes. Two specific point mutations in NLGN3 and NLGN4 genes, involved in autistic spectrum disorder, were further characterized in this experimental system. The R451C allele described in NLGN3, was analyzed with both human NLGN1 (R453C and worm NLG-1 (R437C proteins, and both were not functional in rescuing the osmotic avoidance behavior and the gentle touch response phenotype. The D396X allele described in NLGN4, which produces a truncated protein, was studied with human NLGN1 (D432X and they did not rescue any of the behavioral phenotypes analyzed. In addition, RNAi feeding experiments measuring gentle touch response in wild type strain and worms expressing SID-1 in neurons (which increases the response to dsRNA, both fed with bacteria expressing dsRNA for nlg-1, provided evidence for a postsynaptic in vivo function of neuroligins both in muscle cells and neurons, equivalent to that proposed in mammals. This finding was further confirmed generating transgenic nlg-1 deficient mutants expressing NLG-1 under pan-neuronal (nrx-1 or pan-muscular (myo-3 specific promoters. All these results suggest that the nematode could be used as an in vivo model for studying particular synaptic mechanisms with proteins orthologues of humans involved in pervasive developmental disorders.

  17. Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

    Directory of Open Access Journals (Sweden)

    Antunes Edson

    2008-05-01

    Full Text Available Abstract Background Obesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings. Methods Male Wistar rats were divided into sedentary (SD; trained (TR; sedentary diet (SDD and trained diet (TRD groups. Run training (RT was performed in sessions of 60 min, 5 days/week for 12 weeks (70–80% VO2max. Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx- were measured. Concentration-response curves to acetylcholine (ACh and sodium nitroprusside (SNP were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1 was assessed by Western blotting. Results High caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl. Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml. On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl. Neither high caloric diet nor RT modified NOx- levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM. Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%, but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups or mesenteric rings (10%, for TR and 17%, to TRD groups that was accompanied by up-regulation of SOD-1

  18. GFP-Mutant Human Tau Transgenic Mice Develop Tauopathy Following CNS Injections of Alzheimer's Brain-Derived Pathological Tau or Synthetic Mutant Human Tau Fibrils.

    Science.gov (United States)

    Gibbons, Garrett S; Banks, Rachel A; Kim, Bumjin; Xu, Hong; Changolkar, Lakshmi; Leight, Susan N; Riddle, Dawn M; Li, Chi; Gathagan, Ronald J; Brown, Hannah J; Zhang, Bin; Trojanowski, John Q; Lee, Virginia M-Y

    2017-11-22

    Neurodegenerative proteinopathies characterized by intracellular aggregates of tau proteins, termed tauopathies, include Alzheimer's disease (AD), frontotemporal lobar degeneration (FTLD) with tau pathology (FTLD-tau), and related disorders. Pathological tau proteins derived from human AD brains (AD-tau) act as proteopathic seeds that initiate the templated aggregation of soluble tau upon intracerebral injection into tau transgenic (Tg) and wild-type mice, thereby modeling human tau pathology. In this study, we found that aged Tg mice of both sexes expressing human tau proteins harboring a pathogenic P301L MAPT mutation labeled with green fluorescent protein (T40PL-GFP Tg mouse line) exhibited hyperphosphorylated tau mislocalized to the somatodentritic domain of neurons, but these mice did not develop de novo insoluble tau aggregates, which are characteristic of human AD and related tauopathies. However, intracerebral injections of either T40PL preformed fibrils (PFFs) or AD-tau seeds into T40PL-GFP mice induced abundant intraneuronal pathological inclusions of hyperphosphorylated T40PL-GFP. These injections of pathological tau resulted in the propagation of tau pathology from the injection site to neuroanatomically connected brain regions, and these tau inclusions consisted of both T40PL-GFP and WT endogenous mouse tau. Primary neurons cultured from the brains of neonatal T40PL-GFP mice provided an informative in vitro model for examining the uptake and localization of tau PFFs. These findings demonstrate the seeded aggregation of T40PL-GFP in vivo by synthetic PFFs and human AD-tau and the utility of this system to study the neuropathological spread of tau aggregates. SIGNIFICANCE STATEMENT The stereotypical spread of pathological tau protein aggregates have recently been attributed to the transmission of proteopathic seeds. Despite the extensive use of transgenic mouse models to investigate the propagation of tau pathology in vivo , details of the aggregation

  19. Acrolein-Induced Oxidative Stress and Cell Death Exhibiting Features of Apoptosis in the Yeast Saccharomyces cerevisiae Deficient in SOD1.

    Science.gov (United States)

    Kwolek-Mirek, Magdalena; Zadrąg-Tęcza, Renata; Bednarska, Sabina; Bartosz, Grzegorz

    2015-04-01

    The yeast Saccharomyces cerevisiae is a useful eukaryotic model to study the toxicity of acrolein, an important environmental toxin and endogenous product of lipid peroxidation. The study was aimed at elucidation of the cytotoxic effect of acrolein on the yeast deficient in SOD1, Cu, Zn-superoxide dismutase which is hypersensitive to aldehydes. Acrolein generated within the cell from its precursor allyl alcohol caused growth arrest and cell death of the yeast cells. The growth inhibition involved an increase in production of reactive oxygen species and high level of protein carbonylation. DNA condensation and fragmentation, exposition of phosphatidylserine at the cell surface as well as decreased dynamic of actin microfilaments and mitochondria disintegration point to the induction of apoptotic-type cell death besides necrotic cell death.

  20. [Lower urinary tract dysfunction and neuropathological findings of the neural circuits controlling micturition in familial amyotrophic lateral sclerosis with L106V mutation in the SOD1 gene].

    Science.gov (United States)

    Hineno, Akiyo; Oyanagi, Kiyomitsu; Nakamura, Akinori; Shimojima, Yoshio; Yoshida, Kunihiro; Ikeda, Shu-Ichi

    2016-01-01

    We report lower urinary tract dysfunction and neuropathological findings of the neural circuits controlling micturition in the patients with familial amyotrophic lateral sclerosis having L106V mutation in the SOD1 gene. Ten of 20 patients showed lower urinary tract dysfunction and 5 patients developed within 1 year after the onset of weakness. In 8 patients with an artificial respirator, 6 patients showed lower urinary tract dysfunction. Lower urinary tract dysfunction and respiratory failure requiring an artificial respirator occurred simultaneously in 3 patients. Neuronal loss and gliosis were observed in the neural circuits controlling micturition, such as frontal lobe, thalamus, hypothalamus, striatum, periaqueductal gray, ascending spinal tract, lateral corticospinal tract, intermediolateral nucleus and Onufrowicz' nucleus. Lower urinary tract dysfunction, especially storage symptoms, developed about 1 year after the onset of weakness, and the dysfunction occurred simultaneously with artificial respirator use in the patients.

  1. Production of a Marfan cellular phenotype by expressing a mutant human fibrillin allele on a normal human or murine genetic background

    Energy Technology Data Exchange (ETDEWEB)

    Eldadah, Z.A.; Dietz, H.C. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States); Brenn, T. [Stanford Univ. Medical Center, CA (United States)] [and others

    1994-09-01

    The Marfan Syndrome (MFS) is a heritable disorder of connective tissue caused by defects in fibrillin (FBN1), a 350 kD glycoprotein and principal component of the extracellular microfibril. Previous correlations of mutant transcript level and disease severity suggested a dominant negative model of MFS pathogenesis. To address this hypothesis we assembled an expression construct containing the mutant allele from a patient with severe MFS. This mutation causes skipping of FBN1 exon 2 and a frame shift, leading to a premature termination codon in exon 4. The predicted peptide would thus consist of 55 wild type and 45 missense amino acids. The construct was stably transfected into cultured human and mouse fibroblasts, and several clonal cell populations were established. Human and mouse cells expressing the truncated peptide exhibited markedly diminished fibrillin deposition and disorganized microfibrillar architecture by immunofluorescence. Pulse-chase analysis of these cells demonstrated normal levels of fibrillin synthesis but substantially decreased fibrillin deposition into the extracellular matrix. These data illustrate that expression of a mutant FBN1 allele, on a background of two normal alleles, is sufficient to disrupt normal fibrillin aggregation and reproduce the MFS cellular phenotype. This provides confirmation of a dominant negative model of MFS pathogenesis and may offer mutant allele knockout as a strategy for gene therapy. In addition, these data underscore the importance of the FBN1 amino-terminus in normal multimer formation and suggest that expression of the human extreme 5{prime} FBN1 coding sequence may be sufficient, in isolation, to produce an animal model of MFS. Indeed, transgenic mice harboring this mutant allele have been produced, and phenotype analysis is currently in progress.

  2. Unlike physical exercise, modified environment increases the lifespan of SOD1G93A mice however both conditions induce cellular changes.

    Directory of Open Access Journals (Sweden)

    Yannick N Gerber

    Full Text Available Amyotrophic lateral sclerosis (ALS is characterized by a gradual muscular paralysis resulting from progressive motoneurons death. ALS etiology remains unknown although it has been demonstrated to be a multifactorial disease involving several cellular partners. There is currently no effective treatment. Even if the effect of exercise is under investigation for many years, whether physical exercise is beneficial or harmful is still under debate.We investigated the effect of three different intensities of running exercises on the survival of SOD1(G93A mice. At the early-symptomatic stage (P60, males were isolated and randomly assigned to 5 conditions: 2 sedentary groups ("sedentary" and "sedentary treadmill" placed on the inert treadmill, and 3 different training intensity groups (5 cm/s, 10 cm/s and 21 cm/s; 15 min/day, 5days/week. We first demonstrated that an appropriate "control" of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an 11.6% increase in survival in the "sedentary treadmill" group. Moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. In a second step, we showed that when compared with the proper control, all three running-based training did not modify lifespan of the animals, but result in motoneurons preservation and changes in glial cells activation.We demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of SOD1(G93A. Using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of ALS mice but induce cellular modifications. Our results highlight the critical importance of the control of the environment in ALS studies and may explain discrepancy in the literature regarding the

  3. Potent Sensitisation of Cancer Cells to Anticancer Drugs by a Quadruple Mutant of the Human Deoxycytidine Kinase.

    Directory of Open Access Journals (Sweden)

    Safiatou T Coulibaly

    Full Text Available Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK mutant (G12 that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36 that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC, for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches.

  4. Finding Inhibitors of Mutant Superoxide Dismutase-1 for Amyotrophic Lateral Sclerosis Therapy from Traditional Chinese Medicine

    Directory of Open Access Journals (Sweden)

    Hung-Jin Huang

    2014-01-01

    Full Text Available Superoxide dismutase type 1 (SOD1 mutations cause protein aggregation and decrease protein stability, which are linked to amyotrophic lateral sclerosis (ALS disease. This research utilizes the world’s largest traditional Chinese medicine (TCM database to search novel inhibitors of mutant SOD1, and molecular dynamics (MD simulations were used to analyze the stability of protein that interacted with docked ligands. Docking results show that hesperidin and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG have high affinity to mutant SOD1 and then dopamine. For MD simulation analysis, hesperidin and THSG displayed similar value of RMSD with dopamine, and the migration analysis reveals stable fluctuation at the end of MD simulation time. Interestingly, distance between the protein and ligand has distinct difference, and hesperidin changes the position from initial binding site to the other place. In flexibility of residues analysis, the secondary structure among all complexes does not change, indicating that the structure are not affect ligand binding. The binding poses of hesperidin and THSG are similar to dopamine after molecular simulation. Our result indicated that hesperidin and THSG might be potential lead compound to design inhibitors of mutant SOD1 for ALS therapy.

  5. Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

    Science.gov (United States)

    Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

    2005-01-28

    The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

  6. Mutant p53 protein in serum could be used as a molecular marker in human breast cancer.

    Science.gov (United States)

    Balogh, G A; Mailo, D A; Corte, M M; Roncoroni, P; Nardi, H; Vincent, E; Martinez, D; Cafasso, M E; Frizza, A; Ponce, G; Vincent, E; Barutta, E; Lizarraga, P; Lizarraga, G; Monti, C; Paolillo, E; Vincent, R; Quatroquio, R; Grimi, C; Maturi, H; Aimale, M; Spinsanti, C; Montero, H; Santiago, J; Shulman, L; Rivadulla, M; Machiavelli, M; Salum, G; Cuevas, M A; Picolini, J; Gentili, A; Gentili, R; Mordoh, J

    2006-04-01

    p53 wild-type is a tumor suppressor gene involved in DNA gene transcription or DNA repair mechanisms. When damage to DNA is unrepairable, p53 induces programmed cell death (apoptosis). The mutant p53 gene is the most frequent molecular alteration in human cancer, including breast cancer. Here, we analyzed the genetic alterations in p53 oncogene expression in 55 patients with breast cancer at different stages and in 8 normal women. We measured by ELISA assay the serum levels of p53 mutant protein and p53 antibodies. Immunohistochemistry and RT-PCR using specific p53 primers as well as mutation detection by DNA sequencing were also evaluated in breast tumor tissue. Serological p53 antibody analysis detected 0/8 (0%), 0/4 (0%) and 9/55 (16.36%) positive cases in normal women, in patients with benign breast disease and in breast carcinoma, respectively. We found positive p53 mutant in the sera of 0/8 (0.0%) normal women, 0/4 (0%) with benign breast disease and 29/55 (52.72%) with breast carcinoma. Immunohistochemistry evaluation was positive in 29/55 (52.73%) with mammary carcinoma and 0/4 (0%) with benign breast disease. A very good correlation between p53 mutant protein detected in serum and p53 accumulation by immunohistochemistry (83.3% positive in both assays) was found in this study. These data suggest that detection of mutated p53 could be a useful serological marker for diagnostic purposes.

  7. Cyclopropane fatty acid synthase mutants of probiotic human-derived Lactobacillus reuteri are defective in TNF inhibition.

    Science.gov (United States)

    Jones, Sara E; Whitehead, Kristi; Saulnier, Delphine; Thomas, Carissa M; Versalovic, James; Britton, Robert A

    2011-01-01

    Although commensal microbes have been shown to modulate host immune responses, many of the bacterial factors that mediate immune regulation remain unidentified. Select strains of human-derived Lactobacillus reuteri synthesize immunomodulins that potently inhibit production of the inflammatory cytokine TNF. In this study, genetic and genomic approaches were used to identify and investigate L. reuteri genes required or human TNF immunomodulatory activity. Analysis of membrane fatty acids from multiple L. reuteri strains cultured in MRS medium showed that only TNF inhibitory strains produced the cyclopropane fatty acid (CFA) lactobacillic acid. The enzyme cyclopropane fatty acid synthase is required for synthesis of CFAs such as lactobacillic acid, therefore the cfa gene was inactivated and supernatants from the cfa mutant strain were assayed for TNF inhibitory activity. We found that supernatants from the wild-type strain, but not the cfa mutant, suppressed TNF production by activated THP-1 human monocytoid cells Although this suggested a direct role for lactobacillic acid in immunomodulation, purified lactobacillic acid did not suppress TNF at physiologically relevant concentrations. We further analyzed TNF inhibitory and TNF non-inhibitory strains under different growth conditions and found that lactobacillic acid production did not correlate with TNF inhibition. These results indicate that cfa indirectly contributed to L. reuter immunomodulatory activity and suggest that other mechanisms, such as decreased membrane fluidity or altered expression of immunomodulins, result in the loss of TNF inhibitory activity. By increasing our understanding of immunomodulation by probiotic species, beneficial microbes can be rationally selected to alleviate intestinal inflammation.

  8. ALS-causing profilin-1-mutant forms a non-native helical structure in membrane environments.

    Science.gov (United States)

    Lim, Liangzhong; Kang, Jian; Song, Jianxing

    2017-11-01

    Despite having physiological functions completely different from superoxide dismutase 1 (SOD1), profilin 1 (PFN1) also carries mutations causing amyotrophic lateral sclerosis (ALS) with a striking similarity to that triggered by SOD1 mutants. Very recently, the C71G-PFN1 has been demonstrated to cause ALS by a gain of toxicity and the acceleration of motor neuron degeneration preceded the accumulation of its aggregates. Here by atomic-resolution NMR determination of conformations and dynamics of WT-PFN1 and C71G-PFN1 in aqueous buffers and in membrane mimetics DMPC/DHPC bicelle and DPC micelle, we deciphered that: 1) the thermodynamic destabilization by C71G transforms PFN1 into coexistence with the unfolded state, which is lacking of any stable tertiary/secondary structures as well as restricted ps-ns backbone motions, thus fundamentally indistinguishable from ALS-causing SOD1 mutants. 2) Most strikingly, while WT-PFN1 only weakly interacts with DMPC/DHPC bicelle without altering the native structure, C71G-PFN1 acquires abnormal capacity in strongly interacting with DMPC/DHPC bicelle and DPC micelle, energetically driven by transforming the highly disordered unfolded state into a non-native helical structure, similar to what has been previously observed on ALS-causing SOD1 mutants. Our results imply that one potential mechanism for C71G-PFN1 to initiate ALS might be the abnormal interaction with membranes as recently established for SOD1 mutants. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Comparative Analyses of Cu-Zn Superoxide Dismutase (SOD1) and Thioredoxin Reductase (TrxR) at the mRNA Level between Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) Under Stress Conditions.

    Science.gov (United States)

    Koo, Hyun-Na; Lee, Soon-Gyu; Yun, Seung-Hwan; Kim, Hyun Kyung; Choi, Yong Soo; Kim, Gil-Hah

    2016-01-01

    This study compared stress-induced expression of Cu-Zn superoxide dismutase (SOD1) and thioredoxin reductase (TrxR) genes in the European honeybee Apis mellifera L. and Asian honeybee Apis cerana F. Expression of both SOD1 and TrxR rapidly increased up to 5 h after exposure to cold (4 °C) or heat (37 °C) treatment and then gradually decreased, with a stronger effect induced by cold stress in A. mellifera compared with A. cerana. Injection of stress-inducing substances (methyl viologen, [MV] and H2O2) also increased SOD1 and TrxR expression in both A. mellifera and A. cerana, and this effect was more pronounced with MV than H2O2. Additionally, we heterologously expressed the A. mellifera and A. cerana SOD1 and TrxR proteins in an Escherichia coli expression system, and detection by SDS-PAGE, confirmed by Western blotting using anti-His tag antibodies, revealed bands at 16 and 60 kDa, respectively. Our results show that the expression patterns of SOD1 and TrxR differ between A. mellifera and A. cerana under conditions of low or high temperature as well as oxidative stress. © The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.

  10. Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Daniele Vergara

    2017-01-01

    Full Text Available In this study, we investigated by two-dimensional gel electrophoresis (2-DE and mass spectrometry (MS analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson’s disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson’s disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts.

  11. Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

    Science.gov (United States)

    Gaballo, Antonio; Signorile, Anna; Tanzarella, Paola; Pacelli, Consiglia; Di Paola, Marco

    2017-01-01

    In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson's disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson's disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts. PMID:29138676

  12. Hyperactivity and learning deficits in transgenic mice bearing a human mutant thyroid hormone beta1 receptor gene.

    Science.gov (United States)

    McDonald, M P; Wong, R; Goldstein, G; Weintraub, B; Cheng, S Y; Crawley, J N

    1998-01-01

    Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor beta (TRbeta) gene on chromosome 3, representing a mutation of the ligand-binding domain of the TRbeta gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRbeta gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRbeta gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD.

  13. Hyperactivity and Learning Deficits in Transgenic Mice Bearing a Human Mutant Thyroid Hormone β1 Receptor Gene

    Science.gov (United States)

    McDonald, Michael P.; Wong, Rosemary; Goldstein, Gregory; Weintraub, Bruce; Cheng, Sheue-yann; Crawley, Jacqueline N.

    1998-01-01

    Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor β (TRβ) gene on chromosome 3, representing a mutation of the ligandbinding domain of the TRβ gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRβ gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRβ gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD. PMID:10454355

  14. A Dimeric Mutant of Human Pancreatic Ribonuclease with Selective Cytotoxicity toward Malignant Cells

    Science.gov (United States)

    Piccoli, Renata; di Gaetano, Sonia; de Lorenzo, Claudia; Grauso, Michela; Monaco, Carmen; Spalletti-Cernia, Daniela; Laccetti, Paolo; Cinatl, Jaroslav; Matousek, Josef; D'Alessio, Giuseppe

    1999-07-01

    Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.

  15. [Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

    Science.gov (United States)

    Morozkina, E V; Vavilova, E A; Zatsepin, S S; Klyachko, E V; Yagudin, T A; Chulkin, A M; Dudich, I V; Semenkova, L N; Churilova, I V; Benevolenskii, S V

    2016-01-01

    A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

  16. Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein.

    Science.gov (United States)

    Pype, Stefan; Moechars, Dieder; Dillen, Lieve; Mercken, Marc

    2003-02-01

    Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.

  17. In vivo EPR pharmacokinetic evaluation of the redox status and the blood brain barrier permeability in the SOD1G93A ALS rat model.

    Science.gov (United States)

    Stamenković, Stefan; Pavićević, Aleksandra; Mojović, Miloš; Popović-Bijelić, Ana; Selaković, Vesna; Andjus, Pavle; Bačić, Goran

    2017-07-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting the motor pathways of the central nervous system. Although a number of pathophysiological mechanisms have been described in the disease, post mortem and animal model studies indicate blood-brain barrier (BBB) disruption and elevated production of reactive oxygen species as major contributors to disease pathology. In this study, the BBB permeability and the brain tissue redox status of the SOD1 G93A ALS rat model in the presymptomatic (preALS) and symptomatic (ALS) stages of the disease were investigated by in vivo EPR spectroscopy using three aminoxyl radicals with different cell membrane and BBB permeabilities, Tempol, 3-carbamoyl proxyl (3CP), and 3-carboxy proxyl (3CxP). Additionally, the redox status of the two brain regions previously implicated in disease pathology, brainstem and hippocampus, was investigated by spectrophotometric biochemical assays. The EPR results indicated that among the three spin probes, 3CP is the most suitable for reporting the intracellular redox status changes, as Tempol was reduced in vivo within minutes (t 1/2 =2.0±0.5min), thus preventing reliable kinetic modeling, whereas 3CxP reduction kinetics gave divergent conclusions, most probably due to its membrane impermeability. It was observed that the reduction kinetics of 3CP in vivo, in the head of preALS and ALS SOD1 G93A rats was altered compared to the controls. Pharmacokinetic modeling of 3CP reduction in vivo, revealed elevated tissue distribution and tissue reduction rate constants indicating an altered brain tissue redox status, and possibly BBB disruption in these animals. The preALS and ALS brain tissue homogenates also showed increased nitrilation, superoxide production, lipid peroxidation and manganese superoxide dismutase activity, and a decreased copper-zinc superoxide dismutase activity. The present study highlights in vivo EPR spectroscopy as a reliable tool for the investigation of

  18. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

    International Nuclear Information System (INIS)

    Geller, A.I.; Keyomarsi, K.; Bryan, J.; Pardee, A.B.

    1990-01-01

    The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli β-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses β-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system

  19. Human T-Cell Leukemia Virus I Tax Protein Sensitizes p53-Mutant Cells to DNA Damage

    Science.gov (United States)

    Mihaylova, Valia T.; Green, Allison M.; Khurgel, Moshe; Semmes, Oliver J.; Kupfer, Gary M.

    2018-01-01

    Mutations in p53 are a common cause of resistance of cancers to standard chemotherapy and, thus, treatment failure. Reports have shown that Tax, a human T-cell leukemia virus type I encoded protein that has been associated with genomic instability and perturbation of transcription and cell cycle, sensitizes HeLa cells to UV treatment. The extent to which Tax can sensitize cells and the mechanism by which it exerts its effect are unknown. In this study, we show that Tax sensitizes p53-mutant cells to a broad range of DNA-damaging agents, including mitomycin C, a bifunctional alkylator, etoposide, a topoisomerase II drug, and UV light, but not ionizing radiation, a double-strand break agent, or vinblastine, a tubulin poison. Tax caused hypersensitivity in all p53-deleted cell lines and several, but not all, mutant-expressed p53–containing cell lines, while unexpectedly being protective in p53 wild-type (wt) cells. The effect observed in p53-deleted lines could be reversed for this by transfection of wt p53. We also show that Tax activates a p53-independent proapoptotic program through decreased expression of the retinoblastoma protein and subsequent increased E2F1 expression. The expression of several proapoptotic proteins was also induced by Tax, including Puma and Noxa, culminating in a substantial increase in Bax dimerization. Our results show that Tax can sensitize p53-mutant cells to DNA damage while protecting p53 wt cells, a side benefit that might result in reduced toxicity in normal cells. Such studies hold the promise of a novel adjunctive therapy that could make cancer chemotherapy more effective. PMID:18559532

  20. Systemic injection of AAV9-GDNF provides modest functional improvements in the SOD1G93A ALS rat but has adverse side effects.

    Science.gov (United States)

    Thomsen, G M; Alkaslasi, M; Vit, J-P; Lawless, G; Godoy, M; Gowing, G; Shelest, O; Svendsen, C N

    2017-04-01

    Injecting proteins into the central nervous system that stimulate neuronal growth can lead to beneficial effects in animal models of disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has shown promise in animal and cell models of Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS). Here, systemic AAV9-GDNF was delivered via tail vein injections to young rats to determine whether this could be a safe and functional strategy to treat the SOD1 G93A rat model of ALS and, therefore, translated to a therapy for ALS patients. We found that GDNF administration in this manner resulted in modest functional improvement, whereby grip strength was maintained for longer and the onset of forelimb paralysis was delayed compared to non-treated rats. This did not, however, translate into an extension in survival. In addition, ALS rats receiving GDNF exhibited slower weight gain, reduced activity levels and decreased working memory. Collectively, these results confirm that caution should be applied when applying growth factors such as GDNF systemically to multiple tissues.

  1. Dysregulated expression of death, stress and mitochondrion related genes in the sciatic nerve of presymptomatic SOD1G93A mouse model of Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Chrystian Junqueira Alves

    2015-09-01

    Full Text Available Schwann cells are the main source of paracrine support to motor neurons. Oxidative stress and mitochondrial dysfunction have been correlated to motor neuron death in Amyotrophic Lateral Sclerosis (ALS. Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown. Sciatic nerves of presymptomatic (60-day-old SOD1G93A mice were submitted to a high-density oligonucleotide microarray analysis. DAVID demonstrated the deregulated genes related to death, stress and mitochondrion, which allowed the identification of Cell cycle, ErbB signaling, Tryptophan metabolism and Rig-I-like receptor signaling as the most representative KEGG pathways. The protein-protein interaction networks based upon deregulated genes have identified the top hubs (TRAF2, H2AFX, E2F1, FOXO3, MSH2, NGFR, TGFBR1 and bottlenecks (TRAF2, E2F1, CDKN1B, TWIST1, FOXO3. Schwann cells were enriched from the sciatic nerve of presymptomatic mice using flow cytometry cell sorting. qPCR showed the up regulated (Ngfr, Cdnkn1b, E2f1, Traf2 and Erbb3, H2afx, Cdkn1a, Hspa1, Prdx, Mapk10 and down-regulated (Foxo3, Mtor genes in the enriched Schwann cells. In conclusion, molecular analyses in the presymptomatic sciatic nerve demonstrated the involvement of death, oxidative stress, and mitochondrial pathways in the Schwann cell non-autonomous mechanisms in the early stages of ALS.

  2. A Selective Bottleneck Shapes the Evolutionary Mutant Spectra of Enterovirus A71 during Viral Dissemination in Humans.

