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Sample records for pc mutant caused

  1. Polycystic Kidney Disease in the Medaka (Oryzias latipes) pc Mutant Caused by a Mutation in the Gli-Similar3 (glis3) Gene

    PubMed Central

    Hashimoto, Hisashi; Miyamoto, Rieko; Watanabe, Naoki; Shiba, Dai; Ozato, Kenjiro; Inoue, Chikako; Kubo, Yuko; Koga, Akihiko; Jindo, Tomoko; Narita, Takanori; Naruse, Kiyoshi; Ohishi, Kazuko; Nogata, Keiko; Shin-I, Tadasu; Asakawa, Shuichi; Shimizu, Nobuyoshi; Miyamoto, Tomotsune; Mochizuki, Toshio; Yokoyama, Takahiko; Hori, Hiroshi; Takeda, Hiroyuki; Kohara, Yuji; Wakamatsu, Yuko

    2009-01-01

    Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients. PMID:19609364

  2. Mutants of PC12 cells with altered cyclic AMP responses

    SciTech Connect

    Block, T.; Kon, C.; Breckenridge, B.M.

    1984-10-01

    PCl2 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ..mu..m 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(BETA,..gamma..-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, N/sub s/. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PCl2 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PCl2 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

  3. Neferine attenuates the protein level and toxicity of mutant huntingtin in PC-12 cells via induction of autophagy.

    PubMed

    Wong, Vincent Kam Wai; Wu, An Guo; Wang, Jing Rong; Liu, Liang; Law, Betty Yuen-Kwan

    2015-02-18

    Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington's disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74) in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7)-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

  4. PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor

    PubMed Central

    1986-01-01

    Four mutant PC12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12nnr) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC12nnr cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC12nnr cells closely resembled non-NGF-treated PC12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC12nnr lines differed from PC12 cells in three ways in addition to failure of NGF response: PC12nnr cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC12 cells. The PC12nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC12 cells possess both high- and low-affinity NGF binding sites. The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC12nnr cells compared to PC12 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC12nnr cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity. PMID:3005338

  5. Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

    PubMed Central

    Thompson, L M; Raffioni, S; Wasmuth, J J; Bradshaw, R A

    1997-01-01

    Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to

  6. Pyrin gene and mutants thereof, which cause familial Mediterranean fever

    DOEpatents

    Kastner, Daniel L [Bethesda, MD; Aksentijevichh, Ivona [Bethesda, MD; Centola, Michael [Tacoma Park, MD; Deng, Zuoming [Gaithersburg, MD; Sood, Ramen [Rockville, MD; Collins, Francis S [Rockville, MD; Blake, Trevor [Laytonsville, MD; Liu, P Paul [Ellicott City, MD; Fischel-Ghodsian, Nathan [Los Angeles, CA; Gumucio, Deborah L [Ann Arbor, MI; Richards, Robert I [North Adelaide, AU; Ricke, Darrell O [San Diego, CA; Doggett, Norman A [Santa Cruz, NM; Pras, Mordechai [Tel-Hashomer, IL

    2003-09-30

    The invention provides the nucleic acid sequence encoding the protein associated with familial Mediterranean fever (FMF). The cDNA sequence is designated as MEFV. The invention is also directed towards fragments of the DNA sequence, as well as the corresponding sequence for the RNA transcript and fragments thereof. Another aspect of the invention provides the amino acid sequence for a protein (pyrin) associated with FMF. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. The invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain at the C-terminal of the protein. These mutants include M6801, M694V, K695R, and V726A. Additionally, the invention includes methods for diagnosing a patient at risk for having FMF and kits therefor.

  7. Process optimisation for the biosynthesis of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3 using submerged fermentation.

    PubMed

    Abdullah, Roheena; Zafar, Wajeeha; Nadeem, Muhammad; Iqtedar, Mehwish; Naz, Shagufta; Syed, Quratulain; Kaleem, Afshan

    2015-01-01

    This study deals with optimisation of cultural conditions for enhanced production of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3. Influence of different variables including incubation time, temperature, inoculum size, pH, nitrogen sources and metal ions has been studied. The optimum conditions for cellulase production were incubation period of 72 h, inoculum size 4% incubation temperature 37°C, pH 7, 0.25% ammonium sulphate, 0.2% peptone as inorganic and organic nitrogen source in case of Bacillus PC-BC6. In case of mutant Bacillus N3, optimal conditions were incubation period of 48 h, incubation temperature 37°C, inoculum size 3%, pH 7, 0.2% ammonium chloride and 0.15% yeast extract. Presence of MnSO4 and CaCl2 enhances the enzyme production by Bacillus PC-BC6 and mutant Bacillus N3, respectively. This study was innovative and successful in producing cellulase economically by using cheap indigenous substrate Saccharum spontaneum.

  8. A clinical and molecular review of ubiquitous glucose-6-phosphatase deficiency caused by G6PC3 mutations.

    PubMed

    Banka, Siddharth; Newman, William G

    2013-06-13

    The G6PC3 gene encodes the ubiquitously expressed glucose-6-phosphatase enzyme (G-6-Pase β or G-6-Pase 3 or G6PC3). Bi-allelic G6PC3 mutations cause a multi-system autosomal recessive disorder of G6PC3 deficiency (also called severe congenital neutropenia type 4, MIM 612541). To date, at least 57 patients with G6PC3 deficiency have been described in the literature.G6PC3 deficiency is characterized by severe congenital neutropenia, recurrent bacterial infections, intermittent thrombocytopenia in many patients, a prominent superficial venous pattern and a high incidence of congenital cardiac defects and uro-genital anomalies. The phenotypic spectrum of the condition is wide and includes rare manifestations such as maturation arrest of the myeloid lineage, a normocellular bone marrow, myelokathexis, lymphopaenia, thymic hypoplasia, inflammatory bowel disease, primary pulmonary hypertension, endocrine abnormalities, growth retardation, minor facial dysmorphism, skeletal and integument anomalies amongst others. Dursun syndrome is part of this extended spectrum. G6PC3 deficiency can also result in isolated non-syndromic severe neutropenia. G6PC3 mutations in result in reduced enzyme activity, endoplasmic reticulum stress response, increased rates of apoptosis of affected cells and dysfunction of neutrophil activity.In this review we demonstrate that loss of function in missense G6PC3 mutations likely results from decreased enzyme stability. The condition can be diagnosed by sequencing the G6PC3 gene. A number of G6PC3 founder mutations are known in various populations and a possible genotype-phenotype relationship also exists. G6PC3 deficiency should be considered as part of the differential diagnoses in any patient with unexplained congenital neutropenia.Treatment with G-CSF leads to improvement in neutrophil numbers, prevents infections and improves quality of life. Mildly affected patients can be managed with prophylactic antibiotics. Untreated G6PC3 deficiency can

  9. Wild-type uromodulin prevents NFkB activation in kidney cells, while mutant uromodulin, causing FJHU nephropathy, does not.

    PubMed

    Dinour, Dganit; Ganon, Liat; Nomy, Levin-Iaina; Ron, Rotem; Holtzman, Eliezer J

    2014-06-01

    Uromodulin (Tamm-Horsfall protein) is the most abundant urinary protein in healthy individuals. Despite 60 years of research, its physiological role remains rather elusive. Familial juvenile hyperuricemic nephropathy and medullary cystic kidney disease Type 2 are autosomal dominant tubulointerstitial nephropathies characterized by gouty arthritis and progressive renal insufficiency, caused by uromodulin (UMOD) mutations. The aim of this study was to compare the cellular effects of mutant and wild-type UMOD. Wild-type UMOD cDNA was cloned from human kidney cDNA into pcDNA3 expression vector. A mutant UMOD construct, containing the previously reported mutation, V273, was created by in vitro mutagenesis. Transient and stable transfection studies were performed in human embryonic kidney cells and mouse distal convoluted tubular cells, respectively. Expression was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescence. Oligosaccharide cleavage by glycosidases was performed to characterize different forms of UMOD. Nuclear translocation of P65 and degradation of IκBα and IRAK1 in response to interleukin (IL)-1β were used to evaluate the effects of wild-type and mutant UMOD on the IL-1R-NFκB pathway. The mutant protein was shown to be retained in the endoplasmic reticulum and was not excreted to the cell medium, as opposed to the wild-type protein. NFκB activation in cells expressing mutant UMOD was similar to that of untransfected cells. In contrast, cells over-expressing wild-type UMOD showed markedly reduced NFκB activation. Our findings suggest that UMOD may have a physiologic function related to its inhibitory effect on the NFκB pathway.

  10. Induction of cellular prion protein (PrPc) under hypoxia inhibits apoptosis caused by TRAIL treatment

    PubMed Central

    Lee, Ju-Hee; Moon, Ji-Hong; Kim, Sung-Wook; Lee, You-Jin; Park, Sang-Youel

    2015-01-01

    Hypoxia decreases cytotoxic responses to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. Cellular prion protein (PrPc) is regulated by HIF-1α in neurons. We hypothesized that PrPc is involved in hypoxia-mediated resistance to TRAIL-induced apoptosis. We found that hypoxia induced PrPc protein and inhibited TRAIL-induced apoptosis. Thus silencing of PrPc increased TRAIL-induced apoptosis under hypoxia. Overexpression of PrPc protein using an adenoviral vector inhibited TRAIL-induced apoptosis. In xenograft model in vivo, shPrPc transfected cells were more sensitive to TRAIL-induced apoptosis than in shMock transfected cells. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia. PMID:25742790

  11. Onjisaponin B Derived from Radix Polygalae Enhances Autophagy and Accelerates the Degradation of Mutant α-Synuclein and Huntingtin in PC-12 Cells

    PubMed Central

    Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

    2013-01-01

    Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae—i.e., polygalacic acid, senegenin and onjisaponin B—onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level. PMID:24248062

  12. Impaired mitochondrial function due to familial Alzheimer's disease-causing presenilins mutants via Ca(2+) disruptions.

    PubMed

    Toglia, Patrick; Cheung, King-Ho; Mak, Don-On Daniel; Ullah, Ghanim

    2016-05-01

    Mutants in presenilins (PS1 or PS2) is the major cause of familial Alzheimer's disease (FAD). FAD causing PS mutants affect intracellular Ca(2+) homeostasis by enhancing the gating of inositol trisphosphate (IP3) receptor (IP3R) Ca(2+) release channel on the endoplasmic reticulum, leading to exaggerated Ca(2+) release into the cytoplasm. Using experimental IP3R-mediated Ca(2+) release data, in conjunction with a computational model of cell bioenergetics, we explore how the differences in mitochondrial Ca(2+) uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca(2+). We find that as a result of exaggerated cytosolic Ca(2+) in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca(2+) dependent enzymes that stimulate NADH production. This leads to decreased rates in proton pumping due to diminished membrane potential along with less ATP and enhanced ROS production. These results show that through Ca(2+) signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially to cell death. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    SciTech Connect

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

    2009-08-15

    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.

  14. Differential interaction between iron and mutant alpha-synuclein causes distinctive Parkinsonian phenotypes in Drosophila.

    PubMed

    Zhu, Zhou-Jing; Wu, Ka-Chun; Yung, Wing-Ho; Qian, Zhong-Ming; Ke, Ya

    2016-04-01

    Alpha-synuclein aggregation is the central hallmark of both sporadic and familial Parkinson's disease (PD). Patients with different PD-causing genetic defects of alpha-synuclein usually show distinctive clinical features that are atypical to sporadic PD. Iron accumulation is invariably found in PD. Recent studies showed that mutant and wild-type alpha-synuclein may have differential interaction with iron and mutant alpha-synuclein toxicity could be preferentially exacerbated by iron. We hence hypothesized that iron overload could selectively influence mutant alpha-synuclein toxicity and disease phenotypes. To test the hypothesis, we investigated if Drosophila melanogaster over-expressing A53T, A30P, and wild-type (WT) alpha-synuclein have different responses to iron treatment. We showed that iron treatment induced similar reduction of survival rate in all flies but induced a more severe motor decline in A53T and A30P mutant alpha-synuclein expressing flies, suggesting interaction between mutant alpha-synuclein and iron. Although no significant difference in total head iron content was found among these flies, we demonstrated that iron treatment induced selective DA neuron loss in motor-related PPM3 cluster only in the flies that express A53T and A30P mutant alpha-synuclein. We provided the first in vivo evidence that iron overload could induce distinctive neuropathology and disease phenotypes in mutant but not WT alpha-synuclein expressing flies, providing insights to the cause of clinical features selectively exhibited by mutant alpha-synuclein carriers.

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

    PubMed

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

    2015-06-01

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

  16. Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants

    PubMed Central

    Harper, Marc A.; Chen, Zugen; Toy, Traci; Machado, Iara M. P.; Nelson, Stanley F.; Liao, James C.; Lee, Christopher J.

    2011-01-01

    Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50–100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a “Phenotype Sequencing” approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110–$340. PMID:21364744

  17. Phenotype sequencing: identifying the genes that cause a phenotype directly from pooled sequencing of independent mutants.

    PubMed

    Harper, Marc A; Chen, Zugen; Toy, Traci; Machado, Iara M P; Nelson, Stanley F; Liao, James C; Lee, Christopher J

    2011-02-18

    Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50-100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a "Phenotype Sequencing" approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110-$340.

  18. A Meiotic Uv-Sensitive Mutant That Causes Deletion of Duplications in Neurospora

    PubMed Central

    Newmeyer, Dorothy; Galeazzi, Donna R.

    1978-01-01

    The meiotic-3 (mei-3) mutant of Neurospora crassa has several effects: (1) When homozygous, it almost completely blocks meiosis and ascospore formation, (2) it is sensitive to UV, (3) its growth is inhibited by histidine and, (4) it increases the instability of nontandem duplications. This was shown for duplications produced by five different rearrangements and was demonstrated by two different criteria. The effects on meiosis and duplication instability are expressed strongly at 25°; the effects on sensitivity to UV and to histidine are expressed strongly at 38.5° but only slightly at 25°. Nevertheless, all four effects were shown to be due to a single gene. mei-3 is not allelic with previously reported UV-sensitive mutants.—Two other results were obtained that are not necessarily due to mei-3: (1) A cross involving mei-3 produced a new unlinked meiotic mutant, mei-4, which is not sensitive to UV or histidine, and (2) a burst of several new mutants occurred in a different mei-3 stock, including a partial revertant of mei-3.—mei-3 has previously been shown to cause frequent complete loss of a terminal duplicate segment, beginning exactly at the original rearrangement breakpoint. Possible mechanisms are discussed by which a UV-sensitive mutant could cause such precise deletions. PMID:17248837

  19. Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing

    PubMed Central

    Guthrie, Christine

    2016-01-01

    Pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome uses DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appropriate points in the splicing cycle and repressing it at others. Mutations linked to retinitis pigmentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous in vitro studies of homologous mutations in Saccharomyces cerevisiae show that Prp8-RP mutants cause defects in spliceosome activation. Here we show that a subset of RP mutations in Prp8 also causes defects in the transition between the first and second catalytic steps of splicing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the first and second catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splicing efficiency. The location of the Prp8-RP mutants, at the “hinge” that links the Prp8 Jab1–MPN regulatory “tail” to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing catalysis. PMID:26968627

  20. Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing.

    PubMed

    Mayerle, Megan; Guthrie, Christine

    2016-05-01

    Pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome uses DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appropriate points in the splicing cycle and repressing it at others. Mutations linked to retinitis pigmentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous in vitro studies of homologous mutations in Saccharomyces cerevisiaes how that Prp8-RP mutants cause defects in spliceosome activation. Here we show that a subset of RP mutations in Prp8 also causes defects in the transition between the first and second catalytic steps of splicing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the first and second catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splicing efficiency. The location of the Prp8-RP mutants, at the "hinge" that links the Prp8 Jab1-MPN regulatory "tail" to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing catalysis.

  1. Silicon phthalocyanine Pc 4 and red light causes apoptosis in HIV-infected cells.

    PubMed

    Ben-Hur, E; Oetjen, J; Horowitz, B

    1997-03-01

    The silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), is being studied as a photosensitizer for virus inactivation in red blood cell concentrates (RBCC). The RBCC spiked with cell-free human immunodeficiency virus (HIV) or with HIV actively replicating in the T-lymphocytic cell line CEM can be successfully inactivated (> or = 6 log10) when exposed to 2 microM Pc 4 and 90 J/cm2 red light (600-800 nm). Inactivation of > or = 6 log10 inducible HIV in the latently infected promonocytic cell line U1 occurred at 22.5 J/cm2 (H. Margolis-Nunno et al., Transfusion 36, 743-750, 1996). In order to understand the reason for the increased susceptibility of U1 to photosensitized inactivation we looked for induction of apoptosis by photodynamic treatment (PDT). Agarose gel electrophoresis was used to observe the appearance of a characteristic 180-200 base pair DNA ladder, which can indicate apoptosis. Using this assay it is shown that Pc 4 treatment induced apoptosis in U1 cells in a light dose-dependent manner, starting 30 min after light exposure. Using the ApopTag Plus kit (which attaches a fluorescent label to the 3'-OH ends of the degraded DNA) and flow cytometry, the percentage of cells undergoing apoptosis was quantitated. At 10.5 J/cm2, 3 h after light exposure, about 92.5% of the cells were apoptotic. Under these conditions 99% of the cells eventually die. The CEM cells similarly treated underwent apoptosis at slower kinetics and required higher light doses. Other cell lines latently infected with HIV (ACH-2 and OM 10.1) were as sensitive as U1 to HIV inactivation by Pc 4-PDT (H. Margolis-Nunno et al., Transfusion 36, 743-750, 1996) and underwent apoptosis at a similar kinetic. These results suggest that the enhanced inactivation of HIV in latently infected cells compared to CEM cells by Pc 4-PDT may be due, at least in part, to apoptosis in the former.

  2. Expression and In Vivo Rescue of Human ABCC6 Disease-Causing Mutants in Mouse Liver

    PubMed Central

    Le Saux, Olivier; Fülöp, Krisztina; Yamaguchi, Yukiko; Iliás, Attila; Szabó, Zalán; Brampton, Christopher N.; Pomozi, Viola; Huszár, Krisztina; Arányi, Tamás; Váradi, András

    2011-01-01

    Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application. PMID:21935449

  3. Motility defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations

    PubMed Central

    de Vries, Stefan P. W.; Gupta, Srishti; Baig, Abiyad; L'Heureux, Joanna; Pont, Elsa; Wolanska, Dominika P.; Maskell, Duncan J.

    2015-01-01

    Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome resequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of WT fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of (i) screening multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study. PMID:26385289

  4. [A new rice dwarf1 mutant caused by a frame-shift mutation].

    PubMed

    Chen, Hua-Xia; Zhou, Cheng-Bo; Xing, Yong-Zhong

    2011-04-01

    A dwarf mutant C6PS, which has the similar phenotype as the recessive mutant Dwarf1 (d1), was produced from tissue-cultured plants of Zhonghua 11. In its progeny (T2), the ratio of tall to dwarf plants was in agreement with the expected segregation ratio (3:1) of a single Mendelian inheritance gene, which indicated that the variation of plant height is caused by a single gene. To locate the mutation, C6PS was crossed with Zhenshan 97 and Mudanjiang 8 for producing two F2 populations of F2 (CM) and F2 (CZ), respectively. The plant height in each F2 population also showed the same segregation pattern as that in T2 generation. SSR marker RM430 closely linked to Dwarf1 was preferentially used to genotype the F2 (CZ) population because C6PS showed the similar phenotype to d1 mutant. RM430 was significantly associated with plant height, which indicated that the mutant gene might be D1. Comparative sequencing of D1 between C6PS and Zhonghua 11 showed a 6 bp deletion occurred in the splice site of its ninth exon. The marker C6PS-D1L/R designed on the 6 bp deletion was co-segregated with plant height in T2 generation. The results indicated that C6PS was a new mutant of D1. This mutation led to a 26 bp deletion of the transcript and resulted in a frame-shift mutation and a premature stop codon in C6PS, which could not translate the functional Gα protein. C6PS was weakly sensitive to Brassinolide based on the leaf inclination angle test.

  5. Familial Parkinson mutant alpha-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans.

    PubMed

    Kuwahara, Tomoki; Koyama, Akihiko; Gengyo-Ando, Keiko; Masuda, Mayumi; Kowa, Hisatomo; Tsunoda, Makoto; Mitani, Shohei; Iwatsubo, Takeshi

    2006-01-06

    Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.

  6. Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson's disease

    SciTech Connect

    Kumar, Sonu; Sarkar, Anita; Sundar, Durai

    2009-09-18

    Understanding {alpha}-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson's disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in {alpha}-synuclein acquire {beta}-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of {alpha}-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, {sup 40}Val and {sup 74}Val, which play key roles in {beta}-sheet aggregation in the hydrophobic regions 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D{sub V}74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.

  7. Short stature caused by a mutant growth hormone with an antagonistic effect.

    PubMed

    Takahashi, Y; Kaji, H; Okimura, Y; Goji, K; Abe, H; Chihara, K

    1996-10-01

    The molecular basis of biologically inactive GH remained unclear until recently. We have very recently reported a child with short stature and a mutant GH caused by a single missense mutation in the GH-1 gene, which itself cannot transduce the GH-signal to the cells but can blunt the action of wild-type GH by virtue of its greater affinity for the GH binding protein (GHBP)/GH receptor. Briefly the clinical features of the patient are: At the age of 4.9 years his height was 81.7 cm (-6.1 SD) and bone age was 2 years. The patient's serum insulin-like growth factor-1 (IGF-1) concentration was 34 ng/ml. The basal serum GH concentration ranged from 7.0 to 14.0 ng/ml and peak concentrations after insulin hypoglycemia, arginine and L-dopa were 38.0, 15.0 and 35.0 ng/ml, respectively. A heterozygous single base substitution was identified in the GH-1 gene of the proband, predicted to convert codon 77 from arginine to cysteine. Isoelectric focusing revealed the presence of an abnormal GH peak in addition to a normal GH peak. The affinity of expressed mutant GH to GHBP was approximately 6 times higher than that of wild-type GH. The mutant GH not only failed to stimulate tyrosine phosphorylation by itself, but it also inhibited the activity of wild-type GH when added simultaneously even in a one tenth dose of wild-type GH. The child whom we reported is therefore the first case of short stature caused by mutant GH with an antagonistic effect.

  8. Fibroblast growth factor-23 mutants causing familial tumoral calcinosis are differentially processed.

    PubMed

    Larsson, Tobias; Davis, Siobhan I; Garringer, Holly J; Mooney, Sean D; Draman, Mohamad S; Cullen, Michael J; White, Kenneth E

    2005-09-01

    Familial tumoral calcinosis (TC, OMIM 211900) is a heritable disorder characterized by hyperphosphatemia, normal or elevated serum 1,25-dihydroxyvitamin D, and often severe ectopic calcifications. Two recessive mutations in fibroblast growth factor-23 (FGF23), serine 71/glycine (S71G) and serine 129/phenylalanine (S129F), were identified as causing TC. Herein, we undertook comprehensive biochemical analyses of an extended TC family carrying the S71G FGF23 mutation, which revealed that heterozygous (serine/glycine, S/G) individuals had elevated serum FGF23 C-terminal fragments compared with wild-type (serine/serine, S/S) family members (P < 0.025). To understand the differential processing of FGF23 in TC patients, we transiently expressed S71G as well as S129F FGF23. FGF23 ELISA in tandem with Western analyses revealed increased proteolytic cleavage of mutant FGF23 and a limited secretion of intact protein. Furthermore, S71G and S129F FGF23 carrying mutations that disrupt the furin-like protease RXXR motif in FGF23 rescued the secretion of the intact protein, and both TC mutant proteins harboring the R176Q mutation revealed no altered sensitivity to trypsin compared with the native (R176Q)FGF23. Finally, S71G, but not S129F mutant FGF23, is rescued by temperature. In summary, FGF23 mutations causing TC lead to increased intracellular proteolysis of FGF23, most likely by furin-like proteases, due to conformational changes of the mutant protein. The destabilizing nature of these mutations provides new insight into the pathophysiology of TC and exemplifies the physiological importance of FGF23 in phosphate and vitamin D metabolism.

  9. Structural, Energetic, and Mechanical Perturbations in Rhodopsin Mutant That Causes Congenital Stationary Night Blindness*

    PubMed Central

    Kawamura, Shiho; Colozo, Alejandro T.; Ge, Lin; Müller, Daniel J.; Park, Paul S.-H.

    2012-01-01

    Several point mutations in rhodopsin cause retinal diseases including congenital stationary night blindness and retinitis pigmentosa. The mechanism by which a single amino acid residue substitution leads to dysfunction is poorly understood at the molecular level. A G90D point mutation in rhodopsin causes constitutive activity and leads to congenital stationary night blindness. It is unclear which perturbations the mutation introduces and how they can cause the receptor to be constitutively active. To reveal insight into these mechanisms, we characterized the perturbations introduced into dark state G90D rhodopsin from a transgenic mouse model expressing exclusively the mutant rhodopsin in rod photoreceptor cells. UV-visible absorbance spectroscopy revealed hydroxylamine accessibility to the chromophore-binding pocket of dark state G90D rhodopsin, which is not detected in dark state wild-type rhodopsin but is detected in light-activated wild-type rhodopsin. Single-molecule force spectroscopy suggested that the structural changes introduced by the mutation are small. Dynamic single-molecule force spectroscopy revealed that, compared with dark state wild-type rhodopsin, the G90D mutation decreased energetic stability and increased mechanical rigidity of most structural regions in the dark state mutant receptor. The observed structural, energetic, and mechanical changes in dark state G90D rhodopsin provide insights into the nature of perturbations caused by a pathological point mutation. Moreover, these changed properties observed for dark state G90D rhodopsin are consistent with properties expected for an active state. PMID:22549882

  10. Spinocerebellar ataxia type 13 mutant potassium channel alters neuronal excitability and causes locomotor deficits in zebrafish.

    PubMed

    Issa, Fadi A; Mazzochi, Christopher; Mock, Allan F; Papazian, Diane M

    2011-05-04

    Whether changes in neuronal excitability can cause neurodegenerative disease in the absence of other factors such as protein aggregation is unknown. Mutations in the Kv3.3 voltage-gated K(+) channel cause spinocerebellar ataxia type 13 (SCA13), a human autosomal-dominant disease characterized by locomotor impairment and the death of cerebellar neurons. Kv3.3 channels facilitate repetitive, high-frequency firing of action potentials, suggesting that pathogenesis in SCA13 is triggered by changes in electrical activity in neurons. To investigate whether SCA13 mutations alter excitability in vivo, we expressed the human dominant-negative R420H mutant subunit in zebrafish. The disease-causing mutation specifically suppressed the excitability of Kv3.3-expressing, fast-spiking motor neurons during evoked firing and fictive swimming and, in parallel, decreased the precision and amplitude of the startle response. The dominant-negative effect of the mutant subunit on K(+) current amplitude was directly responsible for the reduced excitability and locomotor phenotype. Our data provide strong evidence that changes in excitability initiate pathogenesis in SCA13 and establish zebrafish as an excellent model system for investigating how changes in neuronal activity impair locomotor control and cause cell death.

  11. A new vaccine escape mutant of hepatitis B virus causes occult infection.

    PubMed

    Ye, Qing; Shang, Shi-Qiang; Li, Wei

    2015-01-01

    There is growing public concern regarding assay sensitivity to HBsAg mutants in clinical diagnosis and vaccine escape. The aim of this study is to introduce a new HBsAg mutant strain. The serum samples were those of patient X at the age of 3 months and 3 years respectively, and of her mother immediately before parturition, which were used to amplify the HBsAg-coding DNA fragments by PCR. The HBsAg DNA sequences were translated into their corresponding amino acid sequences and then aligned in pubmed with nucleotide blast. The sequencing data of S coding regions shows that patient X has been infected by a new HBV variant with an A to C substitution at nt431, resulting in an Asp(GAC)to Ala(GCC) substitution at aa144 of major protein; CC to AA substitution at nt359 and nt360, resulting in an Pro(CCC) to Gln(CAA) substitution at aa120 of pre "a" epitope; A to G substitution at nt491, resulting in an Glu(GAG) to Gly(GGG) substitution at aa164 of post "a" epitope. Three new mutations (S171F, S174N and Q181R) at the antigenic epitopes of HBV presented by HLA class I molecules are found. The HBV mutant strain causes vaccine escape and occult infection.

  12. Deafness in TRβ-mutants is caused by malformation of the tectorial membrane

    PubMed Central

    Winter, Harald; Rüttiger, Lukas; Müller, Marcus; Kuhn, Stephanie; Brandt, Niels; Zimmermann, Ulrike; Hirt, Bernhard; Bress, Andreas; Sausbier, Matthias; Conscience, Aude; Flamant, Frederic; Tian, Yong; Zuo, Jian; Pfister, Markus; Ruth, Peter; Löwenheim, Hubert; Samarut, Jacques; Engel, Jutta; Knipper, Marlies

    2009-01-01

    Thyroid hormone receptor β (TRβ) dysfunction leads to deafness in humans and mice. Deafness in TRβ−/− mutant mice has been attributed to TRβ-mediated control of fast-activating BK current expression in inner hair cells (IHCs). However, normal hearing in young constitutive BKα−/− mutants contradicts this hypothesis. Here we show that mice with hair cell-specific deletion of TRβ after postnatal day (P) 11 have a delay in BKα expression but normal hearing, indicating that the origin of hearing loss in TRβ−/− mutant mice manifested before P11. Analyzing the phenotype of IHCs in constitutive TRβ−/− mice we found normal Ca2+ current amplitudes, exocytosis, and shape of compound action potential waveforms. In contrast, reduced DPOAEs and cochlear microphonics associated with an abnormal structure of the tectorial membrane and enhanced tectorin levels suggest that disturbed mechanical performance is the primary cause of deafness resulting from TRβ deficiency. PMID:19244534

  13. IBMPFD Disease-Causing Mutant VCP/p97 Proteins Are Targets of Autophagic-Lysosomal Degradation.

    PubMed

    Bayraktar, Oznur; Oral, Ozlem; Kocaturk, Nur Mehpare; Akkoc, Yunus; Eberhart, Karin; Kosar, Ali; Gozuacik, Devrim

    2016-01-01

    The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget's Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies.

  14. IBMPFD Disease-Causing Mutant VCP/p97 Proteins Are Targets of Autophagic-Lysosomal Degradation

    PubMed Central

    Bayraktar, Oznur; Akkoc, Yunus; Eberhart, Karin; Kosar, Ali

    2016-01-01

    The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget’s Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies. PMID:27768726

  15. Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population.

    PubMed

    Coffee, Erin M; Yerkes, Laura; Ewen, Elizabeth P; Zee, Tiffany; Tolan, Dean R

    2010-02-01

    Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Delta4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Delta4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.