    Science.gov (United States)

    Huang, Sheng-Wen; Huang, Yi-Hui; Tsai, Huey-Pin; Kuo, Pin-Hwa; Wang, Shih-Min; Liu, Ching-Chuan; Wang, Jen-Ren

    2017-12-01

    RNA viruses accumulate mutations to rapidly adapt to environmental changes. Enterovirus A71 (EV-A71) causes various clinical manifestations with occasional severe neurological complications. However, the mechanism by which EV-A71 evolves within the human body is unclear. Utilizing deep sequencing and haplotype analyses of viruses from various tissues of an autopsy patient, we sought to define the evolutionary pathway by which enterovirus A71 evolves fitness for invading the central nervous system in humans. Broad mutant spectra with divergent mutations were observed at the initial infection sites in the respiratory and digestive systems. After viral invasion, we identified a haplotype switch and dominant haplotype, with glycine at VP1 residue 31 (VP1-31G) in viral particles disseminated into the integumentary and central nervous systems. In vitro viral growth and fitness analyses indicated that VP1-31G conferred growth and a fitness advantage in human neuronal cells, whereas VP1-31D conferred enhanced replication in human colorectal cells. A higher proportion of VP1-31G was also found among fatal cases, suggesting that it may facilitate central nervous system infection in humans. Our data provide the first glimpse of EV-A71 quasispecies from oral tissues to the central nervous system within humans, showing broad implications for the surveillance and pathogenesis of this reemerging viral pathogen. IMPORTANCE EV-A71 continues to be a worldwide burden to public health. Although EV-A71 is the major etiological agent of hand, foot, and mouth disease, it can also cause neurological pulmonary edema, encephalitis, and even death, especially in children. Understanding selection processes enabling dissemination and accurately estimating EV-A71 diversity during invasion in humans are critical for applications in viral pathogenesis and vaccine studies. Here, we define a selection bottleneck appearing in respiratory and digestive tissues. Glycine substitution at VP1 residue 31

  3. Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

    Science.gov (United States)

    Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; González-Valdez, Abigail; Martínez-Rosas, Víctor; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa Angélica; Cuevas-Cruz, Miguel; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site. PMID:27213370

  4. Delayed expression of apoptosis in X-irradiated human leukemic MOLT-4 cells transfected with mutant p53

    International Nuclear Information System (INIS)

    Nakano, Hisako; Yonekawa, Hiromichi; Shinohara, Kunio

    2003-01-01

    The effects of X-rays on cell survival, apoptosis, and long-term response in the development of cell death as measured by the dye exclusion test were studied in human leukemic MOLT-4 cells (p53 wild-type) stably transfected with a mutant p53 cDNA expression vector. Cell survival, as determined from colony-forming ability, was increased in an expression level dependent manner, but the increase was partial even with the highest-expressing clone (B3). This contrasts with the prior observation that cell death and apoptosis in B3 are completely inhibited at 24 h after irradiation with 1.8 Gy of X-rays. The examination of B3 cells incubated for longer than 24 h after X-irradiation showed a delay in the induction of cell death and apoptosis. Western blot analysis revealed that the time required to reach the highest level of wild-type p53 protein in B3 was longer than the time in MOLT-4 and that the p53 may be stabilized by the phosphorylation at Ser-15. These results suggest that the introduction of mutant p53 into MOLT-4 merely delays the development of apoptosis, during which the cells could repair the damage induced by X-rays, and results in the partial increase in cell survival. (author)

  5. Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

    International Nuclear Information System (INIS)

    Kane, W.H.; Devore-Carter, D.; Ortel, T.L.

    1990-01-01

    Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 ± 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 ± 0.012 to 0.124 ± 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 ± 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1,800 ± 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1,700-2,000 units/mg. Immunoprecipitation of [ 35 S]methionine-labeled rHFV revealed a single high molecular mass component. Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. The authors have also expressed a mutant, rHFV-des-B 811-1441 , that lacks a large portion of the highly glycosylated connecting region that is present in factor V. This mutant constitutively expressed 38 ± 7% of the activity of the RVV-V-activated protein. These results suggest that one of the functions of the large connecting region in factor V is to inhibit constitutive procoagulant activity

  6. Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

    Science.gov (United States)

    Doersen, C J; Stanbridge, E J

    1981-04-01

    HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.

  7. Association between single nucleotide polymorphisms in the antioxidant genes CAT, GR and SOD1, erythrocyte enzyme activities, dietary and life style factors and breast cancer risk in a Danish, prospective cohort study

    DEFF Research Database (Denmark)

    Kopp, Tine Iskov; Vogel, Ulla; Dragsted, Lars Ove

    2017-01-01

    Exposure to estrogens and alcohol consumption - the two only well-established risk factors for breast cancer - are capable of causing oxidative stress, which has been linked to progression of breast cancer. Here, five functional polymorphisms in the antioxidant genes SOD1, CAT and GSR were...

  8. Compensatory Motor Neuron Response to Chromatolysis in the Murine hSOD1G93A Model of Amyotrophic Lateral Sclerosis

    Science.gov (United States)

    Riancho, Javier; Ruiz-Soto, Maria; Villagrá, Nuria T.; Berciano, Jose; Berciano, Maria T.; Lafarga, Miguel

    2014-01-01

    We investigated neuronal self-defense mechanisms in a murine model of amyotrophic lateral sclerosis (ALS), the transgenic hSOD1G93A, during both the asymptomatic and symptomatic stages. This is an experimental model of endoplasmic reticulum (ER) stress with severe chromatolysis. As a compensatory response to translation inhibition, chromatolytic neurons tended to reorganize the protein synthesis machinery at the perinuclear region, preferentially at nuclear infolding domains enriched in nuclear pores. This organization could facilitate nucleo-cytoplasmic traffic of RNAs and proteins at translation sites. By electron microscopy analysis, we observed that the active euchromatin pattern and the reticulated nucleolar configuration of control motor neurons were preserved in ALS chromatolytic neurons. Moreover the 5′-fluorouridine (5′-FU) transcription assay, at the ultrastructural level, revealed high incorporation of the RNA precursor 5′-FU into nascent RNA. Immunogold particles of 5′-FU incorporation were distributed throughout the euchromatin and on the dense fibrillar component of the nucleolus in both control and ALS motor neurons. The high rate of rRNA transcription in ALS motor neurons could maintain ribosome biogenesis under conditions of severe dysfunction of proteostasis. Collectively, the perinuclear reorganization of protein synthesis machinery, the predominant euchromatin architecture, and the active nucleolar transcription could represent compensatory mechanisms in ALS motor neurons in response to the disturbance of ER proteostasis. In this scenario, epigenetic activation of chromatin and nucleolar transcription could have important therapeutic implications for neuroprotection in ALS and other neurodegenerative diseases. Although histone deacetylase inhibitors are currently used as therapeutic agents, we raise the untapped potential of the nucleolar transcription of ribosomal genes as an exciting new target for the therapy of some neurodegenerative

  9. Mutant human FUS Is ubiquitously mislocalized and generates persistent stress granules in primary cultured transgenic zebrafish cells.

    Directory of Open Access Journals (Sweden)

    Jamie Rae Acosta

    Full Text Available FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS, a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.

  10. Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

    Science.gov (United States)

    Paiboonsukwong, Kittiphong; Ohbayashi, Fumi; Shiiba, Haruka; Aizawa, Emi; Yamashita, Takayuki; Mitani, Kohnosuke

    2009-11-01

    Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.

  11. Synthesis, purification, and characterization of an Arg152 → Glu site-directed mutant of recombinant human blood clotting factor VII

    International Nuclear Information System (INIS)

    Wildgoose, P.; Kisiel, W.; Berkner, K.L.

    1990-01-01

    Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg 152 -Ile 153 . Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, the authors have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg 152 → Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M r ∼40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX

  12. Synthesis, purification, and characterization of an Arg sub 152 yields Glu site-directed mutant of recombinant human blood clotting factor VII

    Energy Technology Data Exchange (ETDEWEB)

    Wildgoose, P.; Kisiel, W. (Univ. of New Mexico, Albuquerque (USA)); Berkner, K.L. (ZymoGenetics, Inc., Seattle, WA (USA))

    1990-04-03

    Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg{sub 152}-Ile{sub 153}. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, the authors have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg{sub 152} {yields} Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M{sup r}{approx}40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX.

  13. Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

    Directory of Open Access Journals (Sweden)

    Liang Xu

    2017-09-01

    Full Text Available Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26, a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action

  14. Human COQ9 Rescues a coq9 Yeast Mutant by Enhancing Coenzyme Q Biosynthesis from 4-Hydroxybenzoic Acid and Stabilizing the CoQ-Synthome

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    Cuiwen H. He

    2017-07-01

    Full Text Available Coq9 is required for the stability of a mitochondrial multi-subunit complex, termed the CoQ-synthome, and the deamination step of Q intermediates that derive from para-aminobenzoic acid (pABA in yeast. In human, mutations in the COQ9 gene cause neonatal-onset primary Q10 deficiency. In this study, we determined whether expression of human COQ9 could complement yeast coq9 point or null mutants. We found that expression of human COQ9 rescues the growth of the temperature-sensitive yeast mutant, coq9-ts19, on a non-fermentable carbon source and increases the content of Q6, by enhancing Q biosynthesis from 4-hydroxybenzoic acid (4HB. To study the mechanism for the rescue by human COQ9, we determined the steady-state levels of yeast Coq polypeptides in the mitochondria of the temperature-sensitive yeast coq9 mutant expressing human COQ9. We show that the expression of human COQ9 significantly increased steady-state levels of yeast Coq4, Coq6, Coq7, and Coq9 at permissive temperature. Human COQ9 polypeptide levels persisted at non-permissive temperature. A small amount of the human COQ9 co-purified with tagged Coq6, Coq6-CNAP, indicating that human COQ9 interacts with the yeast Q-biosynthetic complex. These findings suggest that human COQ9 rescues the yeast coq9 temperature-sensitive mutant by stabilizing the CoQ-synthome and increasing Q biosynthesis from 4HB. This finding provides a powerful approach to studying the function of human COQ9 using yeast as a model.

  15. An Fgf8 Mouse Mutant Phenocopies Human 22q11 Deletion Syndrome

    OpenAIRE

    Frank, Deborah U.; Fotheringham, Lori K.; Brewer, Judson A.; Muglia, Louis J.; Tristani-Firouzi, Martin; Capecchi, Mario R.; Moon, Anne M.

    2002-01-01

    Deletion of chromosome 22q11, the most common microdeletion detected in humans, is associated with a life-threatening array of birth defects. Although 90% of affected individuals share the same three megabase deletion, their phenotype is highly variable and includes craniofacial and cardiovascular anomalies, hypoplasia or aplasia of the thymus with associated deficiency of T cells, hypocalcemia with hypoplasia or aplasia of the parathyroids, and a variety of central nervous system abnormaliti...

  16. Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells.

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    Michelle A Favila

    2015-12-01

    Full Text Available Leishmania major infection induces robust interleukin-12 (IL12 production in human dendritic cells (hDC, ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG and other phosphoglycan-containing molecules (PGs, making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS responsible for IL12 induction.Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-, or generally deficient for all PGs, (FV1 lpg2-. Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB and Interferon Regulatory Factor (IRF mediated transcription.These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12.

  17. Angiogenesis in Pituitary Adenomas: Human Studies and New Mutant Mouse Models

    Directory of Open Access Journals (Sweden)

    Carolina Cristina

    2014-01-01

    Full Text Available The role of angiogenesis in pituitary tumor development has been questioned, as pituitary tumors have been usually found to be less vascularized than the normal pituitary tissue. Nevertheless, a significantly higher degree of vasculature has been shown in invasive or macropituitary prolactinomas when compared to noninvasive and microprolactinomas. Many growth factors and their receptors are involved in pituitary tumor development. For example, VEGF, FGF-2, FGFR1, and PTTG, which give a particular vascular phenotype, are modified in human and experimental pituitary adenomas of different histotypes. In particular, vascular endothelial growth factor, VEGF, the central mediator of angiogenesis in endocrine glands, was encountered in experimental and human pituitary tumors at different levels of expression and, in particular, was higher in dopamine agonist resistant prolactinomas. Furthermore, several anti-VEGF techniques lowered tumor burden in human and experimental pituitary adenomas. Therefore, even though the role of angiogenesis in pituitary adenomas is contentious, VEGF, making permeable pituitary endothelia, might contribute to adequate temporal vascular supply and mechanisms other than endothelial cell proliferation. The study of angiogenic factor expression in aggressive prolactinomas with resistance to dopamine agonists will yield important data in the search of therapeutical alternatives.

  18. Angiogenesis in pituitary adenomas: human studies and new mutant mouse models.

    Science.gov (United States)

    Cristina, Carolina; Luque, Guillermina María; Demarchi, Gianina; Lopez Vicchi, Felicitas; Zubeldia-Brenner, Lautaro; Perez Millan, Maria Ines; Perrone, Sofia; Ornstein, Ana Maria; Lacau-Mengido, Isabel M; Berner, Silvia Inés; Becu-Villalobos, Damasia

    2014-01-01

    The role of angiogenesis in pituitary tumor development has been questioned, as pituitary tumors have been usually found to be less vascularized than the normal pituitary tissue. Nevertheless, a significantly higher degree of vasculature has been shown in invasive or macropituitary prolactinomas when compared to noninvasive and microprolactinomas. Many growth factors and their receptors are involved in pituitary tumor development. For example, VEGF, FGF-2, FGFR1, and PTTG, which give a particular vascular phenotype, are modified in human and experimental pituitary adenomas of different histotypes. In particular, vascular endothelial growth factor, VEGF, the central mediator of angiogenesis in endocrine glands, was encountered in experimental and human pituitary tumors at different levels of expression and, in particular, was higher in dopamine agonist resistant prolactinomas. Furthermore, several anti-VEGF techniques lowered tumor burden in human and experimental pituitary adenomas. Therefore, even though the role of angiogenesis in pituitary adenomas is contentious, VEGF, making permeable pituitary endothelia, might contribute to adequate temporal vascular supply and mechanisms other than endothelial cell proliferation. The study of angiogenic factor expression in aggressive prolactinomas with resistance to dopamine agonists will yield important data in the search of therapeutical alternatives.

  19. SOD1 Gene +35A/C (exon3/intron3 Polymorphism in Type 2 Diabetes Mellitus among South Indian Population

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    K. Nithya

    2016-01-01

    Full Text Available Superoxide dismutase is an antioxidant enzyme that is involved in defence mechanisms against oxidative stress. Cu/Zn SOD is a variant that is located in exon3/intron3 boundary. The aim of the present study was to investigate whether the Cu/Zn SOD (+35A/C gene polymorphism is associated with the susceptibility to type 2 diabetes mellitus among south Indian population. The study included patients with type 2 diabetes mellitus (n=100 and healthy controls (n=75. DNA was isolated from the blood and genotyping of Cu/Zn SOD gene polymorphism was done by polymerase chain reaction based restriction fragment length polymorphism method. Occurrence of different genotypes and normal (A and mutant (C allele frequencies were determined. The frequency of the three genotypes of the total subjects was as follows: homozygous wild-type A/A (95%, heterozygous genotype A/C (3%, and homozygous mutant C/C (2%. The mutant (C allele and the mutant genotypes (AC/CC were found to be completely absent among the patients with type 2 diabetes mellitus. Absence of mutant genotype (CC shows that the Cu/Zn SOD gene polymorphism may not be associated with the susceptibility to type 2 diabetes mellitus among south Indian population.

  20. Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells

    Science.gov (United States)

    Li, Luyuan; Paz, Ana C.; Wilky, Breelyn A.; Johnson, Britt; Galoian, Karina; Rosenberg, Andrew; Hu, Guozhi; Tinoco, Gabriel; Bodamer, Olaf; Trent, Jonathan C.

    2015-01-01

    Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas. PMID:26368816

  1. Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

    Directory of Open Access Journals (Sweden)

    Luyuan Li

    Full Text Available Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2 were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.

  2. Immortalization of human neural stem cells with the c-myc mutant T58A.

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    Lidia De Filippis

    Full Text Available Human neural stem cells (hNSC represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (v-IhNSC has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs--which do not transform in vitro and are not tumorigenic in vivo--the v-myc gene contains several mutations and recombination elements, whose role(s and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC. T-IhNSCs displayed an enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%. Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in

  3. Altered depression-related behavior and neurochemical changes in serotonergic neurons in mutant R406W human tau transgenic mice.

    Science.gov (United States)

    Egashira, Nobuaki; Iwasaki, Katsunori; Takashima, Akihiko; Watanabe, Takuya; Kawabe, Hideyuki; Matsuda, Tomomi; Mishima, Kenichi; Chidori, Shozo; Nishimura, Ryoji; Fujiwara, Michihiro

    2005-10-12

    Mutant R406W human tau was originally identified in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and causes a hereditary tauopathy that clinically resembles Alzheimer's disease (AD). In the current study, we examined the performance of R406W transgenic (Tg) mice in the forced swimming test, a test with high predictivity of antidepressant efficacy in human depression, and found an enhancement of the immobility time. In contrast, the motor function and anxiety-related emotional response of R406W Tg mice were normal. Furthermore, a selective serotonin reuptake inhibitor (SSRI), fluvoxamine (100 mg/kg, p.o.), significantly reduced this enhancement of the immobility time, whereas a noradrenaline reuptake inhibitor, desipramine, had no effect. In an in vivo microdialysis study, R406W Tg mice exhibited a significantly decreased extracellular 5-hydroxyindoleacetic acid (5-HIAA) level in the frontal cortex and also exhibited a tendency toward a decreased extracellular 5-hydroxytryptamine (5-HT) level. Moreover, fluvoxamine, which reduced the enhancement of the immobility time, significantly increased the extracellular 5-HT level in R406W Tg mice. These results suggest that R406W Tg mice exhibit changes in depression-related behavior involving serotonergic neurons and provide an animal model for investigating AD with depression.

  4. Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank

    Science.gov (United States)

    Ship, Noam J.; Zamble, Deborah B.

    2005-01-01

    The self directed study of a 3D image of a biomolecule stresses the complex nature of the intra- and intermolecular interactions that come together to define its structure. This is made up of a series of in vitro experiments with a wild-type and mutants forms of human carbonic anhydrase II (hCAII) that examine the structure function relationship…

  5. Intrinsic properties of lumbar motor neurones in the adult G127insTGGG superoxide dismutase-1 mutant mouse in vivo: evidence for increased persistent inward currents

    DEFF Research Database (Denmark)

    Meehan, Claire Francesca; Moldovan, Mihai; Marklund, Stefan L.

    2010-01-01

    Aim: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a preferential loss of motoneurones. Previous publications using in vitro neonatal preparations suggest an increased excitability of motoneurones in various superoxide dismutase-1 (SOD1) mutant mice...... of an increased PIC and less spike frequency adaptation which may contribute to excitotoxity of these neurones as the disease progresses....

  6. Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase

    International Nuclear Information System (INIS)

    Wang Yuxia; Ticu Boeck, Andreea; Duysen, Ellen G.; Van Keuren, Margaret; Saunders, Thomas L.; Lockridge, Oksana

    2004-01-01

    Organophosphorus toxicants (OP) include chemical nerve agents and pesticides. The goal of this work was to find out whether an animal could be made resistant to OP toxicity by genetic engineering. The human butyrylcholinesterase (BChE) mutant G117H was chosen for study because it has the unusual ability to hydrolyze OP as well as acetylcholine, and it is resistant to inhibition by OP. Human G117H BChE, under the control of the ROSA26 promoter, was expressed in all tissues of transgenic mice. A stable transgenic mouse line expressed 0.5 μg/ml of human G117H BChE in plasma as well as 2 μg/ml of wild-type mouse BChE. Intestine, kidneys, stomach, lungs, heart, spleen, liver, brain, and muscle expressed 0.6-0.15 μg/g of G117H BChE. Transgenic mice were normal in behavior and fertility. The LD50 dose of echothiophate for wild-type mice was 0.1 mg/kg sc. This dose caused severe cholinergic signs of toxicity and lethality in wild-type mice, but caused no deaths and only mild toxicity in transgenic animals. The mechanism of protection was investigated by measuring acetylcholinesterase (AChE) and BChE activity. It was found that AChE and endogenous BChE were inhibited to the same extent in echothiophate-treated wild type and transgenic mice. This led to the hypothesis that protection against echothiophate toxicity was not explained by hydrolysis of echothiophate. In conclusion, the transgenic G117H BChE mouse demonstrates the factors required to achieve protection from OP toxicity in a vertebrate animal

  7. Construction, characterization, and complementation of a conditional-lethal DNA topoisomerase IIalpha mutant human cell line.

    Science.gov (United States)

    Carpenter, Adam J; Porter, Andrew C G

    2004-12-01

    DNA Topoisomerase IIalpha (topoIIalpha) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIalpha expression can be silenced in all cells by the addition of tetracycline. TopoIIalpha-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIalpha fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIalpha as an indicator for endogenous topoIIalpha dynamics, our analyses provide new evidence that topoIIalpha plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIbeta and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

  8. Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning human peripheral blood T-lymphocytes

    International Nuclear Information System (INIS)

    Hakoda, Masayuki; Akiyama, Mitoshi; Kyoizumi, Seishi; Kobuke, Kyoko; Awa, A.A.

    1987-07-01

    Approximately 80 % of human peripheral blood T-lymphocytes could be cloned in the presence of crude Interleukin-2, phytohemagglutinin, and X-irradiated autologous lymphocytes and Raji B-cells. This modified cloning method was used to measure the in vivo frequency of HGPRT-deficient mutant T-lymphocytes. Repeated experiments using blood from the same individuals revealed that the frequency of mutant cells was almost constant for each individual even though the cloning efficiency of lymphocytes varied somewhat from experiment to experiment. Approximately 80 % of both wild-type unselected and 6-thioguanine-resistant colonies had helper/inducer and about 20 % had suppressor/cytotoxic T-lymphocyte markers. No difference was observed in the distribution of lymphocyte subsets between wild and mutant lymphocyte colonies. (author)

  9. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains

    International Nuclear Information System (INIS)

    LingNah Su; Little, J.B.

    1992-01-01

    A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D o 192 ± 13 vs 127 ± 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. (author)

  10. Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

    Science.gov (United States)

    Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

    2007-05-01

    Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

  11. EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin.

    Science.gov (United States)

    Van Doorslaer, Sabine; Trandafir, Florin; Harmer, Jeffrey R; Moens, Luc; Dewilde, Sylvia

    2014-06-01

    Electron paramagnetic resonance (EPR) data reveal large differences between the ferric ((13)C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo- and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full (13)C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on (13)C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding (13)C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These (13)C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Human stefin B normal and patho-physiological role: molecular and cellular aspects of amyloid-type aggregation of certain EPM1 mutants.

    Directory of Open Access Journals (Sweden)

    Mira ePolajnar

    2012-08-01

    Full Text Available Epilepsies are characterised by abnormal electrophysiological activity of the brain. Among various types of inherited epilepsies different epilepsy syndromes, among them progressive myoclonus epilepsies with features of ataxia and neurodegeneration, are counted. The progressive myoclonus epilepsy of type 1 (EPM1, also known as Unverricht-Lundborg disease presents with features of cerebellar atrophy and increased oxidative stress. It has been found that EPM1 is caused by mutations in human cystatin B gene (human stefin B. We first describe the role of protein aggregation in other neurodegenerative conditions. Protein aggregates appear intraneurally but are also excreted, such as is the case with senile plaques of amyloid- β (Aβ that accumulate in the brain parenchyma and vessel walls. A common characteristic of such diseases is the change of the protein conformation towards β secondary structure that accounts for the strong tendency of such proteins to aggregate and form amyloid fibrils. Second, we describe the patho-physiology of EPM1 and the normal and aberrant roles of stefin B in a mouse model of the disease. Furthermore, we discuss how the increased protein aggregation observed with some of the mutants of human stefin B may relate to the neurodegeneration that occurs in rare EPM1 patients. Our hypothesis (Ceru et al., 2005 states that some of the EPM1 mutants of human stefin B may undergo aggregation in neural cells, thus gaining additional toxic function (apart from loss of normal function. Our in vitro experiments thus far have confirmed that 4 mutants undergo increased aggregation relative to the wild-type protein. It has been shown that the R68X mutant forms amyloid-fibrils very rapidly, even at neutral pH and forms perinuclear inclusions, whereas the G4R mutant exhibits a prolonged lag phase, during which the toxic prefibrillar aggregates accumulate and are scattered more diffusely over the cytoplasm. Initial experiments on the G50E

  13. Replication-dependent 65R→K reversion in human immunodeficiency virus type 1 reverse transcriptase double mutant K65R + L74V

    International Nuclear Information System (INIS)

    Sharma, Prem L.; Nurpeisov, Viktoria; Lee, Kimberly; Skaggs, Sara; Di San Filippo, Christina Amat; Schinazi, Raymond F.

    2004-01-01

    Understanding of the mechanisms of interaction among nucleoside reverse transcriptase inhibitor (NRTI)-selected mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) coding sequence is essential for the design of newer drugs and for enhancing our vision of the structure function relationship among amino acids of the polymerase domain of HIV-1. Although several nucleoside reverse transcriptase inhibitors select RT mutations K65R and L74V, the combination of 65R + 74V is rare in clinics. A novel NRTI (-)-β-D-dioxolane-guanosine (DXG) is known to select in vitro either the 65R or 74V mutant virus (Antimicrob. Agents Chemother. 44 (2000) 1783). These mutations were not selected together during repeated passaging of the HIV-1 in the presence of this drug. To analyze the impact of these RT mutations on viral replication, a double mutant containing K65R + L74V was created by site-directed mutagenesis in a pNL4-3 background. Replication kinetic assays revealed that the mutant K65R + L74V is unstable, and 65R→K reversion occurs during replication of virus in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication kinetic assays in MT-2 cells demonstrated that double mutant 65R + 74V is highly attenuated for replication and the initiation of reversion is related to the increase in RT activity. Additionally, the suppression of viral replication in the presence of DXG or under suboptimal human recombinant interleukin-2 leads to minimal or no 65R→K reversion. These observations provide evidence that 65R→K reversion in the double mutant 65R + 74V is dependent on a specific rate of viral replication in a pNL4-3 background. A similar phenomenon may occur in vivo, which may have implications for treatment management strategies

  14. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Van Rossom, Sofie; Op de Beeck, Ken; Franssens, Vanessa; Swinnen, Erwin; Schepers, Anne; Ghillebert, Ruben; Caldara, Marina; Van Camp, Guy; Winderickx, Joris

    2012-01-01

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  15. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Van Rossom, Sofie [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Op de Beeck, Ken [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Franssens, Vanessa; Swinnen, Erwin [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Schepers, Anne [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Ghillebert, Ruben; Caldara, Marina [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Van Camp, Guy [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Winderickx, Joris, E-mail: guy.vancamp@ua.ac.be, E-mail: joris.winderickx@bio.kuleuven.be [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium)

    2012-07-25

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  16. Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT∗3A, TPMT∗2): Mechanisms for the genetic polymorphism of TPMT activity

    OpenAIRE

    Tai, Hung-Liang; Krynetski, Eugene Y.; Schuetz, Erin G.; Yanishevski, Yuri; Evans, William E.

    1997-01-01

    TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (TPMT∗2 and TPMT∗3A), but the mechanisms for ...

  17. Intrathecal infusion of a Ca(2+)-permeable AMPA channel blocker slows loss of both motor neurons and of the astrocyte glutamate transporter, GLT-1 in a mutant SOD1 rat model of ALS.