  16. Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population

    PubMed Central

    Coffee, Erin M.; Yerkes, Laura; Ewen, Elizabeth P.; Zee, Tiffany

    2010-01-01

    Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Δ4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Δ4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance. PMID:20033295

  17. What can long-lived mutants tell us about mechanisms causing aging and lifespan variation in natural environments?

    PubMed

    Briga, Michael; Verhulst, Simon

    2015-11-01

    Long-lived mutants of model organisms have brought remarkable progress in our understanding of aging mechanisms. However, long-lived mutants are usually maintained in optimal standardized laboratory environments (SLEs), and it is not obvious to what extent insights from long-lived mutants in SLEs can be generalized to more natural environments. To address this question, we reviewed experiments that compared the fitness and lifespan advantage of long-lived mutants relative to wild type controls in SLEs and more challenging environments in various model organisms such as yeast Saccharomyces cerevisiae, the nematode worm Caenorhabditis elegans, the fruitfly Drosophila melanogaster and the mouse Mus musculus. In competition experiments over multiple generations, the long-lived mutants had a lower fitness relative to wild type controls, and this disadvantage was the clearest when the environment included natural challenges such as limited food (N=6 studies). It is well known that most long-lived mutants have impaired reproduction, which provides one reason for the fitness disadvantage. However, based on 12 experiments, we found that the lifespan advantage of long-lived mutants is diminished in more challenging environments, often to the extent that the wild type controls outlive the long-lived mutants. Thus, it appears that information on aging mechanisms obtained from long-lived mutants in SLEs may be specific to such environments, because those same mechanisms do not extend lifespan in more natural environments. This suggests that different mechanisms cause variation in aging and lifespan in SLEs compared to natural populations.

  18. Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets.

    PubMed

    Farrow, Emily G; Davis, Siobhan I; Ward, Leanne M; Summers, Lelia J; Bubbear, Judith S; Keen, Richard; Stamp, Trevor C B; Baker, Laurence R I; Bonewald, Lynda F; White, Kenneth E

    2009-02-01

    We previously demonstrated that the mutations Met1Val (M1V) and the deletion of nucleotides 1484-1490 (1484-1490del) in Dentin matrix protein-1 (DMP1) cause the novel disorder autosomal recessive hypophosphatemic rickets (ARHR), which is associated with elevated fibroblast growth factor-23 (FGF23). To further understand the role of DMP1 in ARHR, we undertook molecular genetic and in vitro expression studies. First, we examined a kindred with a severe hypophosphatemic rickets phenotype and recessive inheritance. Analyses of this family demonstrated that the affected members had elevated serum FGF23 and carried a large, biallelic deletion that removed the majority of DMP1. At a minimum, this deletion encompassed 49 kb between DMP1 exon 3 and an intergenic region 5' to the next telomeric gene, integrin-binding sialoprotein (IBSP). We next performed immunofluorescent studies in cells to understand the effects of the known ARHR mutations on DMP1 cellular processing. These analyses showed that the M1V DMP1 mutant was not sorted to the trans-Golgi network (TGN) and secretory pathway, but filled the entire cytoplasm. In contrast, the 1484-1490del mutant localized to the TGN and was secreted, similar to wild type DMP1. The 1484-1490del mutation replaces the DMP1 18 C-terminal amino acids with 33 non-native residues. Truncation of wild type DMP1 by these native 18 residues followed by Western blot and confocal microscopic analyses demonstrated a wild type expression pattern when compared with the 1484-1490del mutant, indicating that the last 18 residues are not critical for cellular trafficking, but that the 33 additional residues arising from the 1484-1490del mutation likely compromise DMP1 processing. The relationship between DMP1 and FGF23 is unclear. To test endogenous DMP1 response to serum metabolites that also regulate FGF23, UMR-106 cells were treated with 1,25(OH)(2) vitamin D (1x10(-7) M) and showed a 12-fold increase in DMP1 mRNA and protein at 24 h. In summary

  19. Reduction of germ cells in the Odysseus null mutant causes male fertility defect in Drosophila melanogaster.

    PubMed

    Cheng, Ya-Jen; Fang, Shu; Tsaur, Shun-Chern; Chen, Yi-Ling; Fu, Hua-Wen; Patel, Nipam H; Ting, Chau-Ti

    2012-01-01

    Odysseus (OdsH) has been identified as a hybrid male sterility gene between Drosophila mauritiana and D. simulans with accelerated evolutionary rate in both expression and DNA sequence. Loss of a testis-specific expression of OdsH causes male fertility defect in D. melanogaster. Yet, the underlying mechanisms at the cellular level are unknown. In an attempt to identify the possible mechanisms and functional roles of OdsH in spermatogenesis, the cell numbers at different developmental stages during spermatogenesis between the OdsH null mutant and wild-type flies were compared. The results showed that the early developing germ cells, including spermatogonia and spermatocytes, were reduced in the OdsH mutant males. In addition, the number of germline stem cells in aged males was also reduced, presumably due to the disruption of germline stem cell maintenance, which resulted in more severe fertility defect. These results suggest that the function of the enhancement of sperm production by OdsH acted across males of all ages.

  20. Oculomotor instabilities in zebrafish mutant belladonna: a behavioral model for congenital nystagmus caused by axonal misrouting.

    PubMed

    Huang, Ying-Yu; Rinner, Oliver; Hedinger, Patrik; Liu, Shih-Chii; Neuhauss, Stephan C F

    2006-09-27

    A large fraction of homozygous zebrafish mutant belladonna (bel) larvae display a reversed optokinetic response (OKR) that correlates with failure of the retinal ganglion cells to cross the midline and form the optic chiasm. Some of these achiasmatic mutants display strong spontaneous eye oscillations (SOs) in the absence of motion in the surround. The presentation of a stationary grating was necessary and sufficient to evoke SO. Both OKR reversal and SO depend on vision and are contrast sensitive. We built a quantitative model derived from bel fwd (forward) eye behaviors. To mimic the achiasmatic condition, we reversed the sign of the retinal slip velocity in the model, thereby successfully reproducing both reversed OKR and SO. On the basis of the OKR data, and with the support of the quantitative model, we hypothesize that the reversed OKR and the SO can be completely attributed to RGC misrouting. The strong resemblance between the SO and congenital nystagmus (CN) seen in humans with defective retinotectal projections implies that CN, of so far unknown etiology, may be directly caused by a projection defect.

  1. Molecular pathogenesis of Spondylocheirodysplastic Ehlers-Danlos syndrome caused by mutant ZIP13 proteins

    PubMed Central

    Bin, Bum-Ho; Hojyo, Shintaro; Hosaka, Toshiaki; Bhin, Jinhyuk; Kano, Hiroki; Miyai, Tomohiro; Ikeda, Mariko; Kimura-Someya, Tomomi; Shirouzu, Mikako; Cho, Eun-Gyung; Fukue, Kazuhisa; Kambe, Taiho; Ohashi, Wakana; Kim, Kyu-Han; Seo, Juyeon; Choi, Dong-Hwa; Nam, Yeon-Ju; Hwang, Daehee; Fukunaka, Ayako; Fujitani, Yoshio; Yokoyama, Shigeyuki; Superti-Furga, Andrea; Ikegawa, Shiro; Lee, Tae Ryong; Fukada, Toshiyuki

    2014-01-01

    The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13G64D, in which Gly at amino acid position 64 is replaced by Asp, and ZIP13ΔFLA, which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13G64D and ZIP13ΔFLA protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS. PMID:25007800

  2. A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.

    PubMed Central

    Merrill, B J; Holm, C

    1999-01-01

    To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants. PMID:10511542

  3. Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis.

    PubMed

    Zhong, Ruiqin; Morrison, W Herbert; Freshour, Glenn D; Hahn, Michael G; Ye, Zheng-Hua

    2003-06-01

    Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.

  4. Sodium/Iodide Symporter Mutant V270E Causes Stunted Growth but No Cognitive Deficiency.

    PubMed

    Nicola, Juan Pablo; Reyna-Neyra, Andrea; Saenger, Paul; Rodriguez-Buritica, David F; Gamez Godoy, José David; Muzumdar, Radhika; Amzel, L Mario; Carrasco, Nancy

    2015-10-01

    Iodide (I(-)), an essential constituent of the thyroid hormones, is actively accumulated in the thyroid by the Na(+)/I(-) symporter (NIS), a key plasma membrane protein encoded by the slc5a5 gene. Mutations in slc5a5 cause I(-) transport defects (ITDs), autosomal-recessive disorders in which I(-) accumulation is totally or partially impaired, leading to congenital hypothyroidism. The characterization of NIS mutants has yielded significant insights into the molecular mechanism of NIS. This study aimed to determine the basis of a patient's ITD clinical phenotype, by sequencing her slc5a5 gene. Genomic DNA was purified and the slc5a5 gene sequence determined. Functional in vitro studies were performed to characterize the V270E NIS mutant. The index patient was diagnosed with hypothyroidism with minimal radioiodide uptake in a normally located, although enlarged, thyroid gland. We identified a new NIS mutation: V270E. The patient had the compound heterozygous NIS mutation R124H/V270E. R124H NIS has been characterized previously. We show that V270E markedly reduces I(-) uptake via a pronounced (but not total) impairment of the protein's plasma membrane targeting. Remarkably, V270E is intrinsically active. Therefore, a negative charge at position 270 interferes with NIS cell surface trafficking. The patient's minimal I(-) uptake enabled sufficient thyroid hormone biosynthesis to prevent cognitive impairment. A nonpolar residue at position 270, which all members of the SLC5A family have, is required for NIS plasma membrane targeting.

  5. Sodium/Iodide Symporter Mutant V270E Causes Stunted Growth but No Cognitive Deficiency

    PubMed Central

    Nicola, Juan Pablo; Reyna-Neyra, Andrea; Saenger, Paul; Rodriguez-Buritica, David F.; Gamez Godoy, José David; Muzumdar, Radhika; Amzel, L. Mario

    2015-01-01

    Context: Iodide (I−), an essential constituent of the thyroid hormones, is actively accumulated in the thyroid by the Na+/I− symporter (NIS), a key plasma membrane protein encoded by the slc5a5 gene. Mutations in slc5a5 cause I− transport defects (ITDs), autosomal-recessive disorders in which I− accumulation is totally or partially impaired, leading to congenital hypothyroidism. The characterization of NIS mutants has yielded significant insights into the molecular mechanism of NIS. Objective: This study aimed to determine the basis of a patient's ITD clinical phenotype, by sequencing her slc5a5 gene. Design: Genomic DNA was purified and the slc5a5 gene sequence determined. Functional in vitro studies were performed to characterize the V270E NIS mutant. Patient: The index patient was diagnosed with hypothyroidism with minimal radioiodide uptake in a normally located, although enlarged, thyroid gland. Results: We identified a new NIS mutation: V270E. The patient had the compound heterozygous NIS mutation R124H/V270E. R124H NIS has been characterized previously. We show that V270E markedly reduces I− uptake via a pronounced (but not total) impairment of the protein's plasma membrane targeting. Remarkably, V270E is intrinsically active. Therefore, a negative charge at position 270 interferes with NIS cell surface trafficking. The patient's minimal I− uptake enabled sufficient thyroid hormone biosynthesis to prevent cognitive impairment. Conclusions: A nonpolar residue at position 270, which all members of the SLC5A family have, is required for NIS plasma membrane targeting. PMID:26204134

  6. Widespread aggregation of mutant VAPB associated with ALS does not cause motor neuron degeneration or modulate mutant SOD1 aggregation and toxicity in mice

    PubMed Central

    2013-01-01

    Background A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca2+ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations. Results To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice. Conclusion Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration

  7. Widespread aggregation of mutant VAPB associated with ALS does not cause motor neuron degeneration or modulate mutant SOD1 aggregation and toxicity in mice.

    PubMed

    Qiu, Linghua; Qiao, Tao; Beers, Melissa; Tan, Weijia; Wang, Hongyan; Yang, Bin; Xu, Zuoshang

    2013-01-03

    A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca2+ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations. To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice. Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either

  8. Mutant alpha-synuclein causes age-dependent neuropathology in monkey brain.

    PubMed

    Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua; Li, Xiao-Jiang

    2015-05-27

    Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2-3, 7-8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. Copyright © 2015 the authors 0270-6474/15/358345-14$15.00/0.

  9. Mutant Alpha-Synuclein Causes Age-Dependent Neuropathology in Monkey Brain

    PubMed Central

    Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua

    2015-01-01

    Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2–3, 7–8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. PMID:26019347

  10. [Effect of microRNA on proliferation caused by mutant HBx in human hepatocytes].

    PubMed

    Fu, Xiao-yu; Tan, De-ming; Hou, Zhou-hua; Hu, Zhi-liang; Liu, Guo-zhen; Ouyang, Yi; Liu, Fei

    2012-08-01

    To study the effect of micro (mi)RNA on cellular proliferation induced by hepatitis B x protein, HBx, in human liver cells and to investigate the underlying molecular mechanism of this cancer-related effect. The human L02 hepatocyte cell line was stably transfected with HBx (L02/HBx) or an HBx mutant (L02/HBx-d382) that induces higher levels of cellular proliferation. The differential miRNA expression profiles were determined by microarray analysis and confirmed by real-time PCR. Two miRNAs, miR-338-3p and miR-551b, that were found to be significantly down-regulated in the L02/HBx-d382 cells were selected for further study and transfected individually into cells using the lipofectamine procedure. The cell survival rate was analyzed by MTT assay, and cell cycles were assessed by flow cytometry. Expressions of cyclinD1, cyclinG1, and E2F1 were assessed by real-time PCR and Western blotting. Compared with the microarray miRNA profile of L02/pcDNA3.0 cells, six miRNAs were up-regulated and five miRNAs were down-regulated in the L02/HBx-d382 cells, while four miRNAs were up-regulated and 12 were down-regulated in the L02/HBx cells. The microarray results were consistent with real-time PCR results. Transfection of miR-338-3p and miR-551b significantly inhibited the cell survival rates (P less than 0.001) and induced G0/G1 phase cycle arrest. According to MTT results: for L02/HBx-d382 cells, compared with lipofectamine or non-transfected (NC) controls, the t value of miR-338-3p was 10.402, 9.133 and the t value of miR-551b was 8.763, 7.403; for L02/HBx cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 9.105, 8.074 and the t value of miR-551b was 7.673, 7.52. According to flow cytometry results: for L02/HBx-d382 cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 12.173, 11.107 and the t value of miR-551b was 15.364, 13.377; for L02/HBx cells, compared with lipofectamine or NC controls, the t value of miR-338-3p

  11. Analysis of Pseudoxanthoma Elasticum-Causing Missense Mutants of ABCC6 in vivo; Pharmacological Correction of the Mislocalized Proteins

    PubMed Central

    Pomozi, Viola; Brampton, Christopher; Fülöp, Krisztina; Chen, Li-Hsieh; Apana, Ailea; Li, Qiaoli; Uitto, Jouni; Le Saux, Olivier; Váradi, András

    2014-01-01

    Mutations in the ABCC6 gene cause soft tissue calcification in pseudoxanthoma elasticum (PXE) and in some patients generalized arterial calcification of infancy (GACI). PXE is characterized by late-onset and progressive mineralization of elastic fibers in dermal, ocular and cardiovascular tissues. GACI patients present a more severe, often prenatal arterial calcification. We have tested ten frequent disease-causing ABCC6 missense mutants for the transport activity using Sf9 cells, characterized the subcellular localization in MDCKII cells and in mouse liver, and tested the phenotypic rescue in zebrafish. We aimed at identifying mutants with preserved transport activity but with improper plasma membrane localization for rescue by the chemical chaperone 4-phenylbutyrate (4-PBA). Seven of the mutants were transport-competent but mislocalized in mouse liver. The observed divergence in cellular localization of mutants in MDCKII cells vs. mouse liver underlined the limitations of this two-dimensional in vitro cell system. The functionality of ABCC6 mutants was tested in zebrafish and minimal rescue of the morpholino-induced phenotype was found. However, 4-PBA, a drug approved for clinical use, restored the plasma membrane localization of four ABCC6 mutants (R1114P, S1121W, Q1347H, R1314W), suggesting that allele-specific therapy may be useful for selected patients with PXE and GACI. PMID:24352041

  12. A DltA mutant of Haemophilus ducreyi Is partially attenuated in its ability to cause pustules in human volunteers.

    PubMed

    Janowicz, Diane; Leduc, Isabelle; Fortney, Kate R; Katz, Barry P; Elkins, Christopher; Spinola, Stanley M

    2006-02-01

    Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.

  13. Common increase of GATA-3 level in PC-12 cells by three teratogens causing autism spectrum disorders.

    PubMed

    Rout, Ujjwal K; Clausen, Pete

    2009-06-01

    Autism spectrum disorder (ASD) is a disease of neuro-developmental origin of uncertain etiology. The current understanding is that both genetic and environmental factors contribute to the development of ASD. Exposure to valproate, thalidomide and alcohol during gestation are amongst the environmental triggers that are associated with the development of ASD. These teratogens may disturb the ontogeny of the brain by altering the expression pattern of genes that regulate the normal development of the brain. In this study, a neuron-like PC-12 cell model was used to examine the effects of these compounds on the binding potential of 50 different transcription factors to understand the molecular mechanism/s that may be involved in the teratogenesis caused by these agents. Cells in culture were treated with low or high concentrations of teratogens within a range that are reported in the blood of individuals. A pronounced increase in GATA transcription factor binding was observed for all three teratogens. Furthermore, Western blot analysis showed that GATA-3 level in the nuclear fractions was enhanced by each of the three teratogens. Results suggest that altered gene expression pattern due to heightened GATA-3 activities in the fetral brains following exposure to these teratogens may contribute to the development of ASD.

  14. Parkinson's disease-associated mutant VPS35 causes mitochondrial dysfunction by recycling DLP1 complexes.

    PubMed

    Wang, Wenzhang; Wang, Xinglong; Fujioka, Hisashi; Hoppel, Charles; Whone, Alan L; Caldwell, Maeve A; Cullen, Peter J; Liu, Jun; Zhu, Xiongwei

    2016-01-01

    Mitochondrial dysfunction represents a critical step during the pathogenesis of Parkinson's disease (PD), and increasing evidence suggests abnormal mitochondrial dynamics and quality control as important underlying mechanisms. The VPS35 gene, which encodes a key component of the membrane protein-recycling retromer complex, is the third autosomal-dominant gene associated with PD. However, how VPS35 mutations lead to neurodegeneration remains unclear. Here we demonstrate that PD-associated VPS35 mutations caused mitochondrial fragmentation and cell death in cultured neurons in vitro, in mouse substantia nigra neurons in vivo and in human fibroblasts from an individual with PD who has the VPS35(D620N) mutation. VPS35-induced mitochondrial deficits and neuronal dysfunction could be prevented by inhibition of mitochondrial fission. VPS35 mutants showed increased interaction with dynamin-like protein (DLP) 1, which enhanced turnover of the mitochondrial DLP1 complexes via the mitochondria-derived vesicle-dependent trafficking of the complexes to lysosomes for degradation. Notably, oxidative stress increased the VPS35-DLP1 interaction, which we also found to be increased in the brains of sporadic PD cases. These results revealed a novel cellular mechanism for the involvement of VPS35 in mitochondrial fission, dysregulation of which is probably involved in the pathogenesis of familial, and possibly sporadic, PD.

  15. Susceptibility of Maize to Stalk Rot Caused by Fusarium graminearum Deoxynivalenol and Zearalenone Mutants.

    PubMed

    Quesada-Ocampo, L M; Al-Haddad, J; Scruggs, A C; Buell, C R; Trail, F

    2016-08-01

    Fusarium graminearum is a destructive pathogen of cereals that can cause stalk rot in maize. Stalk rot results in yield losses due to impaired grain filling, premature senescence, and lodging, which limits production and harvesting of ears. In addition, mycotoxins can make infected tissues unfit for silage. Our objectives were to evaluate the natural variation in stalk rot resistance among maize inbreds, to establish whether deoxynivalenol (DON)- and zearalenone (ZEA)-deficient strains are pathogenic on a panel of diverse inbreds, and to quantify the accumulation of DON in infected stalk tissue. Wild-type F. graminearum and mycotoxin mutants (DON and ZEA) were used to separately inoculate stalks of 9-week-old plants of 20 inbreds in the greenhouse. Plants were evaluated for lesion area at the inoculation point at 0, 2, 14, and 28 days postinoculation and tissues around lesions were sampled to determine the DON content. Regardless of their ability to produce DON or ZEA, all tested F. graminearum strains caused stalk rot; however, significant differences in disease levels were detected. Among the tested inbreds, Mp717 was resistant to all three F. graminearum strains while Mp317 and HP301 were only partially resistant. Accumulation of DON was significantly lower in infected stalks of the resistant and partially resistant inbreds than the susceptible inbreds. Analysis of the 20 inbreds using data from 17 simple-sequence repeats revealed population structure among the individuals; however, there was no association between genetic clustering and stalk rot resistance. These findings are an additional step toward breeding maize inbreds suitable for planting in fields infested with F. graminearum.

  16. Generation of Pc 1 waves by the ion temperature anisotropy associated with fast shocks caused by sudden impulses

    NASA Technical Reports Server (NTRS)

    Mandt, M. E.; Lee, L. C.

    1991-01-01

    The high correlation of Pc 1 events with magnetospheric compressions is known. A mechanism is proposed which leads to the generation of Pc 1 waves. The interaction of a dynamic pressure pulse with the earth's bow shock leads to the formation of a weak fast-mode shock propagating into the magnetoshealth. The shock wave can pass right through a tangential discontinuity (magnetopause) and into the magnetosphere, without disturbing either of the structures. In a quasiperpendicular geometry, the shock wave exhibits anisotropic heating. This anisotropy drives unstable ion-cyclotron waves which can contribute to the generation of the Pc 1 waves which are detected. The viability of the mechanism is demonstrated with simulations. This mechanism could explain the peak in the occurrence of observed Pc 1 waves in the postnoon sector where a field-aligned discontinuity in the solar wind would most often be parallel to the magnetopause surface due to the average Parker-spiral magnetic-field configuration.

  17. The Maize Low-Phytic Acid Mutant lpa2 Is Caused by Mutation in an Inositol Phosphate Kinase Gene

    PubMed Central

    Shi, Jinrui; Wang, Hongyu; Wu, Yunsheng; Hazebroek, Jan; Meeley, Robert B.; Ertl, David S.

    2003-01-01

    Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P3 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P3, Ins(3,5,6)P3, Ins(3,4,5,6)P4, and Ins(1,2,5,6)P4. The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F2 family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds. PMID:12586875

  18. Different Biochemical Properties Explain Why Two Equivalent Gα Subunit Mutants Cause Unrelated Diseases*

    PubMed Central

    Leyme, Anthony; Marivin, Arthur; Casler, Jason; Nguyen, Lien T.; Garcia-Marcos, Mikel

    2014-01-01

    There is an increasing number of disease-associated Gα mutations identified from genome-wide sequencing campaigns or targeted efforts. Albright's Hereditary Osteodystrophy (AHO) was the first inherited disease associated with loss-of-function mutations in a G protein (Gαs) and other studies revealed gain-of-function Gα mutations in cancer. Here we attempted to solve the apparent quandary posed by the fact that the same mutation in two different G proteins appeared associated with both AHO and cancer. We first confirmed the presence of an inherited Gαs-R265H mutation from a previously described clinical case report of AHO. This mutation is structurally analogous to Gαo-R243H, an oncogenic mutant with increased activity in vitro and in cells due to rapid nucleotide exchange. We found that, contrary to Gαo-R243H, Gαs-R265H activity is compromised due to greatly impaired nucleotide binding in vitro and in cells. We obtained equivalent results when comparing another AHO mutation in Gαs (D173N) with a counterpart cancer mutation in Gαo (D151N). Gαo-R243H binds nucleotides efficiently under steady-state conditions but releases GDP much faster than the WT protein, suggesting diminished affinity for the nucleotide. These results indicate that the same disease-linked mutation in two different G proteins affects a common biochemical feature (nucleotide affinity) but to a different grade depending on the G protein (mild decrease for Gαo and severe for Gαs). We conclude that Gαs-R265H has dramatically impaired nucleotide affinity leading to the loss-of-function in AHO whereas Gαo-R243H has a mild decrease in nucleotide affinity that causes rapid nucleotide turnover and subsequent hyperactivity in cancer. PMID:24982418

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

    PubMed Central

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

    2015-01-01

    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. PMID:26198671

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Human congenital myopathy actin mutants cause myopathy and alter Z-disc structure in Drosophila flight muscle.

    PubMed

    Sevdali, Maria; Kumar, Vikash; Peckham, Michelle; Sparrow, John

    2013-03-01

    Over 190 mutations in the human skeletal muscle α-actin gene, ACTA1 cause congenital actin myopathies. We transgenically expressed six different mutant actins, G15R, I136M, D154N, V163L, V163M and D292V in Drosophila indirect flight muscles and investigated their effects in flies that express one wild type and one mutant actin copy. All the flies were flightless, and the IFMs showed incomplete Z-discs, disorganised actin filaments and 'zebra bodies'. No differences in levels of sarcomeric protein expression were observed, but tropomodulin staining was somewhat disrupted in D164N, V163L, G15R and V163M heterozygotes. A single copy of D292V mutant actin rescued the hypercontractile phenotypes caused by TnI and TnT mutants, suggesting that the D292V mutation interferes with thin filament regulation. Our results show that expression of actin mutations homologous to those in humans in the indirect flight muscles of Drosophila disrupt sarcomere organisation, with somewhat similar phenotypes to those observed in humans. Using Drosophila to study actin mutations may help aid our understanding of congential myopathies caused by actin mutations.

  2. In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

    PubMed Central

    Hendershot, L M; Wei, J Y; Gaut, J R; Lawson, B; Freiden, P J; Murti, K G

    1995-01-01

    BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo. Images PMID:7612964

  3. Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans

    PubMed Central

    Vaccaro, Alexandra; Tauffenberger, Arnaud; Aggad, Dina; Rouleau, Guy; Drapeau, Pierre; Parker, J. Alex

    2012-01-01

    Mutations in the DNA/RNA binding proteins TDP-43 and FUS are associated with Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. Intracellular accumulations of wild type TDP-43 and FUS are observed in a growing number of late-onset diseases suggesting that TDP-43 and FUS proteinopathies may contribute to multiple neurodegenerative diseases. To better understand the mechanisms of TDP-43 and FUS toxicity we have created transgenic Caenorhabditis elegans strains that express full-length, untagged human TDP-43 and FUS in the worm's GABAergic motor neurons. Transgenic worms expressing mutant TDP-43 and FUS display adult-onset, age-dependent loss of motility, progressive paralysis and neuronal degeneration that is distinct from wild type alleles. Additionally, mutant TDP-43 and FUS proteins are highly insoluble while wild type proteins remain soluble suggesting that protein misfolding may contribute to toxicity. Populations of mutant TDP-43 and FUS transgenics grown on solid media become paralyzed over 7 to 12 days. We have developed a liquid culture assay where the paralysis phenotype evolves over several hours. We introduce C. elegans transgenics for mutant TDP-43 and FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening. PMID:22363618

  4. Two novel CRX mutant proteins causing autosomal dominant Leber congenital amaurosis interact differently with NRL.

    PubMed

    Nichols, Lorenzo L; Alur, Ramakrishna P; Boobalan, Elangovan; Sergeev, Yuri V; Caruso, Rafael C; Stone, Edwin M; Swaroop, Anand; Johnson, Mary A; Brooks, Brian P

    2010-06-01

    Leber congenital amaurosis (LCA) is a congenital retinal dystrophy characterized by severe visual loss in infancy and nystagmus. Although most often inherited in an autosomal recessive fashion, rare individuals with mutations in the cone-rod homeobox gene, CRX, have dominant disease. CRX is critical for photoreceptor development and acts synergistically with the leucine-zipper transcription factor, NRL. We report on the phenotype of two individuals with LCA due to novel, de novo CRX mutations, c.G264T(p.K74N) and c.413delT(p.I138fs48), that reduce transactivation in vitro to 10% and 30% of control values, respectively. Whereas the c.413delT(p.I138fs48) mutant allows co-expressed NRL to transactivate independently at its normal, baseline level, the c.G264T(p.K74N) mutant reduces co-expressed NRL transactivation and reduces steady state levels of both proteins. Although both mutant proteins predominantly localize normally to the nucleus, they also both show variable cytoplasmic localization. These observations suggest that some CRX-mediated LCA may result from effects beyond haploinsufficiency, such as the mutant protein interefering with other transcription factors' function. Such patients would therefore not likely benefit from a simple, gene-replacement strategy for their disease.

  5. Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant.

    PubMed

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2014-09-01

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  6. Constant light housing during nursing causes human DSPS (delayed sleep phase syndrome) behaviour in Clock-mutant mice.

    PubMed

    Wakatsuki, Yukako; Kudo, Takashi; Shibata, Shigenobu

    2007-04-01

    Delayed sleep phase syndrome (DSPS) is very often seen among patients with sleep-wake rhythm disorders. Humans with the 3111C allele of the human Clock gene tend to demonstrate a higher evening preference on the morningness-eveningness (ME) preference test. DSPS is thought to be an extreme form of this evening preference. Clock-mutant mice have been proposed as an animal model of evening preference. In this study, we looked at whether constant light (LL) housing of Clock-mutant mice during lactation would result in evening preference and/or DSPS. Housed under light-dark (LD) or constant dark (DD) conditions during the lactation period, both wild-type and Clock-mutant mice did not show a phase-delay in the locomotor activity measured under light-dark conditions, whereas constant light housing during lactation significantly caused a delayed onset. The magnitude of the delay during the light-dark cycle was positively associated with free-running period measured during constant darkness. Among wild, heterozygote, and homozygote pups born from heterozygous dams, only homozygote pups showed a delayed onset. Constant light-housed Clock-mutant mice exhibited a lower number and delayed peak of phospho-MAPK-immunoreactive cells in core regions of the suprachiasmatic nucleus (SCN) compared to light-dark housed wild-type or Clock-mutant mice. Activity onset returned to normal with daily melatonin injection at the lights-off time for 5 days. The present results demonstrate that Clock-mutant mice exposed to constant light during lactation can function as an animal model of DSPS and can be used to gain an understanding of the ethological aspects of DSPS as well as to find medication for its treatment.

  7. A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function

    PubMed Central

    Fuglerud, Bettina M.; Lemma, Roza B.; Wanichawan, Pimthanya; Sundaram, Arvind Y. M.