    Science.gov (United States)

    Yin, Hong Z; Tang, Darryl T; Weiss, John H

    2007-10-01

    Elevated extracellular glutamate, resulting from a loss of astrocytic glutamate transport capacity, may contribute to excitotoxic motor neuron (MN) damage in ALS. Accounting for their high excitotoxic vulnerability, MNs possess large numbers of unusual Ca(2+)-permeable AMPA channels (Ca-AMPA channels), the activation of which triggers mitochondrial Ca(2+) overload and strong reactive oxygen species (ROS) generation. However, the causes of the astrocytic glutamate transport loss remain unexplained. To assess the role of Ca-AMPA channels on the evolution of pathology in vivo, we have examined effects of prolonged intrathecal infusion of the Ca-AMPA channel blocker, 1-naphthyl acetylspermine (NAS), in G93A transgenic rat models of ALS. In wild-type animals, immunoreactivity for the astrocytic glutamate transporter, GLT-1, was particularly strong around ventral horn MNs. However, a marked loss of ventral horn GLT-1 was observed, along with substantial MN damage, prior to onset of symptoms (90-100 days) in the G93A rats. Conversely, labeling with the oxidative marker, nitrotyrosine, was increased in the neuropil surrounding MNs in the transgenic animals. Compared to sham-treated G93A animals, 30-day NAS infusions (starting at 67+/-2 days of age) markedly diminished the loss of both MNs and of astrocytic GLT-1 labeling. These observations are compatible with the hypothesis that activation of Ca-AMPA channels on MNs contributes, likely in part through oxidative mechanisms, to loss of glutamate transporter in surrounding astrocytes.

  18. Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

    Directory of Open Access Journals (Sweden)

    Gastaldelli Michele

    2009-10-01

    Full Text Available Abstract Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5 cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein, which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for

  19. The Overexpression of TDP-43 Protein in the Neuron and Oligodendrocyte Cells Causes the Progressive Motor Neuron Degeneration in the SOD1 G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Lu, Yi; Tang, Chunyan; Zhu, Lei; Li, Jiao; Liang, Huiting; Zhang, Jie; Xu, Renshi

    2016-01-01

    The recent investigation suggested that the TDP-43 protein was closely related to the motor neuron degeneration in amyotrophic lateral sclerosis (ALS), but the pathogenesis contributed to motor neuron degeneration largely remained unknown. Therefore, we detected the alteration of TDP-43 expression and distribution in the adult spinal cord of the SOD1 G93A transgenic mouse model for searching the possible pathogenesis of ALS. We examined the TDP-43 expression and distribution in the different anatomic regions, segments and neural cells in the adult spinal cord at the different stages of the SOD1 wild-type and G93A transgenic model by the fluorescent immunohistochemical technology. We revealed that the amount of TDP-43 positive cell was cervical>lumbar>thoracic segment, that in the ventral horn was more than that in the dorsal horn, a few of TDP-43 protein sparsely expressed and distributed in the other regions, the TDP-43 protein weren't detected in the white matter and the central canal. The TDP-43 protein was mostly expressed and distributed in the nuclear of neuron cells and the cytoplasm of oligodendrocyte cells of the gray matter surrounding the central canal of spinal cord by the granular shape in the SOD1 wild-type and G93A transgenic mice. The amount of TDP-43 positive cell significantly increased at the onset and progression stages of ALS following with the increase of neuron death in spinal cord, particularly in the ventral horn of cervical segment at the progression stage. Our results suggested that the overexpression of TDP-43 protein in the neuron and oligodendrocyte cell causes the progressive motor neuron degeneration in the ALS-like mouse model.

  20. Association between single nucleotide polymorphisms in the antioxidant genes CAT, GR and SOD1, erythrocyte enzyme activities, dietary and life style factors and breast cancer risk in a Danish, prospective cohort study

    DEFF Research Database (Denmark)

    Kopp, Tine Iskov; Vogel, Ulla; Dragsted, Lars Ove

    2017-01-01

    investigated in 703 breast cancer case-control pairs in the Danish, prospective "Diet, Cancer and Health" cohort together with gene-environment interactions between the polymorphisms, enzyme activities and intake of fruits and vegetables, alcohol and smoking in relation to breast cancer risk. Our results...... showed that genetically determined variations in the antioxidant enzyme activities of SOD1, CAT and GSR were not associated with risk of breast cancer per se. However, intake of alcohol, fruit and vegetables, and smoking status interacted with some of the polymorphisms in relation to breast cancer risk...

  1. Effects of mutant human Ki-rasG12C gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

    International Nuclear Information System (INIS)

    Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.; Moore, Joseph E.; Mosley, Libyadda J.; D'Agostino, Ralph B.; Pettenati, Mark J.; Miller, Mark Steven

    2008-01-01

    Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras G12C allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 μg/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras G12C allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 μg/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 μg/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 μg/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras G12C expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models

  2. Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering--A Comparison between Wild-Type Protein and a Hinge Mutant.

    Directory of Open Access Journals (Sweden)

    Saara Laulumaa

    Full Text Available Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS. The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

  3. Potassium channel abnormalities are consistent with early axon degeneration of motor axons in the G127X SOD1 mouse model of amyotrophic lateral sclerosis

    DEFF Research Database (Denmark)

    Maglemose, Rikke; Hedegaard, Anne; Lehnhoff, Janna

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, which selectively affects upper and lower motoneurones. The underlying pathophysiology of the disease is complex but electrophysiological studies of peripheral nerves in ALS patients as well as human autopsy studies indicate...

  4. CARMA2sh and ULK2 control pathogen-associated molecular patterns recognition in human keratinocytes: psoriasis-linked CARMA2sh mutants escape ULK2 censorship.

    Science.gov (United States)

    Scudiero, Ivan; Mazzone, Pellegrino; D'Andrea, Luca E; Ferravante, Angela; Zotti, Tiziana; Telesio, Gianluca; De Rubis, Gabriele; Reale, Carla; Pizzulo, Maddalena; Muralitharan, Shanmugakonar; Vito, Pasquale; Stilo, Romania

    2017-02-23

    The molecular complexes formed by specific members of the family of CARMA proteins, the CARD domain-containing adapter molecule BCL10 and MALT1 (CBM complex) represent a central hub in regulating activation of the pleiotropic transcription factor NF-κB. Recently, missense mutations in CARMA2sh have been shown to cause psoriasis in a dominant manner and with high penetrancy. Here, we demonstrate that in human keratinocytes CARMA2sh plays an essential role in the signal transduction pathway that connects pathogen-associated molecular patterns recognition to NF-κB activation. We also find that the serine/threonine kinase ULK2 binds to and phosphorylates CARMA2sh, thereby inhibiting its capacity to activate NF-κB by promoting lysosomal degradation of BCL10, which is essential for CARMA2sh-mediated NF-κB signaling. Remarkably, CARMA2sh mutants associated with psoriasis escape ULK2 inhibition. Finally, we show that a peptide blocking CARD-mediated BCL10 interactions reduces the capacity of psoriasis-linked CARMA2sh mutants to activate NF-κB. Our work elucidates a fundamental signaling mechanism operating in human keratinocytes and opens to novel potential tools for the therapeutical treatment of human skin disorders.

  5. Diffuse glomerular nodular lesions in diabetic pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha, an inheritant diabetic gene in humans.

    Directory of Open Access Journals (Sweden)

    Satoshi Hara

    Full Text Available Glomerular nodular lesions, known as Kimmelstiel-Wilson nodules, are a pathological hallmark of progressive human diabetic nephropathy. We have induced severe diabetes in pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha (HNF1α P291fsinsC, a maturity-onset diabetes of the young type-3 (MODY3 gene in humans. In this model, glomerular pathology revealed that formation of diffuse glomerular nodules commenced as young as 1 month of age and increased in size and incidence until the age of 10 months, the end of the study period. Immunohistochemistry showed that the nodules consisted of various collagen types (I, III, IV, V and VI with advanced glycation end-product (AGE and Nε-carboxymethyl-lysine (CML deposition, similar to those in human diabetic nodules, except for collagen type I. Transforming growth factor-beta (TGF-β was also expressed exclusively in the nodules. The ultrastructure of the nodules comprised predominant interstitial-type collagen deposition arising from the mesangial matrices. Curiously, these nodules were found predominantly in the deep cortex. However, diabetic pigs failed to show any of the features characteristic of human diabetic nephropathy; e.g., proteinuria, glomerular basement membrane thickening, exudative lesions, mesangiolysis, tubular atrophy, interstitial fibrosis, and vascular hyalinosis. The pigs showed only Armanni-Ebstein lesions, a characteristic tubular manifestation in human diabetes. RT-PCR analysis showed that glomeruli in wild-type pigs did not express endogenous HNF1α and HNF1β, indicating that mutant HNF1α did not directly contribute to glomerular nodular formation in diabetic pigs. In conclusion, pigs harboring the dominant-negative mutant human MODY3 gene showed reproducible and distinct glomerular nodules, possibly due to AGE- and CML-based collagen accumulation. Although the pathology differed in several respects from that of human glomerular nodular lesions, the

  6. Computing Stability Effects of Mutations in Human Superoxide Dismutase 1

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta

    2014-01-01

    Protein stability is affected in several diseases and is of substantial interest in efforts to correlate genotypes to phenotypes. Superoxide dismutase 1 (SOD1) is a suitable test case for such correlations due to its abundance, stability, available crystal structures and thermochemical data......, and physiological importance. In this work, stability changes of SOD1 mutations were computed with five methods, CUPSAT, I-Mutant2.0, I-Mutant3.0, PoPMuSiC, and SDM, with emphasis on structural sensitivity as a potential issue in structure-based protein calculation. The large correlation between experimental...... literature data of SOD1 dimers and monomers (r = 0.82) suggests that mutations in separate protein monomers are mostly additive. PoPMuSiC was most accurate (typical MAE ∼ 1 kcal/mol, r ∼ 0.5). The relative performance of the methods was not very structure-dependent, and the more accurate methods also...

  7. A natural human IgM that binds to gangliosides is therapeutic in murine models of amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Xiaohua Xu

    2015-08-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a devastating, fatal neurological disease that primarily affects spinal cord anterior horn cells and their axons for which there is no treatment. Here we report the use of a recombinant natural human IgM that binds to the surface of neurons and supports neurite extension, rHIgM12, as a therapeutic strategy in murine models of human ALS. A single 200 µg intraperitoneal dose of rHIgM12 increases survival in two independent genetic-based mutant SOD1 mouse strains (SOD1G86R and SOD1G93A by 8 and 10 days, delays the onset of neurological deficits by 16 days, delays the onset of weight loss by 5 days, and preserves spinal cord axons and anterior horn neurons. Immuno-overlay of thin layer chromatography and surface plasmon resonance show that rHIgM12 binds with high affinity to the complex gangliosides GD1a and GT1b. Addition of rHIgM12 to neurons in culture increases α-tubulin tyrosination levels, suggesting an alteration of microtubule dynamics. We previously reported that a single peripheral dose of rHIgM12 preserved neurological function in a murine model of demyelination with axon loss. Because rHIgM12 improves three different models of neurological disease, we propose that the IgM might act late in the cascade of neuronal stress and/or death by a broad mechanism.

  8. Effect of Potent γ-Secretase Modulator in Human Neurons Derived From Multiple Presenilin 1–Induced Pluripotent Stem Cell Mutant Carriers

    Science.gov (United States)

    Liu, Qing; Waltz, Shannon; Woodruff, Grace; Ouyang, Joe; Israel, Mason A.; Herrera, Cheryl; Sarsoza, Floyd; Tanzi, Rudolph E.; Koo, Edward H.; Ringman, John M.; Goldstein, Lawrence S. B.; Wagner, Steven L.; Yuan, Shauna H.

    2015-01-01

    Importance Although considerable effort has been expended developing drug candidates for Alzheimer disease, none have yet succeeded owing to the lack of efficacy or to safety concerns. One potential shortcoming of current approaches to Alzheimer disease drug discovery and development is that they rely primarily on transformed cell lines and animal models that substantially overexpress wild-type or mutant proteins. It is possible that drug development failures thus far are caused in part by the limits of these approaches, which do not accurately reveal how drug candidates will behave in naive human neuronal cells. Objective To analyze purified neurons derived from human induced pluripotent stem cells from patients carrying 3 different presenilin 1 (PS1) mutations and nondemented control individuals in the absence of any overexpression. We tested the efficacy of γ-secretase inhibitor and γ-secretase modulator (GSM) in neurons derived from both normal control and 3 PS1 mutations (A246E, H163R, and M146L). Design, Setting, and Participants Adult human skin biopsies were obtained from volunteers at the Alzheimer Disease Research Center, University of California, San Diego. Cell cultures were treated with γ-secretase inhibitor or GSM. Comparisons of total β-amyloid (Aβ) and Aβ peptides 38, 40, and 42 in the media were made between vehicle- vs drug-treated cultures. Main Outcomes and Measures Soluble Aβ levels in the media were measured by enzyme-linked immunosorbent assay. Results As predicted, mutant PS1 neurons exhibited an elevated Aβ42:Aβ40 ratio (P <.05) at the basal state as compared with the nondemented control neurons. Treatment with a potent non–nonsteroidal anti-inflammatory druglike GSM revealed a new biomarker signature that differs from all previous cell types and animals tested. This new signature was the same in both the mutant and control neurons and consisted of a reduction in Aβ42, Aβ40, and Aβ38 and in the Aβ42:Aβ40 ratio, with no

  9. Synergistic activity of vorinostat combined with gefitinib but not with sorafenib in mutant KRAS human non-small cell lung cancers and hepatocarcinoma.

    Science.gov (United States)

    Jeannot, Victor; Busser, Benoit; Vanwonterghem, Laetitia; Michallet, Sophie; Ferroudj, Sana; Cokol, Murat; Coll, Jean-Luc; Ozturk, Mehmet; Hurbin, Amandine

    2016-01-01

    Development of drug resistance limits the efficacy of targeted therapies. Alternative approaches using different combinations of therapeutic agents to inhibit several pathways could be a more effective strategy for treating cancer. The effects of the approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (gefitinib) or a multi-targeted kinase inhibitor (sorafenib) in combination with a histone deacetylase inhibitor (vorinostat) on cell proliferation, cell cycle distribution, apoptosis, and signaling pathway activation in human lung adenocarcinoma and hepatocarcinoma cells with wild-type EGFR and mutant KRAS were investigated. The effects of the synergistic drug combinations were also studied in human lung adenocarcinoma and hepatocarcinoma cells in vivo. The combination of gefitinib and vorinostat synergistically reduced cell growth and strongly induced apoptosis through inhibition of the insulin-like growth factor-1 receptor/protein kinase B (IGF-1R/AKT)-dependent signaling pathway. Moreover, the gefitinib and vorinostat combination strongly inhibited tumor growth in mice with lung adenocarcinoma or hepatocarcinoma tumor xenografts. In contrast, the combination of sorafenib and vorinostat did not inhibit cell proliferation compared to a single treatment and induced G 2 /M cell cycle arrest without apoptosis. The sorafenib and vorinostat combination sustained the IGF-1R-, AKT-, and mitogen-activated protein kinase-dependent signaling pathways. These results showed that there was synergistic cytotoxicity when vorinostat was combined with gefitinib for both lung adenocarcinoma and hepatocarcinoma with mutant KRAS in vitro and in vivo but that the combination of vorinostat with sorafenib did not show any benefit. These findings highlight the important role of the IGF-1R/AKT pathway in the resistance to targeted therapies and support the use of histone deacetylase inhibitors in combination with EGFR-tyrosine kinase inhibitors, especially for

  10. Focal Transplantation of Human iPSC-Derived Glial-Rich Neural Progenitors Improves Lifespan of ALS Mice

    Directory of Open Access Journals (Sweden)

    Takayuki Kondo

    2014-08-01

    Full Text Available Transplantation of glial-rich neural progenitors has been demonstrated to attenuate motor neuron degeneration and disease progression in rodent models of mutant superoxide dismutase 1 (SOD1-mediated amyotrophic lateral sclerosis (ALS. However, translation of these results into a clinical setting requires a renewable human cell source. Here, we derived glial-rich neural progenitors from human iPSCs and transplanted them into the lumbar spinal cord of ALS mouse models. The transplanted cells differentiated into astrocytes, and the treated mouse group showed prolonged lifespan. Our data suggest a potential therapeutic mechanism via activation of AKT signal. The results demonstrated the efficacy of cell therapy for ALS by the use of human iPSCs as cell source.

  11. Wild-Type, but Not Mutant N296H, Human Tau Restores Aβ-Mediated Inhibition of LTP in Tau−/− mice

    Directory of Open Access Journals (Sweden)

    Mariana Vargas-Caballero

    2017-04-01

    Full Text Available Microtubule associated protein tau (MAPT is involved in the pathogenesis of Alzheimer's disease and many forms of frontotemporal dementia (FTD. We recently reported that Aβ-mediated inhibition of hippocampal long-term potentiation (LTP in mice requires tau. Here, we asked whether expression of human MAPT can restore Aβ-mediated inhibition on a mouse Tau−/− background and whether human tau with an FTD-causing mutation (N296H can interfere with Aβ-mediated inhibition of LTP. We used transgenic mouse lines each expressing the full human MAPT locus using bacterial artificial chromosome technology. These lines expressed all six human tau protein isoforms on a Tau−/− background. We found that the human wild-type MAPT H1 locus was able to restore Aβ42-mediated impairment of LTP. In contrast, Aβ42 did not reduce LTP in slices in two independently generated transgenic lines expressing tau protein with the mutation N296H associated with frontotemporal dementia (FTD. Basal phosphorylation of tau measured as the ratio of AT8/Tau5 immunoreactivity was significantly reduced in N296H mutant hippocampal slices. Our data show that human MAPT is able to restore Aβ42-mediated inhibition of LTP in Tau−/− mice. These results provide further evidence that tau protein is central to Aβ-induced LTP impairment and provide a valuable tool for further analysis of the links between Aβ, human tau and impairment of synaptic function.

  12. Cyclophilin A Levels Dictate Infection Efficiency of Human Immunodeficiency Virus Type 1 Capsid Escape Mutants A92E and G94D ▿

    Science.gov (United States)

    Ylinen, Laura M. J.; Schaller, Torsten; Price, Amanda; Fletcher, Adam J.; Noursadeghi, Mahdad; James, Leo C.; Towers, Greg J.

    2009-01-01

    Cyclophilin A (CypA) is an important human immunodeficiency virus type 1 (HIV-1) cofactor in human cells. HIV-1 A92E and G94D capsid escape mutants arise during CypA inhibition and in certain cell lines are dependent on CypA inhibition. Here we show that dependence on CypA inhibition is due to high CypA levels. Restricted HIV-1 is stable, and remarkably, restriction is augmented by arresting cell division. Nuclear entry is not inhibited. We propose that high CypA levels and capsid mutations combine to disturb uncoating, leading to poor infectivity, particularly in arrested cells. Our data suggest a role for CypA in uncoating the core of HIV-1 to facilitate integration. PMID:19073742

  13. Neuronal glucose metabolism is impaired while astrocytic TCA cycling is unaffected at symptomatic stages in the hSOD1G93A mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Tefera, Tesfaye W; Borges, Karin

    2018-01-01

    Although alterations in energy metabolism are known in ALS, the specific mechanisms leading to energy deficit are not understood. We measured metabolite levels derived from injected [1- 13 C]glucose and [1,2- 13 C]acetate (i.p.) in cerebral cortex and spinal cord extracts of wild type and hSOD1 G93A mice at onset and mid disease stages using high-pressure liquid chromatography, 1 H and 13 C nuclear magnetic resonance spectroscopy. Levels of spinal and cortical CNS total lactate, [3- 13 C]lactate, total alanine and [3- 13 C]alanine, but not cortical glucose and [1- 13 C]glucose, were reduced mostly at mid stage indicating impaired glycolysis. The [1- 13 C]glucose-derived [4- 13 C]glutamate, [4- 13 C]glutamine and [2- 13 C]GABA amounts were diminished at mid stage in cortex and both time points in spinal cord, suggesting decreased [3- 13 C]pyruvate entry into the TCA cycle. Lack of changes in [1,2- 13 C]acetate-derived [4,5- 13 C]glutamate, [4,5- 13 C]glutamine and [1,2- 13 C]GABA levels indicate unchanged astrocytic 13 C-acetate metabolism. Reduced levels of leucine, isoleucine and valine in CNS suggest compensatory breakdown to refill TCA cycle intermediate levels. Unlabelled, [2- 13 C] and [4- 13 C]GABA concentrations were decreased in spinal cord indicating that impaired glucose metabolism contributes to hyperexcitability and supporting the use of treatments which increase GABA amounts. In conclusion, CNS glucose metabolism is compromised, while astrocytic TCA cycling appears to be normal in the hSOD1 G93A mouse model at symptomatic disease stages.

  14. Mutant human torsinA, responsible for early-onset dystonia, dominantly suppresses GTPCH expression, dopamine levels and locomotion in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Noriko Wakabayashi-Ito

    2015-07-01

    Full Text Available Dystonia represents the third most common movement disorder in humans with over 20 genetic loci identified. TOR1A (DYT1, the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. Most cases of DYT1 dystonia are caused by a 3 bp (ΔGAG deletion that results in the loss of a glutamic acid residue (ΔE302/303 in the carboxyl terminal region of torsinA. This torsinAΔE mutant protein has been speculated to act in a dominant-negative manner to decrease activity of wild type torsinA. Drosophila melanogaster has a single torsin-related gene, dtorsin. Null mutants of dtorsin exhibited locomotion defects in third instar larvae. Levels of dopamine and GTP cyclohydrolase (GTPCH proteins were severely reduced in dtorsin-null brains. Further, the locomotion defect was rescued by the expression of human torsinA or feeding with dopamine. Here, we demonstrate that human torsinAΔE dominantly inhibited locomotion in larvae and adults when expressed in neurons using a pan-neuronal promoter Elav. Dopamine and tetrahydrobiopterin (BH4 levels were significantly reduced in larval brains and the expression level of GTPCH protein was severely impaired in adult and larval brains. When human torsinA and torsinAΔE were co-expressed in neurons in dtorsin-null larvae and adults, the locomotion rates and the expression levels of GTPCH protein were severely reduced. These results support the hypothesis that torsinAΔE inhibits wild type torsinA activity. Similarly, neuronal expression of a Drosophila DtorsinΔE equivalent mutation dominantly inhibited larval locomotion and GTPCH protein expression. These results indicate that both torsinAΔE and DtorsinΔE act in a dominant-negative manner. We also demonstrate that Dtorsin regulates GTPCH expression at the post-transcriptional level. This Drosophila model of DYT1 dystonia provides an important tool for studying the differences in the molecular function between the

  15. Human Lysozyme Synergistically Enhances Bactericidal Dynamics and Lowers the Resistant Mutant Prevention Concentration for Metronidazole to Helicobacter pylori by Increasing Cell Permeability

    Directory of Open Access Journals (Sweden)

    Xiaolin Zhang

    2016-10-01

    Full Text Available Metronidazole (MNZ is an effective agent that has been employed to eradicate Helicobacter pylori (H. pylori. The emergence of broad MNZ resistance in H. pylori has affected the efficacy of this therapeutic agent. The concentration of MNZ, especially the mutant prevention concentration (MPC, plays an important role in selecting or enriching resistant mutants and regulating therapeutic effects. A strategy to reduce the MPC that can not only effectively treat H. pylori but also prevent resistance mutations is needed. H. pylori is highly resistant to lysozyme. Lysozyme possesses a hydrolytic bacterial cell wall peptidoglycan and a cationic dependent mode. These effects can increase the permeability of bacterial cells and promote antibiotic absorption into bacterial cells. In this study, human lysozyme (hLYS was used to probe its effects on the integrity of the H. pylori outer and inner membranes using as fluorescent probe hydrophobic 1-N-phenyl-naphthylamine (NPN and the release of aspartate aminotransferase. Further studies using a propidium iodide staining method assessed whether hLYS could increase cell permeability and promote cell absorption. Finally, we determined the effects of hLYS on the bactericidal dynamics and MPC of MNZ in H. pylori. Our findings indicate that hLYS could dramatically increase cell permeability, reduce the MPC of MNZ for H. pylori, and enhance its bactericidal dynamic activity, demonstrating that hLYS could reduce the probability of MNZ inducing resistance mutations.

  16. 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.

  17. Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis

    NARCIS (Netherlands)

    Marissen, W.E.; Kramer, R.A.; Rice, A.; Weldon, W.C.; Niezgoda, M.; Faber, M.; Slootstra, J.W.; Meloen, R.H.; Clijsters-van der Horst, M.; Visser, T.J.; Jongeneelen, M.; Thijsse, S.; Throsby, M.; Kruif, de J.; Rupprecht, C.E.; Dietzschold, B.; Goudsmit, J.; Bakker, A.B.H.

    2005-01-01

    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

  18. Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis

    NARCIS (Netherlands)

    Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

    2005-01-01

    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

  19. Characterization of five new mutants in the carboxyl-terminal domain of human apolipoprotein E: No cosegregation with severe hyperlipidemia

    Energy Technology Data Exchange (ETDEWEB)

    Maagdenberg, A.M.J.M. van den; Bruijn, I.H. de; Hofker, M.H.; Frants, R.R. (Leiden Univ. (Netherlands)); Knijff, P. de; Smelt, A.H.M.; Leuven, J.A.G.; van' t Hooft, F.; Assmann, G.; Havekes, L.M. (Univ. Hospital, Leiden (Netherlands)); Weng, Wei; Funke, H. (Westfalische Wilhelms-Universitaet, Muester (Germany))

    1993-05-01

    Assessment of the apolipoprotein E (apoE) phenotype by isoelectric focusing of both hyperlipidemic and normolipidemic individuals identified five new variants. All mutations were confined to the downstream part of the APOE gene by using denaturing gradient gel electrophoresis (DGGE). Sequence analysis revealed five new mutations causing unique amino acid substitutions in the carboxyl-terminal part of the protein containing the putative lipid-binding domain. Three hyperlipoproteinemic probands were carriers of the APOE*2(Va1236[r arrow]Glu) allele, the APOE*3(Cys112-Arg; Arg251[r arrow]Gly) allele, or the APOE*1(Arg158[r arrow]Cys; Leu252[r arrow]Glu) allele. DGGE of the region encoding the receptor-binding domain was useful for haplotyping the mutations at codons 112 and 158. Family studies failed to demonstrate cosegregation between the new mutations and severe hyperlipoproteinemia, although a number of carriers for the APOE*3(Cys112[r arrow]Arg; Arg251[r arrow]Gly) allele and the APOE*1(Arg158-Cys; Leu252[r arrow]Glu) allele expressed hypertriglyceridemia and/ or hypercholesterolemia. Two other mutant alleles, APOE*4[sup [minus

  20. The VPAC2 agonist peptide histidine isoleucine (PHI) up-regulates glutamate transport in the corpus callosum of a rat model of amyotrophic lateral sclerosis (hSOD1G93A) by inhibiting caspase-3 mediated inactivation of GLT-1a.

    Science.gov (United States)

    Goursaud, Stéphanie; Focant, Marylène C; Berger, Julie V; Nizet, Yannick; Maloteaux, Jean-Marie; Hermans, Emmanuel

    2011-10-01

    Degeneration of corpus callosum appears in patients with amyotrophic lateral sclerosis (ALS) before clinical signs of upper motor neuron death. Considering the ALS-associated impairment of astrocytic glutamate uptake, we have characterized the expression and activity of the glutamate transporter isoforms GLT-1a and GLT-1b in the corpus callosum of transgenic rats expressing a mutated form of the human superoxide dismutase 1 (hSOD1(G93A)). We have also studied the effect of peptide histidine isoleucine (PHI), a vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 2 (VPAC(2)) agonist on glutamate transporters both in vivo and in callosal astrocytes. Before the onset of motor symptoms, the expression of both transporter isoforms was correlated with a constitutive activity of caspase-3. This enzyme participates in the down-regulation of GLT-1 in ALS, and here we demonstrated its involvement in the selective degradation of GLT-1a in the white matter. A single stereotactic injection of PHI into the corpus callosum of symptomatic rats decreased caspase-3 activity and promoted GLT-1a expression and uptake activity. Together, with evidence for a reduced expression of prepro-VIP/PHI mRNA in the corpus callosum of transgenic animals, these data shed light on the modulatory role of the VIP/PHI system on the glutamatergic transmission in ALS.

  1. Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

    Science.gov (United States)

    Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

    2015-09-08

    It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

  2. Synergistic activity of vorinostat combined with gefitinib but not with sorafenib in mutant KRAS human non-small cell lung cancers and hepatocarcinoma

    Directory of Open Access Journals (Sweden)

    Jeannot V

    2016-11-01

    Full Text Available Victor Jeannot,1,2 Benoit Busser,1–3 Laetitia Vanwonterghem,1,2 Sophie Michallet,1,2 Sana Ferroudj,1,2 Murat Cokol,4 Jean-Luc Coll,1,2 Mehmet Ozturk,1,2,5 Amandine Hurbin1,2 1INSERM U1209, Department Cancer Targets and Experimental Therapeutics, Grenoble, France; 2University Grenoble Alpes, Institute for Advanced Biosciences, Grenoble, France; 3Department of Biochemistry, Toxicology and Pharmacology, Grenoble University Hospital, Grenoble, France; 4Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey; 5Faculty of Medicine, Dokuz Eyul University, Izmir Biomedicine and Genome Center, Izmir, Turkey Abstract: Development of drug resistance limits the efficacy of targeted therapies. Alternative approaches using different combinations of therapeutic agents to inhibit several pathways could be a more effective strategy for treating cancer. The effects of the approved epidermal growth factor receptor (EGFR-tyrosine kinase inhibitor (gefitinib or a multi-targeted kinase inhibitor (sorafenib in combination with a histone deacetylase inhibitor (vorinostat on cell proliferation, cell cycle distribution, apoptosis, and signaling pathway activation in human lung adenocarcinoma and hepatocarcinoma cells with wild-type EGFR and mutant KRAS were investigated. The effects of the synergistic drug combinations were also studied in human lung adenocarcinoma and hepatocarcinoma cells in vivo. The combination of gefitinib and vorinostat synergistically reduced cell growth and strongly induced apoptosis through inhibition of the insulin-like growth factor-1 receptor/protein kinase B (IGF-1R/AKT-dependent signaling pathway. Moreover, the gefitinib and vorinostat combination strongly inhibited tumor growth in mice with lung adenocarcinoma or hepatocarcinoma tumor xenografts. In contrast, the combination of sorafenib and vorinostat did not inhibit cell proliferation compared to a single treatment and induced G2/M cell cycle arrest without

  3. A novel humanized mouse model of Huntington disease for preclinical development of therapeutics targeting mutant huntingtin alleles

    DEFF Research Database (Denmark)

    Southwell, Amber L; Skotte, Niels H; Villanueva, Erika B

    2017-01-01

    transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1......Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global......-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both...

  4. Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA

    Directory of Open Access Journals (Sweden)

    Ristic Natalia

    2010-09-01

    Full Text Available Abstract Background The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1 sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants. Results HIV-1 lacking SL1 (NLΔSL1 did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K on day 18 postinfection and C1907T in the SP1 domain (P10L on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1

  5. Hyperactivity and Learning Deficits in Transgenic Mice Bearing a Human Mutant Thyroid Hormone β1 Receptor Gene

    OpenAIRE

    McDonald, Michael P.; Wong, Rosemary; Goldstein, Gregory; Weintraub, Bruce; Cheng, Sheue-yann; Crawley, Jacqueline N.

    1998-01-01

    Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor β (TRβ) gene on chromosome 3, representing a mutation of the ligandbinding domain of the TRβ gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity...

  6. Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

    Science.gov (United States)

    Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

    2011-01-01

    In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

  7. Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

    Science.gov (United States)

    Serebryany, Eugene; Takata, Takumi; Erickson, Erika; Schafheimer, Nathaniel; Wang, Yongting; King, Jonathan A

    2016-06-01

    Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The βγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization. © 2016 The Protein Society.

  8. In vitro evidence for impaired neuroprotective capacities of adult mesenchymal stem cells derived from a rat model of familial amyotrophic lateral sclerosis (hSOD1(G93A)).

    Science.gov (United States)

    Boucherie, Cédric; Caumont, Anne-Sophie; Maloteaux, Jean-Marie; Hermans, Emmanuel

    2008-08-01

    Protection of neurons by stem cells is an attractive challenge in the development of efficient therapies of neurodegenerative diseases. When giving preference to autologous grafts, the bone marrow constitutes a valuable source of adult stem cells. Therefore, we herein studied the acquisition of neuroprotective functions by cultured mesenchymal stem cells (MSCs) exposed to growth factors known to promote the differentiation of neural stem cells into astrocytes. In these conditions, MSCs showed increased transcription and expression of the high-affinity glutamate transporter GLT-1 and functional studies revealed increased aspartate uptake activity. In addition, differentiation was shown to endow the cells with the capacity to respond to riluzole which triggers a robust up-regulation of the GDNF production. In parallel, MSCs derived from the bone marrow of a transgenic rat model of familial ALS (hSOD1(G93A)) were also characterised. Unexpectedly, cells from this rat strain submitted to the differentiation protocol showed modest capacity to take up aspartate and did not respond to the riluzole treatments. These data highlight the neuroprotective potential attributable to MSCs, supporting their use as valuable tools for the treatment of neurodegenerative disorders. However, the cells from the transgenic animal model of ALS appeared deficient in their capacity to gain the neuroprotective properties, raising questions regarding the suitability of autologous stem cell grafts in future therapies against familial forms of this disease.

  9. Production and enzyme engineerinq of human acetylcholinesterase and its mutant derivatives. Midterm report, 15 January 1993-15 July 1994

    Energy Technology Data Exchange (ETDEWEB)

    Shafferman, A.

    1994-07-15

    Specificity determinants of human acetylcholinesterase (HuAChE) towards ligands (substrate and some reversible and irreversible inhibitors) were identified by combination of site-directed mutagenesis, molecular modeling and kinetic studies with enzymes mutated in active center residues Trp86, Glu202, Trp286, Phe295, Phe297, Tyr337, Phe338 and Glu450. Thus, the anionic and hydrophobic subsites as well as the acyl pocket were identified. Enzymes with resistance to OP aging were engineered.The role of N-glycosylation in the function, biosynthesis and stability of HuAChE was examined by site-directed mutagenesis (Asn to GIn substitution) of the three potential N glycosylation sites, Asn265, Asn350 and Asn464. Large scale preparation of recombinant HuAChE was performed utilizing the microcarrier technology. Over 500 milligrams of enzyme was prepared for x-ray crystallography.

  10. Altered methanol embryopathies in embryo culture with mutant catalase-deficient mice and transgenic mice expressing human catalase

    International Nuclear Information System (INIS)

    Miller, Lutfiya; Wells, Peter G.

    2011-01-01

    The mechanisms underlying the teratogenicity of methanol (MeOH) in rodents, unlike its acute toxicity in humans, are unclear, but may involve reactive oxygen species (ROS). Embryonic catalase, although expressed at about 5% of maternal activity, may protect the embryo by detoxifying ROS. This hypothesis was investigated in whole embryo culture to remove confounding maternal factors, including metabolism of MeOH by maternal catalase. C57BL/6 (C57) mouse embryos expressing human catalase (hCat) or their wild-type (C57 WT) controls, and C3Ga.Cg-Catb/J acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 4 mg/ml MeOH or vehicle, and evaluated for functional and morphological changes. hCat and C57 WT vehicle-exposed embryos developed normally. MeOH was embryopathic in C57 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed and turning, whereas hCat embryos were protected. Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to C3H WT controls, suggesting that endogenous ROS are embryopathic. MeOH was more embryopathic in aCat embryos than WT controls, with reduced anterior neuropore closure and head length only in catalase-deficient embryos. These data suggest that ROS may be involved in the embryopathic mechanism of methanol, and that embryonic catalase activity may be a determinant of teratological risk.

  11. Modeling and Docking Studies on Novel Mutants (K71L and T204V of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2014-04-01

    Full Text Available The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO were mutated. 3D mutant models (K71L and T204V using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255, as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy.

  12. Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells.

    Science.gov (United States)

    Yanovsky-Dagan, Shira; Avitzour, Michal; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Mitrani-Rosenbaum, Stella; Levy-Lahad, Ephrat; Birnbaum, Ramon Y; Gepstein, Lior; Epsztejn-Litman, Silvina; Eiges, Rachel

    2015-08-11

    CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Absence of linkage of apparently single gene mediated ADHD with the human syntenic region of the mouse mutant coloboma

    Energy Technology Data Exchange (ETDEWEB)

    Hess, E.J.; Rogan, P.K.; Domoto, M. [Pennsylvania State Univ. College of Medicine, Hershey, PA (United States)] [and others

    1995-12-18

    Attention deficit disorder (ADHD) is a complex biobehavioral phenotype which affects up to 8% of the general population and often impairs social, academic, and job performance. Its origins are heterogeneous, but a significant genetic component is suggested by family and twin studies. The murine strain, coloboma, displays a spontaneously hyperactive phenotype that is responsive to dextroamphetamine and has been proposed as a genetic model for ADHD. Coloboma is a semi-dominant mutation that is caused by a hemizygous deletion of the SNAP-25 and other genes on mouse chromosome 2q. To test the possibility that the human homolog of the mouse coloboma gene(s) could be responsible for ADHD, we have carried out linkage studies with polymorphic markers in the region syntenic to coloboma (20p11-p12). Five families in which the pattern of inheritance of ADHD appears to be autosomal dominant were studied. Segregation analysis of the traits studied suggested that the best fitting model was a sex-influenced, single gene, Mendelian pattern. Several genetic models were evaluated based on estimates of penetrance, phenocopy rate, and allele frequency derived from our patient population and those of other investigators. No significant linkage was detected between the disease locus and markers spanning this chromosome 20 interval. 39 refs., 2 figs., 1 tab.

  14. A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line

    International Nuclear Information System (INIS)

    Suzuki, N.

    1984-01-01

    When cells of a human clonal cell line, RSa, with high sensitivity to UV lethality, were treated with the mutagen, ethyl methanesulfonate, a variant cell strain, UVr-1, was established as a mutant resistant to 254-nm far-ultraviolet radiation (UV). Cell proliferation studies showed that UVr-1 cells survived and actively proliferated at doses of UV-irradiation that greatly suppressed the proliferation of RSa cells. Colony-formation assays also confirmed the increased resistance of UVr-1 cells to UV. The recovery from a UV-induced inhibition in DNA synthesis, as [methyl- 3 H]thymidine uptake into cellular DNA, was more pronounced in UVr-1 cells than in RSa cells. Nevertheless, there was no significant difference in the activity of UV-induced DNA repair synthesis in either cell line, as estimated by the extent of unscheduled DNA synthesis and DNA repair replication. These characteristics of UVr-1 cells are discussed in the light of a previously reported UV-resistant variant, UVr-10, which had an increased DNA repair synthesis activity. (Auth.)

  15. 3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujise

    2004-01-01

    Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

  16. Two mutant alleles of the human cytochrome P-450dbl gene (P450C2D1) associated with genetically deficient metabolism of debrisoquine and other drugs

    International Nuclear Information System (INIS)

    Skoda, R.C.; Gonzalez, F.L.; Demierre, A.; Meyer, R.A.

    1988-01-01

    The debrisoquine polymorphism is a clinically important genetic defect of drug metabolism affecting 5-10% of individuals in Caucasian populations. It is inherited as an autosomal recessive trait. A full-length cDNA for human cytochrome P-450db1, the deficient enzyme (also designated P450IID1 for P450 family II subfamily D isozyme 1), has recently been cloned. Leukocyte DNA from extensive metabolizers (EMs) or poor metabolizers (PMs) of debrisoquine was examined by Southern analysis. Two polymorphic restriction fragments were associated with the PM phenotype when DNAs from 24 unrelated PM and 29 unrelated EM individuals were probed with P-450db1 cDNA after digestion with Xba I restriction endonuclease and Southern blotting. Seventy-five percent of PMs had either the 44-kb or the 11.5-kb fragment or both. Segregation of these restriction fragment length polymorphisms in the families of six PM probands demonstrated that each of the two fragments is allelic with the 29-kb fragment present in all EM individuals and suggests that they identify two independent mutated alleles of the P-450db1 gene (designated P450C2D1). The Xba I 44-kb fragment and 11.5-kb fragment were in linkage disequilibrium with restriction fragment length polymorphisms generated by four and five additional restriction endonucleases, respectively, which can be used to identify the same mutant alleles for the P-450db1 gene

  17. Localization of the homolog of a mouse craniofacial mutant to human chromosome 18q11 and evaluation of linkage to human CLP and CPO

    Energy Technology Data Exchange (ETDEWEB)

    Griffith, A.J.; Burgess, D.L.; Kohrman, D.C.; Yu, J. [Univ. of Michigan, Ann Arbor, MI (United States)] [and others

    1996-06-15

    The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locus ax. To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci. 23 refs., 4 figs., 2 tabs.

  18. Comparative Global Gene Expression Profiles of Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant at Flea and Human Body Temperatures

    Directory of Open Access Journals (Sweden)

    Cristi L. Galindo

    2010-01-01

    Full Text Available Braun/murein lipoprotein (Lpp is involved in inflammatory responses and septic shock. We previously characterized a Δlpp mutant of Yersinia pestis CO92 and found that this mutant was defective in surviving in macrophages and was attenuated in a mouse inhalation model of plague when compared to the highly virulent wild-type (WT bacterium. We performed global transcriptional profiling of WT Y. pestis and its Δlpp mutant using microarrays. The organisms were cultured at 26 and 37 degrees Celsius to simulate the flea vector and mammalian host environments, respectively. Our data revealed vastly different effects of lpp mutation on the transcriptomes of Y. pestis grown at 37 versus 26C. While the absence of Lpp resulted mainly in the downregulation of metabolic genes at 26C, the Y. pestis Δlpp mutant cultured at 37C exhibited profound alterations in stress response and virulence genes, compared to WT bacteria. We investigated one of the stress-related genes (htrA downregulated in the Δlpp mutant relative to WT Y. pestis. Indeed, complementation of the Δlpp mutant with the htrA gene restored intracellular survival of the Y. pestis Δlpp mutant. Our results support a role for Lpp in Y. pestis adaptation to the host environment, possibly via transcriptional activation of htrA.

  19. Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

    International Nuclear Information System (INIS)

    Mill, Christopher P.; Gettinger, Kathleen L.; Riese, David J.

    2011-01-01

    Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.

  20. Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD-mutant leukemia cells.

    Science.gov (United States)

    Mathew, Nimitha R; Baumgartner, Francis; Braun, Lukas; O'Sullivan, David; Thomas, Simone; Waterhouse, Miguel; Müller, Tony A; Hanke, Kathrin; Taromi, Sanaz; Apostolova, Petya; Illert, Anna L; Melchinger, Wolfgang; Duquesne, Sandra; Schmitt-Graeff, Annette; Osswald, Lena; Yan, Kai-Li; Weber, Arnim; Tugues, Sonia; Spath, Sabine; Pfeifer, Dietmar; Follo, Marie; Claus, Rainer; Lübbert, Michael; Rummelt, Christoph; Bertz, Hartmut; Wäsch, Ralph; Haag, Johanna; Schmidts, Andrea; Schultheiss, Michael; Bettinger, Dominik; Thimme, Robert; Ullrich, Evelyn; Tanriver, Yakup; Vuong, Giang Lam; Arnold, Renate; Hemmati, Philipp; Wolf, Dominik; Ditschkowski, Markus; Jilg, Cordula; Wilhelm, Konrad; Leiber, Christian; Gerull, Sabine; Halter, Jörg; Lengerke, Claudia; Pabst, Thomas; Schroeder, Thomas; Kobbe, Guido; Rösler, Wolf; Doostkam, Soroush; Meckel, Stephan; Stabla, Kathleen; Metzelder, Stephan K; Halbach, Sebastian; Brummer, Tilman; Hu, Zehan; Dengjel, Joern; Hackanson, Björn; Schmid, Christoph; Holtick, Udo; Scheid, Christof; Spyridonidis, Alexandros; Stölzel, Friedrich; Ordemann, Rainer; Müller, Lutz P; Sicre-de-Fontbrune, Flore; Ihorst, Gabriele; Kuball, Jürgen; Ehlert, Jan E; Feger, Daniel; Wagner, Eva-Maria; Cahn, Jean-Yves; Schnell, Jacqueline; Kuchenbauer, Florian; Bunjes, Donald; Chakraverty, Ronjon; Richardson, Simon; Gill, Saar; Kröger, Nicolaus; Ayuk, Francis; Vago, Luca; Ciceri, Fabio; Müller, Antonia M; Kondo, Takeshi; Teshima, Takanori; Klaeger, Susan; Kuster, Bernhard; Kim, Dennis Dong Hwan; Weisdorf, Daniel; van der Velden, Walter; Dörfel, Daniela; Bethge, Wolfgang; Hilgendorf, Inken; Hochhaus, Andreas; Andrieux, Geoffroy; Börries, Melanie; Busch, Hauke; Magenau, John; Reddy, Pavan; Labopin, Myriam; Antin, Joseph H; Henden, Andrea S; Hill, Geoffrey R; Kennedy, Glen A; Bar, Merav; Sarma, Anita; McLornan, Donal; Mufti, Ghulam; Oran, Betul; Rezvani, Katayoun; Shah, Omid; Negrin, Robert S; Nagler, Arnon; Prinz, Marco; Burchert, Andreas; Neubauer, Andreas; Beelen, Dietrich; Mackensen, Andreas; von Bubnoff, Nikolas; Herr, Wolfgang; Becher, Burkhard; Socié, Gerard; Caligiuri, Michael A; Ruggiero, Eliana; Bonini, Chiara; Häcker, Georg; Duyster, Justus; Finke, Jürgen; Pearce, Erika; Blazar, Bruce R; Zeiser, Robert

    2018-03-01

    Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD + leukemia cells. This synergized with the allogeneic CD8 + T cell response, leading to long-term survival in six mouse models of FLT3-ITD + AML. Sorafenib-related IL-15 production caused an increase in CD8 + CD107a + IFN-γ + T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD + AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8 + T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

  1. Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

    Science.gov (United States)

    van Lier, Christina J; Tiner, Bethany L; Chauhan, Sadhana; Motin, Vladimir L; Fitts, Eric C; Huante, Matthew B; Endsley, Janice J; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K

    2015-03-01

    We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune

  2. Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus.

    Science.gov (United States)

    Deschamps, Thibaut; Kalamvoki, Maria

    2017-05-01

    Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway. IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets

  3. Promising rice mutants

    International Nuclear Information System (INIS)

    Hakim, L.; Azam, M.A.; Miah, A.J.; Mansur, M.A.; Akanda, H.R.

    1988-01-01

    Two induced mutants namely, Mut NS 1 (tall) and Mut NS 5 (semi-dwarf) derived from rice variety Nizersail were evaluated for various agronomic characters at four locations in Bangladesh. Both the mutants matured about three weeks earlier and yielded significantly higher than the parent variety Nizersail. (author). 3 tabs., 9 refs

  4. Mutant heterosis in rice

    International Nuclear Information System (INIS)

    1987-01-01

    In the variety TKM6 a high yielding semidwarf mutant has been induced. This TKM6 mutant was used in test crosses with a number of other varieties and mutants to examine the extent of heterosis of dwarfs in rice and to select superior crosses. An excerpt of the published data is given. It appears from the backcross of the mutant with its original variety, that an increase in number of productive tillers occurs in the hybrid, leading to a striking grain yield increase, while the semi-dwarf culm length (the main mutant character) reverts to the normal phenotype. In the cross with IR8 on the other hand, there is only a minimal increase in tiller number but a substantial increase in TGW leading to more than 30% yield increase over the better parent

  5. Vitamin C (Vit C) added after irradiation reduces the number and alters the spectrum of CD59- mutants in human/CHO AL cells exposed to high LET carbon ions

    International Nuclear Information System (INIS)

    Vannais, D.B.; Hirai, Y.; Waldren, C.A.; Ueno, A.

    2003-01-01

    Full text: Miyazaki, Watanabe, Kumagai and colleagues discovered the existence in mammalian cells of long-lived radicals (LLR) with half-lives of minutes to hours. They further showed that concentrations of LLR were increased in a dose dependent manner by X-rays; that LLR were transforming and mutagenic but not clastogenic or lethal; that they were scavenged by Vit C but not by DMSO, and that they occured mainly (>99.8%) in proteins from which they escape by atomic tunneling. They also showed that Vit C added after radiation (but not DMSO) eliminated HPRT mutants in human cells exposed to X-rays. Following on their work, we found that Vit C (5 mM) added 30 min after radiation significantly reduced, but did not eliminate, induction of CD59- mutants in human-CHO hybrid AL cells exposed to high LET carbon beam radiation (NIRS-HIMAC, 290 MeV/nucleon, LET 100 KeV/μ: m). Lethality of the carbon beam was not affected by Vit C. DMSO decreased mutation and killing, only when present during radiation. Lycopene, reported to reduce spontaneous mutation, did not affect radiation killing or mutagenesis. Our findings with Vit C for high LET generally support the results reported for X-rays. Analysis of the spectrum of mutations in CD59- mutant cells isolated after carbon beam irradiation (2.5 Gy), indicates a substantial reduction by post-radiation Vit C in mutants with small mutations and those displaying genomic instability, seen as increased levels of translocations. Our results substantiate a role for LLR in radiation mutagenesis and implicate them in radiation-induced genomic instability

  6. Mutant power: using mutant allele collections for yeast functional genomics.

    Science.gov (United States)

    Norman, Kaitlyn L; Kumar, Anuj

    2016-03-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

    Directory of Open Access Journals (Sweden)

    Zeynep A Oztug Durer

    Full Text Available Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1 are one of the causes of familial amyotrophic lateral sclerosis (FALS. Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1 formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

  8. Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of the CCS Cu chaperone.

    Science.gov (United States)

    Carroll, Mark C; Girouard, Jody B; Ulloa, Janella L; Subramaniam, Jamuna R; Wong, Phillip C; Valentine, Joan Selverstone; Culotta, Valeria Cizewski

    2004-04-20

    The Cu- and Zn-containing superoxide dismutase 1 (SOD1) largely obtains Cu in vivo by means of the action of the Cu chaperone CCS. Yet, in the case of mammalian SOD1, a secondary pathway of activation is apparent. Specifically, when human SOD1 is expressed in either yeast or mammalian cells that are null for CCS, the SOD1 enzyme retains a certain degree of activity. This CCS-independent activity is evident with both wild-type and mutant variants of SOD1 that have been associated with familial amyotrophic lateral sclerosis. We demonstrate here that the CCS-independent activation of mammalian SOD1 involves glutathione, particularly the reduced form, or GSH. A role for glutathione in CCS-independent activation was seen with human SOD1 molecules that were expressed in either yeast cells or immortalized fibroblasts. Compared with mammalian SOD1, the Saccharomyces cerevisiae enzyme cannot obtain Cu without CCS in vivo, and this total dependence on CCS involves the presence of dual prolines near the C terminus of the SOD1 polypeptide. Indeed, the insertion of such prolines into human SOD1 rendered this molecule refractory to CCS-independent activation. The possible implications of multiple pathways for SOD1 activation are discussed in the context of SOD1 evolutionary biology and familial amyotrophic lateral sclerosis.

  9. Chemical Genomic Screening of a Saccharomyces cerevisiae Genomewide Mutant Collection Reveals Genes Required for Defense against Four Antimicrobial Peptides Derived from Proteins Found in Human Saliva

    Science.gov (United States)

    Bhatt, Sanjay; Schoenly, Nathan E.; Lee, Anna Y.; Nislow, Corey; Bobek, Libuse A.

    2013-01-01

    To compare the effects of four antimicrobial peptides (MUC7 12-mer, histatin 12-mer, cathelicidin KR20, and a peptide containing lactoferricin amino acids 1 to 11) on the yeast Saccharomyces cerevisiae, we employed a genomewide fitness screen of combined collections of mutants with homozygous deletions of nonessential genes and heterozygous deletions of essential genes. When an arbitrary fitness score cutoffs of 1 (indicating a fitness defect, or hypersensitivity) and −1 (indicating a fitness gain, or resistance) was used, 425 of the 5,902 mutants tested exhibited altered fitness when treated with at least one peptide. Functional analysis of the 425 strains revealed enrichment among the identified deletions in gene groups associated with the Gene Ontology (GO) terms “ribosomal subunit,” “ribosome biogenesis,” “protein glycosylation,” “vacuolar transport,” “Golgi vesicle transport,” “negative regulation of transcription,” and others. Fitness profiles of all four tested peptides were highly similar, particularly among mutant strains exhibiting the greatest fitness defects. The latter group included deletions in several genes involved in induction of the RIM101 signaling pathway, including several components of the ESCRT sorting machinery. The RIM101 signaling regulates response of yeasts to alkaline and neutral pH and high salts, and our data indicate that this pathway also plays a prominent role in regulating protective measures against all four tested peptides. In summary, the results of the chemical genomic screens of S. cerevisiae mutant collection suggest that the four antimicrobial peptides, despite their differences in structure and physical properties, share many interactions with S. cerevisiae cells and consequently a high degree of similarity between their modes of action. PMID:23208710

  10. Structural and functional consequences of binding site mutations in transferrin: crystal structures of the Asp63Glu and Arg124Ala mutants of the N-lobe of human transferrin.

    Science.gov (United States)

    Baker, Heather M; He, Qing-Yu; Briggs, Sara K; Mason, Anne B; Baker, Edward N

    2003-06-17

    Human transferrin is a serum protein whose function is to bind Fe(3+) with very high affinity and transport it to cells, for delivery by receptor-mediated endocytosis. Structurally, the transferrin molecule is folded into two globular lobes, representing its N-terminal and C-terminal halves, with each lobe possessing a high-affinity iron binding site, in a cleft between two domains. Central to function is a highly conserved set of iron ligands, including an aspartate residue (Asp63 in the N-lobe) that also hydrogen bonds between the two domains and an arginine residue (Arg124 in the N-lobe) that binds an iron-bound carbonate ion. To further probe the roles of these residues, we have determined the crystal structures of the D63E and R124A mutants of the N-terminal half-molecule of human transferrin. The structure of the D63E mutant, determined at 1.9 A resolution (R = 0.245, R(free) = 0.261), showed that the carboxyl group still binds to iron despite the larger size of the Glu side chain, with some slight rearrangement of the first turn of alpha-helix residues 63-72, to which it is attached. The structure of the R124A mutant, determined at 2.4 A resolution (R = 0.219, R(free) = 0.288), shows that the loss of the arginine side chain results in a 0.3 A displacement of the carbonate ion, and an accompanying movement of the iron atom. In both mutants, the iron coordination is changed slightly, the principal change being in each case a lengthening of the Fe-N(His249) bond. Both mutants also release iron more readily than the wild type, kinetically and in terms of acid lability of iron binding. We attribute this to more facile protonation of the synergistically bound carbonate ion, in the case of R124A, and to strain resulting from the accommodation of the larger Glu side chain, in the case of D63E. In both cases, the weakened Fe-N(His) bond may also contribute, consistent with protonation of the His ligand being an early intermediate step in iron release, following the

  11. Productive mutants of niger

    International Nuclear Information System (INIS)

    Misra, R.C.

    2001-01-01

    Seeds of six niger (Guizotia abyssinica Cass.) varieties ('GA-10', 'ONS-8', 'IGP-72', 'N-71', 'NB-9' and 'UN-4') were treated with 0.5, 0.75 and 1% ethyl methanesulphonate. After four generations of selection, 29 mutant lines were developed and those were evaluated from 1990-92 during Kharif (July to October) and Rabi (December to March) seasons. Average plant characteristics and yield data of four high yielding mutants along with 'IGP-76' (National Check), GA-10 (Zonal Check) and 'Semiliguda Local' (Local Check) are presented

  12. Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase - Kinetic properties and oligomerization pattern of nine substitution mutants of V106

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Berenstein, Dvora; Munch-Petersen, Birgitte

    2004-01-01

    Information on the regulation and structure-function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106...... and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible...... transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106...