    2017-01-01

    Abstract The transcription factor c-Myb is involved in early differentiation and proliferation of haematopoietic cells, where it operates as a regulator of self-renewal and multi-lineage differentiation. Deregulated c-Myb plays critical roles in leukaemias and other human cancers. Due to its role as a master regulator, we hypothesized it might function as a pioneer transcription factor. Our approach to test this was to analyse a mutant of c-Myb, D152V, previously reported to cause haematopoietic defects in mice by an unknown mechanism. Our transcriptome data from K562 cells indicates that this mutation specifically affects c-Myb's ability to regulate genes involved in differentiation, causing failure in c-Myb's ability to block differentiation. Furthermore, we see a major effect of this mutation in assays where chromatin opening is involved. We show that each repeat in the minimal DNA-binding domain of c-Myb binds to histones and that D152V disrupts histone binding of the third repeat. ATAC-seq data indicates this mutation impairs the ability of c-Myb to cause chromatin opening at specific sites. Taken together, our findings support that c-Myb acts as a pioneer factor and show that D152V impairs this function. The D152V mutant is the first mutant of a transcription factor specifically destroying pioneer factor function. PMID:28472346

  8. A Clonal Genetic Screen for Mutants Causing Defects in Larval Tracheal Morphogenesis in Drosophila

    PubMed Central

    Baer, Magdalena M.; Bilstein, Andreas; Leptin, Maria

    2007-01-01

    The initial establishment of the tracheal network in the Drosophila embryo is beginning to be understood in great detail, both in its genetic control cascades and in its cell biological events. By contrast, the vast expansion of the system during larval growth, with its extensive ramification of preexisting tracheal branches, has been analyzed less well. The mutant phenotypes of many genes involved in this process are probably not easy to reveal, as these genes may be required for other functions at earlier developmental stages. We therefore conducted a screen for defects in individual clonal homozygous mutant cells in the tracheal network of heterozygous larvae using the mosaic analysis with a repressible cell marker (MARCM) system to generate marked, recombinant mitotic clones. We describe the identification of a set of mutants with distinct phenotypic effects. In particular we found a range of defects in terminal cells, including failure in lumen formation and reduced or extensive branching. Other mutations affect cell growth, cell shape, and cell migration. PMID:17603107

  9. Cystic fibrosis-adapted Pseudomonas aeruginosa quorum sensing lasR mutants cause hyperinflammatory responses.

    PubMed

    LaFayette, Shantelle L; Houle, Daniel; Beaudoin, Trevor; Wojewodka, Gabriella; Radzioch, Danuta; Hoffman, Lucas R; Burns, Jane L; Dandekar, Ajai A; Smalley, Nicole E; Chandler, Josephine R; Zlosnik, James E; Speert, David P; Bernier, Joanie; Matouk, Elias; Brochiero, Emmanuelle; Rousseau, Simon; Nguyen, Dao

    2015-07-01

    Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease- dependent cytokine degradation. In subacute pulmonary infections, lasR mutant-infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients.

  10. Plastidic phosphoglucomutase and ADP-glucose pyrophosphorylase mutants impair starch synthesis in rice pollen grains and cause male sterility.

    PubMed

    Lee, Sang-Kyu; Eom, Joon-Seob; Hwang, Seon-Kap; Shin, Dongjin; An, Gynheung; Okita, Thomas W; Jeon, Jong-Seong

    2016-10-01

    To elucidate the starch synthesis pathway and the role of this reserve in rice pollen, we characterized mutations in the plastidic phosphoglucomutase, OspPGM, and the plastidic large subunit of ADP-glucose (ADP-Glc) pyrophosphorylase, OsAGPL4 Both genes were up-regulated in maturing pollen, a stage when starch begins to accumulate. Progeny analysis of self-pollinated heterozygous lines carrying the OspPGM mutant alleles, osppgm-1 and osppgm-2, or the OsAGPL4 mutant allele, osagpl4-1, as well as reciprocal crosses between the wild type (WT) and heterozygotes revealed that loss of OspPGM or OsAGPL4 caused male sterility, with the former condition rescued by the introduction of the WT OspPGM gene. While iodine staining and transmission electron microscopy analyses of pollen grains from homozygous osppgm-1 lines produced by anther culture confirmed the starch null phenotype, pollen from homozygous osagpl4 mutant lines, osagpl4-2 and osagpl4-3, generated by the CRISPR/Cas system, accumulated small amounts of starch which were sufficient to produce viable seed. Such osagpl4 mutant pollen, however, was unable to compete against WT pollen successfully, validating the important role of this reserve in fertilization. Our results demonstrate that starch is mainly polymerized from ADP-Glc synthesized from plastidic hexose phosphates in rice pollen and that starch is an essential requirement for successful fertilization in rice.

  11. Plastidic phosphoglucomutase and ADP-glucose pyrophosphorylase mutants impair starch synthesis in rice pollen grains and cause male sterility

    PubMed Central

    Lee, Sang-Kyu; Eom, Joon-Seob; Hwang, Seon-Kap; Shin, Dongjin; An, Gynheung; Okita, Thomas W.; Jeon, Jong-Seong

    2016-01-01

    To elucidate the starch synthesis pathway and the role of this reserve in rice pollen, we characterized mutations in the plastidic phosphoglucomutase, OspPGM, and the plastidic large subunit of ADP-glucose (ADP-Glc) pyrophosphorylase, OsAGPL4. Both genes were up-regulated in maturing pollen, a stage when starch begins to accumulate. Progeny analysis of self-pollinated heterozygous lines carrying the OspPGM mutant alleles, osppgm-1 and osppgm-2, or the OsAGPL4 mutant allele, osagpl4-1, as well as reciprocal crosses between the wild type (WT) and heterozygotes revealed that loss of OspPGM or OsAGPL4 caused male sterility, with the former condition rescued by the introduction of the WT OspPGM gene. While iodine staining and transmission electron microscopy analyses of pollen grains from homozygous osppgm-1 lines produced by anther culture confirmed the starch null phenotype, pollen from homozygous osagpl4 mutant lines, osagpl4-2 and osagpl4-3, generated by the CRISPR/Cas system, accumulated small amounts of starch which were sufficient to produce viable seed. Such osagpl4 mutant pollen, however, was unable to compete against WT pollen successfully, validating the important role of this reserve in fertilization. Our results demonstrate that starch is mainly polymerized from ADP-Glc synthesized from plastidic hexose phosphates in rice pollen and that starch is an essential requirement for successful fertilization in rice. PMID:27588462

  12. Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

    PubMed Central

    Niehues, Sven; Bussmann, Julia; Steffes, Georg; Erdmann, Ines; Köhrer, Caroline; Sun, Litao; Wagner, Marina; Schäfer, Kerstin; Wang, Guangxia; Koerdt, Sophia N.; Stum, Morgane; RajBhandary, Uttam L.; Thomas, Ulrich; Aberle, Hermann; Burgess, Robert W.; Yang, Xiang-Lei; Dieterich, Daniela; Storkebaum, Erik

    2015-01-01

    Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNAGly aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT. PMID:26138142

  13. Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases.

    PubMed

    Niehues, Sven; Bussmann, Julia; Steffes, Georg; Erdmann, Ines; Köhrer, Caroline; Sun, Litao; Wagner, Marina; Schäfer, Kerstin; Wang, Guangxia; Koerdt, Sophia N; Stum, Morgane; Jaiswal, Sumit; RajBhandary, Uttam L; Thomas, Ulrich; Aberle, Hermann; Burgess, Robert W; Yang, Xiang-Lei; Dieterich, Daniela; Storkebaum, Erik

    2015-07-03

    Dominant mutations in five tRNA synthetases cause Charcot-Marie-Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA(Gly) aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.

  14. Mutant SNAP25B causes myasthenia, cortical hyperexcitability, ataxia, and intellectual disability

    PubMed Central

    Selcen, Duygu; Brengman, Joan

    2014-01-01

    Objective: To identify and characterize the molecular basis of a syndrome associated with myasthenia, cortical hyperexcitability, cerebellar ataxia, and intellectual disability. Methods: We performed in vitro microelectrode studies of neuromuscular transmission, performed exome and Sanger sequencing, and analyzed functional consequences of the identified mutation in expression studies. Results: Neuromuscular transmission at patient endplates was compromised by reduced evoked quantal release. Exome sequencing identified a dominant de novo variant, p.Ile67Asn, in SNAP25B, a SNARE protein essential for exocytosis of synaptic vesicles from nerve terminals and of dense-core vesicles from endocrine cells. Ca2+-triggered exocytosis is initiated when synaptobrevin attached to synaptic vesicles (v-SNARE) assembles with SNAP25B and syntaxin anchored in the presynaptic membrane (t-SNAREs) into an α-helical coiled-coil held together by hydrophobic interactions. Pathogenicity of the Ile67Asn mutation was confirmed by 2 measures. First, the Ca2+ triggered fusion of liposomes incorporating v-SNARE with liposomes containing t-SNAREs was hindered when t-SNAREs harbored the mutant SNAP25B moiety. Second, depolarization of bovine chromaffin cells transfected with mutant SNAP25B or with mutant plus wild-type SNAP25B markedly reduced depolarization-evoked exocytosis compared with wild-type transfected cells. Conclusion: Ile67Asn variant in SNAP25B is pathogenic because it inhibits synaptic vesicle exocytosis. We attribute the deleterious effects of the mutation to disruption of the hydrophobic α-helical coiled-coil structure of the SNARE complex by replacement of a highly hydrophobic isoleucine by a strongly hydrophilic asparagine. PMID:25381298

  15. Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells.

    PubMed

    Chornyy, Sergiy; Parkhomenko, Julia; Chorna, Nataliya

    2007-01-01

    Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.

  16. Cystic fibrosis–adapted Pseudomonas aeruginosa quorum sensing lasR mutants cause hyperinflammatory responses

    PubMed Central

    LaFayette, Shantelle L.; Houle, Daniel; Beaudoin, Trevor; Wojewodka, Gabriella; Radzioch, Danuta; Hoffman, Lucas R.; Burns, Jane L.; Dandekar, Ajai A.; Smalley, Nicole E.; Chandler, Josephine R.; Zlosnik, James E.; Speert, David P.; Bernier, Joanie; Matouk, Elias; Brochiero, Emmanuelle; Rousseau, Simon; Nguyen, Dao

    2015-01-01

    Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease–dependent cytokine degradation. In subacute pulmonary infections, lasR mutant–infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients. PMID:26457326

  17. Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli.

    PubMed Central

    Alix, J H; Guérin, M F

    1993-01-01

    To determine whether the biogenesis of ribosomes in Escherichia coli is the result of the self-assembly of their different constituents or involves the participation of additional factors, we have studied the influence of a chaperone, the product of the gene dnaK, on ribosome assembly in vivo. Using three thermosensitive (ts) mutants carrying the mutations dnaK756-ts, dnaK25-ts, and dnaK103-ts, we have observed the accumulation at nonpermissive temperature (45 degrees C) of ribosomal particles with different sedimentation constants--namely, 45S, 35S, and 25S along with the normal 30S and 50S ribosomal subunits. This is the result of a defect not in thermostability but in ribosome assembly at the nonpermissive temperature. These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature. PMID:8105482

  18. Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

    PubMed Central

    Vishnivetskiy, Sergey. A.; Ostermaier, Martin K.; Singhal, Ankita; Panneels, Valerie; Homan, Kristoff T.; Glukhova, Alisa; Sligar, Stephen G.; Tesmer, John J. G.; Schertler, Gebhard F.X.; Standfuss, Joerg; Gurevich, Vsevolod V.

    2013-01-01

    The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans. PMID:23872075

  19. DICER-LIKE3 Activity in Physcomitrella patens DICER-LIKE4 Mutants Causes Severe Developmental Dysfunction and Sterility

    PubMed Central

    Arif, M. Asif; Fattash, Isam; Cho, Sung Hyun; Beike, Anna K.; Reski, Ralf; Axtell, Michael J.; Frank, Wolfgang

    2012-01-01

    Trans-acting small interfering RNAs (ta-siRNAs) are plant-specific siRNAs released from TAS precursor transcripts after microRNA-dependent cleavage, conversion into double-stranded RNA, and Dicer-dependent phased processing. Like microRNAs (miRNAs), ta-siRNAs direct site-specific cleavage of target RNAs at sites of extensive complementarity. Here, we show that the DICER-LIKE 4 protein of Physcomitrella patens (PpDCL4) is essential for the biogenesis of 21 nucleotide (nt) ta-siRNAs. In ΔPpDCL4 mutants, off-sized 23 and 24-nt ta-siRNAs accumulated as the result of PpDCL3 activity. ΔPpDCL4 mutants display severe abnormalities throughout Physcomitrella development, including sterility, that were fully reversed in ΔPpDCL3/ΔPpDCL4 double-mutant plants. Therefore, PpDCL3 activity, not loss of PpDCL4 function per se, is the cause of the ΔPpDCL4 phenotypes. Additionally, we describe several new Physcomitrella trans-acting siRNA loci, three of which belong to a new family, TAS6. TAS6 loci are typified by sliced miR156 and miR529 target sites and are in close proximity to PpTAS3 loci. PMID:22511605

  20. Reversions to respiratory competence of omnipotent sup45 suppressor mutants may be caused by secondary sup45 mutations.

    PubMed

    Mironova, L N; Samsonova, M G; Zhouravleva, G A; Kulikov, V N; Soom, M J

    1995-02-01

    The molecular nature of the sup45 respiratory deficient omnipotent suppressor, and of three reversions to respiratory competence which removed the suppressor effect of the initial mutation, was examined. All reversions were caused by secondary sup45 mutations which indicates a direct connection between sup45 "respiratory" and "translational" functions. Computer analysis showed the local changes of Sup45 protein characteristics in the suppressor strain and revertants in comparison to the wild-type protein. The distribution of mutant sites in relation to evolutionary conserved, and tentatively functional, regions in the Sup45 protein is discussed.

  1. Common and new acyclovir resistant herpes simplex virus-1 mutants causing bilateral recurrent herpetic keratitis in an immunocompetent patient.

    PubMed

    Pan, Dongli; Kaye, Stephen B; Hopkins, Mark; Kirwan, Ruaidhri; Hart, Ian J; Coen, Donald M

    2014-02-01

    We investigated thymidine kinase (tk) mutants isolated during multiple episodes of recurrent bilateral acyclovir resistant herpes simplex keratitis in an immunocompetent patient. From one eye, we found a single guanine insertion, previously shown to greatly reduce TK expression, and from the other, a previously unidentified substitution, which genetic experiments confirmed confers drug resistance. The substitution, although distant from substrate binding sites, reduced thymidine phosphorylation 10-20-fold, and acyclovir phosphorylation >100-fold. This phenotype should permit reactivation from latency to cause recurrent disease. The results may have implications for the prevalence and prevention of acyclovir resistance in patients with herpes simplex keratitis.

  2. Premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy in Delta1 mutants.

    PubMed

    Schuster-Gossler, Karin; Cordes, Ralf; Gossler, Achim

    2007-01-09

    In vertebrates, skeletal myogenesis is initiated by the generation of myoblasts followed by their differentiation to myocytes and the formation of myofibers. The determination of myoblasts and their differentiation are controlled by muscle regulatory factors that are activated at specific stages during myogenesis. During late embryonic and fetal stages a distinct population of resident proliferating progenitor cells is the major source of myogenic cells. How the differentiation of myoblasts and progenitor cells is regulated is not clear. We show that in mouse embryos the Notch ligand Delta1 (Dll1) controls both differentiation of early myoblasts and maintenance of myogenic progenitor cells. Early dermomyotome-derived myoblasts are determined normally in Dll1 mutant embryos, but their differentiation is accelerated, leading to a transient excess of myotomal muscle fibers. Similarly, migratory hypaxial myogenic cells colonize the limb buds and activate muscle regulatory factor expression normally, but muscle differentiation progresses more rapidly. Resident progenitor cells defined by Pax3/Pax7 expression are formed initially, but they are progressively lost and virtually absent at embryonic day 14.5. Muscle growth declines beginning around embryonic day 12, leading to subsequent severe muscle hypotrophy in hypomorphic Dll1 fetuses. We suggest that premature and excessive differentiation leads to depletion of progenitor cells and cessation of muscle growth, and we conclude that Dll1 provides essential signals that are required to prevent uncontrolled differentiation early and ensure sustained muscle differentiation during development.

  3. Premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy in Delta1 mutants

    PubMed Central

    Schuster-Gossler, Karin; Cordes, Ralf; Gossler, Achim

    2007-01-01

    In vertebrates, skeletal myogenesis is initiated by the generation of myoblasts followed by their differentiation to myocytes and the formation of myofibers. The determination of myoblasts and their differentiation are controlled by muscle regulatory factors that are activated at specific stages during myogenesis. During late embryonic and fetal stages a distinct population of resident proliferating progenitor cells is the major source of myogenic cells. How the differentiation of myoblasts and progenitor cells is regulated is not clear. We show that in mouse embryos the Notch ligand Delta1 (Dll1) controls both differentiation of early myoblasts and maintenance of myogenic progenitor cells. Early dermomyotome-derived myoblasts are determined normally in Dll1 mutant embryos, but their differentiation is accelerated, leading to a transient excess of myotomal muscle fibers. Similarly, migratory hypaxial myogenic cells colonize the limb buds and activate muscle regulatory factor expression normally, but muscle differentiation progresses more rapidly. Resident progenitor cells defined by Pax3/Pax7 expression are formed initially, but they are progressively lost and virtually absent at embryonic day 14.5. Muscle growth declines beginning around embryonic day 12, leading to subsequent severe muscle hypotrophy in hypomorphic Dll1 fetuses. We suggest that premature and excessive differentiation leads to depletion of progenitor cells and cessation of muscle growth, and we conclude that Dll1 provides essential signals that are required to prevent uncontrolled differentiation early and ensure sustained muscle differentiation during development. PMID:17194759

  4. More than one mutant allele causes infantile Tay-Sachs disease in French-Canadians

    PubMed Central

    Hechtman, Peter; Kaplan, Feige; Bayleran, Janet; Boulay, Bernard; Andermann, Eva; de Braekeleer, Marc; Melançon, Serge; Lambert, Marie; Potier, Michel; Gagné, Richard; Kolodny, Edwin; Clow, Carol; Capua, Aniceta; Prevost, Claude; Scriver, Charles

    1990-01-01

    Two Tay-Sachs disease (TSD) patients of French-Canadian origin were shown by Myerowitz and Hogikyan to be homozygous for a 7.6-kb deletion mutation at the 5' end of the hexosaminidase A α-subunit gene. In order to determine whether all French-Canadian TSD patients were homozygotes for the deletion allele and to assess the geographic origins of TSD in this population, we ascertained 12 TSD families of French-Canadian origin and screened for occurrence of mutations associated with infantile TSD. DNA samples were obtained from 12 French-Canadian TSD families. Samples were analyzed using polymerase-chain-reaction (PCR) amplification followed by hybridization to allele-specific oligonucleotides (ASO) or by restriction analysis of PCR products. In some cases Southern analysis of genomic DNA was performed. Eighteen of the 22 independently segregating mutant chromosomes in this sample carried the 7.6-kb deletion mutation at the 5' end of the gene. One chromosome carried the 4-nucleotide insertion in exon 11 (a “Jewish” mutation). In this population no individuals were detected who had the substitution at the splice junction of exon 12 previously identified in Ashkenazi Jews. One chromosome carried an undescribed B1 mutation; this allele came from a parent of non-French-Canadian origin. Patients in three families carried TSD alleles different from any of the above mutations. The 5' deletion mutation clusters in persons originating in southeastern Quebec (Gaspé) and adjacent counties of northern New Brunswick. ImagesFigure 3Figure 4Figure 5Figure 6 PMID:2220821

  5. The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy

    SciTech Connect

    Li, Xiaochuan; Suphamungmee, Worawit; Janco, Miro; Geeves, Michael A.; Marston, Steven B.; Fischer, Stefan; Lehman, William

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Well-known tropomyosin mutants, D175N and E180G are linked to cardiomyopathies. Black-Right-Pointing-Pointer The structural mechanics of D175N and E180G tropomyosins have been investigated. Black-Right-Pointing-Pointer D175N and E180G mutations increase both local and global tropomyosin flexibility. Black-Right-Pointing-Pointer In muscle, this increased flexibility will enhance myosin interactions on actin. Black-Right-Pointing-Pointer Extra myosin interaction can alter cardiac Ca{sup 2+}-switching, leading to dysfunction. -- Abstract: Point mutations targeting muscle thin filament proteins are the cause of a number of cardiomyopathies. In many cases, biological effects of the mutations are well-documented, whereas their structural and mechanical impact on filament assembly and regulatory function is lacking. In order to elucidate molecular defects leading to cardiac dysfunction, we have examined the structural mechanics of two tropomyosin mutants, E180G and D175N, which are associated with hypertrophic cardiomyopathy (HCM). Tropomyosin is an {alpha}-helical coiled-coil dimer which polymerizes end-to-end to create an elongated superhelix that wraps around F-actin filaments of muscle and non-muscle cells, thus modulating the binding of other actin-binding proteins. Here, we study how flexibility changes in the E180G and D175N mutants might affect tropomyosin binding and regulatory motion on F-actin. Electron microscopy and Molecular Dynamics simulations show that E180G and D175N mutations cause an increase in bending flexibility of tropomyosin both locally and globally. This excess flexibility is likely to increase accessibility of the myosin-binding sites on F-actin, thus destabilizing the low-Ca{sup 2+} relaxed-state of cardiac muscle. The resulting imbalance in the on-off switching mechanism of the mutants will shift the regulatory equilibrium towards Ca{sup 2+}-activation of cardiac muscle, as is observed in affected

  6. Iron-mediated aggregation and a localized structural change characterize ferritin from a mutant light chain polypeptide that causes neurodegeneration.

    PubMed

    Baraibar, Martin A; Barbeito, Ana G; Muhoberac, Barry B; Vidal, Ruben

    2008-11-14

    Nucleotide insertions in the ferritin light chain (FTL) polypeptide gene cause hereditary ferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin and iron in the central nervous system. Here we describe for the first time the protein structure and iron storage function of the FTL mutant p.Phe167SerfsX26 (MT-FTL), which has a C terminus altered in sequence and extended in length. MT-FTL polypeptides assembled spontaneously into soluble, spherical 24-mers that were ultrastructurally indistinguishable from those of the wild type. Far-UV CD showed a decrease in alpha-helical content, and 8-anilino-1-naphthalenesulfonate fluorescence revealed the appearance of hydrophobic binding sites. Near-UV CD and proteolysis studies suggested little or no structural alteration outside of the C-terminal region. In contrast to wild type, MT-FTL homopolymers precipitated at much lower iron loading, had a diminished capacity to incorporate iron, and were less thermostable. However, precipitation was significantly reversed by addition of iron chelators both in vitro and in vivo. Our results reveal substantial protein conformational changes localized at the 4-fold pore of MT-FTL homopolymers and imply that the C terminus of the MT-FTL polypeptide plays an important role in ferritin solubility, stability, and iron management. We propose that the protrusion of some portion of the C terminus above the spherical shell allows it to cross-link with other mutant polypeptides through iron bridging, leading to enhanced mutant precipitation by iron. Our data suggest that hereditary ferritinopathy pathogenesis is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates.

  7. A pure chloride channel mutant of CLC-5 causes Dent's disease via insufficient V-ATPase activation.

    PubMed

    Satoh, Nobuhiko; Yamada, Hideomi; Yamazaki, Osamu; Suzuki, Masashi; Nakamura, Motonobu; Suzuki, Atsushi; Ashida, Akira; Yamamoto, Daisuke; Kaku, Yoshitsugu; Sekine, Takashi; Seki, George; Horita, Shoko

    2016-07-01

    Dent's disease is characterized by defective endocytosis in renal proximal tubules (PTs) and caused by mutations in the 2Cl(-)/H(+) exchanger, CLC-5. However, the pathological role of endosomal acidification in endocytosis has recently come into question. To clarify the mechanism of pathogenesis for Dent's disease, we examined the effects of a novel gating glutamate mutation, E211Q, on CLC-5 functions and endosomal acidification. In Xenopus oocytes, wild-type (WT) CLC-5 showed outward-rectifying currents that were inhibited by extracellular acidosis, but E211Q and an artificial pure Cl(-) channel mutant, E211A, showed linear currents that were insensitive to extracellular acidosis. Moreover, depolarizing pulse trains induced a robust reduction in the surface pH of oocytes expressing WT CLC-5 but not E211Q or E211A, indicating that the E211Q mutant functions as a pure Cl(-) channel similar to E211A. In HEK293 cells, E211A and E211Q stimulated endosomal acidification and hypotonicity-inducible vacuolar-type H(+)-ATPase (V-ATPase) activation at the plasma membrane. However, the stimulatory effects of these mutants were reduced compared with WT CLC-5. Furthermore, gene silencing experiments confirmed the functional coupling between V-ATPase and CLC-5 at the plasma membrane of isolated mouse PTs. These results reveal for the first time that the conversion of CLC-5 from a 2Cl(-)/H(+) exchanger into a Cl(-) channel induces Dent's disease in humans. In addition, defective endosomal acidification as a result of insufficient V-ATPase activation may still be important in the pathogenesis of Dent's disease.

  8. A Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming Intracellular Aggregates That Activate Apoptosis.

    PubMed

    Xiao, Xunjun; Jones, Gabrielle; Sevilla, Wednesday A; Stolz, Donna B; Magee, Kelsey E; Haughney, Margaret; Mukherjee, Amitava; Wang, Yan; Lowe, Mark E

    2016-10-28

    Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.

  9. Biochemical defects of mutant nudel alleles causing early developmental arrest or dorsalization of the Drosophila embryo.

    PubMed Central

    LeMosy, E K; Leclerc, C L; Hashimoto, C

    2000-01-01

    The nudel gene of Drosophila is maternally required both for structural integrity of the egg and for dorsoventral patterning of the embryo. It encodes a structurally modular protein that is secreted by ovarian follicle cells. Genetic and molecular studies have suggested that the Nudel protein is also functionally modular, with a serine protease domain that is specifically required for ventral development. Here we describe biochemical and immunolocalization studies that provide insight into the molecular basis for the distinct phenotypes produced by nudel mutations and for the interactions between these alleles. Mutations causing loss of embryonic dorsoventral polarity result in a failure to activate the protease domain of Nudel. Our analyses support previous findings that catalytic activity of the protease domain is required for dorsoventral patterning and that the Nudel protease is auto-activated and reveal an important role for a region adjacent to the protease domain in Nudel protease function. Mutations causing egg fragility and early embryonic arrest result in a significant decrease in extracellular Nudel protein, due to defects in post-translational processing, stability, or secretion. On the basis of these and other studies of serine proteases, we suggest potential mechanisms for the complementary and antagonistic interactions between the nudel alleles. PMID:10628985

  10. Podocyte-Specific Overexpression of Wild Type or Mutant Trpc6 in Mice Is Sufficient to Cause Glomerular Disease

    PubMed Central

    Kairath, Pamela; Carmona-Mora, Paulina; Molina, Jessica; Carpio, J. Daniel; Ruiz, Phillip; Mezzano, Sergio A.; Li, Jing; Wei, Changli; Reiser, Jochen; Young, Juan I.; Walz, Katherina

    2010-01-01

    Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2–3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease. PMID:20877463

  11. A Thermolabile Aldolase A Mutant Causes Fever-Induced Recurrent Rhabdomyolysis without Hemolytic Anemia

    PubMed Central

    Mamoune, Asmaa; Bahuau, Michel; Hamel, Yamina; Serre, Valérie; Pelosi, Michele; Habarou, Florence; Nguyen Morel, Marie-Ange; Boisson, Bertrand; Vergnaud, Sabrina; Viou, Mai Thao; Nonnenmacher, Luc; Piraud, Monique; Nusbaum, Patrick; Vamecq, Joseph; Romero, Norma; Ottolenghi, Chris; Casanova, Jean-Laurent; de Lonlay, Pascale

    2014-01-01

    Aldolase A deficiency has been reported as a rare cause of hemolytic anemia occasionally associated with myopathy. We identified a deleterious homozygous mutation in the ALDOA gene in 3 siblings with episodic rhabdomyolysis without hemolytic anemia. Myoglobinuria was always triggered by febrile illnesses. We show that the underlying mechanism involves an exacerbation of aldolase A deficiency at high temperatures that affected myoblasts but not erythrocytes. The aldolase A deficiency was rescued by arginine supplementation in vitro but not by glycerol, betaine or benzylhydantoin, three other known chaperones, suggesting that arginine-mediated rescue operated by a mechanism other than protein chaperoning. Lipid droplets accumulated in patient myoblasts relative to control and this was increased by cytokines, and reduced by dexamethasone. Our results expand the clinical spectrum of aldolase A deficiency to isolated temperature-dependent rhabdomyolysis, and suggest that thermolability may be tissue specific. We also propose a treatment for this severe disease. PMID:25392908

  12. A thermolabile aldolase A mutant causes fever-induced recurrent rhabdomyolysis without hemolytic anemia.

    PubMed

    Mamoune, Asmaa; Bahuau, Michel; Hamel, Yamina; Serre, Valérie; Pelosi, Michele; Habarou, Florence; Nguyen Morel, Marie-Ange; Boisson, Bertrand; Vergnaud, Sabrina; Viou, Mai Thao; Nonnenmacher, Luc; Piraud, Monique; Nusbaum, Patrick; Vamecq, Joseph; Romero, Norma; Ottolenghi, Chris; Casanova, Jean-Laurent; de Lonlay, Pascale

    2014-11-01

    Aldolase A deficiency has been reported as a rare cause of hemolytic anemia occasionally associated with myopathy. We identified a deleterious homozygous mutation in the ALDOA gene in 3 siblings with episodic rhabdomyolysis without hemolytic anemia. Myoglobinuria was always triggered by febrile illnesses. We show that the underlying mechanism involves an exacerbation of aldolase A deficiency at high temperatures that affected myoblasts but not erythrocytes. The aldolase A deficiency was rescued by arginine supplementation in vitro but not by glycerol, betaine or benzylhydantoin, three other known chaperones, suggesting that arginine-mediated rescue operated by a mechanism other than protein chaperoning. Lipid droplets accumulated in patient myoblasts relative to control and this was increased by cytokines, and reduced by dexamethasone. Our results expand the clinical spectrum of aldolase A deficiency to isolated temperature-dependent rhabdomyolysis, and suggest that thermolability may be tissue specific. We also propose a treatment for this severe disease.

  13. A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle.

    PubMed

    Nishi, Masumi; Yasue, Akihiro; Nishimatu, Shinichirou; Nohno, Tsutomu; Yamaoka, Takashi; Itakura, Mitsuo; Moriyama, Keiji; Ohuchi, Hideyo; Noji, Sumihare

    2002-04-26

    Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.

  14. Motor-Coordinative and Cognitive Dysfunction Caused by Mutant TDP-43 Could Be Reversed by Inhibiting Its Mitochondrial Localization.

    PubMed

    Wang, Wenzhang; Arakawa, Hiroyuki; Wang, Luwen; Okolo, Ogoegbunam; Siedlak, Sandra L; Jiang, Yinfei; Gao, Ju; Xie, Fei; Petersen, Robert B; Wang, Xinglong

    2017-01-04

    Dominant missense mutations in TAR DNA-binding protein 43 (TDP-43) cause amyotrophic lateral sclerosis (ALS), and the cytoplasmic accumulation of TDP-43 represents a pathological hallmark in ALS and frontotemporal lobar degeneration (FTD). Behavioral investigation of the transgenic mouse model expressing the disease-causing human TDP-43 M337V mutant (TDP-43(M337V) mice) is encumbered by premature death in homozygous transgenic mice and a reported lack of phenotype assessed by tail elevation and footprint in hemizygous transgenic mice. Here, using a battery of motor-coordinative and cognitive tests, we report robust motor-coordinative and cognitive deficits in hemizygous TDP-43(M337V) mice by 8 months of age. After 12 months of age, cortical neurons are significantly affected by the mild expression of mutant TDP-43, characterized by cytoplasmic TDP-43 mislocalization, mitochondrial dysfunction, and neuronal loss. Compared with age-matched non-transgenic mice, TDP-43(M337V) mice demonstrate a similar expression of total TDP-43 but higher levels of TDP-43 in mitochondria. Interestingly, a TDP-43 mitochondrial localization inhibitory peptide abolishes cytoplasmic TDP-43 accumulation, restores mitochondrial function, prevents neuronal loss, and alleviates motor-coordinative and cognitive deficits in adult hemizygous TDP-43(M337V) mice. Thus, this study suggests hemizygous TDP-43(M337V) mice as a useful animal model to study TDP-43 toxicity and further consolidates mitochondrial TDP-43 as a novel therapeutic target for TDP-43-linked neurodegenerative diseases. Published by Elsevier Inc.