  13. Formation of multinucleated giant cells and microglial degeneration in rats expressing a mutant Cu/Zn superoxide dismutase gene

    Directory of Open Access Journals (Sweden)

    Streit Wolfgang J

    2007-02-01

    Full Text Available Abstract Background Microglial neuroinflammation is thought to play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS. The purpose of this study was to provide a histopathological evaluation of the microglial neuroinflammatory response in a rodent model of ALS, the SOD1G93A transgenic rat. Methods Multiple levels of the CNS from spinal cord to cerebral cortex were studied in SOD1G93A transgenic rats during three stages of natural disease progression, including presymptomatic, early symptomatic (onset, and late symptomatic (end stage, using immuno- and lectin histochemical markers for microglia, such as OX-42, OX-6, and Griffonia simplicifolia isolectin B4. Results Our studies revealed abnormal aggregates of microglia forming in the spinal cord as early as the presymptomatic stage. During the symptomatic stages there was prominent formation of multinucleated giant cells through fusion of microglial cells in the spinal cord, brainstem, and red nucleus of the midbrain. Other brain regions, including substantia nigra, cranial nerve nuclei, hippocampus and cortex showed normal appearing microglia. In animals during end stage disease at 4–5 months of age virtually all microglia in the spinal cord gray matter showed extensive fragmentation of their cytoplasm (cytorrhexis, indicative of widespread microglial degeneration. Few microglia exhibiting nuclear fragmentation (karyorrhexis indicative of apoptosis were identified at any stage. Conclusion The current findings demonstrate the occurrence of severe abnormalities in microglia, such as cell fusions and cytorrhexis, which may be the result of expression of mutant SOD1 in these cells. The microglial changes observed are different from those that accompany normal microglial activation, and they demonstrate that aberrant activation and degeneration of microglia is part of the pathogenesis of motor neuron disease.

  14. 17-AAG increases autophagic removal of mutant androgen receptor in spinal and bulbar muscular atrophy.

    Science.gov (United States)

    Rusmini, Paola; Simonini, Francesca; Crippa, Valeria; Bolzoni, Elena; Onesto, Elisa; Cagnin, Monica; Sau, Daniela; Ferri, Nicola; Poletti, Angelo

    2011-01-01

    Several types of motorneuron diseases are linked to neurotoxic mutant proteins. These acquire aberrant conformations (misfolding) that trigger deleterious downstream events responsible for neuronal dysfunction and degeneration. The pharmacological removal of misfolded proteins might thus be useful in these diseases. We utilized a peculiar motorneuronal disease model, spinobulbar muscular atrophy (SBMA), in which the neurotoxicity of the protein involved, the mutant androgen receptor (ARpolyQ), can be modulated by its ligand testosterone (T). 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) has already been proven to exert beneficial action in SBMA. Here we demonstrated that 17-AAG exerts its pro-degradative activity on mutant ARpolyQ without impacting on proteasome functions. 17-AAG removes ARpolyQ misfolded species and aggregates by activating the autophagic system. We next analyzed the 17-AAG effects on two proteins (SOD1 and TDP-43) involved in related motorneuronal diseases, such as amyotrophic lateral sclerosis (ALS). In these models 17-AAG was unable to counteract protein aggregation. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Cell-penetrating superoxide dismutase attenuates oxidative stress-induced senescence by regulating the p53-p21Cip1 pathway and restores osteoblastic differentiation in human dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Park YJ

    2012-09-01

    Full Text Available Yoon Jung Choi,1,* Jue Yeon Lee,2,* Chong Pyoung Chung,2 Yoon Jeong Park,1,21Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea; 2Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Human dental pulp stem cells (DPSCs have potential applications in tissue regeneration because of their convenient cell harvesting procedures and multipotent capacity. However, the tissue regenerative potential of DPSCs is known to be negatively regulated by aging in long-term culture and under oxidative stress. With an aim of reducing cellular senescence and oxidative stress in DPSCs, an intracellular delivery system for superoxide dismutase 1 (SOD1 was developed. We conjugated SOD1 with a cell-penetrating peptide known as low-molecular weight protamine (LMWP, and investigated the effect of LMWP-SOD1 conjugates on hydrogen peroxide-induced cellular senescence and osteoblastic differentiation.Results: LMWP-SOD1 significantly attenuated enlarged and flattened cell morphology and increased senescence-associated β-galactosidase activity. Under the same conditions, LMWP-SOD1 abolished activation of the cell cycle regulator proteins, p53 and p21Cip1, induced by hydrogen peroxide. In addition, LMWP-SOD1 reversed the inhibition of osteoblastic differentiation and downregulation of osteogenic gene markers induced by hydrogen peroxide. However, LMWP-SOD1 could not reverse the decrease in odontogenesis caused by hydrogen peroxide.Conclusion: Overall, cell-penetrating LMWP-SOD1 conjugates are effective for attenuation of cellular senescence and reversal of osteoblastic differentiation of DPSCs caused by oxidative stress inhibition. This result suggests potential application in the field of antiaging and tissue engineering to overcome the limitations of senescent stem cells.Keywords: superoxide

  16. Familial hypercholesterolemia and atherosclerosis in cloned minipigs created by DNA transposition of a human PCSK9 gain-of-function mutant

    DEFF Research Database (Denmark)

    Al-Mashhadi, Rozh Husain; Sørensen, Charlotte Brandt; Kragh, Peter M.

    2013-01-01

    dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low...

  17. An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin.

    Science.gov (United States)

    Serebryany, Eugene; Woodard, Jaie C; Adkar, Bharat V; Shabab, Mohammed; King, Jonathan A; Shakhnovich, Eugene I

    2016-09-02

    Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered "amorphous," and in most cases, little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys(32) and Cys(41), was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal β-hairpin and rearrangement of the native β-sheet topology. Specific binding between the extruded hairpin and a distal β-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin*

    Science.gov (United States)

    Serebryany, Eugene; Woodard, Jaie C.; Adkar, Bharat V.; Shabab, Mohammed; King, Jonathan A.; Shakhnovich, Eugene I.

    2016-01-01

    Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered “amorphous,” and in most cases, little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys32 and Cys41, was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro. Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal β-hairpin and rearrangement of the native β-sheet topology. Specific binding between the extruded hairpin and a distal β-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation. PMID:27417136

  19. Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

    DEFF Research Database (Denmark)

    Liu, Dekang; Frederiksen, Jane H.; Liberti, Sascha Emilie

    2017-01-01

    DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other...... organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display...

  20. Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI).

    Science.gov (United States)

    Masuya, Hiroshi; Shimizu, Kunihiko; Sezutsu, Hideki; Sakuraba, Yoshiyuki; Nagano, Junko; Shimizu, Aya; Fujimoto, Naomi; Kawai, Akiko; Miura, Ikuo; Kaneda, Hideki; Kobayashi, Kimio; Ishijima, Junko; Maeda, Takahide; Gondo, Yoichi; Noda, Tetsuo; Wakana, Shigeharu; Shiroishi, Toshihiko

    2005-03-01

    Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.

  1. Photorepair mutants of Arabidopsis

    International Nuclear Information System (INIS)

    Jiang, C.Z.; Yee, J.; Mitchell, D.L.; Britt, A.B.

    1997-01-01

    UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system

  2. Construindo Marcas Mutantes

    Directory of Open Access Journals (Sweden)

    Elizete De Azevedo Kreutz

    2012-09-01

    Full Text Available O presente artigo é o resultado de estudos realizados desde 2000 e busca instrumentalizar os proñssionals para a construção de Marcas Mutantes, que é   uma tendência contemporânea nas estratégias comunicacionais e de branding. Embora esta estratégia ainda não esteja consolidada, observamos que a mesma tem obtido um crescimento constante e tem sido adotadas pelas mais diferentes categorias de marcas e não apenas por aquelas direcionadas aos jovens, ao esporte, ao entretenimento, como era no principia. Com base na Hermenêutica de Profundidade de Thompson (1995, alicerçada nas pesquisas bibliográficas, de intemet, entrevistas e análise semiótica, desenhamos um método de construção de Marcas Mutantes dividido em sete fases. Como resultado, esperamos que este estudo possa auxiliar na compreensão dos processos envolvidos, ao mesmo tempo que provoque a discussão sobreo mesmo e, por consequência, o seu aprimoramento.

  3. [Architecture of the X chromosome, expression of LIM kinase 1, and recombination in the agnostic mutants of Drosophila: a model of human Williams syndrome].

    Science.gov (United States)

    Savvateeva-Popova, E V; Peresleni, A I; Sharagina, L M; Medvedeva, A V; Korochkina, S E; Grigor'eva, I V; Diuzhikova, N A; Popov, A V; Baricheva, E M; Karagodin, D; Heisenberg, M

    2004-06-01

    As the Human Genome and Drosophila Genome Projects were completed, it became clear that functions of human disease-associated genes may be elucidated by studying the phenotypic expression of mutations affecting their structural or functional homologs in Drosophila. Genomic diseases were identified as a new class of human disorders. Their cause is recombination, which takes place at gene-flanking duplicons to generate chromosome aberrations such as deletions, duplications, inversions, and translocations. The resulting imbalance of the dosage of developmentally important genes arises at a frequency of 10(-3) (higher than the mutation rate of individual genes) and leads to syndromes with multiple manifestations, including cognitive defects. Genomic DNA fragments were cloned from the Drosophila melanogaster agnostic locus, whose mutations impair learning ability and memory. As a result, the locus was exactly localized in X-chromosome region 11A containing the LIM kinase 1 (LIMK1) gene (CG1848), which is conserved among many species. Hemizygosity for the LIMK1 gene, which is caused by recombination at neighboring extended repeats, underlies cognitive disorders in human Williams syndrome. LIMK1 is a component of the integrin signaling cascade, which regulates the functions of the actin cytoskeleton, synaptogenesis, and morphogenesis in the developing brain. Immunofluorescence analysis revealed LIMK1 in all subdomains of the central complex and the visual system of Drosophila melanogaster. Like in the human genome, the D. melanogaster region is flanked by numerous repeats, which were detected by molecular genetic methods and analysis of ectopic chromosome pairing. The repeats determined a higher rate of spontaneous and induced recombination. including unequal crossing over, in the agnostic gene region. Hence, the agnostic locus was considered as the first D. melanogaster model suitable for studying the genetic defect associated with Williams syndrome in human.

  4. Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma

    NARCIS (Netherlands)

    Rood, Johannes J M; van Bussel, Mark T J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2016-01-01

    A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was

  5. Polymorphic human (CTAT)n microsatellite provides a conserved linkage marker for mouse mutants causing cleft palate, vestibular defects, obesity and ataxia

    Energy Technology Data Exchange (ETDEWEB)

    Griffith, A.J.; Burgess, D.L.; Kohrman, D. [Univ. of MIchigan, Ann Arbor, MI (United States)] [and others

    1994-09-01

    The Twirler mutation (Tw) causing cleft palate {plus_minus} cleft lip, vestibular defects and obesity is located within 0.5 cM of an ataxia locus (ax) on mouse chromosome 18. We identified a transgene-induced insertional mutation with vestibular and craniofacial defects that appears to be a new allele of Twirler. Mouse DNA flanking the transgene insertion site was isolated from a cosmid library. An evolutionarily conserved, zoo blot positive cosmid subclone was used to probe a human {lambda} genomic library. From the sequence of a highly homologous human {lambda} clone, we designed STS primers and screened a human P1 library. DNA from two positive P1 clones was hybridized with simple sequence probes, and a (CTAT){sub 12} repeat was detected. Analysis of 62 CEPH parents with primers flanking the repeat identified six alleles containing 9 to 14 copies of the repeat, at frequencies of 0.17, 0.17, 0.17, 0.27, 0.15 and 0.07, respectively. The observed heterozygosity was 49/62 with a calculated PIC value of 0.76. This polymorphic microsatellite marker, designated Umi3, was mapped to the predicted conserved human linkage group by analysis of somatic cell hybrid panels. The anticipated short distance between Umi3 and the disease genes will facilitate detection of linkage in small families. We would like to type appropriate human pedigrees with Umi3 in order to identify patients with inherited disorders homologous to the mouse mutations Twirler and ataxia.

  6. An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant.

    Science.gov (United States)

    Zhong, Huailing; Hansen, Kasper B; Boyle, Noel J; Han, Kiho; Muske, Galina; Huang, Xinyan; Egebjerg, Jan; Sánchez, Connie

    2009-10-25

    The human serotonin transporter (hSERT) has primary and allosteric binding sites for escitalopram and R-citalopram. Previous studies have established that the interaction of these two compounds at a low affinity allosteric binding site of hSERT can affect the dissociation of [(3)H]escitalopram from hSERT. The allosteric binding site involves a series of residues in the 10th, 11th, and 12th trans-membrane domains of hSERT. The low affinity allosteric activities of escitalopram and R-citalopram are essentially eliminated in a mutant hSERT with changes in some of these residues, namely A505V, L506F, I507L, S574T, I575T, as measured in dissociation binding studies. We confirm that in association binding experiments, R-citalopram at clinically relevant concentrations reduces the association rate of [(3)H]escitalopram as a ligand to wild type hSERT. We demonstrate that the ability of R-citalopram to reduce the association rate of escitalopram is also abolished in the mutant hSERT (A505V, L506F, I507L, S574T, I575T), along with the expected disruption the low affinity allosteric function on dissociation binding. This suggests that the allosteric binding site mediates both the low affinity and higher affinity interactions between R-citalopram, escitalopram, and hSERT. Our data add an additional structural basis for the different efficacies of escitalopram compared to racemic citalopram reported in animal studies and clinical trials, and substantiate the hypothesis that hSERT has complex allosteric mechanisms underlying the unexplained in vivo activities of its inhibitors.

  7. Isozyme differences in barley mutants

    Energy Technology Data Exchange (ETDEWEB)

    AI-Jibouri, A A.M.; Dham, K M [Department of Botany, Nuclear Research Centre, Baghdad (Iraq)

    1990-01-01

    Full text: Thirty mutants (M{sub 11}) of barley (Hordeum vulgare L.) induced by physical and chemical mutagens were analysed for isozyme composition using polyacrylamide gel electrophoresis. Results show that these mutants were different in the isozymes leucine aminopeptidase, esterase and peroxidase. The differences included the number of forms of each enzyme, relative mobility value and their intensity on the gel. Glutamate oxaloacetate transaminase isozyme was found in six molecular forms and these forms were similar in all mutants. (author)

  8. Isozyme differences in barley mutants

    International Nuclear Information System (INIS)

    AI-Jibouri, A.A.M.; Dham, K.M.

    1990-01-01

    Full text: Thirty mutants (M 11 ) of barley (Hordeum vulgare L.) induced by physical and chemical mutagens were analysed for isozyme composition using polyacrylamide gel electrophoresis. Results show that these mutants were different in the isozymes leucine aminopeptidase, esterase and peroxidase. The differences included the number of forms of each enzyme, relative mobility value and their intensity on the gel. Glutamate oxaloacetate transaminase isozyme was found in six molecular forms and these forms were similar in all mutants. (author)

  9. Detection of single nucleotide polymorphisms in p53 mutation hotspots and expression of mutant p53 in human cell lines using an enzyme-linked electrochemical assay

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Šimková, Eva; Vychodilová, Zdenka; Brázdová, Marie; Fojta, Miroslav

    2009-01-01

    Roč. 21, č. 15 (2009), s. 1723-1729 ISSN 1040-0397 R&D Projects: GA ČR(CZ) GA203/07/1195; GA AV ČR(CZ) IAA400040901; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : enzyme-linked electrochemical assay * SNP typing * p53 mutation Subject RIV: AQ - Safety, Health Protection, Human - Machine Impact factor: 2.630, year: 2009

  10. Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells.

    Science.gov (United States)

    Moguilevsky, N; Varsalona, F; Guillaume, J P; Noyer, M; Gillard, M; Daliers, J; Henichart, J P; Bollen, A

    1995-01-01

    A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

  11. Evaluation of tall rice mutant

    International Nuclear Information System (INIS)

    Hakim, L.; Azam, M.A.; Miah, A.J.; Mansur, M.A.; Akanda, H.R.

    1989-01-01

    One tall mutant (Mut NS1) of rice variety Nizersail was put to multilocation on-farm trial. It showed improvement over the parent in respect of by earlier maturity and higher grain yield at all locations and thus it appears as an improved mutant of Nizersail. (author). 6 refs

  12. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    Energy Technology Data Exchange (ETDEWEB)

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun (Cystic); (UAB); (JHU); (Columbia); (Lilly)

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  13. Human immunodeficiency virus type 1 KK26-27 matrix mutants display impaired infectivity, circularization and integration but not nuclear import

    International Nuclear Information System (INIS)

    Mannioui, Abdelkrim; Nelson, Elisabeth; Schiffer, Cecile

    2005-01-01

    We analyzed the role of human immunodeficiency virus (HIV)-1 matrix protein (MA) during the virus replication afferent phase. Single-round infection of H9 T lymphocytes showed that the combined mutation of MA Lys residues 26-27 in MA reported nuclear localization signal (NLS)-1 [Haffar, O.K., Popov, S., Dubrovsky, L., Agostini, I., Tang, H., Pushkarsky, T., Nadler, S.G., Bukrinsky, M., 2000. Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex. J. Mol. Biol. 299 (2): 359-368] impaired infectivity, abrogated 2-LTR-circle formation and significantly reduced integration. However, the mutation did not affect viral DNA docking to chromatin in either interphasic or mitotic cells, indicating that MA N-terminal basic domain should not represent a major determinant of HIV-1 nuclear import in T lymphocytes. These data point to a previously unreported role of MA in the late, post-chromatin-binding, afferent phase of HIV-1 replication cycle

  14. Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells

    Science.gov (United States)

    Yanovsky-Dagan, Shira; Avitzour, Michal; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Mitrani-Rosenbaum, Stella; Levy-Lahad, Ephrat; Birnbaum, Ramon Y.; Gepstein, Lior; Epsztejn-Litman, Silvina; Eiges, Rachel

    2015-01-01

    Summary CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation. PMID:26190529

  15. Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Shira Yanovsky-Dagan

    2015-08-01

    Full Text Available CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1, frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC lines, we characterized a differentially methylated region (DMR near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation.

  16. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

    DEFF Research Database (Denmark)

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph

    2012-01-01

    chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution...... of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation...... support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation...

  17. Probiotic features of Lactobacillus plantarum mutant strains.

    Science.gov (United States)

    Bove, Pasquale; Gallone, Anna; Russo, Pasquale; Capozzi, Vittorio; Albenzio, Marzia; Spano, Giuseppe; Fiocco, Daniela

    2012-10-01

    In this study, the probiotic potential of Lactobacillus plantarum wild-type and derivative mutant strains was investigated. Bacterial survival was evaluated in an in vitro system, simulating the transit along the human oro-gastro-intestinal tract. Interaction with human gut epithelial cells was studied by assessing bacterial adhesive ability to Caco-2 cells and induction of genes involved in innate immunity. L. plantarum strains were resistant to the combined stress at the various steps of the simulated gastrointestinal tract. Major decreases in the viability of L. plantarum cells were observed mainly under drastic acidic conditions (pH ≤ 2.0) of the gastric compartment. Abiotic stresses associated to small intestine poorly affected bacterial viability. All the bacterial strains significantly adhered to Caco-2 cells, with the ΔctsR mutant strain exhibiting the highest adhesion. Induction of immune-related genes resulted higher upon incubation with heat-inactivated bacteria rather than with live ones. For specific genes, a differential transcriptional pattern was observed upon stimulation with different L. plantarum strains, evidencing a possible role of the knocked out bacterial genes in the modulation of host cell response. In particular, cells from Δhsp18.55 and ΔftsH mutants strongly triggered immune defence genes. Our study highlights the relevance of microbial genetic background in host-probiotic interaction and might contribute to identify candidate bacterial genes and molecules involved in probiosis.

  18. High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

    Science.gov (United States)

    Cancio, Reynel; Silvestri, Romano; Ragno, Rino; Artico, Marino; De Martino, Gabriella; La Regina, Giuseppe; Crespan, Emmanuele; Zanoli, Samantha; Hübscher, Ulrich; Spadari, Silvio; Maga, Giovanni

    2005-01-01

    Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation. PMID:16251294

  19. The Swedish mutant barley collection

    International Nuclear Information System (INIS)

    1989-01-01

    Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

  20. The Swedish mutant barley collection

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1989-07-01

    Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

  1. Early superoxide dismutase alterations during SV40-transformation of human fibroblasts.

    Science.gov (United States)

    Bravard, A; Hoffschir, F; Sabatier, L; Ricoul, M; Pinton, A; Cassingena, R; Estrade, S; Luccioni, C; Dutrillaux, B

    1992-11-11

    The expression of superoxide dismutases (SOD) 1 and 2 was studied in 4 clones of human fibroblasts after their infection by simian virus 40 (SV40), in parallel with the alterations of chromosomes 21 and chromosome 6q arms, carrying the genes that encode for SOD1 and SOD2 respectively. For all clones, a similar scheme with 2 main phases was observed for both chromosome and SOD variations. The first phase, defined as the pre-crisis phase, was characterized by chromosomal instability, but maintenance of normal numbers of chromosome 6q arms and chromosomes 21. The level of SOD2 mRNA was high, while SOD2 activity and immunoreactive protein were low. SOD1 protein and activity were decreased. In the second phase, defined as the post-crisis phase, the accumulation of clonal chromosomal rearrangements led to the loss of 6q arms, while the number of chromosomes 21 remained normal. SOD2 mRNA level was decreased and SOD2 immunoreactive protein and activity remained low. SOD1 protein and activity increased with passages, reaching values similar to those of control cells at late passages. As in established SV40-transformed human fibroblast cell lines, good correlation was found between SOD2 activity and the relative number of 6q arms. These results allow us to reconstruct the sequence of events leading to the decrease of SOD2, a possible tumor-suppressor gene, during the process of SV40-transformation of human fibroblasts.

  2. We Are All Mutants

    Science.gov (United States)

    Timson, David J.

    2017-01-01

    Mutations can cause genetic diseases and the vast majority of these have no effective treatment. They raise some difficult questions on the boundaries of science and social science. Selective breeding to "improve" the human race (eugenics) is often regarded as a Victorian relic or Nazi fantasy. Yet, three fetuses with Down syndrome are…

  3. Mutants of alfalfa mosaic virus

    International Nuclear Information System (INIS)

    Roosien, J.

    1983-01-01

    In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

  4. Root hair mutants of barley

    International Nuclear Information System (INIS)

    Engvild, K.C.; Rasmussen, K.

    2005-01-01

    Barley mutants without root hairs or with short or reduced root hairs were isolated among M 2 seeds of 'Lux' barley (Hordeum vulgare L.) after acidified sodium azide mutagenesis. Root hair mutants are investigated intensively in Arabidopsis where about 40 genes are known. A few root hair mutants are known in maize, rice, barley and tomato. Many plants without root hairs grow quite well with good plant nutrition, and mutants have been used for investigations of uptake of strongly bound nutrients like phosphorus, iron, zinc and silicon. Seed of 'Lux' barley (Sejet Plant Breeding, Denmark) were soaked overnight, and then treated with 1.5-millimolarsodium azide in 0.1 molar sodium phosphate buffer, pH 3, for 2.5 hours according to the IAEA Manual on Mutation Breeding (2nd Ed.). After rinsing in tap water and air-drying, the M 2 seeds were sown in the field the same day. Spikes, 4-6 per M 1 plant, were harvested. The mutation frequency was similar to that obtained with other barley cultivars from which low-phytate mutants were isolated [5]. Seeds were germinated on black filter paper in tap water for 3 or 4 days before scoring for root hair mutants

  5. Grain product of 34 soya mutant lines

    International Nuclear Information System (INIS)

    Salmeron E, J.; Mastache L, A. A.; Valencia E, F.; Diaz V, G. E.; Cervantes S, T.; De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.; Gatica T, M. A.

    2009-01-01

    This work was development with the objective of obtaining information of the agronomic behavior of 34 soya mutant lines (R 4 M 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 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 25 and L 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)

  6. Mutants induced in winter rye (Secale cereale L.): Short straw-mutant No. 2714 and late-senescence mutant

    Energy Technology Data Exchange (ETDEWEB)

    Muszynski, S; Darlewska, M [Department of Plant Breeding and Seed Science, Warsaw Agricultural University, Warsaw (Poland)

    1990-01-01

    Full text: Mutants were induced by treating dormant seeds with ionizing radiation (fast neutrons) or chemicals (N-nitroso-N-ethyl urea or sodium azide). Among several mutants obtained, of special value is the short-straw mutant No. 2714 and a late senescent mutant. (author)

  7. HSPB1 mutations causing hereditary neuropathy in humans disrupt non-cell autonomous protection of motor neurons.

    Science.gov (United States)

    Heilman, Patrick L; Song, SungWon; Miranda, Carlos J; Meyer, Kathrin; Srivastava, Amit K; Knapp, Amy; Wier, Christopher G; Kaspar, Brian K; Kolb, Stephen J

    2017-11-01

    Heat shock protein beta-1 (HSPB1), is a ubiquitously expressed, multifunctional protein chaperone. Mutations in HSPB1 result in the development of a late-onset, distal hereditary motor neuropathy type II (dHMN) and axonal Charcot-Marie Tooth disease with sensory involvement (CMT2F). The functional consequences of HSPB1 mutations associated with hereditary neuropathy are unknown. HSPB1 also displays neuroprotective properties in many neuronal disease models, including the motor neuron disease amyotrophic lateral sclerosis (ALS). HSPB1 is upregulated in SOD1-ALS animal models during disease progression, predominately in glial cells. Glial cells are known to contribute to motor neuron loss in ALS through a non-cell autonomous mechanism. In this study, we examined the non-cell autonomous role of wild type and mutant HSPB1 in an astrocyte-motor neuron co-culture model system of ALS. Astrocyte-specific overexpression of wild type HSPB1 was sufficient to attenuate SOD1(G93A) astrocyte-mediated toxicity in motor neurons, whereas, overexpression of mutHSPB1 failed to ameliorate motor neuron toxicity. Expression of a phosphomimetic HSPB1 mutant in SOD1(G93A) astrocytes also reduced toxicity to motor neurons, suggesting that phosphorylation may contribute to HSPB1 mediated-neuroprotection. These data provide evidence that astrocytic HSPB1 expression may play a central role in motor neuron health and maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. 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...

  9. Mutant genes in pea breeding

    International Nuclear Information System (INIS)

    Swiecicki, W.K.

    1990-01-01

    Full text: Mutations of genes Dpo (dehiscing pods) and A (anthocyanin synthesis) played a role in pea domestication. A number of other genes were important in cultivar development for 3 types of usage (dry seeds, green vegetable types, fodder), e.g. fn, fna, le, p, v, fas and af. New genes (induced and spontaneous), are important for present ideotypes and are registered by the Pisum Genetics Association (PGA). Comparison of a pea variety ideotype with the variation available in gene banks shows that breeders need 'new' features. In mutation induction experiments, genotype, mutagen and method of treatment (e.g. combined or fractionated doses) are varied for broadening the mutation spectrum and selecting more genes of agronomic value. New genes are genetically analysed. In Poland, some mutant varieties with the gene afila were registered, controlling lodging by a shorter stem and a higher number of internodes. Really non-lodging pea varieties could strongly increase seed yield. But the probability of detecting a major gene for lodging resistance is low. Therefore, mutant genes with smaller influence on plant architecture are sought, to combine their effect by crossing. Promising seem to be the genes rogue, reductus and arthritic as well as a number of mutant genes not yet genetically identified. The gene det for terminal inflorescence - similarly to Vicia faba - changes plant development. Utilisation of assimilates and ripening should be better. Improvement of harvest index should give higher seed yield. A number of genes controlling disease resistance are well known (eg. Fw, Fnw, En, mo and sbm). Important in mass screening of resistance are closely linked gene markers. Pea gene banks collect respective lines, but mutants induced in highly productive cultivars would be better. Inducing gene markers sometimes seems to be easier than transfer by crossing. Mutation induction in pea breeding is probably more important because a high number of monogenic features are

  10. Defective glycinergic synaptic transmission in zebrafish motility mutants

    Directory of Open Access Journals (Sweden)

    Hiromi Hirata

    2010-01-01

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

  11. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    Science.gov (United States)

    Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

    2009-01-01

    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. PMID:20161699

  12. An extra early mutant of pigeonpea

    International Nuclear Information System (INIS)

    Ravikesavan, R.; Kalaimagal, T.; Rathnaswamy, R.

    2001-01-01

    The redgram (Cajanus cajan (L.) Huth) variety 'Prabhat DT' was gamma irradiated with 100, 200, 300 and 400 Gy doses. Several mutants have been identified viz., extra early mutants, monostem mutants, obcordifoliate mutants and bi-stigmatic mutants. The extra early mutant was obtained when treated with 100 Gy dose. The mutant was selfed and forwarded from M 2 to M 4 generation. In the M 4 generation the mutant line was raised along with the parental variety. Normal cultural practices were followed and the biometrical observations were recorded. It was observed that for the characters viz., total number of branches per plant, number of pods per plants, seeds per pod, 100 seed weight and seed yield per plant there was no difference between the mutant and parent variety. Whereas, regarding the days to flowering and maturity the mutants were earlier than the parents. The observation was recorded from two hundred plants each. The mutant gives the same yield in 90 days as that of the parent variety in 107 days, which make it an economic mutant

  13. 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…

  14. Mutants dissecting development and behaviour in drosophila

    International Nuclear Information System (INIS)

    Joshi, Adita; Chandrashekaran, Shanti; Sharma, R.P.

    2005-01-01

    We have traced in this paper the progress in Drosophila genetics research from the 1960s, at the IARI, spearheaded by the visionary insight of M. S. Swaminathan. The work started with the study of indirect effect of radiation and the synergistic interaction of physical and chemical mutagens on chromosomal and genetic changes. This paved the way for the study of single gene mutants in dissecting developmental and behavioural processes. New genes discovered by us have been shown to encode conserved cell signalling molecules controlling developmental and behavioural pathways. With the complete sequencing of the Drosophila genome, in the year 2000, mounting evidence for the homology between Drosophila and human genes controlling genetic disorders became available. This has led to the fly becoming an indispensable tool for studying human diseases as well as a model to test for drugs and pharmaceuticals against human diseases and complex behavioural processes. For example wingless in Drosophila belongs to the conserved Wnt gene family and aberrant WNT signalling is linked to a range of human diseases, most notably cancer. Inhibition as well as activation of WNT signalling form the basis of an effective therapy for some cancers as well as several other clinical conditions. Recent experiments have shown that WNTs might also normally participate in self-renewal, proliferation or differentiation of stem cells and altering WNT signalling might be beneficial to the use of stem cells for therapeutic means. Likewise, the stambhA mutant of Drosophila which was discovered for its temperature-dependent paralytic behaviour is the fly homologue of Phospholipase Cβ. Phospholipase C mediated G protein signalling plays a central role in vital processes controlling epilepsy, vision, taste, and olfaction in animals. Proteins of the G-signalling pathway are of intense research interest since many human diseases involve defects in G-protein signalling pathways. In fact, approximately 50

  15. Dwarf mutant of rice variety Seratus Malam

    International Nuclear Information System (INIS)

    Mugiono, P. S.; Soemanggono, A.M.R.

    1989-01-01

    Full text: Seeds of 'Seratus Malam', a local tall upland variety with long panicles and high yield potential were irradiated with 10-50 krad gamma rays in 1983. From 50,000 M 2 plants, 130 semidwarf mutants and 1 dwarf mutant were selected. The dwarf mutant M-362 was obtained from the 10 krad treatment. The mutant shows about 50% reduction in plant height, but also in number of productive tillers. Thus the yield per plant is also significantly less. However, the mutant gene is not allelic to DGWG and therefore may be useful in cross breeding. (author)

  16. Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.

    Science.gov (United States)

    Lim, K-H; Chang, Y-C; Chiang, Y-H; Lin, H-C; Chang, C-Y; Lin, C-S; Huang, L; Wang, W-T; Gon-Shen Chen, C; Chou, W-C; Kuo, Y-Y

    2016-10-07

    CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

  17. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia

    Directory of Open Access Journals (Sweden)

    Chao Liu

    2015-08-01

    Full Text Available Apolipoprotein C-II (APOC2 is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

  18. PNRI mutant variety: Cordyline 'Afable'

    International Nuclear Information System (INIS)

    Aurigue, Fernando B.

    2012-01-01

    Cordyline 'Afable', registered by the Philippine Nuclear Research Institute as NSIC 2009 Or-83, is an induced mutant developed from Cordyline 'Kiwi' by treating stem cuttings with acute gamma radiation from a Cobalt-60 source. The new mutant is identical to Cordyline 'Kiwi' in growth habit but differs in foliage color, and exhibits field resistance to Phytophthora sp., a fungus that causes leaf blight and rot in Ti plants. Results of this mutation breeding experiment showed that leaf color was altered by gamma irradiation and resistance to fungal diseases was improved. It also demonstrated how mutations that occur in nature may be generated artificially. Propagation of cordyline 'Afable' is true-to-type by vegetative propagation methods, such as separation of suckers and offshoots, shoot tip cutting, and top cutting. Aside from landscaping material, terrarium or dish-garden plant, it is ideal as containerized plant for indoor and outdoor use. The leaves or shoots may be harvested as cut foliage for flower arrangements. (author)

  19. Gamma ray induced mutants in Coleus

    International Nuclear Information System (INIS)

    Vasudevan, K.; Jos, J.S.

    1988-01-01

    The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M 1 V 1 generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m 2 area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing

  20. Gamma ray induced mutants in Coleus

    Energy Technology Data Exchange (ETDEWEB)

    Vasudevan, K; Jos, J S [Central Tuber Crops Research Institute, Trivandrum, Kerala (India)

    1988-07-01

    The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M{sub 1}V{sub 1} generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m{sup 2} area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing.

  1. EMS mutant spectra generated by multi-parameter flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Keysar, Stephen B. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Fox, Michael H., E-mail: michael.fox@colostate.edu [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States)

    2009-12-01

    The CHO A{sub L} cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59{sup -} regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59{sup -} clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

  2. Phenotypic characterization of adenovirus type 12 temperature-sensitive mutants in productive infection and transformation.

    Science.gov (United States)

    Hama, S; Kimura, G

    1980-01-01

    Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxylamine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for "initiation" of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutation in H12ts505 is required to maintain at least some aspects of the transformed state.

  3. 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

    2018-06-01

    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.

  4. Studies on reduced height mutants in rice

    International Nuclear Information System (INIS)

    Narahari, P.; Bhagwat, S.G.

    1984-01-01

    Two cross-bred derivatives of the mutant TR5xTR17 and TR21 continued to show promise and were advanced to wider scale testing. TR5 was found to carry a semi-dwarfing gene different from that in IR8. New semi-dwarf mutants were screened from M 2 through M 4 from two separate radiation experiments. The gibberellin response of seedlings of mutant and tester strains was evaluated and crosses of tester stocks and mutant semi-dwarfs were made for genetic analyses. (author)

  5. Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kabashi, Edor; Agar, Jeffrey N; Taylor, David M; Minotti, Sandra; Durham, Heather D

    2004-06-01

    Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.

  6. Disturbed secretion of mutant adiponectin associated with the metabolic syndrome.

    Science.gov (United States)

    Kishida, Ken; Nagaretani, Hiroyuki; Kondo, Hidehiko; Kobayashi, Hideki; Tanaka, Sachiyo; Maeda, Norikazu; Nagasawa, Azumi; Hibuse, Toshiyuki; Ohashi, Koji; Kumada, Masahiro; Nishizawa, Hitoshi; Okamoto, Yoshihisa; Ouchi, Noriyuki; Maeda, Kazuhisa; Kihara, Shinji; Funahashi, Tohru; Matsuzawa, Yuji

    2003-06-20

    Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.

  7. Genetic fingerprinting of mutant rose cultivars

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, S; Prasad, K V; Singh, K P; Singh, A.P. [Division of Floriculture and Landscaping, Indian Agricultural Research Institute, Pusa, New Delhi (India)], E-mail: kvprasad66@gmail.com

    2008-07-01

    Six rose mutants evolved at the Indian Agricultural Research Institute, New Delhi from four parent cultivars were characterized based on RAPD markers. Contrary to the earlier findings our effort has conclusively proven that the RAPD markers are indeed robust tools to discern the mutants from their parents. Among 40 primers screened, 7 primers produced inconsistent banding pattern. The number of polymorphic bands varied between 4 (OPA 14) and 10 (OPA1) with an average of 6.5 bands per primer. The percentage polymorphism ranged from 62.5 (OPM 9) to 100 percent (OPA 1). Most of the primers produced monomorphic bands between parent and mutant rose cultivars. When primer OPA 2 was used a specific band of 2.5 kb was noticed in mutant cv. Pusa Urmil and cv. Pusa Abhishek but was absent in parent cv. Jantar Mantar. A polymorphic band of 750 bp was noticed in the parent Kiss of Fire and helped in differentiating the parent from its mutant when amplified with OPK 3. Primer OPS 16 produced discriminatory band of 800 bp in mutant cv. Pink Sport of Montezuma while it was absent in its parent cv. Montezuma. Another specific band of 650 bp was present in parent cv. Montezuma and absent in its mutant cv. Pink Sport of Montezuma signifying the uniqueness of the mutant. Primer OPM 5 brought out distinct polymorphism among the parent Jantar Mantar and its three mutants with absence of a specific band of 1.5 kb in the parent. The four parents and 6 mutants were divided into four distinct groups in the Dendogram constructed by UPGMA method. The most genetically similar cultivar among the 10 cultivars analyzed are Montezuma and its pink sport of Montezuma whereas Abhisarika a mutant of cv. Kiss of Fire was distinctly different and formed a separate cluster. (author)

  8. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    Compared to the wild CC-124, these mutants are characterized by a decrease in chlorophyll a & b content and an increase in carotenoids. The lowest decrease in chlorophyll a was 3 to 4 folds, while the highest increase in carotenoids was 2 to 4 folds. The result of bio-test, using the resulting pigment mutant of C. reinhardtii ...

  9. Characterization of mitomycin-C-sensitive mouse lymphoma L5178Y cell mutants

    International Nuclear Information System (INIS)

    Inaba, Hiroko; Shiomi, Naoko; Shiomi, Tadahiro; Sato, Koki; Yoshida, Michihiro.

    1985-01-01

    Twenty-six mutants showing high sensitivity to mytomicin-C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Twenty-five of the mutants were 5 - 10 times more sensitive to MMC than were parental cells, and showed normal sensitivity to U.V. light and x-rays. From a complementation analysis, 5 mutants (MC s ) isolated from independently mutagenized cell populations were classified into two groups. These mutants possessed recessive character for MMC-sensitivity and there were at least two genes involved in the MMC-sensitivity. As for DNA-damaging factors, such as photoadducts of 8-methoxypsoralen (8-MOP) and 3-carbethoxysoralen (3-CPs), MC s mutants showed higher sensitivity to photoadducts of 8-MOP than to (3-CPs). MC s mutants were also highly sensitive to a DNA cross-linking agent, cisplatin. Characterization of the sensitivity of mouse MC s mutants was analogous to that of Fanconi's anemia (FA)-derived cells. Low concentrations (10 ng/ml) of MMC induced chromosome aberration in a high incidence in mouse MC s cells, as well as in FA cells. The frequency of MMC-induced chromosome aberrations was normal in hybrid cells between normal human diploid somatic cells and mouse mutants and between FA cells and mouse wild cells, and hereditary deficiency became normal by hybrization. (Namekawa, K.)

  10. Andrographolide induces degradation of mutant p53 via activation of Hsp70.

    Science.gov (United States)

    Sato, Hirofumi; Hiraki, Masatsugu; Namba, Takushi; Egawa, Noriyuki; Baba, Koichi; Tanaka, Tomokazu; Noshiro, Hirokazu

    2018-05-22

    The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein (Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

  11. Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: Sequence analysis of an enzymatically amplified mutant HRAS allele

    International Nuclear Information System (INIS)

    Stevens, C.W.; Manoharan, T.H.; Fahl, W.E.

    1988-01-01

    Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[α]pyrene (±)anti-7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC → TA codon 12 transversions that have been commonly observed in human tumors

  12. A mutant of a mutant of a mutant of a ...: Irradiation of progressive radiation-induced mutants in a mutation-breeding programme with Chrysanthenum morifolium RAM

    International Nuclear Information System (INIS)

    Broertjes, C.; Koene, P.; Veen, J.W.H. van.

    1980-01-01

    Radiation-induced sports in Chrysanthemum morifolium RAM. have been reported for several years. It has become an everyday practice to produce flower-colour mutants from outstanding cross-breeding products, even before they are distributed for the commercial production of cut flowers. One of the most successful and recent examples is that of cv. Horim, of which hundreds of mutants were produced by successive use of radiation-induced mutants in the mutation-breeding programme. Over about 4 years a variety of flower-colour mutants was obtained, not only largely including the outstanding characteristics of the original cultivar but sometimes even with an appreciable improvement in quality and yield. It is expected that the latter types, the Miros group, will soon completely supersede the spontaneous or raditation-induced Horim sports and mutants and take over the leading position of the Horim group in the production of all-year-round (AYR) cut-flowers. (orig.)

  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. Therapeutic Targeting of Spliceosomal-Mutant Acquired Bone Marrow Failure Disorders

    Science.gov (United States)

    2017-05-01

    spliceosomal mutant cells . This effort has also highlighted a requirement for innate immune signaling in SF3B1-mutant MDS and has implicated a few specific...relative to single-mutant cells (Figure 5A). As innate immune signaling has been implicated in MDS pathogenesis (Basiorka et al., 2016; Fang et al...Sato et al., 2005; Tang et al., 2008; Vink et al., 2013; Xin et al., 2017). Here, we observed that SF3B1K700E/+ human lymphoid leukemia cells (NALM-6

  15. Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae

    International Nuclear Information System (INIS)

    Gazdag, Emerich Mihai; Cirstea, Ion Cristian; Breitling, Reinhard; Lukeš, Julius; Blankenfeldt, Wulf; Alexandrov, Kirill

    2010-01-01

    The structures of two new crystal forms of human Cu/Zn superoxide dismutase produced in the eukaryotic expression host L. tarentolae are reported. The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30 mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5 mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research

  16. Early ripening, productive soybean mutant variety suitable for combine harvesting

    International Nuclear Information System (INIS)

    Krausse, G.W.

    1989-01-01

    Between 1979 and 1981 seeds of the early ripening Swedish variety ''Fiskeby V'' were treated with N-nitroso-N-methylurea, sodium azide or gamma rays. Induced mutants were selected which are characterized by longer stalk, higher insertion point of the lowest pods and improved grain yield as well as sufficiently early maturity. Starting from 60 000 treated seeds, 6 mutant lines were developed, and in 1988 one variety named ''Dorado'' was released. Under optimal agrotechnical conditions in fields of 2-10 ha ''Dorado'' achieved grain yields between 1,5 and 2,3 t/ha during the last four years. It is intended to grow this variety in the middle and southern districts of the German Democratic Republic, primarily for human consumption. A technology for growing this variety was recommended

  17. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

    OpenAIRE

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates f...

  18. Induction of Mutants in Durum Wheat

    International Nuclear Information System (INIS)

    AL-Ubaidi, M.; Ibrahim, I.; AL-Hadithi, A.

    2002-01-01

    This investigation presents a breeding program for induction and development of a new genotype of durum wheat, resistant to lodging with high yield, by irradiation durum wheat hybrids (F2) with gamma rays 100 Gy, during 1990-1997 cultivation seasons. This program involves: induction of variability, selection evaluation of the mutants at three locations: Twaitha (Baghdad) Latifya ( Babylon) and Swari (Kutt). All mutants showed resistance to lodging and there was a significant reduction in plant height. Mutant SIXIZ-22 surpassed other mutants and its origin in lodging resistance and plant height (83.5,82.8 and 89.4 cm) in the three locations at generation M5 and M6, respectively. Also, there were significant differences between mutant and their origin in the number of spikes/M 2 and grain yild during the two successive generation. On the other hand, mutant IZxCO-105 surpassed other mutants in the number of spikes/M 2 (231.8,242.3 and 292) and grain yield (4336,3376 and 5232 kg/ha) in all testing location, respectively . (authors) 14 refs., 4 tabs

  19. Spectrum of induced floral mutants in Petunia

    International Nuclear Information System (INIS)

    Padmaja, V.; Sudhakar, P.

    1987-01-01

    A total of six floral mutants of garden Petunia isolated from the populations raised from the seed treatment with γ-rays, 2, 4-D and sodium azide are described. Five of the mutants viz. stellata, Campyloflora, Rubriflora mixed, Grandiflora and Albiflora mixed originated as segregants in M 2 generation while the chimeral floral phenotype was expressed in M 1 generation itself. Breeding behaviour of these horticulturally interesting altered floral phenotypes were studied in subsequent generations and appropriate conclusions were drawn regarding mode of inheritance of the mutant traits. 15 refs., 4 figures, 1 table. (author)

  20. A Neisseria meningitidis fbpABC mutant is incapable of using nonheme iron for growth.

    Science.gov (United States)

    Khun, H H; Kirby, S D; Lee, B C

    1998-05-01

    The neisserial fbpABC locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an fbpABC mutant in Neisseria meningitidis by insertional inactivation of fbpABC with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of fbpABC in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway.

  1. Voronoi-based spatial analysis reveals selective interneuron changes in the cortex of FALS mice.

    Science.gov (United States)

    Minciacchi, Diego; Kassa, Roman M; Del Tongo, Claudia; Mariotti, Raffaella; Bentivoglio, Marina

    2009-01-01

    The neurodegenerative disease amyotrophic lateral sclerosis affects lower motoneurons and corticospinal cells. Mice expressing human mutant superoxide dismutase (SOD)1 provide widely investigated models of the familial form of disease, but information on cortical changes in these mice is still limited. We here analyzed the spatial organization of interneurons characterized by parvalbumin immunoreactivity in the motor, somatosensory, and visual cortical areas of SOD1(G93A) mice. Cell number and sociological spatial behavior were assessed by digital charts of cell location in cortical samples, cell counts, and generation of two-dimensional Voronoi diagrams. In end-stage SOD1-mutant mice, an increase of parvalbumin-containing cortical interneurons was found in the motor and somatosensory areas (about 35% and 20%, respectively) with respect to wild-type littermates. Changes in cell spatial distribution, as documented by Voronoi-derived coefficients of variation, indicated increased tendency of parvalbumin cells to aggregate into clusters in the same areas of the SOD1-mutant cortex. Counts and coefficients of variation of parvalbumin cells in the visual cortex gave instead similar results in SOD1-mutant and wild-type mice. Analyses of motor and somatosensory areas in presymptomatic SOD1-mutant mice provided findings very similar to those obtained at end-stage, indicating early changes of interneurons in these cortical areas during the pathology. Altogether the data reveal in the SOD1-mutant mouse cortex an altered architectonic pattern of interneurons, which selectively affects areas involved in motor control. The findings, which can be interpreted as pathogenic factors or early disease-related adaptations, point to changes in the cortical regulation and modulation of the motor circuit during motoneuron disease.

  2. Semi-dwarf mutants for rice improvement

    International Nuclear Information System (INIS)

    Othman, Ramli; Osman, Mohammad; Ibrahim, Rusli

    1990-01-01

    Full text: MARDI and the National University of Malaysia embarked on a programme to induce resistance against blast in rice in 1978. MARDI also obtained semi dwarf mutants of cvs 'Mahsuri', 'Muda', 'Pongsu seribu' and 'Jarum Mas', which are under evaluation. The popular local rice variety 'Manik' was subjected to gamma irradiation (15-40 krad) and 101 promising semidwarf mutants have been obtained following selection in M 2 -M 6 . 29 of them show grain yields of 6.0-7.3 t/ha, compared with 5.7t for 'Manik'. Other valuable mutants were found showing long grain, less shattering, earlier maturity, and glutinous endosperm. One mutant, resistant to brown plant hopper yields 6.3t/ha. (author)

  3. X-rays sensitive mammalian cell mutant

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi

    1982-01-01

    A phenomenon that in x-ray-sensitive mammalian-cell mutants, cellular death due to x-ray radiation was not increased by caffeine, but on the contrary, the dead cells were resuscitated by it was discussed. The survival rate of mutant cells increased by caffein in a low concentration. This suggested that caffeine may have induced some mechanism to produce x-ray resistant mutant cells. Postirradiation treatment with caffeine increased considerably the survival rate of the mutant cells, and this suggested the existence of latent caffeine-sensitive potentially lethal damage repair system. This system, after a few hours, is thought to be substituted by caffeine-resistant repair system which is induced by caffeine, and this may be further substituted by x-ray-resistant repair system. The repair system was also induced by adenine. (Ueda, J.)

  4. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    acer

    2014-01-08

    Jan 8, 2014 ... aquatic ecosystems were studied. In the present ... logy and photosynthesis research (Stolbov, 1995;. Pedersen ... Microalgal strain and cultivation conditions ..... evaluated for their ecotoxicological effects using 124y-1 mutant.

  5. Deleterious effects of lymphocytes at the early stage of neurodegeneration in an animal model of amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Nakatsuji Yuji

    2011-02-01

    Full Text Available Abstract Background Non-neuronal cells, such as microglia and lymphocytes, are thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS. Previous studies have demonstrated neuroprotective effects of lymphocytes at the end stage of ALS, partly through induction of alternatively activated microglia (M2 microglia, which are neuroprotective. In this study, we investigated the role of lymphocytes in the early stage of the disease using an animal model of inherited ALS. Methods We established a transgenic mouse line overexpressing the familial ALS-associated G93A-SOD1 mutation (harboring a single amino acid substitution of glycine to alanine at codon 93 with depletion of the Rag2 gene (mSOD1/RAG2-/- mice, an animal model of inherited ALS lacking mature lymphocytes. Body weights, clinical scores and motor performance (hanging wire test of mSOD1/RAG2-/- mice were compared to those of mutant human SOD1 transgenic mice (mSOD1/RAG2+/+ mice. Activation of glial cells in the spinal cords of these mice was determined immunohistochemically, and the expression of mRNA for various inflammatory and anti-inflammatory molecules was evaluated. Results Clinical onset in mSOD1/RAG2-/- mice was significantly delayed, and the number of lectin-positive cells in spinal cord was increased at the early stage of disease when compared to mSOD1/RAG2+/+ mice. Quantitative RT-PCR confirmed that mRNA for Ym1, an M2 microglial-related molecule, was significantly increased in mSOD1/RAG2-/- mouse spinal cords at the early disease stage. Conclusions Compared with mSOD1/RAG2+/+ mice, mSOD1/RAG2-/- mice displayed delayed onset and increased M2 microglial activation at the early stage of disease. Thus, lymphocytes at the early pathological phase of ALS display a deleterious effect via inhibition of M2 microglial activation.

  6. Molecular analysis of waxy mutants in rice

    International Nuclear Information System (INIS)

    Yatou, O.; Amano, E.

    1990-01-01

    Full text: The 'waxy' gene is a structural gene coding a glycosyl transferase which synthesises amylose in the endosperm tissue. 'Non-waxy' rice cultivars have an active gene and their amylose content is 18-25% depending upon gene performance and modifier genes. In 'waxy' rice, no amylose is found because the enzyme is absent. In mutants induced by gamma rays, neutrons, EI or EMS, amylose content ranged from 0 to 20%, i.e. there are intermediate phenotypes as well. Some of them had the same amount of the enzyme as a 'non-waxy' cultivar, even fully 'waxy' mutants showed a certain amount of the enzyme. This suggests that in mutants there may be no structural change in the enzyme gene but the enzyme produced might be less active. By molecular analysis of the mutants' genes it was found that only two mutants induced by thermal neutrons show structural alterations, the changes in other mutants are either too small to be detected by Southern analysis or are outside the structural gene in question. (author)

  7. Commercialization Of Orchid Mutants For Floriculture Industry

    International Nuclear Information System (INIS)

    Sakinah Ariffin; Zaiton Ahmad

    2014-01-01

    Orchids are the main contributors to cut flower industry in Malaysia with an existing good market and a huge business potential. Orchid industry has been established in Malaysia since 1960s but only started to develop and expand since 1980s. Continuous development of new orchid varieties is essential to meet customers' demands. Orchid mutagenesis research using gamma irradiation at Malaysian Nuclear Agency has successfully generated a number of new orchid varieties with commercial potentials. Therefore, Nuclear Malaysia has collaborated with an industrial partner, Hexagon Green Sdn Bhd (HGSB), to carry out commercialization research on these mutants under a Technofund project entitled 'Pre-Commercialization of Mutant Orchids for Cut Flowers Industry' from July 2011 to July 2014. Through this collaboration, Dendrobium orchid mutant plants developed by Nuclear Malaysia were transferred to HGSB's commercial orchid nursery at Bukit Changgang Agrotechnology Park, Banting, Selangor, for mass-propagation. The activities include evaluations on plant growth performance, flower quality, post harvest and market potential of these mutants. Mutants with good field performance have been identified and filed for Plant Variety Protection (PVP) with Department of Agriculture Malaysia. This paper describes outputs from this collaboration and activities undertaken in commercializing these mutants. (author)

  8. Oncogenic Signaling by Leukemia-Associated Mutant Cbl Proteins

    Science.gov (United States)

    Nadeau, Scott; An, Wei; Palermo, Nick; Feng, Dan; Ahmad, Gulzar; Dong, Lin; Borgstahl, Gloria E. O.; Natarajan, Amarnath; Naramura, Mayumi; Band, Vimla; Band, Hamid

    2013-01-01

    Members of the Cbl protein family (Cbl, Cbl-b, and Cbl-c) are E3 ubiquitin ligases that have emerged as critical negative regulators of protein tyrosine kinase (PTK) signaling. This function reflects their ability to directly interact with activated PTKs and to target them as well as their associated signaling components for ubiquitination. Given the critical roles of PTK signaling in driving oncogenesis, recent studies in animal models and genetic analyses in human cancer have firmly established that Cbl proteins function as tumor suppressors. Missense mutations or small in-frame deletions within the regions of Cbl protein that are essential for its E3 activity have been identified in nearly 5% of leukemia patients with myelodysplastic/myeloproliferative disorders. Based on evidence from cell culture studies, in vivo models and clinical data, we discuss the potential signaling mechanisms of mutant Cbl-driven oncogenesis. Mechanistic insights into oncogenic Cbl mutants and associated animal models are likely to enhance our understanding of normal hematopoietic stem cell homeostasis and provide avenues for targeted therapy of mutant Cbl-driven cancers. PMID:23997989

  9. From one body mutant to one cell mutant. A progress of radiation breeding in crops

    International Nuclear Information System (INIS)

    Nagatomi, Shigeki

    1996-01-01

    An effective method was established to obtain non-chimeral mutants with wide spectrum of flower colors, regenerated from floral organs on which mutated sectors were come out on chronic irradiated plants. By this way, six mutant varieties of flower colors have been selected from one pink flower of chrysanthemum, and cultivated for cut-flower production. By the same method, 3 mutant varieties with small and spray type flowers were selected in Eustoma. Mutant varieties such as a rust disease resistant in sugarcane, 6 dwarfs in Cytisus and pure-white mushroom in velvet shank have been selected successively for short period. (J.P.N.)

  10. Gamma-radiation Mutagenesis in Genetically Unstable Barley Mutants. Pt. 2. Comparison of Various Mutants

    International Nuclear Information System (INIS)

    Balchiuniene, L.