  15. Temperature Sensitivity Caused by Mutant Release Factor 1 Is Suppressed by Mutations That Affect 16S rRNA Maturation

    PubMed Central

    Kaczanowska, Magdalena; Rydén-Aulin, Monica

    2004-01-01

    To study the effect of slow termination on the protein synthesizing machinery, we isolated suppressors to a temperature-sensitive release factor 1 (RF1). Of 26 independent clones, five complementation groups have been identified, two of which are presented here. The first mutation disrupts a base pair in the transcription terminator stem for the rplM-rpsI operon, which encodes ribosomal proteins L13 and S9. We have found that this leads to readthrough of the terminator and that lower levels of transcript (compared to the results seen with the wild type) are found in the cell. This probably leads to decreased expression of the two proteins. The second mutation is a small deletion of the yrdC open reading frame start site, and it is not likely that the protein is expressed. Both mutant strains show an increased accumulation of 17S rRNA (immature 16S rRNA). Maturation of 16S rRNA is dependent on proper assembly of the ribosomal proteins, a process that is disturbed when proteins are missing. The function of the YrdC protein is not known, but it is able to bind to double-stranded RNA; therefore, we suggest that it is an assembly factor important for 30S subunit biogenesis. On the basis of our findings, we propose that lesser amounts of S9 or a lack of YrdC causes the maturation defect. We have shown that as a consequence of the maturation defect, fewer 70S ribosomes and polysomes are formed. This and other results suggest that it is the lowered concentration of functional ribosomes that suppresses the temperature sensitivity caused by the mutant RF1. PMID:15126466

  16. Mutant huntingtin's interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington's disease.

    PubMed

    Shirendeb, Ulziibat P; Calkins, Marcus J; Manczak, Maria; Anekonda, Vishwanath; Dufour, Brett; McBride, Jodi L; Mao, Peizhong; Reddy, P Hemachandra

    2012-01-15

    The purpose of this study was to investigate the link between mutant huntingtin (Htt) and neuronal damage in relation to mitochondria in Huntington's disease (HD). In an earlier study, we determined the relationship between mutant Htt and mitochondrial dynamics/synaptic viability in HD patients. We found mitochondrial loss, abnormal mitochondrial dynamics and mutant Htt association with mitochondria in HD patients. In the current study, we sought to expand on our previous findings and further elucidate the relationship between mutant Htt and mitochondrial and synaptic deficiencies. We hypothesized that mutant Htt, in association with mitochondria, alters mitochondrial dynamics, leading to mitochondrial fragmentation and defective axonal transport of mitochondria in HD neurons. In this study, using postmortem HD brains and primary neurons from transgenic BACHD mice, we identified mutant Htt interaction with the mitochondrial protein Drp1 and factors that cause abnormal mitochondrial dynamics, including GTPase Drp1 enzymatic activity. Further, using primary neurons from BACHD mice, for the first time, we studied axonal transport of mitochondria and synaptic degeneration. We also investigated the effect of mutant Htt aggregates and oligomers in synaptic and mitochondrial deficiencies in postmortem HD brains and primary neurons from BACHD mice. We found that mutant Htt interacts with Drp1, elevates GTPase Drp1 enzymatic activity, increases abnormal mitochondrial dynamics and results in defective anterograde mitochondrial movement and synaptic deficiencies. These observations support our hypothesis and provide data that can be utilized to develop therapeutic targets that are capable of inhibiting mutant Htt interaction with Drp1, decreasing mitochondrial fragmentation, enhancing axonal transport of mitochondria and protecting synapses from toxic insults caused by mutant Htt.

  17. Structural and functional analysis of the ASM p.Ala359Asp mutant that causes acid sphingomyelinase deficiency.

    PubMed

    Acuña, Mariana; Castro-Fernández, Víctor; Latorre, Mauricio; Castro, Juan; Schuchman, Edward H; Guixé, Victoria; González, Mauricio; Zanlungo, Silvana

    2016-10-21

    Niemann-Pick disease (NPD) type A and B are recessive hereditary disorders caused by deficiency in acid sphingomyelinase (ASM). The p.Ala359Asp mutation has been described in several patients but its functional and structural effects in the protein are unknown. In order to characterize this mutation, we modeled the three-dimensional ASM structure using the recent available crystal of the mammalian ASM as a template. We found that the p.Ala359Asp mutation is localized in the hydrophobic core and far from the sphingomyelin binding site. However, energy function calculations using statistical potentials indicate that the mutation causes a decrease in ASM stability. Therefore, we investigated the functional effect of the p.Ala359Asp mutation in ASM expression, secretion, localization and activity in human fibroblasts. We found a 3.8% residual ASM activity compared to the wild-type enzyme, without changes in the other parameters evaluated. These results support the hypothesis that the p.Ala359Asp mutation causes structural alterations in the hydrophobic environment where ASM is located, decreasing its enzymatic activity. A similar effect was observed in other previously described NPDB mutations located outside the active site of the enzyme. This work shows the first full size ASM mutant model describe at date, providing a complete analysis of the structural and functional effects of the p.Ala359Asp mutation over the stability and activity of the enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons

    PubMed Central

    Trushina, Eugenia; Rana, Sandeep; McMurray, Cynthia T; Hua, Duy H

    2009-01-01

    Background Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-β (Aβ) aggregates in cell and mouse models representing Alzheimer's Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD. Results TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons. Conclusion We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD. PMID:19586540

  19. Effect of trehalose on the properties of mutant {gamma}PKC, which causes spinocerebellar ataxia type 14, in neuronal cell lines and cultured Purkinje cells.

    PubMed

    Seki, Takahiro; Abe-Seki, Nana; Kikawada, Takahiro; Takahashi, Hideyuki; Yamamoto, Kazuhiro; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2010-10-22

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation, which induces apoptotic cell death. The disaccharide trehalose has been reported to inhibit aggregate formation and to alleviate symptoms in cellular and animal models of Huntington disease, Alzheimer disease, and prion disease. Here, we show that trehalose can be incorporated into SH-SY5Y cells and reduces the aggregation of mutant γPKC-GFP, thereby inhibiting apoptotic cell death in SH-SY5Y cells and primary cultured Purkinje cells (PCs). Trehalose acts by directly stabilizing the conformation of mutant γPKC without affecting protein turnover. Trehalose was also found to alleviate the improper development of dendrites in PCs expressing mutant γPKC-GFP without aggregates but not in PCs with aggregates. In PCs without aggregates, trehalose improves the mobility and translocation of mutant γPKC-GFP, probably by inhibiting oligomerization and thereby alleviating the improper development of dendrites. These results suggest that trehalose counteracts various cellular dysfunctions that are triggered by mutant γPKC in both neuronal cell lines and primary cultured PCs by inhibiting oligomerization and aggregation of mutant γPKC.

  20. The Role of Protein Denaturation Energetics and Molecular Chaperones in the Aggregation and Mistargeting of Mutants Causing Primary Hyperoxaluria Type I

    PubMed Central

    Riverol, Debora; Yunta, Cristina; Albert, Armando; Salido, Eduardo; Pey, Angel L.

    2013-01-01

    Primary hyperoxaluria type I (PH1) is a conformational disease which result in the loss of alanine:glyoxylate aminotransferase (AGT) function. The study of AGT has important implications for protein folding and trafficking because PH1 mutants may cause protein aggregation and mitochondrial mistargeting. We herein describe a multidisciplinary study aimed to understand the molecular basis of protein aggregation and mistargeting in PH1 by studying twelve AGT variants. Expression studies in cell cultures reveal strong protein folding defects in PH1 causing mutants leading to enhanced aggregation, and in two cases, mitochondrial mistargeting. Immunoprecipitation studies in a cell-free system reveal that most mutants enhance the interactions with Hsc70 chaperones along their folding process, while in vitro binding experiments show no changes in the interaction of folded AGT dimers with the peroxisomal receptor Pex5p. Thermal denaturation studies by calorimetry support that PH1 causing mutants often kinetically destabilize the folded apo-protein through significant changes in the denaturation free energy barrier, whereas coenzyme binding overcomes this destabilization. Modeling of the mutations on a 1.9 Å crystal structure suggests that PH1 causing mutants perturb locally the native structure. Our work support that a misbalance between denaturation energetics and interactions with chaperones underlie aggregation and mistargeting in PH1, suggesting that native state stabilizers and protein homeostasis modulators are potential drugs to restore the complex and delicate balance of AGT protein homeostasis in PH1. PMID:24205397

  1. A missense mutation P136L in the arylsulfatase A gene causes instability and loss of activity of the mutant enzyme.

    PubMed

    Kafert, S; Heinisch, U; Zlotogora, J; Gieselmann, V

    1995-02-01

    Metachromatic leukodystrophy is a lysosomal storage disease caused by deficiency of arylsulfatase A. Sequencing of the arylsulfatase A genes of an Ashkenazi Jewish patient suffering from the severe late infantile form of the disease revealed a point mutation in exon 2 causing proline 136 to be substituted by leucine. The patient was homozygous for this mutation. Studies on Ltk- cells stably expressing the mutant enzyme show that the mutation causes complete loss of enzyme activity and rapid degradation in an early biosynthetic compartment.

  2. Brassinosteroid deficiency due to truncated steroid 5alpha-reductase causes dwarfism in the lk mutant of pea.

    PubMed

    Nomura, Takahito; Jager, Corinne E; Kitasaka, Yukiko; Takeuchi, Keiichi; Fukami, Motohiro; Yoneyama, Koichi; Matsushita, Yasuhiko; Nyunoya, Hiroshi; Takatsuto, Suguru; Fujioka, Shozo; Smith, Jennifer J; Kerckhoffs, L Huub J; Reid, James B; Yokota, Takao

    2004-08-01

    The endogenous brassinosteroids in the dwarf mutant lk of pea (Pisum sativum) were quantified by gas chromatography-selected ion monitoring. The levels of castasterone, 6-deoxocastasterone, and 6-deoxotyphasterol in lk shoots were reduced 4-, 70-, and 6-fold, respectively, compared with those of the wild type. The fact that the application of brassinolide restored the growth of the mutant indicated that the dwarf mutant lk is brassinosteroid deficient. Gas chromatography-selected ion monitoring analysis of the endogenous sterols in lk shoots revealed that the levels of campestanol and sitostanol were reduced 160- and 10-fold, respectively, compared with those of wild-type plants. These data, along with metabolic studies, showed that the lk mutant has a defect in the conversion of campest-4-en-3-one to 5alpha-campestan-3-one, which is a key hydrogenation step in the synthesis of campestanol from campesterol. This defect is the same as that found in the Arabidopsis det2 mutant and the Ipomoea nil kbt mutant. The pea gene homologous to the DET2 gene, PsDET2, was cloned, and it was found that the lk mutation would result in a putative truncated PsDET2 protein. Thus it was concluded that the short stature of the lk mutant is due to a defect in the steroidal 5alpha-reductase gene. This defect was also observed in the callus induced from the lk mutant. Biosynthetic pathways involved in the conversion of campesterol to campestanol are discussed in detail.

  3. Mutant HSPB1 overexpression in neurons is sufficient to cause age-related motor neuronopathy in mice

    PubMed Central

    Srivastava, Amit K.; Renusch, Samantha R.; Naiman, Nicole E.; Gu, Shuping; Sneh, Amita; Arnold, W. David; Sahenk, Zarife; Kolb, Stephen J.

    2012-01-01

    The small heat shock protein HSPB1 is a multifunctional, α-crystallin-based protein that has been shown to be neuroprotective in animal models of motor neuron disease and peripheral nerve injury. Missense mutations in HSPB1 result in axonal Charcot-Marie-Tooth disease with minimal sensory involvement (CMT2F) and distal hereditary motor neuropathy type 2 (dHMN-II). These disorders are characterized by a selective loss of motor axons in peripheral nerve resulting in distal muscle weakness and often severe disability. To investigate the pathogenic mechanisms of HSPB1 mutations in motor neurons in vivo, we have developed and characterized transgenic PrP-HSPB1 and PrP-HSPB1(R136W) mice. These mice express the human HSPB1 protein throughout the nervous system including in axons of peripheral nerve. Although both mouse strains lacked obvious motor deficits, the PrP-HSPB1(R136W) mice developed an age-dependent motor axonopathy. Mutant mice showed axonal pathology in spinal cord and peripheral nerve with evidence of impaired neurofilament cytoskeleton, associated with organelle accumulation. Accompanying these findings, increases in the number of Schmidt-Lanterman incisures, as evidence of impaired axon-Schwann cell interactions, were present. These observations suggest that overexpression of HSPB1(R136W) in neurons is sufficient to cause pathological and electrophysiological changes in mice that are seen in patients with hereditary motor neuropathy. PMID:22521462

  4. Transgenic expression of the N525S-tuberin variant in Tsc2 mutant (Eker) rats causes dominant embryonic lethality

    PubMed Central

    Shiono, Masatoshi; Kobayashi, Toshiyuki; Takahashi, Riichi; Ueda, Masatsugu; Ishioka, Chikashi; Hino, Okio

    2014-01-01

    The Tsc2 product, tuberin, negatively regulates the mTOR pathway. We have exploited the Eker (Tsc2-mutant) rat system to analyse various Tsc2 mutations. Here, we focus on the N525S-Tsc2 variant (NSM), which is known to cause distinct symptoms in patients even though normal suppression of mTOR is observed. Unexpectedly, we were repeatedly unable to generate viable rats carrying the NSM transgene. Genotypic analysis revealed that most of the embryos carrying the transgene died around embryonic day after 14.5—similar to the stage of lethality observed for Eker homozygotes. Thus, the NSM transgene appeared to have a dominant lethal effect in our rat model. Further, no significant differences were observed for various signal transduction molecules in transiently expressed NSM cells compared to WT. These results indicate that a non-mTOR pathway, critical for embryogenesis, is being regulated by tuberin, providing a link between tuberin expression and the severity of Tsc2 mutation-related pathogenesis. PMID:25088526

  5. Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

    PubMed

    Firsov, D; Schild, L; Gautschi, I; Mérillat, A M; Schneeberger, E; Rossier, B C

    1996-12-24

    The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

  6. Mutant GlialCAM Causes Megalencephalic Leukoencephalopathy with Subcortical Cysts, Benign Familial Macrocephaly, and Macrocephaly with Retardation and Autism

    PubMed Central

    López-Hernández, Tania; Ridder, Margreet C.; Montolio, Marisol; Capdevila-Nortes, Xavier; Polder, Emiel; Sirisi, Sònia; Duarri, Anna; Schulte, Uwe; Fakler, Bernd; Nunes, Virginia; Scheper, Gert C.; Martínez, Albert; Estévez, Raúl; van der Knaap, Marjo S.

    2011-01-01

    Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We recently showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule that is also called HepaCAM and is encoded by HEPACAM, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different HEPACAM mutations. Ten patients with the classical, deteriorating phenotype had two mutations, and 18 patients with the improving phenotype had one mutation. Most parents with a single mutation had macrocephaly, indicating dominant inheritance. In some families with dominant HEPACAM mutations, the clinical picture and magnetic resonance imaging normalized, indicating that HEPACAM mutations can cause benign familial macrocephaly. In other families with dominant HEPACAM mutations, patients had macrocephaly and mental retardation with or without autism. Further experiments demonstrated that GlialCAM and MLC1 both localize in axons and colocalize in junctions between astrocytes. GlialCAM is additionally located in myelin. Mutant GlialCAM disrupts the localization of MLC1-GlialCAM complexes in astrocytic junctions in a manner reflecting the mode of inheritance. In conclusion, GlialCAM is required for proper localization of MLC1. HEPACAM is the second gene found to be mutated in MLC. Dominant HEPACAM mutations can cause either macrocephaly and mental retardation with or without autism or benign familial macrocephaly. PMID:21419380

  7. Congenital Cataract Causing Mutants of αA-Crystallin/sHSP Form Aggregates and Aggresomes Degraded through Ubiquitin-Proteasome Pathway

    PubMed Central

    Raju, Ilangovan; Abraham, Edathara C.

    2011-01-01

    Background Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy. Methodology/Principal Findings YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control. Conclusions/Significance Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells

  8. RNA polymerase III mutants in TFIIFα-like C37 that cause terminator readthrough with no decrease in transcription output

    PubMed Central

    Rijal, Keshab; Maraia, Richard J.

    2013-01-01

    How eukaryotic RNA polymerases switch from elongation to termination is unknown. Pol III subunits Rpc53 and Rpc37 (C53/37) form a heterodimer homologous to TFIIFβ/α. C53/37 promotes efficient termination and together with C11 also mediates pol III recycling in vitro. We previously developed Schizosaccharomyces pombe strains that report on two pol III termination activities: RNA oligo(U) 3′-end cleavage, and terminator readthrough. We randomly mutagenized C53 and C37 and isolated many C37 mutants with terminator readthrough but no comparable C53 mutants. The majority of C37 mutants have strong phenotypes with up to 40% readthrough and map to a C-terminal tract previously localized near Rpc2p in the pol III active center while a minority represent a distinct class with weaker phenotype, less readthrough and 3′-oligo(U) lengthening. Nascent pre-tRNAs released from a terminator by C37 mutants have shorter 3′-oligo(U) tracts than in cleavage-deficient C11 double mutants indicating RNA 3′-end cleavage during termination. We asked whether termination deficiency affects transcription output in the mutants in vivo both by monitoring intron-containing nascent transcript levels and 14C-uridine incorporation. Surprisingly, multiple termination mutants have no decrease in transcript output relative to controls. These data are discussed in context of current models of pol III transcription. PMID:23093604

  9. JAK3 mutants transform hematopoietic cells through JAK1 activation, causing T-cell acute lymphoblastic leukemia in a mouse model.

    PubMed

    Degryse, Sandrine; de Bock, Charles E; Cox, Luk; Demeyer, Sofie; Gielen, Olga; Mentens, Nicole; Jacobs, Kris; Geerdens, Ellen; Gianfelici, Valentina; Hulselmans, Gert; Fiers, Mark; Aerts, Stein; Meijerink, Jules P; Tousseyn, Thomas; Cools, Jan

    2014-11-13

    JAK3 is a tyrosine kinase that associates with the common γ chain of cytokine receptors and is recurrently mutated in T-cell acute lymphoblastic leukemia (T-ALL). We tested the transforming properties of JAK3 pseudokinase and kinase domain mutants using in vitro and in vivo assays. Most, but not all, JAK3 mutants transformed cytokine-dependent Ba/F3 or MOHITO cell lines to cytokine-independent proliferation. JAK3 pseudokinase mutants were dependent on Jak1 kinase activity for cellular transformation, whereas the JAK3 kinase domain mutant could transform cells in a Jak1 kinase-independent manner. Reconstitution of the IL7 receptor signaling complex in 293T cells showed that JAK3 mutants required receptor binding to mediate downstream STAT5 phosphorylation. Mice transplanted with bone marrow progenitor cells expressing JAK3 mutants developed a long-latency transplantable T-ALL-like disease, characterized by an accumulation of immature CD8(+) T cells. In vivo treatment of leukemic mice with the JAK3 selective inhibitor tofacitinib reduced the white blood cell count and caused leukemic cell apoptosis. Our data show that JAK3 mutations are drivers of T-ALL and require the cytokine receptor complex for transformation. These results warrant further investigation of JAK1/JAK3 inhibitors for the treatment of T-ALL.

  10. Brassinosteroid Deficiency Due to Truncated Steroid 5α-Reductase Causes Dwarfism in the lk Mutant of Pea1

    PubMed Central

    Nomura, Takahito; Jager, Corinne E.; Kitasaka, Yukiko; Takeuchi, Keiichi; Fukami, Motohiro; Yoneyama, Koichi; Matsushita, Yasuhiko; Nyunoya, Hiroshi; Takatsuto, Suguru; Fujioka, Shozo; Smith, Jennifer J.; Kerckhoffs, L. Huub J.; Reid, James B.; Yokota, Takao

    2004-01-01

    The endogenous brassinosteroids in the dwarf mutant lk of pea (Pisum sativum) were quantified by gas chromatography-selected ion monitoring. The levels of castasterone, 6-deoxocastasterone, and 6-deoxotyphasterol in lk shoots were reduced 4-, 70-, and 6-fold, respectively, compared with those of the wild type. The fact that the application of brassinolide restored the growth of the mutant indicated that the dwarf mutant lk is brassinosteroid deficient. Gas chromatography-selected ion monitoring analysis of the endogenous sterols in lk shoots revealed that the levels of campestanol and sitostanol were reduced 160- and 10-fold, respectively, compared with those of wild-type plants. These data, along with metabolic studies, showed that the lk mutant has a defect in the conversion of campest-4-en-3-one to 5α-campestan-3-one, which is a key hydrogenation step in the synthesis of campestanol from campesterol. This defect is the same as that found in the Arabidopsis det2 mutant and the Ipomoea nil kbt mutant. The pea gene homologous to the DET2 gene, PsDET2, was cloned, and it was found that the lk mutation would result in a putative truncated PsDET2 protein. Thus it was concluded that the short stature of the lk mutant is due to a defect in the steroidal 5α-reductase gene. This defect was also observed in the callus induced from the lk mutant. Biosynthetic pathways involved in the conversion of campesterol to campestanol are discussed in detail. PMID:15286289

  11. Special AT-rich Binding Protein-2 (SATB2) Differentially Affects Disease-causing p63 Mutant Proteins*

    PubMed Central

    Chung, Jacky; Grant, R. Ian; Kaplan, David R.; Irwin, Meredith S.

    2011-01-01

    p63, a p53 family member, is critical for proper skin and limb development and directly regulates gene expression in the ectoderm. Mice lacking p63 exhibit skin and craniofacial defects including cleft palate. In humans p63 mutations are associated with several distinct developmental syndromes. p63 sterile-α-motif domain, AEC (ankyloblepharon-ectodermal dysplasia-clefting)-associated mutations are associated with a high prevalence of orofacial clefting disorders, which are less common in EEC (ectrodactyly-ectodermal dysplasia-clefting) patients with DNA binding domain p63 mutations. However, the mechanisms by which these mutations differentially influence p63 function remain unclear, and interactions with other proteins implicated in craniofacial development have not been identified. Here, we show that AEC p63 mutations affect the ability of the p63 protein to interact with special AT-rich binding protein-2 (SATB2), which has recently also been implicated in the development of cleft palate. p63 and SATB2 are co-expressed early in development in the ectoderm of the first and second branchial arches, two essential sites where signaling is required for craniofacial patterning. SATB2 attenuates p63-mediated gene expression of perp (p53 apoptosis effector related to PMP-22), a critical downstream target gene during development, and specifically decreases p63 perp promoter binding. Interestingly, AEC but not EEC p63 mutations affect the ability of p63 to interact with SATB2 and the inhibitory effects of SATB2 on p63 transactivation of perp are most pronounced for AEC-associated p63 mutations. Our findings reveal a novel gain-of-function property of AEC-causing p63 mutations and identify SATB2 as the first p63 binding partner that differentially influences AEC and EEC p63 mutant proteins. PMID:21965674

  12. Identification of p38 MAPK and JNK as new targets for correction of Wilson disease‐causing ATP7B mutants

    PubMed Central

    Chesi, Giancarlo; Hegde, Ramanath N.; Iacobacci, Simona; Concilli, Mafalda; Parashuraman, Seetharaman; Festa, Beatrice Paola; Polishchuk, Elena V.; Di Tullio, Giuseppe; Carissimo, Annamaria; Montefusco, Sandro; Canetti, Diana; Monti, Maria; Amoresano, Angela; Pucci, Piero; van de Sluis, Bart; Lutsenko, Svetlana

    2016-01-01

    Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans‐Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum‐retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c‐Jun N‐terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7BH1069Q (as well as that of several other WD‐causing mutants) from the endoplasmic reticulum to the trans‐Golgi network compartment, in recovery of its Cu‐dependent trafficking, and in reduction of intracellular Cu levels. Conclusion: Our findings indicate p38 and c‐Jun N‐terminal kinase as intriguing targets for correction of WD‐causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD. (Hepatology 2016;63:1842‐1859) PMID:26660341

  13. Identification of p38 MAPK and JNK as new targets for correction of Wilson disease-causing ATP7B mutants.

    PubMed

    Chesi, Giancarlo; Hegde, Ramanath N; Iacobacci, Simona; Concilli, Mafalda; Parashuraman, Seetharaman; Festa, Beatrice Paola; Polishchuk, Elena V; Di Tullio, Giuseppe; Carissimo, Annamaria; Montefusco, Sandro; Canetti, Diana; Monti, Maria; Amoresano, Angela; Pucci, Piero; van de Sluis, Bart; Lutsenko, Svetlana; Luini, Alberto; Polishchuk, Roman S

    2016-06-01

    Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans-Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum-retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c-Jun N-terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7B(H1069Q) (as well as that of several other WD-causing mutants) from the endoplasmic reticulum to the trans-Golgi network compartment, in recovery of its Cu-dependent trafficking, and in reduction of intracellular Cu levels. Our findings indicate p38 and c-Jun N-terminal kinase as intriguing targets for correction of WD-causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD. (Hepatology 2016;63:1842-1859). © 2015 by The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

  14. Unraveling of the E-helices and Disruption of 4-Fold Pores Are Associated with Iron Mishandling in a Mutant Ferritin Causing Neurodegeneration

    SciTech Connect

    Baraibar, Martin A.; Muhoberac, Barry B.; Garringer, Holly J.; Hurley, Thomas D.; Vidal, Ruben

    2010-03-12

    Mutations in the coding sequence of the ferritin light chain (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy, which is characterized by the presence of intracellular inclusion bodies containing the mutant FTL polypeptide and by abnormal accumulation of iron in the brain. Here, we describe the x-ray crystallographic structure and report functional studies of ferritin homopolymers formed from the mutant FTL polypeptide p.Phe167SerfsX26, which has a C terminus that is altered in amino acid sequence and length. The structure was determined and refined to 2.85 {angstrom} resolution and was very similar to the wild type between residues Ile-5 and Arg-154. However, instead of the E-helices normally present in wild type ferritin, the C-terminal sequences of all 24 mutant subunits showed substantial amounts of disorder, leading to multiple C-terminal polypeptide conformations and a large disruption of the normally tiny 4-fold axis pores. Functional studies underscored the importance of the mutant C-terminal sequence in iron-induced precipitation and revealed iron mishandling by soluble mutant FTL homopolymers in that only wild type incorporated iron when in direct competition in solution with mutant ferritin. Even without competition, the amount of iron incorporation over the first few minutes differed severalfold. Our data suggest that disruption at the 4-fold pores may lead to direct iron mishandling through attenuated iron incorporation by the soluble form of mutant ferritin and that the disordered C-terminal polypeptides may play a major role in iron-induced precipitation and formation of ferritin inclusion bodies in hereditary ferritinopathy.

  15. Improved plasma membrane expression of the trafficking defective P344R mutant of muscle, skeletal, receptor tyrosine kinase (MuSK) causing congenital myasthenic syndrome.

    PubMed

    Milhem, Reham M; Al-Gazali, Lihadh; Ali, Bassam R

    2015-03-01

    Muscle, skeletal, receptor tyrosine kinase (MuSK) is a key organizer at the postsynaptic membrane and critical for proper development and maintenance of the neuromuscular junction. Mutations in MUSK result in congenital myasthenic syndrome (CMS). We hypothesized that the CMS-causing missense mutation (P344R), found within the cysteine-rich domain of the protein, will affect its conformational tertiary structure. Consequently, the protein will misfold, get retained in the endoplasmic reticulum (ER) and lose its biological function through degradation by the highly conserved ER associated degradation (ERAD) machinery. We report that P344R-MuSK mutant is trafficking-deficient when expressed at 37°C in HeLa, COS-7 and HEK293 cell lines. It colocalized with the ER marker calnexin in contrast to wild-type MuSK which localized to the plasma membrane. The N-glycosylation status of P344R-MuSK is that of an immature and not properly post-translationally modified protein. Inhibition of protein synthesis showed that the P344R mutant's half-life is shorter than wild-type MuSK protein. Proteasomal inhibition resulted in the stabilization of the mutant protein. The mutant protein is highly ubiquitinated compared to wild-type confirming targeting for proteasomal degradation. The mutant showed around 50% of its in vivo autophosphorylation activity. P344R-MuSK mutant's trafficking defect is correctable by culturing the expressing cells at 27°C. Moreover, chemical compounds namely 2.5% glycerol, 1% dimethyl sulfoxide, 10 μM thapsigargin and 1 μM curcumin improved the maturation and exit of the mutant protein from the ER. These findings open perspectives for potential therapeutic intervention for patients with CMS harboring the P344R-MuSK mutation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.

    PubMed

    Komorisono, Masahiko; Ueguchi-Tanaka, Miyako; Aichi, Ikuko; Hasegawa, Yasuko; Ashikari, Motoyuki; Kitano, Hidemi; Matsuoka, Makoto; Sazuka, Takashi

    2005-08-01

    Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.

  17. Recovery of Nonpathogenic Mutant Bacteria from Tumors Caused by Several Agrobacterium tumefaciens Strains: a Frequent Event?▿

    PubMed Central

    Llop, Pablo; Murillo, Jesús; Lastra, Beatriz; López, María M.

    2009-01-01

    We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor. PMID:19700547

  18. Mirror movement-like defects in startle behavior of zebrafish dcc mutants are caused by aberrant midline guidance of identified descending hindbrain neurons.

    PubMed

    Jain, Roshan A; Bell, Hannah; Lim, Amy; Chien, Chi-Bin; Granato, Michael

    2014-02-19

    Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns.

  19. Mirror Movement-Like Defects in Startle Behavior of Zebrafish dcc Mutants Are Caused by Aberrant Midline Guidance of Identified Descending Hindbrain Neurons

    PubMed Central

    Jain, Roshan A.; Bell, Hannah; Lim, Amy; Chien, Chi-Bin

    2014-01-01

    Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns. PMID:24553931

  20. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W

    PubMed Central

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2014-01-01

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213

  1. Wing Defects in Drosophila xenicid Mutant Clones Are Caused by C-Terminal Deletion of Additional Sex Combs (Asx)

    PubMed Central

    Bischoff, Kara; Ballew, Anna C.; Simon, Michael A.; O'Reilly, Alana M.

    2009-01-01

    Background The coordinated action of genes that control patterning, cell fate determination, cell size, and cell adhesion is required for proper wing formation in Drosophila. Defects in any of these basic processes can lead to wing aberrations, including blisters. The xenicid mutation was originally identified in a screen designed to uncover regulators of adhesion between wing surfaces [1]. Principal Findings Here, we demonstrate that expression of the βPS integrin or the patterning protein Engrailed are not affected in developing wing imaginal discs in xenicid mutants. Instead, expression of the homeotic protein Ultrabithorax (Ubx) is strongly increased in xenicid mutant cells. Conclusion Our results suggest that upregulation of Ubx transforms cells from a wing blade fate to a haltere fate, and that the presence of haltere cells within the wing blade is the primary defect leading to the adult wing phenotypes observed. PMID:19956620

  2. Structural Mutants of the Spindle Pole Body Cause Distinct Alteration of Cytoplasmic Microtubules and Nuclear Dynamics in Multinucleated Hyphae

    PubMed Central

    Lang, Claudia; Grava, Sandrine; Finlayson, Mark; Trimble, Rhonda; Philippsen, Peter

    2010-01-01

    In the multinucleate fungus Ashbya gossypii, cytoplasmic microtubules (cMTs) emerge from the spindle pole body outer plaque (OP) in perpendicular and tangential directions. To elucidate the role of cMTs in forward/backward movements (oscillations) and bypassing of nuclei, we constructed mutants potentially affecting cMT nucleation or stability. Hyphae lacking the OP components AgSpc72, AgNud1, AgCnm67, or the microtubule-stabilizing factor AgStu2 grew like wild- type but showed substantial alterations in the number, length, and/or nucleation sites of cMTs. These mutants differently influenced nuclear oscillation and bypassing. In Agspc72Δ, only long cMTs were observed, which emanate tangentially from reduced OPs; nuclei mainly moved with the cytoplasmic stream but some performed rapid bypassing. Agnud1Δ and Agcnm67Δ lack OPs; short and long cMTs emerged from the spindle pole body bridge/half-bridge structures, explaining nuclear oscillation and bypassing in these mutants. In Agstu2Δ only very short cMTs emanated from structurally intact OPs; all nuclei moved with the cytoplasmic stream. Therefore, long tangential cMTs promote nuclear bypassing and short cMTs are important for nuclear oscillation. Our electron microscopy ultrastructural analysis also indicated that assembly of the OP occurs in a stepwise manner, starting with AgCnm67, followed by AgNud1 and lastly AgSpc72. PMID:20053682

  3. RETINAL GUANYLYL CYCLASE ISOZYME 1 IS THE PREFERENTIAL IN VIVO TARGET FOR CONSTITUTIVELY ACTIVE GCAP1 MUTANTS CAUSING CONGENITAL DEGENERATION OF PHOTORECEPTORS

    PubMed Central

    Olshevskaya, Elena V.; Peshenko, Igor V.; Savchenko, Andrey B.; Dizhoor, Alexander M.

    2012-01-01

    Two calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) activate cGMP synthesis in photoreceptor by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) to expedite recovery, but calcium-insensitive constitutively active GCAP1 mutants cause photoreceptor degeneration in human patients and in transgenic mice. Although GCAP1 and GCAP2 can both activate RetGC1 and RetGC2 in vitro, we find that GCAP1 selectively regulates RetGC1 in vivo. Furthermore, elimination of RetGC1 but not RetGC2 isozyme reverses abnormal calcium sensitivity of cGMP synthesis and rescues mouse rods in transgenic mice expressing GCAP1 mutants causing photoreceptor disease. Rods expressing mutant GCAP1 not only survive in the absence of RetGC1, but also remain functional, albeit with reduced electroretinography (ERG) amplitudes typical of RetGC1−/− genotype. The rod ERG recovery from a strong flash, only slightly affected in both RetGC1−/− and RetGC2−/− mice, becomes very slow in RetGC1−/− but not RetGC2−/− mice when GCAP2 is not available to provide Ca2+ feedback to the remaining RetGC isozyme. The intrinsic biochemical properties of RetGC and GCAP determined in vitro do not explain the observed phenomena. Instead, our results argue that there must be a cellular mechanism that limits GCAP1 access to RetGC2 and makes RetGC1 isozyme a preferential target for the disease-causing GCAP1 mutants. A more general conclusion from our findings is that non-discriminatory interactions between homologous effector enzymes and their regulatory proteins permitted by their intrinsic biochemical properties can be effectively restricted in a living photoreceptor. PMID:22623665

  4. Loss of Tropomodulin4 in the zebrafish mutant träge causes cytoplasmic rod formation and muscle weakness reminiscent of nemaline myopathy.

    PubMed

    Berger, Joachim; Tarakci, Hakan; Berger, Silke; Li, Mei; Hall, Thomas E; Arner, Anders; Currie, Peter D

    2014-12-01

    Nemaline myopathy is an inherited muscle disease that is mainly diagnosed by the presence of nemaline rods in muscle biopsies. Of the nine genes associated with the disease, five encode components of striated muscle sarcomeres. In a genetic zebrafish screen, the mutant träge (trg) was isolated based on its reduction in muscle birefringence, indicating muscle damage. Myofibres in trg appeared disorganised and showed inhomogeneous cytoplasmic eosin staining alongside malformed nuclei. Linkage analysis of trg combined with sequencing identified a nonsense mutation in tropomodulin4 (tmod4), a regulator of thin filament length and stability. Accordingly, although actin monomers polymerize to form thin filaments in the skeletal muscle of tmod4(trg) mutants, thin filaments often appeared to be dispersed throughout myofibres. Organised myofibrils with the typical striation rarely assemble, leading to severe muscle weakness, impaired locomotion and early death. Myofibrils of tmod4(trg) mutants often featured thin filaments of various lengths, widened Z-disks, undefined H-zones and electron-dense aggregations of various shapes and sizes. Importantly, Gomori trichrome staining and the lattice pattern of the detected cytoplasmic rods, together with the reactivity of rods with phalloidin and an antibody against actinin, is reminiscent of nemaline rods found in nemaline myopathy, suggesting that misregulation of thin filament length causes cytoplasmic rod formation in tmod4(trg) mutants. Although Tropomodulin4 has not been associated with myopathy, the results presented here implicateTMOD4 as a novel candidate for unresolved nemaline myopathies and suggest that the tmod4(trg) mutant will be a valuable tool to study human muscle disorders. © 2014. Published by The Company of Biologists Ltd.

  5. Loss of Tropomodulin4 in the zebrafish mutant träge causes cytoplasmic rod formation and muscle weakness reminiscent of nemaline myopathy

    PubMed Central

    Berger, Joachim; Tarakci, Hakan; Berger, Silke; Li, Mei; Hall, Thomas E.; Arner, Anders; Currie, Peter D.

    2014-01-01

    Nemaline myopathy is an inherited muscle disease that is mainly diagnosed by the presence of nemaline rods in muscle biopsies. Of the nine genes associated with the disease, five encode components of striated muscle sarcomeres. In a genetic zebrafish screen, the mutant träge (trg) was isolated based on its reduction in muscle birefringence, indicating muscle damage. Myofibres in trg appeared disorganised and showed inhomogeneous cytoplasmic eosin staining alongside malformed nuclei. Linkage analysis of trg combined with sequencing identified a nonsense mutation in tropomodulin4 (tmod4), a regulator of thin filament length and stability. Accordingly, although actin monomers polymerize to form thin filaments in the skeletal muscle of tmod4trg mutants, thin filaments often appeared to be dispersed throughout myofibres. Organised myofibrils with the typical striation rarely assemble, leading to severe muscle weakness, impaired locomotion and early death. Myofibrils of tmod4trg mutants often featured thin filaments of various lengths, widened Z-disks, undefined H-zones and electron-dense aggregations of various shapes and sizes. Importantly, Gomori trichrome staining and the lattice pattern of the detected cytoplasmic rods, together with the reactivity of rods with phalloidin and an antibody against actinin, is reminiscent of nemaline rods found in nemaline myopathy, suggesting that misregulation of thin filament length causes cytoplasmic rod formation in tmod4trg mutants. Although Tropomodulin4 has not been associated with myopathy, the results presented here implicateTMOD4 as a novel candidate for unresolved nemaline myopathies and suggest that the tmod4trg mutant will be a valuable tool to study human muscle disorders. PMID:25288681

  6. High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production.

    PubMed

    Blum, Ailisa; Benfield, Aurélie H; Stiller, Jiri; Kazan, Kemal; Batley, Jacqueline; Gardiner, Donald M

    2016-05-01

    Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  7. Mutant superoxide dismutase 1 (SOD1), a cause of amyotrophic lateral sclerosis, disrupts the recruitment of SMN, the spinal muscular atrophy protein to nuclear Cajal bodies.

    PubMed

    Kariya, Shingo; Re, Diane B; Jacquier, Arnaud; Nelson, Katelyn; Przedborski, Serge; Monani, Umrao R

    2012-08-01

    Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are among the most common motor neuron diseases to afflict the human population. A deficiency of the survival of motor neuron (SMN) protein causes SMA and is also reported to be an exacerbating factor in the development of ALS. However, pathways linking the two diseases have yet to be defined and it is not clear precisely how the pathology of ALS is aggravated by reduced SMN or whether mutant proteins underlying familial forms of ALS interfere with SMN-related biochemical pathways to exacerbate the neurodegenerative process. In this study, we show that mutant superoxide dismutase-1 (SOD1), a cause of familial ALS, profoundly alters the sub-cellular localization of the SMN protein, preventing the formation of nuclear 'gems' by disrupting the recruitment of the protein to Cajal bodies. Overexpressing the SMN protein in mutant SOD1 mice, a model of familial ALS, alleviates this phenomenon, most likely in a cell-autonomous manner, and significantly mitigates the loss of motor neurons in the spinal cord and in culture dishes. In the mice, the onset of the neuromuscular phenotype is delayed and motor function enhanced, suggestive of a therapeutic benefit for ALS patients treated with agents that augment the SMN protein. Nevertheless, this finding is tempered by an inability to prolong survival, a limitation most likely imposed by the inexorable denervation that characterizes ALS and eventually disrupts the neuromuscular synapses even in the presence of increased SMN.

  8. Discordant measures of androgen-binding kinetics in two mutant androgen receptors causing mild or partial androgen insensitivity, respectively.

    PubMed

    Shkolny, D L; Beitel, L K; Ginsberg, J; Pekeles, G; Arbour, L; Pinsky, L; Trifiro, M A

    1999-02-01

    We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (Kd) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k(diss)) for both androgens. In contrast, the MAIS subject's hAR (R871G) has k(diss) values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR's ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects' genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function. An increased k(diss) despite a normal Kd for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.

  9. Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer’s Disease-Causing Mutants in Presenilins

    PubMed Central

    Mak, Don-On Daniel; Cheung, King-Ho; Toglia, Patrick; Foskett, J. Kevin; Ullah, Ghanim

    2015-01-01

    Familial Alzheimer’s disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ release channels resulting in enhanced IP3R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP3R activity is considered to be the main reason behind the upregulation of intracellular Ca2+ signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca2+ signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP3R channel activity records obtained under optimal Ca2+ and multiple IP3 concentrations to gain deeper insights into the enhancement of IP3R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP3R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP3R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP3 and Ca2+ concentrations and quantify the sensitivity of IP3R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP3 and Ca2+ and that very small amount of IP3 is required to stimulate IP3R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP3 concentrations. We further demonstrate with simulations that the relatively longer time spent by IP3R in the H mode leads to the observed higher frequency of local Ca2+ signals, which can account for the more frequent global Ca2+ signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca2+ signals in cells expressing FAD-causing

  10. Detection of quantitative trait loci causing abnormal spermatogenesis and reduced testis weight in the small testis (Smt) mutant mouse.

    PubMed

    Bolor, Hasbaira; Wakasugi, Noboru; Zhao, Wei Dong; Ishikawa, Akira

    2006-04-01

    The small testis (Smt) mutant mouse is characterized by a small testis of one third to one half the size of a normal testis, and its spermatogenesis is mostly arrested at early stages of meiosis, although a small number of spermatocytes at the late prophase of meiosis and a few spermatids can sometimes be seen. We performed quantitative trait locus (QTL) analysis of these spermatogenic traits and testis weight using 221 F2 males obtained from a cross between Smt and MOM (Mus musculus molossinus) mice. At the genome-wide 5% level, we detected two QTLs affecting meiosis on chromosomes 4 and 13, and two QTLs for paired testis weight as a percentage of body weight on chromosomes 4 and X. In addition, we found several QTLs for degenerated germ cells and multinuclear giant cells on chromosomes 4, 7 and 13. Interestingly, for cell degeneration, the QTL on chromosome 13 interacted epistatically with the QTL on chromosome 4. These results reveal polygenic participation in the abnormal spermatogenesis and small testis size in the Smt mutant.

  11. A Novel Lamin A Mutant Responsible for Congenital Muscular Dystrophy Causes Distinct Abnormalities of the Cell Nucleus.

    PubMed

    Barateau, Alice; Vadrot, Nathalie; Vicart, Patrick; Ferreiro, Ana; Mayer, Michèle; Héron, Delphine; Vigouroux, Corinne; Buendia, Brigitte

    2017-01-01

    A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by LMNA. LMNA mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous LMNA p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient's skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient's mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other LMNA mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.

  12. Reduced immunogenicity of Arabidopsis hgl1 mutant N-glycans caused by altered accessibility of xylose and core fucose epitopes.

    PubMed

    Kaulfürst-Soboll, Heidi; Rips, Stephan; Koiwa, Hisashi; Kajiura, Hiroyuki; Fujiyama, Kazuhito; von Schaewen, Antje

    2011-07-01

    Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry β1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-β1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins.

  13. The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase, is caused by a deletion in the VRN1 gene.

    PubMed

    Shitsukawa, Naoki; Ikari, Chihiro; Shimada, Sanae; Kitagawa, Satoshi; Sakamoto, Koichi; Saito, Hiroyuki; Ryuto, Hiromichi; Fukunishi, Nobuhisa; Abe, Tomoko; Takumi, Shigeo; Nasuda, Shuhei; Murai, Koji

    2007-04-01

    The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase (mvp), was induced by nitrogen ion-beam treatment and was identified by its inability to transit from the vegetative to reproductive phase. In our previous study, we showed that WAP1 (wheat APETALA1) is a key gene in the regulatory pathway that controls phase transition from vegetative to reproductive growth in common wheat. WAP1 is an ortholog of the VRN1 gene that is responsible for vernalization insensitivity in einkorn wheat. The mvp mutation resulted from deletion of the VRN1 coding and promoter regions, demonstrating that WAP1/VRN1 is an indispensable gene for phase transition in wheat. Expression analysis of flowering-related genes in mvp plants indicated that wheat GIGANTIA (GI), CONSTANS (CO) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) genes either act upstream of or in a different pathway to WAP1/VRN1.

  14. A Novel Lamin A Mutant Responsible for Congenital Muscular Dystrophy Causes Distinct Abnormalities of the Cell Nucleus

    PubMed Central

    Barateau, Alice; Vadrot, Nathalie; Vicart, Patrick; Ferreiro, Ana; Mayer, Michèle; Héron, Delphine; Vigouroux, Corinne

    2017-01-01

    A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by LMNA. LMNA mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous LMNA p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient’s skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient’s mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other LMNA mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype. PMID:28125586

  15. Immunization with a highly attenuated replication-competent herpes simplex virus type 1 mutant, HF10, protects mice from genital disease caused by herpes simplex virus type 2.

    PubMed

    Luo, Chenhong; Goshima, Fumi; Kamakura, Maki; Mutoh, Yoshifumi; Iwata, Seiko; Kimura, Hiroshi; Nishiyama, Yukihiro

    2012-01-01

    Genital herpes is an intractable disease caused mainly by herpes simplex virus (HSV) type 2 (HSV-2), and is a major concern in public health. A previous infection with HSV type 1 (HSV-1) enhances protection against primary HSV-2 infection to some extent. In this study, we evaluated the ability of HF10, a naturally occurring replication-competent HSV-1 mutant, to protect against genital infection in mice caused by HSV-2. Subcutaneous inoculation of HF10-immunized mice against lethal infection by HSV-2, and attenuated the development of genital ulcer diseases. Immunization with HF10 inhibited HSV-2 replication in the mouse vagina, reduced local inflammation, controlled emergence of neurological dysfunctions of HSV-2 infection, and increased survival. In HF10-immunized mice, we observed rapid and increased production of interferon-γ in the vagina in response to HSV-2 infection, and numerous CD4(+) and a few CD8(+) T cells localized to the infective focus. CD4(+) T cells invaded the mucosal subepithelial lamina propria. Thus, the protective effect of HF10 was related to induction of cellular immunity, mediated primarily by Th1 CD4(+) cells. These data indicate that the live attenuated HSV-1 mutant strain HF10 is a promising candidate antigen for a vaccine against genital herpes caused by HSV-2.

  16. Iron Loading-Induced Aggregation and Reduction of Iron Incorporation in Heteropolymeric Ferritin Containing a Mutant Light Chain that Causes Neurodegeneration

    PubMed Central

    Muhoberac, Barry B.; Baraibar, Martin A.; Vidal, Ruben

    2010-01-01

    Hereditary Ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a

  17. Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration.

    PubMed

    Muhoberac, Barry B; Baraibar, Martin A; Vidal, Ruben

    2011-04-01

    Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a

  18. The Cataract-linked Mutant Connexin50D47A Causes Endoplasmic Reticulum Stress in Mouse Lenses.

    PubMed

    Berthoud, Viviana M; Minogue, Peter J; Lambert, Paul A; Snabb, Joseph I; Beyer, Eric C

    2016-08-19

    Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive αB-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1α or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2α (2.4- and 3.3-fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9- and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival.

  19. Two types of albino mutants in desert and migratory locusts are caused by gene defects in the same signaling pathway.

    PubMed

    Sugahara, Ryohei; Tanaka, Seiji; Jouraku, Akiya; Shiotsuki, Takahiro

    2017-04-15

    Albinism is caused by mutations in the genes involved in melanin production. Albino nymphs of Locusta migratoria and Schistocerca gregaria reared under crowded conditions are uniformly creamy-white in color. However, nothing is known about the molecular mechanisms underlying this phenomenon in locusts. The albino strain of L. migratoria is known to lack the dark-color-inducing neuropeptide corazonin (Crz). In this study, we report that this albino strain has a 10-base-pair deletion in the gene LmCRZ, which encodes Crz. This mutation was found to cause a frame-shift, resulting in a null mutation in Crz. On the other hand, the albino strain of S. gregaria is known to have an intact Crz. This strain was found to possess a single-nucleotide substitution in the middle of the Crz receptor-encoding gene, SgCRZR, which caused a nonsense mutation, resulting in a truncated receptor. Silencing of SgCRZR in wild-type S. gregaria nymphs greatly reduced the area and intensity of their black patterning, suggesting that the functional defect of SgCRZR likely causes the albinism. The expression level of SgCRZR in the albino S. gregaria was comparable to that in the wild type. Unlike the wild type, the albino strain of this locust did not show a phase-dependent shift in a morphometric trait controlled by Crz. From these results, we conclude that the mutations in LmCRZ and SgCRZR are responsible for the albinism in L. migratoria and S. gregaria, respectively, indicating that the two types of albinism are caused by different genetic defects in the same Crz signaling pathway.

  20. Inositol Hexaphosphate Down-regulates both Constitutive and Ligand-Induced Mitogenic and Cell Survival Signaling, and Causes Caspase-Mediated Apoptotic Death of Human Prostate Carcinoma PC-3 cells

    PubMed Central

    Gu, Mallikarjuna; Raina, Komal; Agarwal, Chapla; Agarwal, Rajesh

    2009-01-01

    Constitutively active mitogenic and pro-survival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). EGF and IGF-1 are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both MAPK- and Akt-mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF-1)-induced biological responses and associated signaling cascades in advanced and androgen-independent human PCa PC-3 cells. Treatment of PC-3 cells with 2 mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC-3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand-induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs ERK1/2, JNK1/2 and p38 under constitutive and ligand-activated conditions. Under similar condition, IP6 also inhibited AP-1 DNA binding activity and decreased nuclear levels of both phospho and total c-Fos and c-Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGFR or IGF-1R pathway-mediated sustained growth promoting and survival signaling cascades in advanced and androgen-independent human PCa PC-3 cells, which might have translational implications in advanced human PCa control and management. PMID:19544333

  1. Caffeic Acid Phenethyl Ester Causes p21Cip1 Induction, Akt Signaling Reduction, and Growth Inhibition in PC-3 Human Prostate Cancer Cells

    PubMed Central

    Lin, Hui-Ping; Jiang, Shih Sheng; Chuu, Chih-Pin

    2012-01-01

    Caffeic acid phenethyl ester (CAPE) treatment suppressed proliferation, colony formation, and cell cycle progression in PC-3 human prostate cancer cells. CAPE decreased protein expression of cyclin D1, cyclin E, SKP2, c-Myc, Akt1, Akt2, Akt3, total Akt, mTOR, Bcl-2, Rb, as well as phosphorylation of Rb, ERK1/2, Akt, mTOR, GSK3α, GSK3β, PDK1; but increased protein expression of KLF6 and p21Cip1. Microarray analysis indicated that pathways involved in cellular movement, cell death, proliferation, and cell cycle were affected by CAPE. Co-treatment of CAPE with chemotherapeutic drugs vinblastine, paclitaxol, and estramustine indicated synergistic suppression effect. CAPE administration may serve as a potential adjuvant therapy for prostate cancer. PMID:22347457

  2. Snyder-Robinson Syndrome: Rescuing the Disease-Causing Effect of G56S mutant by Small Molecule Binding

    NASA Astrophysics Data System (ADS)

    Zhang, Zhe; Martiny, Virginie; Lagorce, David; Alexov, Emil; Miteva, Maria; Clemson University Team; Université Paris Diderot Team

    2013-03-01

    Snyder-Robinson Syndrome (SRS) is an X-linked mental retardation disorder, which is caused by defects in a particular gene coding for the spermine synthase (SMS) protein. Among the missense mutations known to be disease-causing is the G56S, which is positioned at the interface of the SMS homo-dimer. Previous computational and experimental investigations have shown that G56S mutation destabilizes the homo-dimer and thus greatly reduces the SMS enzymatic activity. In this study, we explore the possibility of mitigating the effect of G56S mutation by binding small molecules to suitable pockets around the mutation site. It is done by combined efforts of molecular dynamics simulations and in silico screening. The binding of selected molecules was calculated to fully compensate the effect of the mutation and rescue the wild type dimer affinity. This work was supported by NIH, NLM grant. No. 1R03LM009748

  3. AID induces double-strand breaks at immunoglobulin switch regions and c-MYC causing chromosomal translocations in yeast THO mutants.

    PubMed

    Ruiz, José F; Gómez-González, Belén; Aguilera, Andrés

    2011-02-01

    Transcription of the switch (S) regions of immunoglobulin genes in B cells generates stable R-loops that are targeted by Activation Induced Cytidine Deaminase (AID), triggering class switch recombination (CSR), as well as translocations with c-MYC responsible for Burkitt's lymphomas. In Saccharomyces cerevisiae, stable R-loops are formed co-transcriptionally in mutants of THO, a conserved nuclear complex involved in mRNP biogenesis. Such R-loops trigger genome instability and facilitate deamination by human AID. To understand the mechanisms that generate genome instability mediated by mRNP biogenesis impairment and by AID, we devised a yeast chromosomal system based on different segments of mammalian S regions and c-MYC for the analysis of chromosomal rearrangements in both wild-type and THO mutants. We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks (DSBs) which in turn cause chromosomal translocations via Non-Homologous End Joining (NHEJ). AID-induced translocations were strongly enhanced in yeast THO null mutants, consistent with the idea that AID-mediated DSBs depend on R-loop formation. Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID-mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.

  4. Peripheral Nerve Demyelination Caused by a Mutant Rho GTPase Guanine Nucleotide Exchange Factor, Frabin/FGD4

    PubMed Central

    Stendel, Claudia ; Roos, Andreas ; Deconinck, Tine ; Pereira, Jorge ; Castagner, François ; Niemann, Axel ; Kirschner, Janbernd ; Korinthenberg, Rudolf ; Ketelsen, Uwe-Peter ; Battaloglu, Esra ; Parman, Yesim ; Nicholson, Garth ; Ouvrier, Robert ; Seeger, Jürgen ; Jonghe, Peter De ; Weis, Joachim ; Krüttgen, Alexander ; Rudnik-Schöneborn, Sabine ; Bergmann, Carsten ; Suter, Ueli ; Zerres, Klaus ; Timmerman, Vincent ; Relvas, João B. ; Senderek, Jan 

    2007-01-01

    GTPases of the Rho subfamily are widely involved in the myelination of the vertebrate nervous system. Rho GTPase activity is temporally and spatially regulated by a set of specific guanine nucleotide exchange factors (GEFs). Here, we report that disruption of frabin/FGD4, a GEF for the Rho GTPase cell-division cycle 42 (Cdc42), causes peripheral nerve demyelination in patients with autosomal recessive Charcot-Marie-Tooth (CMT) neuropathy. These data, together with the ability of frabin to induce Cdc42-mediated cell-shape changes in transfected Schwann cells, suggest that Rho GTPase signaling is essential for proper myelination of the peripheral nervous system. PMID:17564972

  5. Peripheral nerve demyelination caused by a mutant Rho GTPase guanine nucleotide exchange factor, frabin/FGD4.

    PubMed

    Stendel, Claudia; Roos, Andreas; Deconinck, Tine; Pereira, Jorge; Castagner, Francois; Niemann, Axel; Kirschner, Janbernd; Korinthenberg, Rudolf; Ketelsen, Uwe-Peter; Battaloglu, Esra; Parman, Yesim; Nicholson, Garth; Ouvrier, Robert; Seeger, Jürgen; De Jonghe, Peter; Weis, Joachim; Krüttgen, Alexander; Rudnik-Schöneborn, Sabine; Bergmann, Carsten; Suter, Ueli; Zerres, Klaus; Timmerman, Vincent; Relvas, João B; Senderek, Jan

    2007-07-01

    GTPases of the Rho subfamily are widely involved in the myelination of the vertebrate nervous system. Rho GTPase activity is temporally and spatially regulated by a set of specific guanine nucleotide exchange factors (GEFs). Here, we report that disruption of frabin/FGD4, a GEF for the Rho GTPase cell-division cycle 42 (Cdc42), causes peripheral nerve demyelination in patients with autosomal recessive Charcot-Marie-Tooth (CMT) neuropathy. These data, together with the ability of frabin to induce Cdc42-mediated cell-shape changes in transfected Schwann cells, suggest that Rho GTPase signaling is essential for proper myelination of the peripheral nervous system.

  6. Cold-aggravated pain in humans caused by a hyperactive NaV1.9 channel mutant

    PubMed Central

    Leipold, Enrico; Hanson-Kahn, Andrea; Frick, Miya; Gong, Ping; Bernstein, Jonathan A.; Voigt, Martin; Katona, Istvan; Oliver Goral, R.; Altmüller, Janine; Nürnberg, Peter; Weis, Joachim; Hübner, Christian A.; Heinemann, Stefan H.; Kurth, Ingo

    2015-01-01

    Gain-of-function mutations in the human SCN11A-encoded voltage-gated Na+ channel NaV1.9 cause severe pain disorders ranging from neuropathic pain to congenital pain insensitivity. However, the entire spectrum of the NaV1.9 diseases has yet to be defined. Applying whole-exome sequencing we here identify a missense change (p.V1184A) in NaV1.9, which leads to cold-aggravated peripheral pain in humans. Electrophysiological analysis reveals that p.V1184A shifts the voltage dependence of channel opening to hyperpolarized potentials thereby conferring gain-of-function characteristics to NaV1.9. Mutated channels diminish the resting membrane potential of mouse primary sensory neurons and cause cold-resistant hyperexcitability of nociceptors, suggesting a mechanistic basis for the temperature dependence of the pain phenotype. On the basis of direct comparison of the mutations linked to either cold-aggravated pain or pain insensitivity, we propose a model in which the physiological consequence of a mutation, that is, augmented versus absent pain, is critically dependent on the type of NaV1.9 hyperactivity. PMID:26645915

  7. Cleft Palate Defect of Dlx1/2−/− Mutant Mice is Caused by Lack of Vertical Outgrowth in the Posterior Palate

    PubMed Central

    Jeong, Juhee; Cesario, Jeffry; Zhao, Yangu; Burns, Lorel; Westphal, Heiner; Rubenstein, John L. R.

    2014-01-01

    Background Mice lacking the activities of Dlx1 and Dlx2 (Dlx1/2−/−) exhibit cleft palate, one of the most common human congenital defects, but the etiology behind this phenotype has been unknown. Therefore, we analyzed the morphological, cellular, and molecular changes caused by inactivation of Dlx1 and Dlx2 as related to palate development. Results Dlx1/2−/− mutants exhibited lack of vertical growth in the posterior palate during the earliest stage of palatogenesis. We attributed this growth deficiency to reduced cell proliferation. Expression of a cell cycle regulator Ccnd1 was specifically down-regulated in the same region. Previous studies established that the epithelial-mesenchymal signaling loop involving Shh, Bmp4 and Fgf10 is important for cell proliferation and tissue growth during palate development. This signaling loop was disrupted in Dlx1/2−/− palate. Interestingly, however, the decreases in Ccnd1 expression and mitosis in Dlx1/2−/− mutants were independent of this signaling loop. Finally, Dlx1/2 activity was required for normal expression of several transcription factor genes whose mutation results in palate defects. Conclusions The functions of Dlx1 and Dlx2 are crucial for the initial formation of the posterior palatal shelves, and that the Dlx genes lie upstream of multiple signaling molecules and transcription factors important for later stages of palatogenesis. PMID:22972697

  8. Differential Phospholipid Substrates and Directional Transport by ATP-binding Cassette Proteins ABCA1, ABCA7, and ABCA4 and Disease-causing Mutants*♦

    PubMed Central

    Quazi, Faraz; Molday, Robert S.

    2013-01-01

    ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation. PMID:24097981

  9. SI PC104 Performance Test Report

    SciTech Connect

    Montelongo, S

    2005-12-16

    The Spectral Instruments (SI) PC104 systems associated with the SI-1000 CCD camera exhibited intermittent power problems during setup, test and operations which called for further evaluation and testing. The SI PC104 System is the interface between the SI-1000 CCD camera and its associated Diagnostic Controller (DC). As such, the SI PC104 must be a reliable, robust system capable of providing consistent performance in various configurations and operating conditions. This SI PC104 system consists of a stackable set of modules designed to meet the PC104+ Industry Standard. The SI PC104 System consists of a CPU module, SI Camera card, Media converter card, Video card and a I/O module. The root cause of power problems was identified as failing solder joints at the LEMO power connector attached to the SI Camera Card. The recommended solution was to provide power to the PC104 system via a PC104+ power supply module configured into the PC104 stack instead of thru the LEMO power connector. Test plans (2) were developed to test SI PC104 performance and identify any outstanding issues noted during extended operations. Test Plan 1 included performance and image acquisition tests. Test Plan 2 verified performance after implementing recommendations. Test Plan 2 also included verifying integrity of system files and driver installation after bootup. Each test plan was implemented to fully test against each set of problems noted. Test Plan presentations and Test Plan results are attached as appendices. Anticipated test results will show successful operation and reliable performance of the SI PC104 system receiving its power via a PC104 power supply module. A SI PC104 Usage Recommendation Memo will be sent out to the SI PC104 User Community. Recommendation memo(s) are attached as appendices.

  10. Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome.

    PubMed

    Goldman, Robert D; Shumaker, Dale K; Erdos, Michael R; Eriksson, Maria; Goldman, Anne E; Gordon, Leslie B; Gruenbaum, Yosef; Khuon, Satya; Mendez, Melissa; Varga, Renée; Collins, Francis S

    2004-06-15

    Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.

  11. Novel mutation in the AVPR2 gene in a Danish male with nephrogenic diabetes insipidus caused by ER retention and subsequent lysosomal degradation of the mutant receptor

    PubMed Central

    Nejsum, Lene N.; Christensen, Tomas M.; Robben, Joris H.; Milligan, Graeme; Deen, Peter M. T.; Bichet, Daniel G.