    1995-01-01

    Spontaneous and gamma-induced mutability was compared in two groups of genetically unstable barley ear structure mutants - tweaky spike (tw) and branched ear (be). Instability in different loci causes different levels of spontaneous and gamma-induced mutability. A high spontaneous level of chlorophyll mutations is peculiar to be-ust mutants. It is suggested that the high level of induced chlorophyll mutations in allelic tw mutants is a result of better surviving of chlorophyll mutation carriers in the genotypical-physiological environment created by mutant tw alleles. (author). 6 refs., 2 tabs

  11. Rescuing mutant CFTR: a multi-task approach to a better outcome in treating cystic fibrosis.

    Science.gov (United States)

    Amaral, Margarida D; Farinha, Carlos M

    2013-01-01

    Correcting multiple defects of mutant CFTR with small molecule compounds has been the goal of an increasing number of recent Cystic Fibrosis (CF) drug discovery programmes. However, the mechanism of action (MoA) by which these molecules restore mutant CFTR is still poorly understood, in particular of CFTR correctors, i.e., compounds rescuing to the cells surface the most prevalent mutant in CF patients--F508del-CFTR. However, there is increasing evidence that to fully restore the multiple defects associated with F508del-CFTR, different small molecules with distinct corrective properties may be required. Towards this goal, a better insight into MoA of correctors is needed and several constraints should be addressed. The methodological approaches to achieve this include: 1) testing the combined effect of compounds with that of other (non-pharmacological) rescuing strategies (e.g., revertants or low temperature); 2) assessing effects in multiple cellular models (non-epithelial vs epithelial, non-human vs human, immortalized vs primary cultures, polarized vs non polarized, cells vs tissues); 3) assessing compound effects on isolated CFTR domains (e.g., compound binding by surface plasmon resonance, assessing effects on domain folding and aggregation); and finally 4) assessing compounds specificity in rescuing different CFTR mutants and other mutant proteins. These topics are reviewed and discussed here so as to provide a state-of-the art review on how to combine multiple ways of rescuing mutant CFTR to the ultimate benefit of CF patients.

  12. Gamma radiation induced mutant for improved yield components in sunflower

    International Nuclear Information System (INIS)

    Elangovan, M.

    2001-01-01

    Sunflower has become an important oilseed in the Indian vegetable oil pool following its introduction from Russia in 1969. It can be used for all quality products useful to humans. The need for genetic variability and new useful gene sources has necessitated that sunflower breeders and geneticists utilize a wide range of germplasm in their breeding programmes. The induction of mutations in sunflower by physical and chemical mutagens has been practiced quite intensively in the last two decades. The results recorded to date suggest that utilization of mutagenesis could be a great advantage in improving the sunflower crop. An induced mutation programme was undertaken to generate variability in the variety 'Morden' using gamma rays. The certified and genetically pure seeds were irradiated with 50, 100, and 150 Gy gamma rays and used for further studies. Selection in M 2 generations, raised from different treatments, revealed the presence of an erectophylly leaf mutant from 50 Gy treatment. The isolated mutant showed improved yield components like head diameter, 100- seed weight and yield per plant. The mutant was a plant with short petiole length and erect leaves. This type of leaf get sunlight throughout the day. From morning to afternoon, the first half of the leaf gets sunlight, and from afternoon to evening the second half of the leaf gets sunlight. As a result of getting sunlight the whole day, the plant had more photosynthetic products and grew vigorously. Plant height, head diameter and 100-seed weight had direct effect on seed yield, and the number of leaves and stem diameter influenced the seed yield indirectly. In the M 3 generation, the mutant showed an almost two-fold increase over the parent variety for all investigated characters, except that of the yield per plant where there was a three-fold increase. The present investigation has shown that there are remarkable possibilities of increasing the yield components in sunflower by induced mutations

  13. Breeding cultivars of barley and mustard containing biochemical mutants

    Energy Technology Data Exchange (ETDEWEB)

    Oram, R N [Division of Plant industry, CSIRO, Canberra (Australia)

    1990-01-01

    Full text: The inactivation of dominant and co-dominant alleles is becoming increasingly important in changing the composition of seed carbohydrates, protein, oil, fibre and secondary products to suit modern food and feed technologies. In barley, breeding lines adapted to south-eastern Australian conditions have been developed containing a waxy endosperm from the Japanese variety 'Sumire Mochi', the high lysine gene lys from cv. 'Hiproly' of Ethiopia, and the induced high lysine mutant gene lys 3a from 'Risoe 1508'. The improved mutant lines yield 12-34% less than the highest yielding feed barley. The lys and lys 3a alleles suppress the formation of prolamins, the waxy allele inhibits the formation of amylose. It seems difficult to modify the background genotype to fully compensate for the reduction of major storage carbohydrate or protein compounds. However, waxy barleys have uses in some human foods and a premium can be paid to producers. The grain of the provisionally-patented waxy cultivar Wasiro is suitable for pearling. It contains 5% {beta}-glucan (soluble fibre) and therefore should be as effective as oat bran for reducing blood cholesterol. In Indian mustard (Brassica juncea), three cultivars differing in date of maturity, each containing the spontaneous mutant alleles for low erucic acid levels in the seed oil, have been developed to produce a high quality, mildly flavoured cooking/salad oil. The concentration of glucosinolates in the seed meal must be reduced to make it palatable and non-toxic to pigs and poultry. Three B. juncea lines were treated in up to four successive generations with gamma rays or EMS. 60,000 seed samples were analysed in subsequent generations. Two induced mutants with reduced glucosinolate concentrations are now available besides 4 naturally-occurring sources with only little reduced yields. Recombination may give a high-yielding low erucic acid and low glucosinolate variety of B. juncea. (author)

  14. Breeding cultivars of barley and mustard containing biochemical mutants

    International Nuclear Information System (INIS)

    Oram, R.N.

    1990-01-01

    Full text: The inactivation of dominant and co-dominant alleles is becoming increasingly important in changing the composition of seed carbohydrates, protein, oil, fibre and secondary products to suit modern food and feed technologies. In barley, breeding lines adapted to south-eastern Australian conditions have been developed containing a waxy endosperm from the Japanese variety 'Sumire Mochi', the high lysine gene lys from cv. 'Hiproly' of Ethiopia, and the induced high lysine mutant gene lys 3a from 'Risoe 1508'. The improved mutant lines yield 12-34% less than the highest yielding feed barley. The lys and lys 3a alleles suppress the formation of prolamins, the waxy allele inhibits the formation of amylose. It seems difficult to modify the background genotype to fully compensate for the reduction of major storage carbohydrate or protein compounds. However, waxy barleys have uses in some human foods and a premium can be paid to producers. The grain of the provisionally-patented waxy cultivar Wasiro is suitable for pearling. It contains 5% β-glucan (soluble fibre) and therefore should be as effective as oat bran for reducing blood cholesterol. In Indian mustard (Brassica juncea), three cultivars differing in date of maturity, each containing the spontaneous mutant alleles for low erucic acid levels in the seed oil, have been developed to produce a high quality, mildly flavoured cooking/salad oil. The concentration of glucosinolates in the seed meal must be reduced to make it palatable and non-toxic to pigs and poultry. Three B. juncea lines were treated in up to four successive generations with gamma rays or EMS. 60,000 seed samples were analysed in subsequent generations. Two induced mutants with reduced glucosinolate concentrations are now available besides 4 naturally-occurring sources with only little reduced yields. Recombination may give a high-yielding low erucic acid and low glucosinolate variety of B. juncea. (author)

  15. Influence of the protein kinase C activator phorbol myristate acetate on the intracellular activity of antibiotics against hemin- and menadione-auxotrophic small-colony variant mutants of Staphylococcus aureus and their wild-type parental strain in human THP-1 cells.

    Science.gov (United States)

    Garcia, Laetitia G; Lemaire, Sandrine; Kahl, Barbara C; Becker, Karsten; Proctor, Richard A; Tulkens, Paul M; Van Bambeke, Françoise

    2012-12-01

    In a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700-3711, 2012), we evaluated the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistant Staphylococcus aureus strain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, its hemB mutant, and the genetically complemented strain in PMA-activated cells and against the menD strain in both activated and nonactivated cells. This effect was inhibited when cells were incubated with N-acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H(2)O(2). In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition of N-acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H(2)O(2). Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.

  16. 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)

  17. Nutritional evaluation of cereal mutants

    International Nuclear Information System (INIS)

    1977-01-01

    An advisory group of experts, comprising nutritionists, analysts and plant breeders, discussed the desirability of nutritional goals for plant breeding and attempted to specify the deficiencies of various cereal crops in essential nutrients. It considered the plant factors influencing the value for human and animal nutrition and the feasibility of improving these by genetic and plant breeding methods. Methods of assaying nutritional quality were discussed, particularly in relation to the need for rapid, inexpensive methods capable of being used as screening procedures in plant breeding programmes. The proceedings contain 9 scientific papers and a conclusion and recommendations, including a review of the chemical cuzymatic, microbiological and animal assay techniques that are available

  18. Officially released mutant varieties in China

    International Nuclear Information System (INIS)

    Liu, L.; Van Zanten, L.; Shu, Q.Y.; Maluszynski, M.

    2004-01-01

    The use of mutation techniques for crop improvement in China has a long and well-established tradition of more than 50 years. As the result of intensive research in many institutes dealing with application of nuclear technologies more than 620 cultivars of 44 crop species have been released. Numerous mutant varieties have been grown on a large scale bringing significant economic impact, sustaining crop production and greatly contributing to increase of food production also in stress prone areas of the country. However, there is still missing information not only on the number of mutant varieties released in particular crop species but also on mutagens applied, selection approaches and on the use of mutants in cross breeding. Numerous Chinese scientists collected and systematized this information. Results of their work were often published in local scientific journals in the Chinese language and as such were unavailable to breeders from other countries. Having this in mind, we requested Dr. Liu Luxiang, the Director of the Department of Plant Mutation Breeding and Genetics, Institute for Application of Atomic Energy, Chinese Academy of Agricultural Sciences in Beijing to help us in finding as much information as possible on mutant varieties officially released in China. The data has been collected in close collaboration with his colleagues from various institutions all over the country and then evaluated, edited and prepared for publication by our team responsible for the FAO/IAEA Database of Officially Released Mutant Varieties. We would like to thank all Chinese colleagues who contributed to this list of Chinese mutant varieties. We hope that this publication will stimulate plant breeders in China to collect more information on released mutant varieties and especially on the use of mutated genes in cross breeding. (author)

  19. Development of high yielding mutants in lentil

    International Nuclear Information System (INIS)

    Rajput, M.A.; Sarwar, G.; Siddiqui, K.A.

    2001-01-01

    Full text: Lentil (Lens culinaris Medik.) locally known as Masoor, is the second most important rabi pulse crop, after chickpea, in Pakistan. It is cultivated on an area of over 63,400 ha, which constitutes about 4.83% of the total area under pulses. The annual production of the crop is 28,200 tones with an average yield of 445 kg/ha. Yield at the national level is very low, about one-half of the world's yield, which is mainly due to non-availability of high yield potential genotypes. Keeping in view the importance of mutants in developing a large number of new varieties, an induced mutations programme was initiated at AEARC, Tandojam during 1987-88, to develop high yielding varieties in lentil. For this, seeds of two lentil varieties, 'Masoor-85' and 'ICARDA-8' had been irradiated with gamma-rays ranging from 100-600 Gy in NIAB, Faisalabad during 1990. Selections were made in M2 on the basis of earliness, plant height, branches/plant and 100 grain weight. After confirming these mutants in M3 they were promoted in station yield trials and studied continuously for three consecutive years (1993- 1995). Overall results revealed that these mutants have consistent improvement of earliness in flowering and maturity. Plant height also increased in all mutant lines except AEL 23/40/91 where reduction in this attribute was observed as compared to parent variety. Mutant lines AEL 49/20/91 and AEL 13/30/91 showed improvement in 100 grain weight. The improvement of some agronomic characters enhanced the yield of mutant lines in comparison to parent varieties (Masoor-85 and ICARDA-8). The diversity in yield over the respective parents was computed from 6.94 to 60.12%. From these encouraging results it is hoped that mutant lines like AEL 12/30/91 and AEL 49/20/91 may serve as potential lentil genotypes in future. (author)

  20. Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression.

    Science.gov (United States)

    Yue, Xuetian; Zhang, Cen; Zhao, Yuhan; Liu, Juan; Lin, Alan W; Tan, Victor M; Drake, Justin M; Liu, Lianxin; Boateng, Michael N; Li, Jun; Feng, Zhaohui; Hu, Wenwei

    2017-08-15

    Tumor suppressor p53 is frequently mutated in human cancer. Mutant p53 often promotes tumor progression through gain-of-function (GOF) mechanisms. However, the mechanisms underlying mutant p53 GOF are not well understood. In this study, we found that mutant p53 activates small GTPase Rac1 as a critical mechanism for mutant p53 GOF to promote tumor progression. Mechanistically, mutant p53 interacts with Rac1 and inhibits its interaction with SUMO-specific protease 1 (SENP1), which in turn inhibits SENP1-mediated de-SUMOylation of Rac1 to activate Rac1. Targeting Rac1 signaling by RNAi, expression of the dominant-negative Rac1 (Rac1 DN), or the specific Rac1 inhibitor NSC23766 greatly inhibits mutant p53 GOF in promoting tumor growth and metastasis. Furthermore, mutant p53 expression is associated with enhanced Rac1 activity in clinical tumor samples. These results uncover a new mechanism for Rac1 activation in tumors and, most importantly, reveal that activation of Rac1 is an unidentified and critical mechanism for mutant p53 GOF in tumorigenesis, which could be targeted for therapy in tumors containing mutant p53. © 2017 Yue et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase.

    Science.gov (United States)

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D; Steinbach, Peter J; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G; Gärtner, Jutta

    2012-08-01

    Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone. © 2012 Wiley Periodicals, Inc.

  2. NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

    Science.gov (United States)

    Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

    2018-05-02

    K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

  3. The research progress on plant mutant germplasm resources in China

    International Nuclear Information System (INIS)

    He Cexi; Ji Linzhen; Zhao Shirong

    1991-07-01

    Mutants induced by nuclear radiation or other mutagens are new artificial germplasm resources. Some mutants have been applied in plant breeding and great achievements have been reached. The status and progress on the collection, identification and utilization of mutants in China are introduced. A proposal for developing mutant germplasm resources with good agronomic characters is suggested

  4. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-01-01

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs

  5. A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid.

    Directory of Open Access Journals (Sweden)

    Man Hei Cheng

    Full Text Available Cataract is the most common cause of visual loss in humans. A spontaneously occurred, autosomal dominant mouse mutant Secc, which displayed combined features of small eye, cataract and closed eyelid was discovered in our laboratory. In this study, we identified the mutation and characterized the cataract phenotype of this novel Secc mutant. The Secc mutant mice have eyelids that remain half-closed throughout their life. The mutant lens has a significant reduction in size and with opaque spots clustered in the centre. Histological analysis showed that in the core region of the mutant lens, the fiber cells were disorganized and clefts and vacuoles were observed. The cataract phenotype was evident from new born stage. We identified the Secc mutation by linkage analysis using whole genome microsatellite markers and SNP markers. The Secc locus was mapped at chromosome 1 flanked by SNPs rs3158129 and rs13475900. Based on the chromosomal position, the candidate cataract locus γ-crystallin gene cluster (Cryg was investigated by sequencing. A single base deletion (299delG in exon 3 of Cryga which led to a frame-shift of amino acid sequence from position 91 was identified. As a result of this mutation, the sequences of the 3rd and 4th Greek-key motifs of the γA-crystallin are replaced with an unrelated C-terminal peptide of 75 residues long. Coincidentally, the point mutation generated a HindIII restriction site, allowing the identification of the CrygaSecc mutant allele by RFLP. Western blot analysis of 3-week old lenses showed that the expression of γ-crystallins was reduced in the CrygaSecc mutant. Furthermore, in cell transfection assays using CrygaSecc mutant cDNA expression constructs in 293T, COS-7 and human lens epithelial B3 cell lines, the mutant γA-crystallins were enriched in the insoluble fractions and appeared as insoluble aggregates in the transfected cells. In conclusion, we have demonstrated that the Secc mutation leads to the

  6. Mutant IDH1 Promotes Glioma Formation In Vivo

    Directory of Open Access Journals (Sweden)

    Beatrice Philip

    2018-05-01

    Full Text Available Summary: Isocitrate dehydrogenase 1 (IDH1 is the most commonly mutated gene in grade II–III glioma and secondary glioblastoma (GBM. A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease. : Philip et al. show that mutant IDH1 cooperates with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote gliomagenesis in vivo in a mouse model of glioma. These tumors resemble proneural human mutant IDH1 glioblastoma and exhibit enhanced sensitivity to PARP inhibition in combination with chemotherapy. Keywords: IDH1, Cdkn2a, Atrx, Pten, glioma, mouse model, RCAS/TVA

  7. Bacterio-opsin mutants of Halobacterium halobium

    Science.gov (United States)

    Betlach, Mary; Pfeifer, Felicitas; Friedman, James; Boyer, Herbert W.

    1983-01-01

    The bacterio-opsin (bop) gene of Halobacterium halobium R1 has been cloned with about 40 kilobases of flanking genomic sequence. The 40-kilobase segment is derived from the (G+C)-rich fraction of the chromosome and is not homologous to the major (pHH1) or minor endogenous covalently closed circular DNA species of H. halobium. A 5.1-kilobase Pst I fragment containing the bop gene was subcloned in pBR322 and a partial restriction map was determined. Defined restriction fragments of this clone were used as probes to analyze the defects associated with the bop gene in 12 bacterio-opsin mutants. Eleven out of 12 of the mutants examined had inserts ranging from 350 to 3,000 base pairs either in the bop gene or up to 1,400 base pairs upstream. The positions of the inserts were localized to four regions in the 5.1-kilobase genomic fragment: within the gene (one mutant), in a region that overlaps the 5′ end of the gene (seven mutants), and in two different upstream regions (three mutants). Two revertants of the mutant with the most distal insert had an additional insert in the same region. The polar effects of these inserts are discussed in terms of inactivation of a regulatory gene or disruption of part of a coordinately expressed operon. Given the defined nature of the bop mRNA—i.e., it has a 5′ leader sequence of three ribonucleotides—these observations indicate that the bop mRNA might be processed from a large mRNA transcript. Images PMID:16593291

  8. Chlorophyll mutants in Phaseolus vulgaris (L.) Savi

    International Nuclear Information System (INIS)

    Svetleva, D.; Petkova, S.

    1991-01-01

    Three-year investigations were conducted on chlorophyll mutants of three type: viridissima, claroviridis, flavoviridis, viridocostata and xanthomarginata produced post gamma irradiation ( 60 Co, 8 krad, 280 rad/min). Cell division rate in spectrum and in quantity of induced aberrations was found to have no significant differences with the control. Chlorophyll mutations compared to the control are less developed and their productive characters are less manifested. Cell division rate and the quantity of induced aberrations have no relation to the elements of productivity in the mutants investigated. 3 tabs., 12 refs

  9. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    International Nuclear Information System (INIS)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

    2009-01-01

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  10. Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate.

    Science.gov (United States)

    Bao, Yanqing; Tian, Mingxing; Li, Peng; Liu, Jiameng; Ding, Chan; Yu, Shengqing

    2017-04-04

    Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 10 5 colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.

  11. Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA.

    Science.gov (United States)

    Zhao, Carolyn Y; Grinkevich, Vera V; Nikulenkov, Fedor; Bao, Wenjie; Selivanova, Galina

    2010-05-01

    Expression of mutant p53 correlates with poor prognosis in many tumors, therefore strategies aimed at reactivation of mutant p53 are likely to provide important benefits for treatment of tumors that are resistant to chemotherapy and radiotherapy. We have previously identified and characterized a small molecule RITA which binds p53 and induces a conformational change which prevents the binding of p53 to several inhibitors, including its own destructor MDM2. In this way, RITA rescues the tumor suppression function of wild type p53. Here, we demonstrate that RITA suppressed the growth and induced apoptosis in human tumor cell lines of a diverse origin carrying mutant p53 proteins. RITA restored transcriptional transactivation and transrepression function of several hot spot p53 mutants. The ability of RITA to rescue the activity of different p53 mutants suggests its generic mechanism of action. Thus, RITA is a promising lead for the development of anti-cancer drugs that reactivate the tumor suppressor function of p53 in cancer cells irrespective whether they express mutant or wild type p53.

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

    Science.gov (United States)

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

    2015-07-01

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

  13. Purification, gene cloning, gene expression, and mutants of Dps from the obligate anaerobe Porphyromonas gingivalis.

    Science.gov (United States)

    Ueshima, Junichi; Shoji, Mikio; Ratnayake, Dinath B; Abe, Kihachiro; Yoshida, Shinichi; Yamamoto, Kenji; Nakayama, Koji

    2003-03-01

    The periodontopathogen Porphyromonas gingivalis is an obligate anaerobe that is devoid of catalase but exhibits a relatively high degree of resistance to peroxide stress. In the present study, we demonstrate that P. gingivalis contains a Dps homologue that plays an important role in the protection of cells from peroxide stress. The Dps protein isolated from P. gingivalis displayed a ferritin-like spherical polymer consisting of 19-kDa subunits. Molecular cloning and sequencing of the gene encoding this protein revealed that it had a high similarity in nucleotide and amino acid sequences to Dps proteins from other species. The expression of Dps was significantly increased by exposure of P. gingivalis to atmospheric oxygen in an OxyR-dependent manner, indicating that it is regulated by the reactive oxygen species-regulating gene oxyR. The Dps-deficient mutants, including the dps single mutant and the ftn dps double mutant, showed no viability loss upon exposure to atmospheric oxygen for 6 h. In contrast to the wild type, however, these mutants exhibited the high susceptibility to hydrogen peroxide, thereby disrupting the viability. On the other hand, no significant difference in sensitivity to mitomycin C and metronidazole was observed between the wild type and the mutants. Furthermore, the dps single mutant, compared with the wild type, showed a lower viability in infected human umbilical vein endothelial cells.

  14. Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents

    International Nuclear Information System (INIS)

    Hoy, C.A.; Thompson, L.H.; Mooney, C.L.; Salazar, E.P.

    1985-01-01

    DNA repair-deficient mutants from five genetic complementation groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6- to 3-fold hypersensitivity compared to the wild-type cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity. Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15- to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measurements showed that this compound produced few DNA interstrand cross-links but numerous strand breaks. Therefore, the extreme hypersensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross-links. Mutant UV5 was only 1- to 4-fold hypersensitive to all the compounds. Although the initial number of cross-links was similar for the three cell lines, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes

  15. Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans.

    Science.gov (United States)

    Katoh, Toshihiko; Katayama, Takane; Tomabechi, Yusuke; Nishikawa, Yoshihide; Kumada, Jyunichi; Matsuzaki, Yuji; Yamamoto, Kenji

    2016-10-28

    Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. 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.

  17. Molecular Genetic Identification Of Some Flax Mutants

    International Nuclear Information System (INIS)

    AMER, I.M.; MOUSTAFA, H.A.M.

    2009-01-01

    Five flax genotypes (Linum usitatissimum L.) i.e., commercial cultivar Sakha 2, the mother variety Giza 4 and three mutant types induced by gamma rays, were screened for their salinity tolerance in field experiments (salinity concentration was 8600 and 8300 ppm for soil and irrigation water, respectively). Mutation 6 was the most salt tolerant as compared to the other four genotypes.RAPD technique was used to detect some molecular markers associated with salt tolerance in flax (Mut 6), RAPD-PCR results using 12 random primers exhibited 149 amplified fragments; 91.9% of them were polymorphic and twelve molecular markers (8.1%) for salt tolerant (mutant 6) were identified with molecular size ranged from 191 to 4159 bp and only eight primers successes to amplify these specific markers. Concerning the other mutants, Mut 15 and Mut 25 exhibited 4.3% and 16.2% specific markers, respectively. The induced mutants exhibited genetic similarity to the parent variety were about 51%, 58.3% and 61.1% for Mut 25, Mut 6 and Mut 15, respectively. These specific markers (SM) are used for identification of the induced mutations and it is important for new variety registration.

  18. Induced mutants for cereal grain protein improvement

    International Nuclear Information System (INIS)

    1982-01-01

    Out of 17 papers and one summary presented, six dealing with the genetic improvement of seed protein using ionizing radiations fall within the INIS subject scope. Other topics discussed were non-radiation induced mutants used for cereal grain protein improvement

  19. Male sterile mutant in Vigna radiata

    International Nuclear Information System (INIS)

    Pande, Kalpana; Raghuvanshi, S.S.

    1987-01-01

    Single and combined treatment of γ-rays and 0.25 per cent EMS were tried on Vigna radiata variety K851. A male sterile mutant was isolated in M 2 generation. Experiments indicated male sterility to be recessive and monogenic in nature. 6 figures. (author)

  20. Phanerochaete mutants with enhanced ligninolytic activity

    International Nuclear Information System (INIS)

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

    1994-01-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organo pollutants in soils and aqueous media. Most of the organic compounds are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, bio pulping, bio bleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated, or are hyper producers or super secretors of key enzymes under enriched conditions. Through UV-light and γ-ray mutagenesis, we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants, 76UV, produced 272 U of lignin peroxidases enzyme activity/L after 9 d under high nitrogen (although the parent strain does not produce this enzyme under these conditions). The mutant and the parent strains produced up to 54 and 62 U/L, respectively, of the enzyme activity under low nitrogen growth conditions during this period. In some experiments, the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 d

  1. The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter*

    Science.gov (United States)

    Geisler, Christoph; Kotu, Varshika; Sharrow, Mary; Rendić, Dubravko; Pöltl, Gerald; Tiemeyer, Michael; Wilson, Iain B. H.; Jarvis, Donald L.

    2012-01-01

    Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac1 flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac1 mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac1 Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac1 mutant. These results validate the Drosophila nac1 mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency. PMID:22745127

  2. Distinct Rayleigh scattering from hot spot mutant p53 proteins reveals cancer cells.

    Science.gov (United States)

    Jun, Ho Joon; Nguyen, Anh H; Kim, Yeul Hong; Park, Kyong Hwa; Kim, Doyoun; Kim, Kyeong Kyu; Sim, Sang Jun

    2014-07-23

    The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  4. Increased somatic cell mutant frequency in atomic bomb survivors

    International Nuclear Information System (INIS)

    Hakoda, Masayuki; Akiyama, Mitoshi; Kyoizumi, Seishi; Awa, A.A.; Yamakido, Michio; Otake, Masanori.

    1988-05-01

    Frequencies of mutant T-cells in peripheral blood, which are deficient in the activity of hypoxanthine guanine phosphoribosyltransferase (HPRT) were determined for atomic bomb survivors by direct clonal assay using a previously reported method. Results from 30 exposed survivors (exposed to more than 1 rad) and 17 age- and sex-matched controls (exposed to less than 1 rad) were analyzed. The mean mutant frequency (Mf) in the exposed (5.2 x 10 -6 ; range 0.8 - 14.4 x 10 -6 ) was significantly higher than in controls (3.4 x 10 -6 ; range 1.3 - 9.3 x 10 -6 ), a fact not attributable to lower nonmutant cell cloning efficiencies in the exposed group since cell cloning efficiencies were virtually identical in both groups. An initial analysis of the data did not reveal a significant correlation between individual Mfs and individual radiation dose estimates when the latter were defined by the original, tentative estimates (T65D), even though there was a significant positive correlation of Mfs with individual frequency of lymphocytes bearing chromosome aberration. However, reanalysis using the newer revised individual dose estimates (DS86) for 27 exposed survivors and 17 controls did reveal a significant but shallow positive correlation between T-cell Mf values and individual exposure doses. These results indicate that HPRT mutation in vivo in human T-cells could be detected in these survivors 40 years after the presumed mutational event. (author)

  5. 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.