    2011-01-01

    Mutations in the arginine vasopressin receptor 2 (AVPR2) gene can cause X-linked nephrogenic diabetes insipidus (NDI) characterized by the production of large amounts of urine and an inability to concentrate urine in response to the antidiuretic hormone vasopressin. We have identified a novel mutation in the AVPR2 gene (L170P) located in the fourth transmembrane domain in a Danish NDI male. Analysis of the mutant receptor in Madin-Darby Canine Kidney cell culture revealed that AVPR2-L170P was retained in the endoplasmic reticulum, and the expression was dramatically downregulated compared to wild-type AVPR2. Inhibition of the lysosome resulted in increased intracellular accumulation of AVPR2-L170P, indicating that AVPR2-L170P is downregulated via the lysosome. Inhibition of the proteasome resulted in plasma membrane localization of AVPR2-L170P, although the overall levels of AVPR2-L170P were unchanged. PMID:21629670

  12. Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

    PubMed

    Ferone, Giustina; Thomason, Helen A; Antonini, Dario; De Rosa, Laura; Hu, Bing; Gemei, Marica; Zhou, Huiqing; Ambrosio, Raffaele; Rice, David P; Acampora, Dario; van Bokhoven, Hans; Del Vecchio, Luigi; Koster, Maranke I; Tadini, Gianluca; Spencer-Dene, Bradley; Dixon, Michael; Dixon, Jill; Missero, Caterina

    2012-03-01

    Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, which is characterized by cleft palate and severe defects of the skin, is an autosomal dominant disorder caused by mutations in the gene encoding transcription factor p63. Here, we report the generation of a knock-in mouse model for AEC syndrome (p63(+/L514F) ) that recapitulates the human disorder. The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment. These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes. In parallel, a defective stem cell compartment is observed in humans affected by AEC syndrome and in Fgfr2b(-/-) mice. Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation. These findings establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells and provide mechanistic insights into the pathogenesis of AEC syndrome. Copyright © 2012 EMBO Molecular Medicine.

  13. Astrocyte pathology in a human neural stem cell model of frontotemporal dementia caused by mutant TAU protein

    PubMed Central

    Hallmann, Anna-Lena; Araúzo-Bravo, Marcos J.; Mavrommatis, Lampros; Ehrlich, Marc; Röpke, Albrecht; Brockhaus, Johannes; Missler, Markus; Sterneckert, Jared; Schöler, Hans R.; Kuhlmann, Tanja; Zaehres, Holm; Hargus, Gunnar

    2017-01-01

    Astroglial pathology is seen in various neurodegenerative diseases including frontotemporal dementia (FTD), which can be caused by mutations in the gene encoding the microtubule-associated protein TAU (MAPT). Here, we applied a stem cell model of FTD to examine if FTD astrocytes carry an intrinsic propensity to degeneration and to determine if they can induce non-cell-autonomous effects in neighboring neurons. We utilized CRISPR/Cas9 genome editing in human induced pluripotent stem (iPS) cell-derived neural progenitor cells (NPCs) to repair the FTD-associated N279K MAPT mutation. While astrocytic differentiation was not impaired in FTD NPCs derived from one patient carrying the N279K MAPT mutation, FTD astrocytes appeared larger, expressed increased levels of 4R-TAU isoforms, demonstrated increased vulnerability to oxidative stress and elevated protein ubiquitination and exhibited disease-associated changes in transcriptome profiles when compared to astrocytes derived from one control individual and to the isogenic control. Interestingly, co-culture experiments with FTD astrocytes revealed increased oxidative stress and robust changes in whole genome expression in previously healthy neurons. Our study highlights the utility of iPS cell-derived NPCs to elucidate the role of astrocytes in the pathogenesis of FTD. PMID:28256506

  14. Inappropriate translation inhibition and P-body formation cause cold-sensitivity in tryptophan-auxotroph yeast mutants.

    PubMed

    Ballester-Tomás, Lidia; Prieto, Jose A; Alepuz, Paula; González, Asier; Garre, Elena; Randez-Gil, Francisca

    2017-02-01

    In response to different adverse conditions, most eukaryotic organisms, including Saccharomyces cerevisiae, downregulate protein synthesis through the phosphorylation of eIF2α (eukaryotic initiation factor 2α) by Gcn2, a highly conserved protein kinase. Gcn2 also controls the translation of Gcn4, a transcription factor involved in the induction of amino acid biosynthesis enzymes. Here, we have studied the functional role of Gcn2 and Gcn2-regulating proteins, in controlling translation during temperature downshifts of TRP1 and trp1 yeast cells. Our results suggest that neither cold-instigated amino acid limitation nor Gcn2 are involved in the translation suppression at low temperature. However, loss of TRP1 causes increased eIF2α phosphorylation, Gcn2-dependent polysome disassembly and overactivity of Gcn4, which result in cold-sensitivity. Indeed, knock-out of GCN2 improves cold growth of trp1 cells. Likewise, mutation of several Gcn2-regulators and effectors results in cold-growth effects. Remarkably, we found that Hog1, the osmoresponsive MAPK, plays a role in the regulatory mechanism of Gcn2-eIF2α. Finally, we demonstrated that P-body formation responds to a downshift in temperature in a TRP1-dependent manner and is required for cold tolerance.

  15. Structures of the troponin core domain containing the cardiomyopathy-causing mutants studied by small-angle X-ray scattering

    PubMed Central

    Matsuo, Tatsuhito; Takeda, Soichi; Oda, Toshiro; Fujiwara, Satoru

    2015-01-01

    Troponin (Tn), consisting of three subunits, TnC, TnI, and TnT, is a protein in the thin filaments in muscle, and, together with another thin-filament protein tropomyosin (Tm), plays a major role in regulation of muscle contraction. Various mutations of Tn cause familial hypertrophic cardiomyopathy. These mutations are directly related to aberrations in this regulatory mechanism. Here we focus on the mutations E244D and K247R of TnT, which reside in the middle of the pathway of the Ca2+-binding signal from TnC to Tm. These mutations induce an increase in the maximum tension of cardiac muscle without changes in Ca2+-sensitivity. As a first step toward elucidating the molecular mechanism underlying this functional aberration, we carried out small-angle X-ray scattering experiments on the Tn core domain containing the wild type subunits and those containing the mutant TnT in the absence and presence of Ca2+. Changes in the overall shape induced by the mutations were detected for the first time by the changes in the radius of gyration and the maximum dimension between the wild type and the mutants. Analysis of the scattering curves by model calculations shows that TnC adopts a dumbbell structure regardless of the mutations, and that the mutations change the distributions of the conformational ensembles so that the flexible N- and C-terminal regions of TnT become close to the center of the whole moelcule. This suggests, since these regions are related to the Tn-Tm interactions, that alteration of the Tn-Tm interactions induced by the mutations causes the functional aberration. PMID:27493864

  16. Biomimicry 1: PC.

    PubMed

    Cumberland, D C; Gunn, J; Malik, N; Holt, C M

    1998-01-01

    The surface properties of stents can be modified by coating them, for example with a polymer. Phosphorylcoline (PC) is the major component of the outer layer of the cell membrane. The haemo- and biocompatibility of a PC-containing polymer is thus based on biomimicry, and has been confirmed by several experiments showing much reduced thrombogenicity of PC-coated surfaces, and porcine coronary artery implants showing no sign of adverse effect. Clinical experience with the PC-coated BiodivYsio appears favourable. The PC coating can be tailored for take up and controlled elution of various drugs for stent-based local delivery, a property which is being actively explored.

  17. The role of the p53 protein in nitrosative stress-induced apoptosis of PC12 rat pheochromocytoma cells.

    PubMed

    Varga, Judit; Bátor, Judit; Péter, Márton; Árvai, Zita; Pap, Marianna; Sétáló, György; Szeberényi, József

    2014-10-01

    PC12 rat pheochromocytoma cells are widely used to investigate signaling pathways. The p143p53PC12 cell line expresses a Val143Ala mutant p53 protein that is less capable of binding to the p53 consensus site in DNA than its wild-type counterpart. Nitric oxide (NO), depending on its concentration, is able to activate several signal transduction pathways. We used sodium nitroprusside (SNP), an NO donor compound, to analyze NO-induced cellular stress in order to clarify the mechanism and role of nitrosative stress in pathological processes, including inflammation and cancer. SNP caused cell death when applied at a concentration of 400 μM, p143p53PC12 cells showing higher sensitivity than wild-type PC12 cells. The mechanisms leading to the increased SNP-sensitivity of p143p53PC12 cells were then investigated. The 400-μM SNP treatment caused stress kinase activation, phosphorylation of the eukaryotic initiation factor eIF2α and p53 protein, proteolytic activation of protein kinase R, caspase-9, and caspase-3, p53 stabilization, CHOP induction, cytochrome c release from mitochondria, and a decline in the level of the Bcl-2 protein in both cell lines. All these SNP-induced changes were more robust and/or permanent in cells with the mutant p53 protein. We thus conclude that (1) the main cause of the SNP-induced apoptosis of PC12 cells is the repression of the bcl-2 gene, evoked through p53 stabilization, stress kinase activation, and CHOP induction; (2) the higher SNP sensitivity of p143p53PC12 cells is the consequence of the stronger and earlier activation of the intrinsic apoptotic pathway.

  18. Use of a high-throughput screen to identify Leptospira mutants unable to colonize the carrier host or cause disease in the acute model of infection.

    PubMed

    Marcsisin, Renee A; Bartpho, Thanatchaporn; Bulach, Dieter M; Srikram, Amporn; Sermswan, Rasana W; Adler, Ben; Murray, Gerald L

    2013-10-01

    The molecular basis for leptospirosis infection and colonization remains poorly understood, with no efficient methods available for screening libraries of mutants for attenuation. We analysed the attenuation of leptospiral transposon mutants in vivo using a high-throughput method by infecting animals with pooled sets of transposon mutants. A total of 95 mutants was analysed by this method in the hamster model of acute infection, and one mutant was identified as attenuated (M1233, lb058 mutant). All virulence factors identified in Leptospira to date have been characterized in the acute model of infection, neglecting the carrier host. To address this, a BALB/c mouse colonization model was established. The lb058 mutant and two mutants defective in LPS synthesis were colonization deficient in the mouse model. By applying the high-throughput screening method, a further five colonization-deficient mutants were identified for the mouse model; these included two mutants in genes encoding proteins with a predicted role in iron uptake (LB191/HbpA and LB194). Two attenuated mutants had transposon insertions in either la0589 or la2786 (encoding proteins of unknown function). The final attenuated mutant had an unexpected deletion of genes la0969-la0975 at the point of transposon insertion. This is the first description of defined, colonization-deficient mutants in a carrier host for Leptospira. These mutants were either not attenuated or only weakly attenuated in the hamster model of acute leptospirosis, thus illustrating that different factors that may be required in the carrier and acute models of leptospiral infection. High-throughput screening can reduce the number of animals used in virulence studies and increase the capacity to screen mutants for attenuation, thereby enhancing the likelihood of detecting unique virulence factors. A comparison of virulence factors required in the carrier and acute models of infection will help to unravel colonization and dissemination

  19. COOH-terminal collagen Q (COLQ) mutants causing human deficiency of endplate acetylcholinesterase impair the interaction of ColQ with proteins of the basal lamina.

    PubMed

    Arredondo, Juan; Lara, Marian; Ng, Fiona; Gochez, Danielle A; Lee, Diana C; Logia, Stephanie P; Nguyen, Joanna; Maselli, Ricardo A

    2014-05-01

    Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ with MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL.

  20. COOH-Terminal Collagen Q (COLQ) Mutants Causing Human Deficiency of Endplate Acetylcholinesterase Impair the Interaction of ColQ with Proteins of the Basal Lamina

    PubMed Central

    Arredondo, Juan; Lara, Marian; Ng, Fiona; Gochez, Danielle A.; Lee, Diana C.; Logia, Stephanie P.; Nguyen, Joanna; Maselli, Ricardo A.

    2014-01-01

    Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ to MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL. PMID:24281389

  1. Antibiotic resistance in Mycobacterium tuberculosis: peroxidase intermediate bypass causes poor isoniazid activation by the S315G mutant of M. tuberculosis catalase-peroxidase (KatG).

    PubMed

    Suarez, Javier; Ranguelova, Kalina; Schelvis, Johannes P M; Magliozzo, Richard S

    2009-06-12

    KatG (catalase-peroxidase) in Mycobacterium tuberculosis is responsible for activation of isoniazid (INH), a pro-drug used to treat tuberculosis infections. Resistance to INH is a global health problem most often associated with mutations in the katG gene. The origin of INH resistance caused by the KatG[S315G] mutant enzyme is examined here. Overexpressed KatG[S315G] was characterized by optical, EPR, and resonance Raman spectroscopy and by studies of the INH activation mechanism in vitro. Catalase activity and peroxidase activity with artificial substrates were moderately reduced (50 and 35%, respectively), whereas the rates of formation of oxyferryl heme:porphyrin pi-cation radical and the decay of heme intermediates were approximately 2-fold faster in KatG[S315G] compared with WT enzyme. The INH binding affinity for the resting enzyme was unchanged, whereas INH activation, measured by the rate of formation of an acyl-nicotinamide adenine dinucleotide adduct considered to be a bactericidal molecule, was reduced by 30% compared with WT KatG. INH resistance is suggested to arise from a redirection of catalytic intermediates into nonproductive reactions that interfere with oxidation of INH. In the resting mutant enzyme, a rapid evolution of 5-c heme to 6-c species occurred in contrast with the behavior of WT KatG and KatG[S315T] and consistent with greater flexibility at the heme edge in the absence of the hydroxyl of residue 315. Insights into the effects of mutations at residue 315 on enzyme structure, peroxidation kinetics, and specific interactions with INH are presented.

  2. Expression of mutant CHMP2B, an ESCRT-III component involved in frontotemporal dementia, causes eye deformities due to Notch misregulation in Drosophila

    PubMed Central

    Cheruiyot, Abigael; Lee, Jin-A; Gao, Fen-Biao; Ahmad, S. Tariq

    2014-01-01

    Endosomal sorting complexes required for transport (ESCRTs) mediate sorting of ubiquitinated membrane proteins into multivesicular bodies en route to lysosomes for degradation. A mutation in CHMP2B (CHMP2BIntron5, an ESCRT-III component) that is associated with a hereditary form of frontotemporal dementia (FTD3) disrupts the endosomal-lysosomal pathway and causes accumulation of autophagosomes and multilamellar structures. We previously demonstrated that expression of CHMP2BIntron5 in the Drosophila eye using GMR-Gal4 causes misregulation of the Toll receptor pathway. Here, we show that ectopic expression of CHMP2BIntron5 using eyeless-Gal4 (ey>CHMP2BIntron5), a driver with different spatiotemporal expression attributes than GMR-Gal4 in the Drosophila eye, causes eye deformities when compared to expression of wild-type CHMP2B (CHMP2BWT) and the Drosophila homologue of CHMP2B (CG4618). In addition, ey>CHMP2BIntron5 flies showed defects in photoreceptor cell patterning and phototactic behavior. Furthermore, ey>CHMP2BIntron5 flies showed accumulation of Notch in enlarged endosomes and up-regulation of Notch activity. Partial loss of Notch activity in ey>CHMP2BIntron5 flies significantly rescued eye deformities, photoreceptor patterning defect, and phototactic behavior defect, indicating that these defects are primarily due to Notch misregulation. These results demonstrate that CHMP2BIntron5 preferentially affects different receptor signaling pathways in a cellular and developmental context-dependent manner.—Cheruiyot, A., Lee, J-A., Gao, F-B., Ahmad, S. T. Expression of mutant CHMP2B, an ESCRT-III component involved in frontotemporal dementia, causes eye deformities due to Notch misregulation in Drosophila. PMID:24158394

  3. Human P301L-Mutant Tau Expression in Mouse Entorhinal-Hippocampal Network Causes Tau Aggregation and Presynaptic Pathology but No Cognitive Deficits

    PubMed Central

    Harris, Julie A.; Koyama, Akihiko; Maeda, Sumihiro; Ho, Kaitlyn; Devidze, Nino; Dubal, Dena B.; Yu, Gui-Qiu; Masliah, Eliezer; Mucke, Lennart

    2012-01-01

    Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer’s disease (AD). It can occur before significant Aβ deposition and appears to “spread” into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here. PMID:23029293

  4. Cuticular Defects in Oryza sativa ATP-binding Cassette Transporter G31 Mutant Plants Cause Dwarfism, Elevated Defense Responses and Pathogen Resistance.

    PubMed

    Garroum, Imène; Bidzinski, Przemyslaw; Daraspe, Jean; Mucciolo, Antonio; Humbel, Bruno M; Morel, Jean-Benoit; Nawrath, Christiane

    2016-06-01

    The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense.

  5. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2017-04-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2017. Published by Elsevier Inc.

  6. PC-FACS.

    PubMed

    Davis, Mellar P

    2017-07-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2017. Published by Elsevier Inc.

  7. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2016-11-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2016. Published by Elsevier Inc.

  8. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2016-07-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2016. Published by Elsevier Inc.

  9. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2016-10-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2016. Published by Elsevier Inc.

  10. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2017-05-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2017. Published by Elsevier Inc.

  11. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2016-12-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2016. Published by Elsevier Inc.

  12. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2017-03-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2017. Published by Elsevier Inc.

  13. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2017-02-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2016. Published by Elsevier Inc.

  14. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2016-06-01

    PC-FACS(FastArticleCriticalSummaries for Clinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PC-FACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pc-facs@aahpm.org. Copyright © 2016. Published by Elsevier Inc.

  15. Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: Mutations in the 25K FP gene may be caused by DNA replication errors

    Treesearch

    David S. Bischoff; James M. Slavicek

    1997-01-01

    We previously demonstrated that polyhedron formation (PF) mutants arise at a high frequency during serial passage of the Lymantria dispar nucleopolyhedrovirus (LdMNPV) in the L. dispar 652Y cell line (J.M. Slavicek, N. Hayes-Plazolles, and M.E. Kelly, Biol. Control 5:251-261, 1995). Most of these PF mutants...

  16. shk1-D, a dwarf Arabidopsis mutant caused by activation of the CYP72C1 gene, has altered brassinosteroid levels.

    PubMed

    Takahashi, Naoki; Nakazawa, Miki; Shibata, Kyomi; Yokota, Takao; Ishikawa, Akie; Suzuki, Kumiko; Kawashima, Mika; Ichikawa, Takanari; Shimada, Hiroaki; Matsui, Minami

    2005-04-01

    Brassinosteroids (BRs) are plant steroidal hormones that regulate plant growth and development. An Arabidopsis dwarf mutant, shrink1-D (shk1-D), was isolated and the phenotype was shown to be caused by activation of the CYP72C1 gene. CYP72C1 is a member of the cytochrome P450 monooxygenase gene family similar to BAS1/CYP734A1 that regulates BR inactivation. shk1-D has short hypocotyls in both light and dark, and short petioles and siliques. The seeds are also shortened along the longitudinal axis indicating CYP72C1 controls cell elongation. The expression of CPD, TCH4 and BAS1 were altered in CYP72C1 overexpression transgenic lines and endogenous levels of castasterone, 6-deoxocastasterone and 6-deoxotyphasterol were also altered. Unlike BAS1/CYP734A1 the expression of CYP72C1 was not changed by application of exogenous brassinolide. We propose that CYP72C1 controls BR homeostasis by modulating the concentration of BRs.

  17. Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner

    PubMed Central

    Lachance-Touchette, Pamela; Choudhury, Mayukh; Stoica, Ana; Di Cristo, Graziella; Cossette, Patrick

    2014-01-01

    Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X) that were found in a cohort of French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1flox/flox mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1−/− GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D) induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic

  18. Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner.

    PubMed

    Lachance-Touchette, Pamela; Choudhury, Mayukh; Stoica, Ana; Di Cristo, Graziella; Cossette, Patrick

    2014-01-01

    Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X) that were found in a cohort of French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1 (flox/flox) mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1 (-/-) GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D) induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic

  19. The proteinopathy of D169G and K263E mutants at the RNA Recognition Motif (RRM) domain of tar DNA binding protein (tdp43) causing neurological disorders: A computational study.

    PubMed

    Bhandare, Vishwambhar Vishnu; Ramaswamy, Amutha

    2017-03-22

    One of the multitasking proteins, transactive response DNA-binding protein 43 (tdp43) plays a key role in RNA regulation and the two pathogenic mutations such as D169G and K263E, located at the RNA Recognition Motif (RRM) of tdp43, are reported to cause neurological disorders such as Amyotrophic Lateral Sclerosis (ALS) and Fronto Temporal Lobar Degeneration (FTLD). As the exploration of the proteinopathy demands both structural and functional characterization of mutants, a comparative analysis on the wild type and mutant tdp43 (D169G and K263E) and their complexes with RNA have been performed using computational approaches. Molecular dynamics simulations revealed comparatively stable mutant structures compared to wild type tdp43. Both mutants show lesser binding affinity towards RNA molecule when compared to the wild type tdp43. Some of the observed features, including the increased solvent accessible surface area, conformational flexibility as well as unfolding of tdp43 and the altered RNA conformation in tp43-RNA complex, reveal the susceptibility of these mutants to induce conformational changes in tdp43 for a possible aggregation in the cytoplasm. Particularly, the enhanced aggregation propensity of both mutants also evidences the higher probability of cytoplasmic aggregation of tdp43 mutants. Hence, the present analysis highlighting the structural and functional aspects of wild and mutant tdp43 will form the basis to gain insight into the proteinopathy of tdp43 and the related structure based drug discovery. Thus, tdp43 can be used as target to develop novel therapeutic approaches or drug designing.

  20. Hyperactivity of the Arabidopsis cryptochrome (cry1) L407F mutant is caused by a structural alteration close to the cry1 ATP-binding site.

    PubMed

    Orth, Christian; Niemann, Nils; Hennig, Lars; Essen, Lars-Oliver; Batschauer, Alfred

    2017-08-04

    Plant cryptochromes (cry) act as UV-A/blue light receptors. The prototype, Arabidopsis thaliana cry1, regulates several light responses during the life cycle, including de-etiolation, and is also involved in regulating flowering time. The cry1 photocycle is initiated by light absorption by its FAD chromophore, which is most likely fully oxidized (FADox) in the dark state and photoreduced to the neutral flavin semiquinone (FADH°) in its lit state. Cryptochromes lack the DNA-repair activity of the closely related DNA photolyases, but they retain the ability to bind nucleotides such as ATP. The previously characterized L407F mutant allele of Arabidopsis cry1 is biologically hyperactive and seems to mimic the ATP-bound state of cry1, but the reason for this phenotypic change is unclear. Here, we show that cry1L407F can still bind ATP, has less pronounced photoreduction and formation of FADH° than wild-type cry1, and has a dark reversion rate 1.7 times lower than that of the wild type. The hyperactivity of cry1L407F is not related to a higher FADH° occupancy of the photoreceptor but is caused by a structural alteration close to the ATP-binding site. Moreover, we show that ATP binds to cry1 in both the dark and the lit states. This binding was not affected by cry1's C-terminal extension, which is important for signal transduction. Finally, we show that a recently discovered chemical inhibitor of cry1, 3-bromo-7-nitroindazole, competes for ATP binding and thereby diminishes FADH° formation, which demonstrates that both processes are important for cry1 function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Pale-green phenotype of atl31atl6 double mutant leaves is caused by disruption of 5-aminolevulinic acid biosynthesis in Arabidopsis thaliana.

    PubMed

    Maekawa, Shugo; Takabayashi, Atsushi; Huarancca Reyes, Thais; Yamamoto, Hiroko; Tanaka, Ayumi; Sato, Takeo; Yamaguchi, Junji

    2015-01-01

    Arabidopsis ubiquitin ligases ATL31 and homologue ATL6 control the carbon/nitrogen nutrient and pathogen responses. A mutant with the loss-of-function of both atl31 and atl6 developed light intensity-dependent pale-green true leaves, whereas the single knockout mutants did not. Plastid ultrastructure and Blue Native-PAGE analyses revealed that pale-green leaves contain abnormal plastid structure with highly reduced levels of thylakoid proteins. In contrast, the pale-green leaves of the atl31/atl6 mutant showed normal Fv/Fm. In the pale-green leaves of the atl31/atl6, the expression of HEMA1, which encodes the key enzyme for 5-aminolevulinic acid synthesis, the rate-limiting step in chlorophyll biosynthesis, was markedly down-regulated. The expression of key transcription factor GLK1, which directly promotes HEMA1 transcription, was also significantly decreased in atl31/atl6 mutant. Finally, application of 5-aminolevulinic acid to the atl31/atl6 mutants resulted in recovery to a green phenotype. Taken together, these findings indicate that the 5-aminolevulinic acid biosynthesis step was inhibited through the down-regulation of chlorophyll biosynthesis-related genes in the pale-green leaves of atl31/atl6 mutant.

  2. Pale-Green Phenotype of atl31 atl6 Double Mutant Leaves Is Caused by Disruption of 5-Aminolevulinic Acid Biosynthesis in Arabidopsis thaliana

    PubMed Central

    Maekawa, Shugo; Takabayashi, Atsushi; Huarancca Reyes, Thais; Yamamoto, Hiroko; Tanaka, Ayumi; Sato, Takeo; Yamaguchi, Junji

    2015-01-01

    Arabidopsis ubiquitin ligases ATL31 and homologue ATL6 control the carbon/nitrogen nutrient and pathogen responses. A mutant with the loss-of-function of both atl31 and atl6 developed light intensity-dependent pale-green true leaves, whereas the single knockout mutants did not. Plastid ultrastructure and Blue Native-PAGE analyses revealed that pale-green leaves contain abnormal plastid structure with highly reduced levels of thylakoid proteins. In contrast, the pale-green leaves of the atl31/atl6 mutant showed normal Fv/Fm. In the pale-green leaves of the atl31/atl6, the expression of HEMA1, which encodes the key enzyme for 5-aminolevulinic acid synthesis, the rate-limiting step in chlorophyll biosynthesis, was markedly down-regulated. The expression of key transcription factor GLK1, which directly promotes HEMA1 transcription, was also significantly decreased in atl31/atl6 mutant. Finally, application of 5-aminolevulinic acid to the atl31/atl6 mutants resulted in recovery to a green phenotype. Taken together, these findings indicate that the 5-aminolevulinic acid biosynthesis step was inhibited through the down-regulation of chlorophyll biosynthesis-related genes in the pale-green leaves of atl31/atl6 mutant. PMID:25706562

  3. dBRWD3 Regulates Tissue Overgrowth and Ectopic Gene Expression Caused by Polycomb Group Mutations

    PubMed Central

    Shih, Hsueh-Tzu; Chen, Wei-Yu; Liu, Kwei-Yan; Shih, Zong-Siou; Chen, Yi-Jyun; Hsieh, Paul-Chen; Kuo, Kuan-Lin; Huang, Kuo-How; Hsu, Pang-Hung; Liu, Ya-Wen; Tsai, Yu-Chen; Wu, June-Tai

    2016-01-01

    To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations. PMID:27588417

  4. PC-SPES (PDQ)

    MedlinePlus

    ... contain prescription medicines . It was taken off the market and is no longer being made (see Question ... Serenoa repens ) PC-SPES was taken off the market because some batches were found to contain prescription ...

  5. Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element.

    PubMed Central

    Schartl, M; Hornung, U; Gutbrod, H; Volff, J N; Wittbrodt, J

    1999-01-01

    The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus. PMID:10545466

  6. Peak Pc Prediction in Conjunction Analysis: Conjunction Assessment Risk Analysis. Pc Behavior Prediction Models

    NASA Technical Reports Server (NTRS)

    Vallejo, J.J.; Hejduk, M.D.; Stamey, J. D.

    2015-01-01

    Satellite conjunction risk typically evaluated through the probability of collision (Pc). Considers both conjunction geometry and uncertainties in both state estimates. Conjunction events initially discovered through Joint Space Operations Center (JSpOC) screenings, usually seven days before Time of Closest Approach (TCA). However, JSpOC continues to track objects and issue conjunction updates. Changes in state estimate and reduced propagation time cause Pc to change as event develops. These changes a combination of potentially predictable development and unpredictable changes in state estimate covariance. Operationally useful datum: the peak Pc. If it can reasonably be inferred that the peak Pc value has passed, then risk assessment can be conducted against this peak value. If this value is below remediation level, then event intensity can be relaxed. Can the peak Pc location be reasonably predicted?

  7. The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the high pigment-1 mutant phenotype.

    PubMed

    Lieberman, Michal; Segev, Orit; Gilboa, Nehama; Lalazar, Avraham; Levin, Ilan

    2004-05-01

    A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 ( hp-1) and the high pigment-1(w) ( hp-1(w)) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 ( hp-2), high pigment-2(j) ( hp-2(j)) and dark green ( dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1(w) mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A(931)-to-T(931) base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1(w) mutant, on the other hand, a single G(2392)-to-A(2392) transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F(2) population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25mutant phenotypes.

  8. PC-FACS.

    PubMed

    Abernethy, Amy P

    2012-03-01

    PC-FACS (Fast Article Critical Summaries for Clinicians in Palliative Care), an electronic publication of the American Academy of Hospice and Palliative Medicine, provides palliative care clinicians with concise summaries of the most important findings from more than 50 medical and scientific journals. Each month, structured summaries and insightful commentaries on 6-10 articles help palliative care clinicians stay on top of the research that is critical to contemporary practice. PC-FACS is free to AAHPM members and members can earn up to 3 CME credits quarterly. Following are excerpts from recent issues, and comments from readers are welcomed at resources@aahpm.org.

  9. PC-FACS.

    PubMed

    Zhukovsky, Donna S

    2012-04-01

    PC-FACS (Fast Article Critical Summaries for Clinicians in Palliative Care), an electronic publication of the American Academy of Hospice and Palliative Medicine, provides palliative care clinicians with concise summaries of the most important findings from more than 50 medical and scientific journals. Each month, structured summaries and insightful commentaries on 6-10 articles help palliative care clinicians stay on top of the research that is critical to contemporary practice. PC-FACS is free to AAHPM members. Following are excerpts from recent issues, and comments from readers are welcomed at resources@aahpm.org.

  10. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

  11. slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8.

    PubMed

    Ikeda, A; Ueguchi-Tanaka, M; Sonoda, Y; Kitano, H; Koshioka, M; Futsuhara, Y; Matsuoka, M; Yamaguchi, J

    2001-05-01

    The rice slender mutant (slr1-1) is caused by a single recessive mutation and results in a constitutive gibberellin (GA) response phenotype. The mutant elongates as if saturated with GAs. In this mutant, (1) elongation was unaffected by an inhibitor of GA biosynthesis, (2) GA-inducible alpha-amylase was produced by the aleurone layers without gibberellic acid application, and (3) endogenous GA content was lower than in the wild-type plant. These results indicate that the product of the SLR1 gene is an intermediate of the GA signal transduction pathway. SLR1 maps to OsGAI in rice and has significant homology with height-regulating genes, such as RHT-1Da in wheat, D8 in maize, and GAI and RGA in Arabidopsis. The GAI gene family is likely to encode transcriptional factors belonging to the GRAS gene superfamily. DNA sequence analysis revealed that the slr1-1 mutation is a single basepair deletion of the nuclear localization signal domain, resulting in a frameshift mutation that abolishes protein production. Furthermore, introduction of a 6-kb genomic DNA fragment containing the wild-type SLR1 gene into the slr1-1 mutant restored GA sensitivity to normal. These results indicate that the slr1-1 mutant is caused by a loss-of-function mutation of the SLR1 gene, which is an ortholog of GAI, RGA, RHT, and D8. We also succeeded in producing GA-insensitive dwarf rice by transforming wild-type rice with a modified SLR1 gene construct that has a 17-amino acid deletion affecting the DELLA region. Thus, we demonstrate opposite GA response phenotypes depending on the type of mutations in SLR1.

  12. Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance

    PubMed Central

    Miao, Jianqiang; Cai, Meng; Dong, Xue; Liu, Li; Lin, Dong; Zhang, Can; Pang, Zhili; Liu, Xili

    2016-01-01

    The potential for oxathiapiprolin resistance in Phytophthora capsici was evaluated. The baseline sensitivities of 175 isolates to oxathiapiprolin were initially determinated and found to conform to a unimodal curve with a mean EC50 value of 5.61 × 10-4 μg/ml. Twelve stable oxathiapiprolin-resistant mutants were generated by fungicide adaptation in two sensitive isolates, LP3 and HNJZ10. The fitness of the LP3-mutants was found to be similar to or better than that of the parental isolate LP3, while the HNJZ10-mutants were found to have lost the capacity to produce zoospores. Taken together these results suggest that the risk of P. capsici developing resistance to oxathiapiprolin is moderate. Comparison of the PcORP1 genes in the LP3-mutants and wild-type parental isolate, which encode the target protein of oxathiapiprolin, revealed that a heterozygous mutation caused the amino acid substitution G769W. Transformation and expression of the mutated PcORP1-769W allele in the sensitive wild-type isolate BYA5 confirmed that the mutation in PcORP1 was responsible for the observed oxathiapiprolin resistance. Finally diagnostic tests including As-PCR and CAPs were developed to detect the oxathiapiprolin resistance resulting from the G769W point mutation in field populations of P. capsici. PMID:27199944

  13. Beware the PC Police.

    ERIC Educational Resources Information Center

    Cheney, Lynne V.

    1992-01-01

    Decries the prevalence of "racism,""sexual harassment," and "speciesism" labels applied to supposed violators of "political correctness" (PC). When freedom of speech on college campuses is held hostage to political correctness, K-12 education is bound to be affected. This new brand of McCarthyism cheapens…

  14. Computational diagnosis of protein conformational diseases: short molecular dynamics simulations reveal a fast unfolding of r-LDL mutants that cause familial hypercholesterolemia.

    PubMed

    Cuesta-López, S; Falo, F; Sancho, J

    2007-01-01

    The molecular basis of conformational diseases frequently resides in mutant proteins constituting a subset of the vast mutational space. While the subtleties of protein structure point to molecular dynamics (MD) techniques as promising tools for an efficient exploration of such a space, the average size of proteins and the time scale of unfolding events make this goal difficult with present computational capabilities. We show here, nevertheless, that an efficient approach is already feasible for modular proteins. Familial hypercholesterolemia (FH) is a conformational disease linked to mutations in the gene encoding the low density lipoprotein receptor. A high percentage of these mutations has been found in the seven small modular binding repeats of the receptor. Taking advantage of its small size, we have performed an in depth MD study of the fifth binding repeat. Fast unfolding dynamics have been observed in the absence of a structural bound calcium ion, which agrees with its reported essential role in the stability of the module. In addition, several mutations detected in FH patients have been analyzed, starting from the native conformation. Our results indicate that in contrast with the wild type protein and an innocuous control mutant, disease-related mutants experience, in short simulation times (2-8 ns), gross departures from the native state that lead to unfolded conformations and, in some cases, to binding site desorganization deriving in calcium release. Computational diagnosis of mutations leading to conformational diseases seems thus feasible, at least for small or modular pathogenic proteins.

  15. Impaired Recruitment of Grk6 and β-Arrestin2 Causes Delayed Internalization and Desensitization of a WHIM Syndrome-Associated CXCR4 Mutant Receptor

    PubMed Central

    McCormick, Peter J.; Segarra, Marta; Gasperini, Paola; Gulino, A. Virginia; Tosato, Giovanna

    2009-01-01

    WHIM (warts, hypogammaglobulinemia, infections, and myelokatexis) syndrome is a rare immunodeficiency syndrome linked to heterozygous mutations of the chemokine receptor CXCR4 resulting in truncations of its cytoplasmic tail. Leukocytes from patients with WHIM syndrome display impaired CXCR4 internalization and enhanced chemotaxis in response to its unique ligand SDF-1/CXCL12, which likely contribute to the clinical manifestations. Here, we investigated the biochemical mechanisms underlying CXCR4 deficiency in WHIM syndrome. We report that after ligand activation, WHIM-associated mutant CXCR4 receptors lacking the carboxy-terminal 19 residues internalize and activate Erk 1/2 slower than wild-type (WT) receptors, while utilizing the same trafficking endocytic pathway. Recruitment of β-Arrestin 2, but not β-Arrestin 1, to the active WHIM-mutant receptor is delayed compared to the WT CXCR4 receptor. In addition, while both kinases Grk3 and Grk6 bind to WT CXCR4 and are critical to its trafficking to the lysosomes, Grk6 fails to associate with the WHIM-mutant receptor whereas Grk3 associates normally. Since β-Arrestins and Grks play critical roles in phosphorylation and internalization of agonist-activated G protein-coupled receptors, these results provide a molecular basis for CXCR4 dysfunction in WHIM syndrome. PMID:19956569

  16. The rice semi-dwarf mutant sd37, caused by a mutation in CYP96B4, plays an important role in the fine-tuning of plant growth.

    PubMed

    Zhang, Jie; Liu, Xiaoqiang; Li, Shuyu; Cheng, Zhukuan; Li, Chuanyou

    2014-01-01

    Plant cytochrome P450 has diverse roles in developmental processes and in the response to environmental cues. Here, we characterized the rice (Oryza sativa L ssp. indica cultivar 3037) semi-dwarf mutant sd37, in which the gene CYP96B4 (Cytochrome P450 96B subfamily) was identified and confirmed as the target by map-based cloning and a complementation test. A point mutation in the SRS2 domain of CYP96B4 resulted in a threonine to lysine substitution in the sd37 mutant. Examination of the subcellular localization of the protein revealed that SD37 was ER-localized protein. And SD37 was predominantly expressed in the shoot apical meristem and developing leaf and root maturation zone but not in the root apical meristem. The sd37 leaves, panicles, and seeds were smaller than those of the wild type. Histological analysis further revealed that a decrease in cell number in the mutant, specifically in the shoots, was the main cause of the dwarf phenotype. Microarray analysis demonstrated that the expression of several cell division-related genes was disturbed in the sd37 mutant. In addition, mutation or strongly overexpression of SD37 results in dwarf plants but moderate overexpression increases plant height. These data suggest that CYP96B4 may be an important regulator of plant growth that affects plant height in rice.

  17. [Dry eye syndrome and the PC screen].

    PubMed

    Moldovan, Iulia; Stan, Cristina; Marc, Alexandra

    2013-01-01

    To study the correlation between PC screen exposure of over 8 hours and Dry Eye Syndrome in 18-25 years-old students. This is a cross-sectional, cohort clinical study, carried out in March 2012 - February 2013. All subjects completed a questionnaire, underwent a slit lamp examination and measurement of visual acuity. Among the 59 participants of this study, 26 were EXPOSED (> 8 hours of PC screen exposure = EXPOSED) and 33 were NONEXPOSED. The 18-25 - year old participants who were exposed over 8 hours to the PC screen had a relative risk of 5,5 to develop Dry Eye Syndrome, compared to NONEXPOSED participants. Results indicate that Dry Eye Syndrome incidence and intensity of symptoms had increased proportionally with the hours of exposure. Tear Film Breakup Time, the Ocular Protection Index and the PC Ocular Protection Index decreased with the hours of PC screen exposure, suggesting a behavioral change in the EXPOSED participants. Exposure of over 8 hours to the PC screen caused Dry Eye Syndrome in 18-25 - year old students, with a relative risk of 5,5.

  18. PDGFRA-mutant syndrome.

    PubMed

    Ricci, Riccardo; Martini, Maurizio; Cenci, Tonia; Carbone, Arnaldo; Lanza, Paola; Biondi, Alberto; Rindi, Guido; Cassano, Alessandra; Larghi, Alberto; Persiani, Roberto; Larocca, Luigi M

    2015-07-01

    Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

  19. Genome sequencing of the Trichoderma reesei QM9136 mutant identifies a truncation of the transcriptional regulator XYR1 as the cause for its cellulase-negative phenotype

    DOE PAGES

    Lichius, Alexander; Bidard, Frédérique; Buchholz, Franziska; ...

    2015-04-20

    Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a.

  20. Easy PC Astronomy

    NASA Astrophysics Data System (ADS)

    Duffett-Smith, Peter

    1996-11-01

    Easy PC Astronomy is the perfect book for everyone who wants to make easy and accurate astronomical calculations. The author supplies a simple but powerful script language called AstroScript on a disk, ready to use on any IBM PC-type computer. Equipped with this software, readers can compute complex but interesting astronomical results within minutes: from the time of moonrise or moonset anywhere in the world on any date, to the display of a lunar or solar eclipse on the computer screen--all within a few minutes of opening the book! The Sky Graphics feature of the software displays a detailed image of the sky as seen from any point on earth--at any time in the future or past--showing the constellations, planets, and a host of other features. Readers need no expert knowledge of astronomy, math or programming; the author provides full details of the calculations and formulas, which the reader can absorb or ignore as desired, and a comprehensive glossary of astronomical terms. Easy PC Astronomy is of immediate practical use to beginning and advanced amateur astronomers, students at all levels, science teachers, and research astronomers. Peter Duffett-Smith is at the Cavendish Laboratory of the University of Cambridge and is the author of Astronomy with Your Personal Computer (Cambridge University Press, 1990) and Practical Astronomy with Your Calculator (Cambridge University Press, 1989).

  1. Different Mutant/Wild-Type p53 Combinations Cause a Spectrum of Increased Invasive Potential in Nonmalignant Immortalized Human Mammary Epithelial Cells1

    PubMed Central

    Junk, Damian J; Vrba, Lukas; Watts, George S; Oshiro, Marc M; Martinez, Jesse D; Futscher, Bernard W

    2008-01-01

    Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease. PMID:18472962

  2. Iron Inefficiency in Maize Mutant ys1 (Zea mays L. cv Yellow-Stripe) Is Caused by a Defect in Uptake of Iron Phytosiderophores.

    PubMed Central

    Von Wiren, N.; Mori, S.; Marschner, H.; Romheld, V.

    1994-01-01

    To determine the Fe inefficiency factors in the maize mutant ys1 (Zea mays L. cv Yellow Stripe), root exudates of Fe-inefficient ys1 and of two Fe-efficient maize cultivars (Alice, WF9) were collected in axenic nutrient solution cultures. Analysis by thin-layer chromatography and high-performance liquid chromatography revealed that under Fe deficiency ys1 released the phytosiderophore 2[prime]-deoxymugineic acid (DMA) in quantities similar to those of Alice and WF9. Under nonaxenic conditions, DMA released by plants of all three cultivars was rapidly decomposed by microorganisms in the nutrient solution. Uptake experiments with 59Fe-labeled DMA, purified from root exudates of either Fe-deficient Alice or ys1 plants, showed up to 20 times lower uptake and translocation of 59Fe in ys1 than in Alice or WF9 plants. The presence of microorganisms during preculture and short-term uptake experiments had no significant effect on uptake and translocation rates of 59Fe in Alice and ys1 plants. We conclude that Fe inefficiency in the maize mutant ys1 is the result of a defect in the uptake system for Fe-phytosiderophores. PMID:12232304

  3. Intracellular Transport, Assembly, and Degradation of Wild-Type and Disease-linked Mutant Gap Junction Proteins

    PubMed Central

    VanSlyke, Judy K.; Deschenes, Suzanne M.; Musil, Linda S.

    2000-01-01

    More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot–Marie–Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t1/2 < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway. PMID:10848620

  4. Ionizing radiation causes greater DNA base damage in radiation-sensitive mutant M10 cells than in parent mouse lymphoma L5178Y cells

    SciTech Connect

    Mori, T. |; Dizdaroglu, M.

    1994-10-01

    DNA base damage in radiation-sensitive mutant M10 cells and parent mouse lymphoma L5178Y cells was studied. Cells were exposed to ionizing radiation in the dose range of 48 to 400 Gy. Chromatin was isolated from cells and analyzed by gas chromatography-mass spectrometry. Ten DNA base products were identified and quantified. A dose-dependent formation of the products was observed. The yields of products in M10 cells were up to threefold greater than in L5178Y cells. Of the products measured, formamidopyrimidines had the highest difference in their yields between the two cell lines. The greater initial DNA base damage in M10 cells may play a role in their hypersensitivity to ionizing radiation. 41 refs., 2 figs., 1 tab.

  5. Chemical chaperone treatment reduces intracellular accumulation of mutant collagen IV and ameliorates the cellular phenotype of a COL4A2 mutation that causes haemorrhagic stroke.

    PubMed

    Murray, Lydia S; Lu, Yinhui; Taggart, Aislynn; Van Regemorter, Nicole; Vilain, Catheline; Abramowicz, Marc; Kadler, Karl E; Van Agtmael, Tom

    2014-01-15

    Haemorrhagic stroke accounts for ∼20% of stroke cases and porencephaly is a clinical consequence of perinatal cerebral haemorrhaging. Here, we report the identification of a novel dominant G702D mutation in the collagen domain of COL4A2 (collagen IV alpha chain 2) in a family displaying porencephaly with reduced penetrance. COL4A2 is the obligatory protein partner of COL4A1 but in contrast to most COL4A1 mutations, the COL4A2 mutation does not lead to eye or kidney disease. Analysis of dermal biopsies from a patient and his unaffected father, who also carries the mutation, revealed that both display basement membrane (BM) defects. Intriguingly, defective collagen IV incorporation into the dermal BM was observed in the patient only and was associated with endoplasmic reticulum (ER) retention of COL4A2 in primary dermal fibroblasts. This intracellular accumulation led to ER stress, unfolded protein response activation, reduced cell proliferation and increased apoptosis. Interestingly, the absence of ER retention of COL4A2 and ER stress in cells from the unaffected father indicate that accumulation and/or clearance of mutant COL4A2 from the ER may be a critical modifier for disease development. Our analysis also revealed that mutant collagen IV is degraded via the proteasome. Importantly, treatment of patient cells with a chemical chaperone decreased intracellular COL4A2 levels, ER stress and apoptosis, demonstrating that reducing intracellular collagen accumulation can ameliorate the cellular phenotype of COL4A2 mutations. Importantly, these data highlight that manipulation of chaperone levels, intracellular collagen accumulation and ER stress are potential therapeutic options for collagen IV diseases including haemorrhagic stroke.

  6. A Distorted Circadian Clock Causes Early Flowering and Temperature-Dependent Variation in Spike Development in the Eps-3Am Mutant of Einkorn Wheat

    PubMed Central

    Gawroński, Piotr; Ariyadasa, Ruvini; Himmelbach, Axel; Poursarebani, Naser; Kilian, Benjamin; Stein, Nils; Steuernagel, Burkhard; Hensel, Goetz; Kumlehn, Jochen; Sehgal, Sunish Kumar; Gill, Bikram S.; Gould, Peter; Hall, Anthony; Schnurbusch, Thorsten

    2014-01-01

    Viable circadian clocks help organisms to synchronize their development with daily and seasonal changes, thereby providing both evolutionary fitness and advantage from an agricultural perspective. A high-resolution mapping approach combined with mutant analysis revealed a cereal ortholog of Arabidopsis thaliana LUX ARRHYTHMO/PHYTOCLOCK 1 (LUX/PCL1) as a promising candidate for the earliness per se 3 (Eps-3Am) locus in einkorn wheat (Triticum monococcum L.). Using delayed fluorescence measurements it was shown that Eps-3Am containing einkorn wheat accession KT3-5 had a distorted circadian clock. The hypothesis was subsequently confirmed by performing a time course study on central and output circadian clock genes, which showed arrhythmic transcript patterns in KT3-5 under constant ambient conditions, i.e., constant light and temperature. It was also demonstrated that variation in spikelet number between wild-type and mutants is sensitive to temperature, becoming negligible at 25°. These observations lead us to propose that the distorted clock is causative for both early flowering and variation in spike size and spikelet number, and that having a dysfunctional LUX could have neutral, or even positive, effects in warmer climates. To test the latter hypothesis we ascertained sequence variation of LUX in a range of wheat germplasm. We observed a higher variation in the LUX sequence among accessions coming from the warmer climate and a unique in-frame mutation in early-flowering Chinese T. turgidum cultivar ‘Tsing Hua no. 559.’ Our results emphasize the importance of the circadian clock in temperate cereals as a promising target for adaptation to new environments. PMID:24443443

  7. Conditional Expression of Parkinson disease-related Mutant α-synuclein in the Midbrain Dopaminergic Neurons causes Progressive Neurodegeneration and Degradation of Transcription Factor Nuclear Receptor Related 1

    PubMed Central

    Lin, Xian; Parisiadou, Loukia; Sgobio, Carmelo; Liu, Guoxiang; Yu, Jia; Sun, Lixin; Shim, Hoon; Gu, Xing-Long; Luo, Jing; Long, Cai-Xia; Ding, Jinhui; Mateo, Yolanda; Sullivan, Patricia H.; Wu, Ling-Gang; Goldstein, David S.; Lovinger, David; Cai, Huaibin

    2012-01-01

    α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD. PMID:22764233

  8. Rough Endoplasmic Reticulum (rER) Trafficking Errors by Different Classes of Mutant DSPP Cause the Dominant Negative Effects in both Dentinogenesis Imperfecta and Dentin Dysplasia by Entrapping Normal DSPP

    PubMed Central

    von Marschall, Zofia; Mok, Seeun; Phillips, Matthew D.; McKnight, Dianalee A.; Fisher, Larry W.

    2012-01-01

    Families with nonsyndromic dentinogenesis imperfecta (DGI) and the milder, dentin dysplasia (DD), have mutations in one allele of the dentin sialophosphoprotein (DSPP) gene. Because loss of a single Dspp allele in mice (and likely, humans) causes no dental phenotype, the mechanism(s) underling the dominant-negative effects were investigated. DSPP mutations occur in three classes. (The first class, the mid-leader missense mutation, Y6D, was not investigated in this report.) All other 5' mutations of DSPP result in changes/loss in the first three amino acids (IPV) of mature DSPP or, for A15V, some retention of the hydrophobic leader sequence. All of this second class of mutations caused mutant DSPP to be retained in the rER of transfected HEK293 cells. Trafficking out of the rER by co-expressed normal DSPP was reduced in a dose-responsive manner, probably due to formation of Ca2+-dependent complexes with the retained mutant DSPP. IPV-like sequences begin many secreted Ca2+-binding proteins, and changing the third amino acid to the charged aspartate (D) in three other acidic proteins also caused increased rER accumulation. Both the leader-retaining A15V and the long string of hydrophobic amino acids resulting from all known frameshift mutations within the 3'-encoded Ca2+-binding repeat domain (third class of mutations) caused retention by association of the mutant proteins with rER membranes. More 5' frameshift mutations result in longer mutant hydrophobic domains but the milder phenotype, DD, probably due to lower effectiveness of the remaining, shorter Ca2+-binding domain in capturing normal DSPP protein within the rER. This study presents evidence of a shared underlying mechanism of capturing of normal DSPP by two different classes of DSPP mutations and offers an explanation for the mild (DD-II) versus severe (DGI-II & III) nonsyndromic dentin phenotypes. Evidence is also presented that many acidic, Ca2+-binding proteins may use the same IPV-like receptor

  9. Changes in growth kinetics of stamen filaments cause inefficient pollination in massugu2, an auxin insensitive, dominant mutant of Arabidopsis thaliana.

    PubMed

    Tashiro, Satoko; Tian, Chang-en; Watahiki, Masaaki K; Yamamoto, Kotaro T

    2009-10-01

    We investigated the physiological and molecular basis of lower fecundity of massugu2 (msg2), which is a dominant mutant of an auxin primary response gene, IAA19, in Arabidopsis thaliana. By measuring the length of all stamens and pistils in inflorescences and the reference growth rate of pistils, we constructed growth curves of pistils and stamens between stages 12 and 15 of flower development. Pistil growth was found to consist of a single exponential growth, while stamen growth consisted of three exponential phases. During the second exponential phase, the growth rate of stamen filaments was approximately 10 times greater than the growth rates in the other two phases. Consequently, stamens whose growth was initially retarded grew longer than the pistil, putting pollen grains on the stigma. msg2-1 stamens, on the other hand, exhibited a less obvious growth increase, resulting in less frequent contact between anthers and stigma. MSG2 was expressed in the stamen filaments and its expression almost coincided with the second growth phase. Stamen filaments appeared to elongate by cell elongation rather than cell division in the epidermal cell file. Considering that MSG2 is likely to be a direct target of the auxin F-box receptors, MSG2 may be one of the master genes that control the transient growth increase of stamen filaments.

  10. Thymoquinone, a bioactive component of black caraway seeds, causes G1 phase cell cycle arrest and apoptosis in triple-negative breast cancer cells with mutant p53.

    PubMed

    Sutton, Kimberly M; Greenshields, Anna L; Hoskin, David W

    2014-01-01

    Thymoquinone (TQ) from black caraway seeds has several anticancer activities; however, its effect on triple-negative breast cancer (TNBC) cells that lack functional tumor suppressor p53 is not known. Here, we explored the growth inhibitory effect of TQ on 2 TNBC cell lines with mutant p53. Cell metabolism assays showed that TQ inhibited TNBC cell growth without affecting normal cell growth. Flow cytometric analyses of TQ-treated TNBC cells showed G1 phase cell cycle arrest and apoptosis characterized by the loss of mitochondrial membrane integrity. Western blots of lysates from TQ-treated TNBC cells showed cytochrome c and apoptosis-inducing factor in the cytoplasm, as well as caspase-9 activation consistent with the mitochondrial pathway of apoptosis. Caspase-8 was also activated in TQ-treated TNBC cells, although the mechanism of activation is not clear at this time. Importantly, TQ-induced apoptosis was only partially inhibited by zVAD-fmk, indicating a role for caspase-independent effector molecules. Poly(ADP-ribose) polymerase cleavage and increased γH2AX, as well as reduced Akt phosphorylation and decreased expression of X-linked inhibitor of apoptosis, were evident in TQ-treated cells. Finally, TQ enhanced cisplatin- and docetaxel-induced cytotoxicity. These findings suggest that TQ could be useful in the management of TNBC, even when functional p53 is absent.

  11. Multifunctional human transcriptional coactivator protein PC4 is a substrate of Aurora kinases and activates the Aurora enzymes.

    PubMed

    Dhanasekaran, Karthigeyan; Kumari, Sujata; Boopathi, Ramachandran; Shima, Hiroki; Swaminathan, Amrutha; Bachu, Mahesh; Ranga, Udaykumar; Igarashi, Kazuhiko; Kundu, Tapas K

    2016-03-01

    Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase-dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase-mediated phosphorylation.

  12. PC index and magnetic substorms

    NASA Astrophysics Data System (ADS)

    Troshichev, Oleg; Janzhura, Alexander; Sormakov, Dmitry; Podorozhkina, Nataly

    PC index is regarded as a proxy of the solar wind energy that entered into the magnetosphere as distinct from the AL and Dst indices, which are regarded as characteristics of the energy that realize in the magnetosphere in form of substorm and magnetic storms. This conclusion is based on results of analysis of relationships between the polar cap magnetic activity (PC-index) and parameters of the solar wind, on the one hand, relationships between changes of PC and development of magnetospheric substorms (AL-index) and magnetic storms (Dst-index), on the other hand. This paper describes in detail the following main results which demonstrate a strong connection between the behavior of PC and development of magnetic disturbances in the auroral zone: (1) magnetic substorms are preceded by the РС index growth (isolated and extended substorms) or long period of stationary PC (postponed substorms), (2) the substorm sudden onsets are definitely related to such PC signatures as leap and reverse, which are indicative of sharp increase of the PC growth rate, (3) substorms generally start to develop when the PC index exceeds the threshold level ~ 1.5±0.5 mV/m, irrespective of the substorm growth phase duration and type of substorm, (4) linear dependency of AL values on PC is typical of all substorm events irrespective of type and intensity of substorm.

  13. Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants.

    PubMed

    Chin, Stephanie; Hung, Maurita; Bear, Christine E

    2017-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.

  14. Altered neuronal mitochondrial coenzyme A synthesis in neurodegeneration with brain iron accumulation caused by abnormal processing, stability, and catalytic activity of mutant pantothenate kinase 2.

    PubMed

    Kotzbauer, Paul T; Truax, Adam C; Trojanowski, John Q; Lee, Virginia M-Y

    2005-01-19

    Mutations in the pantothenate kinase 2 (PANK2) gene have been identified in patients with neurodegeneration with brain iron accumulation (NBIA; formerly Hallervorden-Spatz disease). However, the mechanisms by which these mutations cause neurodegeneration are unclear, especially given the existence of multiple pantothenate kinase genes in humans and multiple PanK2 transcripts with potentially different subcellular localizations. We demonstrate that PanK2 protein is localized to mitochondria of neurons in human brain, distinguishing it from other pantothenate kinases that do not possess mitochondrial-targeting sequences. PanK2 protein translated from the most 5' start site is sequentially cleaved at two sites by the mitochondrial processing peptidase, generating a long-lived 48 kDa mature protein identical to that found in human brain extracts. The mature protein catalyzes the initial step in coenzyme A (CoA) synthesis but displays feedback inhibition in response to species of acyl CoA rather than CoA itself. Some, but not all disease-associated point mutations result in significantly reduced catalytic activity. The most common mutation, G521R, results in marked instability of the intermediate PanK2 isoform and reduced production of the mature isoform. These results suggest that NBIA is caused by altered neuronal mitochondrial lipid metabolism caused by mutations disrupting PanK2 protein levels and catalytic activity.

  15. Substitution of tryptophan 89 with tyrosine switches the DNA binding mode of PC4.

    PubMed

    Huang, Jinguang; Zhao, Yanxiang; Liu, Huaian; Huang, Dan; Cheng, Xiankun; Zhao, Wensheng; Taylor, Ian A; Liu, Junfeng; Peng, You-Liang

    2015-03-05

    PC4, a well-known general transcription cofactor, has multiple functions in transcription and DNA repair. Residue W89, is engaged in stacking interactions with DNA in PC4, but substituted by tyrosine in some PC4 orthologous proteins. In order to understand the consequences and reveal the molecular details of this substitution we have determined the crystal structures of the PC4 orthologue MoSub1 and a PC4 W89Y mutant in complex with DNA. In the structure of MoSub1-DNA complex, Y74 interacts directly with a single nucleotide of oligo DNA. By comparison, the equivalent residue, W89 in wild type PC4 interacts with two nucleotides and the base of the second nucleotide has distinct orientation relative to that of the first one. A hydrophobic patch around W89 that favours interaction with two nucleotides is not formed in the PC4 W89Y mutant. Therefore, the change of the surface hydrophobicity around residue 89 results in a difference between the modes of DNA interaction. These results indicate that the conserved Y74 in MoSub1 or W89 in PC4, are not only key residues in making specific interactions with DNA but also required to determine the DNA binding mode of PC4 proteins.

  16. Expression of mutant CHMP2B, an ESCRT-III component involved in frontotemporal dementia, causes eye deformities due to Notch misregulation in Drosophila.

    PubMed

    Cheruiyot, Abigael; Lee, Jin-A; Gao, Fen-Biao; Ahmad, S Tariq

    2014-02-01

    Endosomal sorting complexes required for transport (ESCRTs) mediate sorting of ubiquitinated membrane proteins into multivesicular bodies en route to lysosomes for degradation. A mutation in CHMP2B (CHMP2B(Intron5), an ESCRT-III component) that is associated with a hereditary form of frontotemporal dementia (FTD3) disrupts the endosomal-lysosomal pathway and causes accumulation of autophagosomes and multilamellar structures. We previously demonstrated that expression of CHMP2B(Intron5) in the Drosophila eye using GMR-Gal4 causes misregulation of the Toll receptor pathway. Here, we show that ectopic expression of CHMP2B(Intron5) using eyeless-Gal4 (ey>CHMP2B(Intron5)), a driver with different spatiotemporal expression attributes than GMR-Gal4 in the Drosophila eye, causes eye deformities when compared to expression of wild-type CHMP2B (CHMP2B(WT)) and the Drosophila homologue of CHMP2B (CG4618). In addition, ey>CHMP2B(Intron5) flies showed defects in photoreceptor cell patterning and phototactic behavior. Furthermore, ey>CHMP2B(Intron5) flies showed accumulation of Notch in enlarged endosomes and up-regulation of Notch activity. Partial loss of Notch activity in ey>CHMP2B(Intron5) flies significantly rescued eye deformities, photoreceptor patterning defect, and phototactic behavior defect, indicating that these defects are primarily due to Notch misregulation. These results demonstrate that CHMP2B(Intron5) preferentially affects different receptor signaling pathways in a cellular and developmental context-dependent manner.

  17. Wanted: A Solid, Reliable PC

    ERIC Educational Resources Information Center

    Goldsborough, Reid

    2004-01-01

    This article discusses PC reliability, one of the most pressing issues regarding computers. Nearly a quarter century after the introduction of the first IBM PC and the outset of the personal computer revolution, PCs have largely become commodities, with little differentiating one brand from another in terms of capability and performance. Most of…

  18. NASA PC software evaluation project

    NASA Technical Reports Server (NTRS)

    Dominick, Wayne D. (Editor); Kuan, Julie C.

    1986-01-01

    The USL NASA PC software evaluation project is intended to provide a structured framework for facilitating the development of quality NASA PC software products. The project will assist NASA PC development staff to understand the characteristics and functions of NASA PC software products. Based on the results of the project teams' evaluations and recommendations, users can judge the reliability, usability, acceptability, maintainability and customizability of all the PC software products. The objective here is to provide initial, high-level specifications and guidelines for NASA PC software evaluation. The primary tasks to be addressed in this project are as follows: to gain a strong understanding of what software evaluation entails and how to organize a structured software evaluation process; to define a structured methodology for conducting the software evaluation process; to develop a set of PC software evaluation criteria and evaluation rating scales; and to conduct PC software evaluations in accordance with the identified methodology. Communication Packages, Network System Software, Graphics Support Software, Environment Management Software, General Utilities. This report represents one of the 72 attachment reports to the University of Southwestern Louisiana's Final Report on NASA Grant NGT-19-010-900. Accordingly, appropriate care should be taken in using this report out of context of the full Final Report.