  6. PNRI mutant variety: sansevieria 'Sword of Ibe'

    International Nuclear Information System (INIS)

    Aurigue, Fernando B.

    2011-01-01

    Sansevieria 'Sword of Ibe,' registered by the Philippine Nuclear Research Institute as NSIC 2008 Or-66, is a chlorophyll mutant of Sansevieria trifasciata 'Moonshine' developed by treating its suckers or shoots arising from a rhizome with acute gamma radiation from a Cobalt-60 source. The new mutant is identical in growth habit and vigor to Sansevieria 'Moonshine,' also known as Moonglow. Results of this mutation breeding experiment showed that leaf color and flowering were altered by gamma irradiation without changing the other characteristics of the plant. Propagation is true-to-type by separation of sucker and top cutting. The plant is recommended for use as landscaping material and as pot plant for indoor and outdoor use. The leaves may be harvested as cut foliage for Japanese flower arrangements. (author)

  7. Serine:glyoxylate aminotransferase mutant of barley

    International Nuclear Information System (INIS)

    Blackwell, R.; Murray, A.; Joy, K.; Lea, P.

    1987-01-01

    A photorespiratory mutant of barley (LaPr 85/84), deficient in both of the major peaks of serine:glyoxylate aminotransferase activity detected in the wild type, also lacks serine:pyruvate and asparagine:glyoxylate aminotransferase activities. Genetic analysis of the mutation demonstrated that these three activities are all carried on the same enzyme. The mutant, when placed in air, accumulated a large pool of serine, showed the expected rate (50%) of ammonia release during photorespiration but produced CO 2 at twice the wild type rate when it was fed [ 14 C] glyoxylate. Compared with the wild type, LaPr 85/84 exhibited abnormal transient changes in chlorophyll a fluorescence when the CO 2 concentration of the air was altered, indicating that the rates of the fluorescence quenching mechanisms were affected in vivo by the lack of this enzyme

  8. Intact interval timing in circadian CLOCK mutants.

    Science.gov (United States)

    Cordes, Sara; Gallistel, C R

    2008-08-28

    While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval-timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/- and -/- mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing.

  9. Probing the mechanism of insulin fibril formation with insulin mutants.

    Science.gov (United States)

    Nielsen, L; Frokjaer, S; Brange, J; Uversky, V N; Fink, A L

    2001-07-27

    The molecular basis of insulin fibril formation was investigated by studying the structural properties and kinetics of fibril formation of 20 different human insulin mutants at both low pH (conditions favoring monomer/dimer) and at pH 7.4 (conditions favoring tetramer/hexamer). Small-angle X-ray scattering showed insulin to be monomeric in 20% acetic acid, 0.1 M NaCl, pH 2. The secondary structure of the mutants was assessed using far-UV circular dichroism, and the tertiary structure was determined using near-UV circular dichroism, quenching of intrinsic fluorescence by acrylamide and interactions with the hydrophobic probe 1-anilino-8-naphthalene-sulfonic acid (ANS). The kinetics of fibril formation were monitored with the fluorescent dye, Thioflavin T. The results indicate that the monomer is the state from which fibrils arise, thus under some conditions dissociation of hexamers may be rate limiting or partially rate limiting. The insulin mutants were found to retain substantial nativelike secondary and tertiary structure under all conditions studied. The results suggest that fibril formation of the insulin mutants is controlled by specific molecular interactions that are sensitive to variations in the primary structure. The observed effects of several mutations on the rate of fibril formation are inconsistent with a previously suggested model for fibrillation [Brange, J., Whittingham, J., Edwards, D., Youshang, Z., Wollmer, A., Brandenburg, D., Dodson, G., and Finch, J. (1997) Curr. Sci. 72, 470-476]. Two surfaces on the insulin monomer are identified as potential interacting sites in insulin fibrils, one consisting of the residues B10, B16, and B17 and the other consisting of at least the residues A8 and B25. The marked increase in the lag time for fibril formation with mutations to more polar residues, as well as mutations to charged residues, demonstrates the importance of both hydrophobic and electrostatic interactions in the initial stages of fibrillation

  10. The application of shortened upper leaf mutant in barley breeding

    International Nuclear Information System (INIS)

    Jin Hua

    2004-01-01

    The shortened upper leaf mutant was induced from Fuji Nigo by γ-ray irradiation. Fuji Nigo, the mutant, cross-cut F 1 , F 2 and back-cross F 1 , F 2 were used to analyze mutant heredity by comparative study. The yield, chlorophyll content, light intensity, dry matter of mutant were investigated. The results showed that (1) the mutant character was controlled by a couple of nuclear genes which were partial dominance; (2) the transmittance of the mutant colony was better than that of Fuji Nigo and bottom dry matter was much more than that of Fuji Nigo; (3) under the condition of high fertilizer and high plant population , the yield of mutant was higher than that of Fuji Nigo; (4) the content of chlorophyll a in the mutant was higher than that in Fuji Nigo

  11. ''Fushi'' - excellent mutant germplasm for peanut improvement

    International Nuclear Information System (INIS)

    Jiang, X.; Zhou, Y.

    1989-01-01

    Full text: The mutant line ''Fushi'' was selected following seed treatment of the variety ''Shi Xuan 64'' in 1960 with 32 P. Many good peanut varieties were developed using ''Fushi'' in cross-breeding (ref. Mutation Breeding Newsletter No. 30 (July 1987) p. 2-3). In the past 10 years, planting areas of these varieties added up to 3,3 million ha in South China, peanut production was increased by more than 500 000 t valued 500 million Yuan. (author)

  12. Radiation induced early maturing mutants in barley

    International Nuclear Information System (INIS)

    Kumar, R.; Chauhan, S.V.S.; Sharma, R.P.

    1978-01-01

    In M 2 generation, two early maturing plants were screened from a single spike progeny of a plant obtained from 20 kR of gamma-ray irradiation of a six-rowed barley (Hordeum vulgare L. var. Jyoti). Their true breeding nature was confirmed in M 3 generation. These mutants flower and mature 38 and 22 days earlier than those of control. (auth.)

  13. Multivariate analysis for selecting apple mutants

    International Nuclear Information System (INIS)

    Faedi, W.; Bagnara, G.L.; Rosati, P.; Cecchini, M.

    1992-01-01

    The mutlivariate analysis of four year records on several vegetative and productive traits of twenty-one apple mutants (3 of 'Jonathan', 3 of 'Ozark Gold', 14 of 'Mollie's Delicious', 1 of 'Neipling's Early Stayman)' induced by gamma radiations showed that observation of some traits of one-year-old shoots is the most efficient way to reveal compact growing apple mutants. In particular, basal cross-section area, total length and leaf area resulted the most appropriate parameters, while internode length together with conopy height and width are less appropriate. The most interesting mutants we found are: one of 'Mollie's Delicious for the best balance among tree and fruit traits and for high skin color; one of 'Neipling's Early Stayman' with an earlier and more extensively red colored apple than the original clone. (author)

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

    Indian Academy of Sciences (India)

    Srabani Taraphder

    We present in this article a detailed analysis of representative structures and proton transfer activity of .... cal molecular dynamics simulations to identify potential unfolding ... clustering parameters to carry out K-means cluster- ing of different ...

  15. Estrogen Receptor Mutants/Variants in Human Breast Cancer.

    Science.gov (United States)

    1997-12-01

    a recipient of a USAMRMC Postdoctoral Fellowship Award. The authors thank Caroline Cumins -Leygue and Helen Bergen for labora- tory assistance with...of their ER status as determined by ligand binding assay. It should be stressed that an additional band can be observed in most of the samples. This

  16. Radiation studies in Cajanus cajan: meiotic behaviour in some M/sub 2/ mutants

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, S.S.N.; Akhaury, S.B. (Ranchi Univ. (India). Dept. of Botany)

    1982-01-01

    A qualitative study of the mutants produced in M/sub 2/ generation has been made. The mutants were classified as: (1) chlorophyll mutant, (2) morphological mutant, (3) pollen mutant, (4) semi-sterile and (5) sterile mutant. Cytological investigations of pollen mutants, sterile and semi-sterile mutants have revealed that these mutants generally arise at higher dose levels (20 Kr and 25 Kr).

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

    Indian Academy of Sciences (India)

    2012-08-07

    Aug 7, 2012 ... and mutants induced with X-ray, UV or chemical mutagens. ... We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature ..... mutants in yeast by selection for constitutive behavior in pig-.

  18. Biological changes in Barley mutants resistant to powdery mildew disease

    International Nuclear Information System (INIS)

    Amer, I. M.; Fahim, M. M.; Moustafa, N. A.

    2012-12-01

    physiological studies showed that all kinds of chlorophyll (a), (b) and (a + b) content in infected plant were decreased while, the carotenes pigment were increased. Infection generally reduced total sugars content of all resistant mutants. Infected resistant mutant showed more phenols content and peroxidase, polyphenoloxidase activities than healthy ones of the mutants. (Author)

  19. Transgenic mice display hair loss and regrowth overexpressing mutant Hr gene.

    Science.gov (United States)

    Zhu, Kuicheng; Xu, Cunshuan; Zhang, Jintao; Chen, Yingying; Liu, Mengduan

    2017-10-30

    Mutations in the hairless (Hr) gene in both mice and humans have been implicated in the development of congenital atrichia, but the role of Hr in skin and hair follicle (HF) biology remains unknown. Here, we established transgenic mice (TG) overexpressing mutant Hr to investigate its specific role in the development of HF. Three transgenic lines were successfully constructed, and two of them (TG3 and TG8) displayed a pattern of hair loss and regrowth with alternation in the expression of HR protein. The mutant Hr gene inhibited the expression of the endogenous gene in transgenic individuals, which led to the development of alopecia. Interestingly, the hair regrew with the increase in the endogenous expression levels resulting from decreased mutant Hr expression. The findings of our study indicate that the changes in the expression of Hr result in hair loss or regrowth.

  20. Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

    Directory of Open Access Journals (Sweden)

    David J Speca

    2010-08-01

    Full Text Available The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt, disrupts the homolog of the Caenorhabditis elegans (C. elegans unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

  1. Gamma-radiation Mutagenesis in Genetically Unstable Barley Mutants. Pt. 1. Chlorophyll Mutations in Allelic tw Mutants and Their Revertants

    International Nuclear Information System (INIS)

    Vaitkuniene, V.

    1995-01-01

    Genotypical environment is an essential factor determining the mutability of mutants of the same type. Decreased chlorophyll mutant frequency was a common characteristic of all tested tw type (tw, tw 1 , tw 2 ) mutants induced in barley c. 'Auksiniai II'. The mutability of all the tested revertants was close to that of the initial c. 'Auksiniai II'. (author). 9 refs., 2 tabs

  2. High yielding mutants of blackgram variety 'PH-25'

    International Nuclear Information System (INIS)

    Misra, R.C.; Mohapatra, B.D.; Panda, B.S.

    2001-01-01

    Seeds of blackgram (Vigna mungo L.) variety 'PH-5' were treated with chemical mutagens ethyl methanesulfonate (EMS), nitrosoguanidine (NG), maleic hydrazide (MH) and sodium azide (NaN 3 ), each at 3 different concentrations. Thirty six mutant lines developed from mutagenic treatments along with parent varieties were tested in M 4 generation. The mutants showed wide variation in most of the traits and multivariante D 2 analysis showed genetic divergence among themselves. Twenty of the thirty mutants showed genetic divergence from parent. Ten selected high yielding mutants were tested in M 5 . Yield and other productive traits of five high yielding mutants in M 4 and M 5 are presented

  3. Canine degenerative myelopathy: a model of human amyotrophic lateral sclerosis.

    Science.gov (United States)

    Nardone, Raffaele; Höller, Yvonne; Taylor, Alexandra C; Lochner, Piergiorgio; Tezzon, Frediano; Golaszewski, Stefan; Brigo, Francesco; Trinka, Eugen

    2016-02-01

    Canine degenerative myelopathy (CDM) represents a unique naturally occurring animal model for human amyotrophic lateral sclerosis (ALS) because of similar clinical signs, neuropathologic findings, and involvement of the superoxide dismutase 1 (SOD1) mutation. A definitive diagnosis can only be made postmortem through microscopic detection of axonal degeneration, demyelination and astroglial proliferation, which is more severe in the dorsal columns of the thoracic spinal cord and in the dorsal portion of the lateral funiculus. Interestingly, the muscle acetylcholine receptor complexes are intact in CDM prior to functional impairment, thus suggesting that muscle atrophy in CDM does not result from physical denervation. Moreover, since sensory involvement seems to play an important role in CDM progression, a more careful investigation of the sensory pathology in ALS is also warranted. The importance of SOD1 expression remains unclear, while oxidative stress and denatured ubiquinated proteins appear to play a crucial role in the pathogenesis of CDM. In this updated narrative review we performed a systematic search of the published studies on CDM that may shed light on the pathophysiological mechanisms of human ALS. A better understanding of the factors that determine the disease progression in CDM may be beneficial for the development of effective treatments for ALS. Copyright © 2015 Elsevier GmbH. All rights reserved.

  4. Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Phuoc T Tran

    Full Text Available KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with Kras(G12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

  5. Mutant p53 interactions with supercoiled DNA

    Czech Academy of Sciences Publication Activity Database

    Brázdová, Marie; Němcová, Kateřina; Činčárová, Lenka; Šebest, Peter; Pivoňková, Hana; Brázda, Václav; Fojta, Miroslav; Paleček, Emil

    2007-01-01

    Roč. 24, č. 6 (2007), s. 639-640 ISSN 0739-1102. [Alban 2007: The 15th Conversation . 19.06.2007-23.06.2007, Albany] R&D Projects: GA MŠk(CZ) 1K04119; GA ČR(CZ) GP204/06/P369; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : mutant p53 * supercoiled DNA * cancer Subject RIV: BO - Biophysics

  6. Radiation induced desynaptic mutants in barley

    International Nuclear Information System (INIS)

    Srivastava, H.M.

    1974-01-01

    Spontaneous occurrence of asynapsis and desynapsis has been frequently reported in a number of crop plants (Beadle 1930, 1933; Beasley and Brown 1942; Li et al. 1945; Magoon et al. 1961; Miller 1963) and other angiospermic texa (Calarier 1955; Chennaveraiah and Krisnappa 1968; Ehrenberg 1949; Johnson 1941, 1944; Roy and Jha 1958). However, there are only a few reports of induced asynapsis or desynapsis (Gottschalk and Baquar 1971; Martini and Bozzini 1966). The present paper deals with the morphology and meiotic behavior of gamma-ray induced barley mutants showing high degree of desynapsis resulting in partial to complete sterility. (author)

  7. Molecular mechanism of action of pharmacoperone rescue of misrouted GPCR mutants: the GnRH receptor.

    Science.gov (United States)

    Janovick, Jo Ann; Patny, Akshay; Mosley, Ralph; Goulet, Mark T; Altman, Michael D; Rush, Thomas S; Cornea, Anda; Conn, P Michael

    2009-02-01

    The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D(98) and K(121). This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E(90)-K(121)) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D(98) mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.

  8. Overexpression of mutant ataxin-3 in mouse cerebellum induces ataxia and cerebellar neuropathology.

    Science.gov (United States)

    Nóbrega, Clévio; Nascimento-Ferreira, Isabel; Onofre, Isabel; Albuquerque, David; Conceição, Mariana; Déglon, Nicole; de Almeida, Luís Pereira

    2013-08-01

    Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a fatal, dominant neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Clinical manifestations include cerebellar ataxia and pyramidal signs culminating in severe neuronal degeneration. Currently, there is no therapy able to modify disease progression. In the present study, we aimed at investigating one of the most severely affected brain regions in the disorder--the cerebellum--and the behavioral defects associated with the neuropathology in this region. For this purpose, we injected lentiviral vectors encoding full-length human mutant ataxin-3 in the mouse cerebellum of 3-week-old C57/BL6 mice. We show that circumscribed expression of human mutant ataxin-3 in the cerebellum mediates within a short time frame--6 weeks, the development of a behavioral phenotype including reduced motor coordination, wide-based ataxic gait, and hyperactivity. Furthermore, the expression of mutant ataxin-3 resulted in the accumulation of intranuclear inclusions, neuropathological abnormalities, and neuronal death. These data show that lentiviral-based expression of mutant ataxin-3 in the mouse cerebellum induces localized neuropathology, which is sufficient to generate a behavioral ataxic phenotype. Moreover, this approach provides a physiologically relevant, cost-effective and time-effective animal model to gain further insights into the pathogenesis of MJD and for the evaluation of experimental therapeutics of MJD.

  9. Loss of anti-Bax function in Gerstmann-Sträussler-Scheinker syndrome-associated prion protein mutants.

    Directory of Open Access Journals (Sweden)

    Julie Jodoin

    2009-08-01

    Full Text Available Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD-associated prion protein (PrP mutants that are unable to generate cytosolic PrP (CyPrP. To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS-associated PrP mutants. These PrP mutants contained their respective methionine ((M or valine ((V at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V, D202N(V, P102L(V and Q217R(V retained, whereas the P102L(M, P105L(V, Y145stop(M and Q212P(M PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L(V, none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

  10. Induction of drought tolerant mutants of rice

    International Nuclear Information System (INIS)

    El-Hissewy, A.A.; Abd Allah, A.

    2001-01-01

    The ultimate goal of crop breeding is to develop varieties with a high yield potential and desirable agronomic characteristics. In Egypt, the most important qualities sought by breeders have been high yield potential, resistance to major diseases and insects, and improved grain and eating quality. However, breeding efforts should concentrate on varieties with the potential to minimize yield losses under unfavorable conditions such as drought, and to maximize yields when conditions are favorable. Rice (Oryza sativa L.) in Egypt is completely irrigated and a significant portion of the rice cultivated area is subject to water deficit resulting from an inadequate or insufficient irrigation supply. Drought tolerance is a complex trait in that it results from the interaction of histological and physiological characters of plant with environmental factors, both above-ground and under-ground. Accordingly, root characters are closely related to drought tolerance. Little attention has been paid in Egyptian breeding programs to root characters and their relation to shoot characters. Furthermore, induced mutations are considered as one of the most important methods to induce useful mutants, especially with improved root characters, to overcome the drought problem. The present investigation aimed to study the effect of different doses of gamma rays on several characters of three Egyptian rice varieties, i.e. 'Giza 171', 'Giza 175' and 'Giza 176' and to induce one or more mutants possessing drought tolerance

  11. 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.

  12. Flower morphology of Dendrobium Sonia mutants

    International Nuclear Information System (INIS)

    Sakinah Ariffin; Azhar Mohamad; Affrida Abu Hassan; Zaiton Ahmad; Mohd Nazir Basiran

    2010-01-01

    Dendrobium Sonia is a commercial hybrid which is popular as cut flower and potted plant in Malaysia. Variability in flower is important for new variety to generate more demands and choices in selection. Mutation induction is a tool in creating variability for new flower color and shape. In vitro cultures of protocorm-like bodies (PLBs) were exposed to gamma ray at dose 35 Gy. Phenotypic characteristics of the flower were observed at fully bloomed flower with emphasis on shape and color. Approximately 2000 regenerated irradiated plants were observed and after subsequent flowering, 100 plants were finally selected for further evaluation. Most of the color and shape changes are expressed in different combinations of petal, sepal and lip of the flower. In this work, 11 stable mutants were found different at flower phenotype as compared to control. Amongst these, four mutant varieties with commercial potential has been named as Dendrobium 'SoniaKeenaOval', Dendrobium 'SoniaKeenaRadiant', Dendrobium 'SoniaKeenaHiengDing' and Dendrobium 'Sonia KeenaAhmadSobri'. In this paper, variations in flower morphology and flower color were discussed, giving emphasis on variations in flower petal shape. (author)

  13. High yielding rice mutants for West Bengal

    International Nuclear Information System (INIS)

    Debnath, A.R.; Sen, S.

    1980-01-01

    Four high yielding mutants with specific genetic corrections of the simply inherited characters were developed from IR-8 through X-irradiation. Recurrent selections of the promising isolates were made under diverse agro-climatic conditions in Winter and Summer seasons of West Bengal. The isolates CNM 6 and CNM 25 belonging to early maturity group and CNM 20 and CNM 31, to mid-early maturity group were finally selected at X 5 generation on the basis of their resistance qualities, maturity period and grain yield. They were evaluated upto X 10 qeneration at multi-locations as Pre-release and Minikit Varieties at State level. They were also placed at the National Screening Nursery (NSN) for screening against multiple diseases and pests at the National level. CNM 6 is reported to be promising in IRTP nurseries. It is reported that CNM 25 (IET 5646) ranked 2nd on the basis of average grain yield, CNM 20 (IET 5937) and CNM 31 (IET 5936) were resistant to diseases and with yield comparable to Jaya. These four productive mutants of superior types are widely accepted. CNM 6 is recommended for cultivation in Bankura and Birbhum districts and CNM 25 and CNM 31 in the different agro-climatic zones of West Bengal. (author)

  14. Elevated expression of ribosomal protein genes L37, RPP-1, and S2 in the presence of mutant p53.

    Science.gov (United States)

    Loging, W T; Reisman, D

    1999-11-01

    The wild-type p53 protein is a DNA-binding transcription factor that activates genes such as p21, MDM2, GADD45, and Bax that are required for the regulation of cell cycle progression or apoptosis in response to DNA damage. Mutant forms of p53, which are transforming oncogenes and are expressed at high levels in tumor cells, generally have a reduced binding affinity for the consensus DNA sequence. Interestingly, some p53 mutants that are no longer effective at binding to the consensus DNA sequence and transactivating promoters containing this target site have acquired the ability to transform cells in culture, in part through their ability to transactivate promoters of a number of genes that are not targets of the wild-type protein. Certain p53 mutants are therefore considered to be gain-of-function mutants and appear to be promoting proliferation or transforming cells through their ability to alter the expression of novel sets of genes. Our goal is to identify genes that have altered expression in the presence of a specific mutant p53 (Arg to Trp mutation at codon 248) protein. Through examining differential gene expression in cells devoid of p53 expression and in cells that express high levels of mutant p53 protein, we have identified three ribosomal protein genes that have elevated expression in response to mutant p53. Consistent with these findings, the overexpression of a number of ribosomal protein genes in human tumors and evidence for their contribution to oncogenic transformation have been reported previously, although the mechanism leading to this overexpression has remained elusive. We show results that indicate that expression of these specific ribosomal protein genes is increased in the presence of the R248W p53 mutant, which provides a mechanism for their overexpression in human tumors.

  15. 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. (Novartis)

    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.

  16. Serrated leaf mutant in mungbean (Vigna radiata (L) Wilczek)

    International Nuclear Information System (INIS)

    Malik, I.A.; Ghulam, Sarwar; Yousaf, Ali; Saleem, M.

    1988-01-01

    Dry dormant seeds of mungbean (Vigna radiata (L) Wilczek) were treated with gamma rays (15, 30 and 60 kR). The serrated leaf mutation was noticed in M 2 of cultivar Pak 32 treated with 60 kR. Cf 14 plants, 3 showed the altered leaf structure and the others were normal. The feature of this mutant was the deep serration of leaflet margins. The mutant had large thick leaflets with prominent venation. The mutant bred true in the M 3 and successive generation. Details of the morphological characteristics of the mutant are presented. The mutant exhibited slower growth particularly during the early stages of development, flowered later and attained shorter height. There was an increase in the number of pods, in seed weight and in seed protein content, but number of seed per pod was considerably reduced. The seed coat colour showed a change from green to yellowish green. In the mutant's flowers the stamina were placed much below the stigma level and the stigma sometimes protruded the corolla. Outcrossing of 4% recorded in some of the mutant lines revealed a reduced cleistogamy. The low number of seeds per pod in the mutant could be due to reduced pollen fertility. The mutant behaved as monogenic recessive. The symbols SL/sl are proposed for this allelic pair. The mutant may have use as a green manure crop because of its large foliage and for the breeders as a genetic marker

  17. Characterization of a Weak Allele of Zebrafish cloche Mutant

    Science.gov (United States)

    Ma, Ning; Huang, Zhibin; Chen, Xiaohui; He, Fei; Wang, Kun; Liu, Wei; Zhao, Linfeng; Xu, Xiangmin; Liao, Wangjun; Ruan, Hua; Luo, Shenqiu; Zhang, Wenqing

    2011-01-01

    Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche 172 (clo 172) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo 172 mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo 172 mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo s5 mutant. In contrast, primitive myeloid cells were totally lost in clo 172 mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo 172 mutant, confirmed by the dramatic decrease of lyc in clo 172 runx1w84x double mutant. Collectively, the clo 172 mutant is a weak allele compared to the clo s5 mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene. PMID:22132109

  18. Enhanced hippocampal long-term potentiation and fear memory in Btbd9 mutant mice.

    Directory of Open Access Journals (Sweden)

    Mark P DeAndrade

    Full Text Available Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS, a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS.

  19. Enhanced hippocampal long-term potentiation and fear memory in Btbd9 mutant mice.

    Science.gov (United States)

    DeAndrade, Mark P; Zhang, Li; Doroodchi, Atbin; Yokoi, Fumiaki; Cheetham, Chad C; Chen, Huan-Xin; Roper, Steven N; Sweatt, J David; Li, Yuqing

    2012-01-01

    Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS), a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS.

  20. Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine.

    Science.gov (United States)

    Ghanavatian, Parisa; Khalifeh, Khosrow; Jafarian, Vahab

    2016-12-01

    Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala 19 can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. Screening of drugs inhibiting in vitro oligomerization of Cu/Zn-superoxide dismutase with a mutation causing amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Itsuki Anzai

    2016-08-01

    Full Text Available Dominant mutations in Cu/Zn-superoxide dismutase (SOD1 gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS. A major pathological hallmark of this disease is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. Recently, we have reproduced the formation of SOD1 oligomers abnormally cross-linked via disulfide bonds in a test tube. Using our in vitro model of SOD1 oligomerization, therefore, we screened 640 FDA-approved drugs for inhibiting the oligomerization of SOD1 proteins, and three effective classes of chemical compounds were identified. Those hit compounds will provide valuable information on the chemical structures for developing a novel drug candidate suppressing the abnormal oligomerization of mutant SOD1 and possibly curing the disease.

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

    International Nuclear Information System (INIS)

    Upchurch, R.G.; Walker, D.C.; Rollins, J.A.; Ehrenshaft, M.; Daub, M.E.

    1991-01-01

    The authors have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus

  3. Temperature sensitive riboflavin mutants of Penicillium vermiculatum Dangeard

    International Nuclear Information System (INIS)

    Mitra, J.; Chaudhari, K.L.

    1974-01-01

    Two temperature sensitive UV induced riboflavin mutants rib 1 and rib 6 have been physiologically and genetically characterized. The two mutants behave differently with regard to their temperature sensitivity. The rib 1 mutant exhibits a leaky growth in minimal medium between 15 0 C and 30 0 C but grows well when the medium is supplemented with riboflavin. At 35 0 C the growth response of the mutant is at its max. and at 40 0 C and below 15 0 C it ceases to grow. The rib 6 mutant which is red brown in colour shows wild type character at temp. below 25 0 C in minimal medium but requires riboflavin at 30 0 C and above. Heterokaryotic analysis revealed the nonallelic nature of the two temperature mutants. Genetic tests of allelic relationship between riboflavin markers by crossing were also done. (author)

  4. Some mutants in maize obtained by irradiation with thermal neutrons

    International Nuclear Information System (INIS)

    Diaconu, P.

    1993-01-01

    Irradiation was carried out at the Bucharest Institute of Atomic Physics and the National Laboratory Brookhaven, USA. A description is given of 22 genic mutants affecting leaf color, plant size, and branching capacity. Characteristics related to pollen fertility and the vegetative period were affected in all the mutants. Improvement of pollen fertility was attempted over four generations without success. The maize mutants obtained by irradiation may be considered as being without practical significance. (author). 7 figs., 1 tab. 11 ref