  19. Wanted: A Solid, Reliable PC

    ERIC Educational Resources Information Center

    Goldsborough, Reid

    2004-01-01

    This article discusses PC reliability, one of the most pressing issues regarding computers. Nearly a quarter century after the introduction of the first IBM PC and the outset of the personal computer revolution, PCs have largely become commodities, with little differentiating one brand from another in terms of capability and performance. Most of…

  20. Muscle dystrophy-causing ΔK32 lamin A/C mutant does not impair functions of nucleoplasmic LAP2α - lamin A/C complexes in mice

    PubMed Central

    Pilat, Ursula; Dechat, Thomas; Bertrand, Anne T; Woisetschläger, Nikola; Gotic, Ivana; Spilka, Rita; Biadasiewicz, Katarzyna; Bonne, Gisèle; Foisner, Roland

    2015-01-01

    Summary A-type lamins are components of the nuclear lamina, a filamentous network of the nuclear envelope in metazoans that supports nuclear architecture. In addition, lamin A/C can also be found in the nuclear interior. This nucleoplasmic lamin pool is soluble in physiological buffer, depends on the presence of the lamin-binding protein, Lamina-associated polypeptide 2α (LAP2α) and regulates cell cycle progression in tissue progenitor cells. ΔK32 mutations in A-type lamins cause severe congenital muscle disease in humans and a muscle maturation defect in LmnaΔK32/ΔK32 knock-in mice. At molecular level, mutant ΔK32 lamin A/C protein levels were reduced and all mutant lamin A/C was soluble and mislocalized to the nucleoplasm. To test the role of LAP2α in nucleoplasmic ΔK32 lamin A/C regulation and functions, we deleted LAP2α in LmnaΔK32/ΔK32 knock-in mice. In double mutant mice the LmnaΔK32/ΔK32- linked muscle defect was unaffected. LAP2α interacted with mutant lamin A/C, but unlike wild-type lamin A/C, the intranuclear localization of ΔK32 lamin A/C was not affected by loss of LAP2α. In contrast, loss of LAP2α in LmnaΔK32/ΔK32 mice impaired the regulation of tissue progenitor cells like in lamin A/C wild type animals. These data indicate that a LAP2α-independent assembly defect of ΔK32 lamin A/C is predominant for the mouse pathology, while the LAP2α-linked functions of nucleoplasmic lamin A/C in the regulation of tissue progenitor cells are not affected in LmnaΔK32/ΔK32 mice. PMID:23444379

  1. Anxiety, memory impairment, and locomotor dysfunction caused by a mutant thyroid hormone receptor α1 can be ameliorated by T3 treatment

    PubMed Central

    Venero, César; Guadaño-Ferraz, Ana; Herrero, Ana Isabel; Nordström, Kristina; Manzano, Jimena; de Escobar, Gabriella Moreale; Bernal, Juan; Vennström, Björn

    2005-01-01

    The transcriptional properties of unliganded thyroid hormone receptors are thought to cause the misdevelopment during hypothyroidism of several functions essential for adult life. To specifically determine the role of unliganded thyroid hormone receptor α1 (TRα1) in neuronal tissues, we introduced a mutation into the mouse TRα1 gene that lowers affinity to thyroid hormone (TH) 10-fold. The resulting heterozygous mice exhibit several distinct neurological abnormalities: extreme anxiety, reduced recognition memory, and locomotor dysfunction. The anxiety and memory deficiencies were relieved by treatment with high levels of TH in adulthood, an effect that correlated with a normalization of GABAergic inhibitory interneurons in the hippocampal CA1 region. In contrast, a post-natal TH treatment was necessary and sufficient for ameliorating the adult locomotor dysfunction. Here, the hormone treatment normalized the otherwise delayed cerebellar development. The data thus identify two novel and distinct functions of an unliganded TRα1 during development and adulthood, respectively. PMID:16131613

  2. Intravitreal injection of ciliary neurotrophic factor (CNTF) causes peripheral remodeling and does not prevent photoreceptor loss in canine RPGR mutant retina.

    PubMed

    Beltran, William A; Wen, Rong; Acland, Gregory M; Aguirre, Gustavo D

    2007-04-01

    Ciliary neurotrophic factor (CNTF) rescues photoreceptors in several animal models of retinal degeneration and is currently being evaluated as a potential treatment for retinitis pigmentosa in humans. This study was conducted to test whether CNTF prevents photoreceptor cell loss in XLPRA2, an early onset canine model of X-linked retinitis pigmentosa caused by a frameshift mutation in RPGR exon ORF15. Four different treatment regimens of CNTF were tested in XLPRA2 dogs. Under anesthesia, the animals received at different ages an intravitreal injection of 12 microg of CNTF in the left eye. The right eye served as a control and was injected with a similar volume of phosphate buffered saline (PBS). Ocular examinations were performed regularly during the treatment periods. At termination, the dogs were euthanatized, eyes collected and the retinas were processed for embedding in optimal cutting temperature (OCT) medium. The outer nuclear layer (ONL) thickness was evaluated on H&E sections and values in both CNTF- and PBS-treated eyes were compared. Morphologic alterations in the peripheral retina were characterized by immunohistochemistry using cell-specific markers. Cell proliferation in the retinas was examined on semi-thin plastic sections, and by BrdU pulse-labeling and Ki67 immunohistochemistry on cryosections. All CNTF-treated eyes showed early clinical signs of corneal epitheliopathy, subcapsular cataracts and uveitis. No statistically significant difference in ONL thickness was seen between the CNTF- and PBS-injected eyes. Prominent retinal remodeling that consisted in an abnormal increase in the number of rods, and in misplacement of some rods, cones, bipolar and Müller cells, was observed in the peripheral retina of CNTF-treated eyes. This was only seen when CNTF was in injected before the age at which the canine retina reaches full maturation. In XLPRA2 dogs, intravitreal injections of CNTF failed to prevent photoreceptors from undergoing cell death in the

  3. Intravitreal injection of ciliary neurotrophic factor (CNTF) causes peripheral remodeling and does not prevent photoreceptor loss in canine RPGR mutant retina

    PubMed Central

    Beltran, William A.; Wen, Rong; Acland, Gregory M.; Aguirre, Gustavo D.

    2009-01-01

    Ciliary neurotrophic factor (CNTF) rescues photoreceptors in several animal models of retinal degeneration and is currently being evaluated as a potential treatment for retinitis pigmentosa in humans. This study was conducted to test whether CNTF prevents photoreceptor cell loss in XLPRA2, an early onset canine model of X-linked retinitis pigmentosa caused by a frameshift mutation in RPGR exon ORF15. Four different treatment regimens of CNTF were tested in XLPRA2 dogs. Under anesthesia, the animals received at different ages an intravitreal injection of 12 μg of CNTF in the left eye. The right eye served as a control and was injected with a similar volume of phosphate buffered saline (PBS). Ocular examinations were performed regularly during the treatment periods. At termination, the dogs were euthanatized, eyes collected and the retinas were processed for embedding in optimal cutting temperature (OCT) medium. The outer nuclear layer (ONL) thickness was evaluated on H&E sections and values in both CNTF- and PBS-treated eyes were compared. Morphologic alterations in the peripheral retina were characterized by immunohistochemistry using cell-specific markers. Cell proliferation in the retinas was examined on semi-thin plastic sections, and by BrdU pulse-labeling and Ki67 immunohistochemistry on cryosections. All CNTF-treated eyes showed early clinical signs of corneal epitheliopathy, subcapsular cataracts and uveitis. No statistically significant difference in ONL thickness was seen between the CNTF- and PBS-injected eyes. Prominent retinal remodeling that consisted in an abnormal increase in the number of rods, and in misplacement of some rods, cones, bipolar and Müller cells, was observed in the peripheral retina of CNTF-treated eyes. This was only seen when CNTF was in injected before the age at which the canine retina reaches full maturation. In XLPRA2 dogs, intravitreal injections of CNTF failed to prevent photoreceptors from undergoing cell death in the

  4. Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells

    PubMed Central

    1992-01-01

    Cyclin proteins form complexes with members of the p34cdc2 kinase family and they are essential components of the cell cycle regulatory machinery. They are thought to determine the timing of activation, the subcellular distribution, and/or the substrate specificity of cdc2- related kinases, but their precise mode of action remains to be elucidated. Here we report the cloning and sequencing of avian cyclin B2. Based on the use of monospecific antibodies raised against bacterially expressed protein, we also describe the subcellular distribution of cyclin B2 in chick embryo fibroblasts and in DU249 hepatoma cells. By indirect immunofluorescence microscopy we show that cyclin B2 is cytoplasmic during interphase of the cell cycle, but undergoes an abrupt translocation to the cell nucleus at the onset of mitotic prophase. Finally, we have examined the phenotypic consequences of expressing wild-type and mutated versions of avian cyclin B2 in HeLa cells. We found that expression of cyclin B2 carrying a mutation at arginine 32 (to serine) caused HeLa cells to arrest in a pseudomitotic state. Many of the arrested cells displayed multiple mitotic spindles, suggesting that the centrosome cycle had continued in spite of the cell cycle arrest. PMID:1532584

  5. Overexpression of metallothionein-I, a copper-regulating protein, attenuates intracellular copper dyshomeostasis and extends lifespan in a mouse model of amyotrophic lateral sclerosis caused by mutant superoxide dismutase-1.

    PubMed

    Tokuda, Eiichi; Okawa, Eriko; Watanabe, Shunsuke; Ono, Shin-Ichi

    2014-03-01

    Over 170 mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS), a lethal motor neuron disease. Although the molecular properties of SOD1 mutants differ considerably, we have recently shown that intracellular copper dyshomeostasis is a common pathogenic feature of different SOD1 mutants. Thus, the potentiation of endogenous copper regulation could be a therapeutic strategy. In this study, we investigated the effects of the overexpression of metallothionein-I (MT-I), a major copper-regulating protein, on the disease course of a mouse model of ALS (SOD1(G93A)). Using double transgenic techniques, we found that the overexpression of MT-I in SOD1(G93A) mice significantly extended the lifespan and slowed disease progression, but the effects on disease onset were modest. Genetically induced MT-I normalized copper dyshomeostasis in the spinal cord without influencing SOD1 enzymatic activity. The overexpression of MT-I in SOD1(G93A) mice markedly attenuated the pathological features of the mice, including the death of motor neurons, the degeneration of ventral root axons, the atrophy of skeletal muscles, and the activation of glial cells. Double transgenic mice also showed a decreased level of SOD1 aggregates within the glial cells of the spinal cord. Furthermore, the overexpression of MT-I in SOD1(G93A) mice reduced the number of spheroid-shaped astrocytes cleaved by active caspase-3. We concluded that therapeutic strategies aimed at the potentiation of copper regulation by MT-I could be of benefit in cases of ALS caused by SOD1 mutations.

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

    SciTech Connect

    Griffith, A.J.; Burgess, D.L.; Kohrman, D.

    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.

  7. Higher Susceptibility of Mast-Cell-Deficient W/WV Mutant Mice to Brain Thromboembolism and Mortality Caused by Intravenous Injection of India Ink

    PubMed Central

    Kitamura, Y.; Taguchi, T.; Yokoyama, M.; Inoue, M.; Yamatodani, A.; Asano, H.; Koyama, T.; Kanamaru, A.; Hatanaka, K.; Wershil, B. K.; Galli, S. J.

    1986-01-01

    of WBB6F1-+/+ mice injected with ink, and examination of their tissues in 1-μ sections, indicated that intravenous ink did not cause substantial mast cell degranulation. As a result, the possibility that mast cells protect +/+ mice from the adverse effects of intravenous ink by a mechanism other than degranulation and release of heparin, or that the differences in the response of W/WV or S1/S1d mice and their +/+ littermates are due to defects other than their lack of mast cells, cannot be excluded. ImagesFigure 2Figure 4 PMID:3513601

  8. Tetrahymena mutants with short telomeres.

    PubMed Central

    Ahmed, S; Sheng, H; Niu, L; Henderson, E

    1998-01-01

    Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30 degrees, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation. PMID:9755196

  9. CtBP Levels Control Intergenic Transcripts, PHO/YY1 DNA Binding, and PcG Recruitment to DNA

    PubMed Central

    Basu, Arindam; Atchison, Michael L.

    2013-01-01

    Carboxy-terminal binding protein (CtBP) is a well-known corepressor of several DNA binding transcription factors in Drosophila as well as in mammals. CtBP is implicated in Polycomb Group (PcG) complex-mediated transcriptional repression because it can bind to some PcG proteins, and mutation of the ctbp gene in flies results in lost PcG protein recruitment to Polycomb Response Elements (PREs) and lost PcG repression. However, the mechanism of reduced PcG DNA binding in CtBP mutant backgrounds is unknown. We show here that in a Drosophila CtBP mutant background, intergenic transcripts are induced across several PRE sequences and this corresponds to reduced DNA binding by PcG proteins Pleiohomeotic (PHO) and Polycomb (Pc), and reduced trimethylation of histone H3 on lysine 27, a hallmark of PcG repression. Restoration of CtBP levels by expression of a CtBP transgene results in repression of intergenic transcripts, restored PcG binding, and elevated trimethylation of H3 on lysine 27. Our results support a model in which CtBP regulates expression of intergenic transcripts that controls DNA binding by PcG proteins and subsequent histone modifications and transcriptional activity. PMID:20082324

  10. 76 FR 1990 - Airworthiness Directives; Pilatus Aircraft Ltd. Models PC-6, PC-6-H1, PC-6-H2, PC-6/350, PC-6/350...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-12

    ... requirement for the ``Mild Corrosion Severity Zone''. In addition, some editorial changes have been made for... PC- 6 Aircraft Maintenance Manual (AMM) Chapter 5 limitations which have included the same repetitive... Aircraft Ltd. Pilatus PC-6 Aircraft Maintenance Manual, dated November 30, 2008 (referenced as revision...

  11. The Caenorhabditis elegans PcG-like gene sop-2 regulates the temporal and sexual specificities of cell fates.

    PubMed

    Cai, Qingchun; Sun, Yinyan; Huang, Xinxin; Guo, Cong; Zhang, Yuxia; Zhu, Zuoyan; Zhang, Hong

    2008-03-01

    How spatial, temporal, and sexual specific cues are integrated to specify distinct cell fates during multicellular organism development is largely unknown. Here we demonstrate that the Caenorhabditis elegans PcG-like gene sop-2 determines the temporal and sexual specificities of a row of hypodermal seam cells, in addition to specifying their positional identities. Loss-of-function of sop-2 causes premature expression of adult fates at larval stages. sop-2 acts upstream of lin-29 in the heterochronic pathway and genetically interacts with other heterochronic genes in specifying the temporal fates of seam cells at different larval stages. We show that the number of ALG-1-containing P bodies is increased in seam cells in sop-2 mutants. Furthermore, the microRNA-mediated repression of a heterochronic gene reporter is enhanced in sop-2 mutants. Mutations in sop-2 also cause partial hermaphrodite-to-male sexual transformations. The homeotic transformations, heterochronic defects, and sexual transformations can occur concomitantly in sop-2 mutants. In summary, our studies reveal that sop-2 integrates spatial, temporal, and sexual cues during C. elegans development.

  12. Reduction of trophic support enhances apoptosis in PC12 cells expressing Alzheimer's APP mutation and sensitizes cells to staurosporine-induced cell death.

    PubMed

    Leutz, Steffen; Steiner, Barbara; Marques, Celio A; Haass, Christian; Müller, Walter E; Eckert, Anne

    2002-06-01

    Mutations in the amyloid precursor protein (APP) gene are known as causative factors in the pathogenesis of early-onset familial Alzheimer's disease (FAD). In this study, the influence of the Swedish double-mutation form of APP (APPsw; KM670/671NL) on apoptosis regulation in PC12 cells was investigated. APPsw-transfected PC12 cells were compared with wild-type APP (APPwt)-expressing and vector-transfected PC12 cells with regard to their susceptibility to cell death induced by the reduction of trophic support or by additional treatment with staurosporine. Expression of APPsw markedly enhanced the level of apoptotic PC12 cells induced by serum reduction. A similar hypersensitivity of APPsw-expressing PC12 cells could be detected after differentiation with nerve growth factor under serum-reduced conditions. Likewise, the expression of APPsw rendered PC12 cells more vulnerable to staurosporine but only under serum-reduced conditions. This APPsw-effect disappeared in high serum-containing medium. Thus, expression of APPsw seems to enhance cellular sensitivity not in general but after the reduction of trophic factors probably by causing oxidative stress. This, in turn, may sensitize cells to secondary apoptotic stimuli. Moreover, the mutation-specific increase in vulnerability to cell death was only seen at the stage of apoptotic nuclei, but not using methods measuring cell death by determining metabolic activity or membrane integrity. Therefore, the expression of APPsw seems to affect specifically apoptotic cell death rather than overall cell death in vitro. Our study further emphasizes the pathogenic role of mutant APP and may provide new insights in the mechanisms underlying the massive neurodegeneration in brain from patients bearing the APPsw mutation.

  13. A novel homozygous no-stop mutation in G6PC gene from a Chinese patient with glycogen storage disease type Ia.

    PubMed

    Gu, Lei-Lei; Li, Xin-Hua; Han, Yue; Zhang, Dong-Hua; Gong, Qi-Ming; Zhang, Xin-Xin

    2014-02-25

    Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive genetic disorder resulting in hypoglycemia, hepatomegaly and growth retardation. It is caused by mutations in the G6PC gene encoding Glucose-6-phosphatase. To date, over 80 mutations have been identified in the G6PC gene. Here we reported a novel mutation found in a Chinese patient with abnormal transaminases, hypoglycemia, hepatomegaly and short stature. Direct sequencing of the coding region and splicing-sites in the G6PC gene revealed a novel no-stop mutation, p.*358Yext*43, leading to a 43 amino-acid extension of G6Pase. The expression level of mutant G6Pase transcripts was only 7.8% relative to wild-type transcripts. This mutation was not found in 120 chromosomes from 60 unrelated healthy control subjects using direct sequencing, and was further confirmed by digestion with Rsa I restriction endonuclease. In conclusion, we revealed a novel no-stop mutation in this study which expands the spectrum of mutations in the G6PC gene. The molecular genetic analysis was indispensable to the diagnosis of GSD-Ia for the patient.

  14. KSC-98pc1133

    NASA Image and Video Library

    1998-09-18

    KENNEDY SPACE CENTER, FLA. -- The Spartan solar-observing deployable spacecraft is placed inside the payload canister in the Multi-Payload Processing Facility at KSC. Spartan is one of the payloads for the STS-95 mission, scheduled to launch Oct. 29. Spartan is a solar physics spacecraft designed to perform remote sensing of the hot outer layers of the sun's atmosphere or corona. The objective of the observations is to investigate the mechanisms causing the heating of the solar corona and the acceleration of the solar wind which originates in the corona. Other research payloads include the Hubble Space Telescope Orbital Systems Test Platform, the International Extreme Ultraviolet Hitchhiker, and the SPACEHAB single module with experiments on space flight and the aging process

  15. KSC-98pc1132

    NASA Image and Video Library

    1998-09-18

    KENNEDY SPACE CENTER, FLA. -- The Spartan solar-observing deployable spacecraft is suspended above the payload canister in the Multi-Payload Processing Facility at KSC. Spartan is one of the payloads for the STS-95 mission, scheduled to launch Oct. 29. Spartan is a solar physics spacecraft designed to perform remote sensing of the hot outer layers of the sun's atmosphere or corona. The objective of the observations is to investigate the mechanisms causing the heating of the solar corona and the acceleration of the solar wind which originates in the corona. Other research payloads include the Hubble Space Telescope Orbital Systems Test Platform, the International Extreme Ultraviolet Hitchhiker, and the SPACEHAB single module with experiments on space flight and the aging process

  16. KSC-98pc1131

    NASA Image and Video Library

    1998-09-18

    KENNEDY SPACE CENTER, FLA. -- The Spartan solar-observing deployable spacecraft is lifted from its work stand to move it to a payload canister in the Multi-Payload Processing Facility at KSC. Spartan is one of the payloads for the STS-95 mission, scheduled to launch Oct. 29. Spartan is a solar physics spacecraft designed to perform remote sensing of the hot outer layers of the sun's atmosphere or corona. The objective of the observations is to investigate the mechanisms causing the heating of the solar corona and the acceleration of the solar wind which originates in the corona. Other research payloads include the Hubble Space Telescope Orbital Systems Test Platform, the International Extreme Ultraviolet Hitchhiker, and the SPACEHAB single module with experiments on space flight and the aging process

  17. The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

    SciTech Connect

    Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.; Volkert, Michael R.

    2004-02-01

    Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

  18. [Proteasome degradation of protein C and plasmin inhibitor mutants: molecular mechanism of congenital protein deficiency].

    PubMed

    Nishio, Miwako; Koyama, Takatoshi; Hirosawa, Shinsaku

    2009-08-01

    In many inherited disorders, protein deficiency is one of the major aetiologies, but the molecular and cellular mechanisms remain unclear. We investigated the intracellular degradation of mutant proteins, using naturally occurring PC and PI mutants that lead to congenital deficiencies. We have shown that proteasomes are very important for the degradation of PC and PI mutants, irrespective of the presence or absence of N-glycosylation moieties. Furthermore, mannose trimming after glucose removal is very important for initiation of the degradation. Inhibition of glucose trimming of the mutant proteins accelerated degradation by the proteasomes, and initiation of the degradation occurs after mannose trimming of the middle chain of N-linked glycosylation by mannosidase I. The binding of molecular chaperons influenced by the presence of N-glycosylation moieties may affect the efficient degradation of the mutant proteins. Cotransfection of endoplasmic reticulum (ER) degradation enhancing alpha-mannosidase like protein (EDEM) accelerated the degradation of N-glycosylated PC. The mutant PC or PI molecules were ubiquitin-independently degraded by proteasomes. Autophagy does not appear to contribute to the degradation of PC and PI mutants. These findings might help to elucidate the molecular mechanisms and potential treatments of congenital deficiencies of proteins in a system of coagulation and fibrinolysis.

  19. Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid beta-peptide toxicity: central roles of superoxide production and caspase activation.

    PubMed

    Guo, Q; Sebastian, L; Sopher, B L; Miller, M W; Ware, C B; Martin, G M; Mattson, M P

    1999-03-01

    Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Overexpression of PS1 mutations in cultured PC12 cells increases their vulnerability to apoptosis-induced trophic factor withdrawal and oxidative insults. We now report that primary hippocampal neurons from PS1 mutant knock-in mice, which express the human PS1M146V mutation at normal levels, exhibit increased vulnerability to amyloid beta-peptide toxicity. The endangering action of mutant PS1 was associated with increased superoxide production, mitochondrial membrane depolarization, and caspase activation. The peroxynitrite-scavenging antioxidant uric acid and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone protected hippocampal neurons expressing mutant PS1 against cell death induced by amyloid beta-peptide. Increased oxidative stress may contribute to the pathogenic action of PS1 mutations, and antioxidants may counteract the adverse property of such AD-linked mutations.

  20. Remote estimation of phycocyanin (PC) for inland waters coupled with YSI PC fluorescence probe.

    PubMed

    Song, Kaishan; Li, Lin; Tedesco, Lenore; Clercin, Nicole; Hall, Bob; Li, Shuai; Shi, Kun; Liu, Dawei; Sun, Ying

    2013-08-01

    Nuisance cyanobacterial blooms degrade water resources through accelerated eutrophication, odor generation, and production of toxins that cause adverse effects on human health. Quick and effective methods for detecting cyanobacterial abundance in drinking water supplies are urgently needed to compliment conventional laboratory methods, which are costly and time consuming. Hyperspectral remote sensing can be an effective approach for rapid assessment of cyanobacterial blooms. Samples (n=250) were collected from five drinking water sources in central Indiana (CIN), USA, and South Australia (SA), which experience nuisance cyanobacterial blooms. In situ hyperspectral data were used to develop models by relating spectral signal with handheld fluorescence probe (YSI 6600 XLM-SV) measured phycocyanin (PC in cell/ml), a proxy pigment unique for indicating the presence of cyanobacteria. Three-band model (TBM), which is effective for chlorophyll-a estimates, was tuned to quantify cyanobacteria coupled with the PC probe measured cyanobacteria. As a comparison, two band model proposed by Simis et al. (Limnol Oceanogr, 50(11): 237-245, 2005; denoted as SM05) was paralleled to evaluate TBM model performance. Our observation revealed a high correlation between measured and estimated PC for SA dataset (R (2) =0.96; range: 534-20,200 cell/ml) and CIN dataset (R (2) =0.88; range: 1,300-44,500 cell/ml). The potential of this modeling approach for imagery data were assessed by simulated ESA/Centinel3/OLCI spectra, which also resulted in satisfactory performance with the TBM for both SA dataset (RMSE % =26.12) and CIN dataset (RMSE % =34.49). Close relationship between probe-measured PC and laboratory measured cyanobacteria biovolume was observed (R (2) =0.93, p<0.0001) for the CIN dataset, indicating a stable performance for PC probe. Based on our observation, field spectroscopic measurement coupled with PC probe measurements can provide quantitative cyanobacterial bloom

  1. Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

    SciTech Connect

    Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori

    2014-03-07

    Highlights: • Dioctanoyl-PC (diC8PC) supported growth of a yeast mutant defective in PC synthesis. • diC8PC was converted to PC species containing longer acyl residues in the mutant. • Both acyl residues of diC8PC were replaced by longer fatty acids in vitro. • This system will contribute to the elucidation of the acyl chain remodeling of PC. - Abstract: A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of {sup 13}C-labeled diC8PC ((methyl-{sup 13}C){sub 3}-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-{sup 13}C){sub 3}-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.

  2. D2PC sensitivity analysis

    SciTech Connect

    Lombardi, D.P.

    1992-08-01

    The Chemical Hazard Prediction Model (D2PC) developed by the US Army will play a critical role in the Chemical Stockpile Emergency Preparedness Program by predicting chemical agent transport and dispersion through the atmosphere after an accidental release. To aid in the analysis of the output calculated by D2PC, this sensitivity analysis was conducted to provide information on model response to a variety of input parameters. The sensitivity analysis focused on six accidental release scenarios involving chemical agents VX, GB, and HD (sulfur mustard). Two categories, corresponding to conservative most likely and worst case meteorological conditions, provided the reference for standard input values. D2PC displayed a wide variety of sensitivity to the various input parameters. The model displayed the greatest overall sensitivity to wind speed, mixing height, and breathing rate. For other input parameters, sensitivity was mixed but generally lower. Sensitivity varied not only with parameter, but also over the range of values input for a single parameter. This information on model response can provide useful data for interpreting D2PC output.

  3. Spontaneous Pneumocystis carinii pneumonia in immunodeficient mutant scid mice. Natural history and pathobiology.

    PubMed Central

    Roths, J. B.; Marshall, J. D.; Allen, R. D.; Carlson, G. A.; Sidman, C. L.

    1990-01-01

    The opportunistic pathogen Pneumocystis carinii (Pc) poses a major clinical health problem in individuals with immune deficiency, including those patients with human immunodeficiency (HIV)-associated acquired immune deficiency disease (AIDS). Heretofore, in vivo investigations of the biology of Pc and pathogenesis of pneumocystosis have generally employed steroid-induced immune suppression with antibiotic prophylaxis and protein deprivation. This approach has many drawbacks, chief among them being the widespread, multiple interacting effects caused by the inducing agents. Athymic (nude) mice and rats have been used, but are less than ideal, as the immune defect primarily affects T lymphocytes. This article describes the natural history, pathobiology, and environmental effects on Pc pneumonitis in nonaxenically housed mice homozygous for the autosomal recessive mutation 'severe combined immunodeficiency' (scid), which almost totally lack both cell-mediated and antibody-mediated immune functions. The predictability, unequivocal expression, high morbidity, and well-defined genetic basis make scid/scid mutant mice the model of choice for in vivo studies of spontaneous pneumocystosis. Images Figure 3 Figure 6 PMID:2349968

  4. Spontaneous Pneumocystis carinii pneumonia in immunodeficient mutant scid mice. Natural history and pathobiology.

    PubMed

    Roths, J B; Marshall, J D; Allen, R D; Carlson, G A; Sidman, C L

    1990-05-01

    The opportunistic pathogen Pneumocystis carinii (Pc) poses a major clinical health problem in individuals with immune deficiency, including those patients with human immunodeficiency (HIV)-associated acquired immune deficiency disease (AIDS). Heretofore, in vivo investigations of the biology of Pc and pathogenesis of pneumocystosis have generally employed steroid-induced immune suppression with antibiotic prophylaxis and protein deprivation. This approach has many drawbacks, chief among them being the widespread, multiple interacting effects caused by the inducing agents. Athymic (nude) mice and rats have been used, but are less than ideal, as the immune defect primarily affects T lymphocytes. This article describes the natural history, pathobiology, and environmental effects on Pc pneumonitis in nonaxenically housed mice homozygous for the autosomal recessive mutation 'severe combined immunodeficiency' (scid), which almost totally lack both cell-mediated and antibody-mediated immune functions. The predictability, unequivocal expression, high morbidity, and well-defined genetic basis make scid/scid mutant mice the model of choice for in vivo studies of spontaneous pneumocystosis.

  5. Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans.

    PubMed

    Lam, Minh; Jou, Paul C; Lattif, Ali A; Lee, Yoojin; Malbasa, Christi L; Mukherjee, Pranab K; Oleinick, Nancy L; Ghannoum, Mahmoud A; Cooper, Kevin D; Baron, Elma D

    2011-01-01

    The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis.

  6. Identifying representative drug resistant mutants of HIV

    PubMed Central

    2015-01-01

    Background Drug resistance is one of the most important causes for failure of anti-AIDS treatment. During therapy, multiple mutations accumulate in the HIV genome, eventually rendering the drugs ineffective in blocking replication of the mutant virus. The huge number of possible mutants precludes experimental analysis to explore the molecular mechanisms of resistance and develop improved antiviral drugs. Results In order to solve this problem, we have developed a new algorithm to reveal the most representative mutants from the whole drug resistant mutant database based on our newly proposed unified protein sequence and 3D structure encoding method. Mean shift clustering and multiple regression analysis were applied on genotype-resistance data for mutants of HIV protease and reverse transcriptase. This approach successfully chooses less than 100 mutants with the highest resistance to each drug out of about 10K in the whole database. When considering high level resistance to multiple drugs, the numbers reduce to one or two representative mutants. Conclusion This approach for predicting the most representative mutants for each drug has major importance for experimental verification since the results provide a small number of representative sequences, which will be amenable for in vitro testing and characterization of the expressed mutant proteins. PMID:26678327

  7. The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments

    PubMed Central

    1996-01-01

    The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH- terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates. PMID:8947550

  8. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

    PubMed Central

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

    1995-01-01

    An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants. PMID:7770517

  9. Reduced immobilizing properties of isoflurane and nitrous oxide in mutant mice lacking the N-methyl-D-aspartate receptor GluR(epsilon)1 subunit are caused by the secondary effects of gene knockout.

    PubMed

    Petrenko, Andrey B; Yamakura, Tomohiro; Kohno, Tatsuro; Sakimura, Kenji; Baba, Hiroshi

    2010-02-01

    Until recently, the N-methyl-D-aspartate (NMDA) receptor was considered to possibly mediate the immobility produced by inhaled anesthetics such as isoflurane and nitrous oxide. However, new evidence suggests that the role of this receptor in abolition of the movement response may be less important than previously thought. To provide further evidence supporting or challenging this view, we examined the anesthetic potencies of isoflurane and nitrous oxide in genetically modified animals with established NMDA receptor dysfunction caused by GluRepsilon1 subunit knockout. The immobilizing properties of inhaled anesthetics in mice quantitated by the minimum alveolar anesthetic concentration (MAC) were evaluated using the classic tail clamp method. Compared with wild-type controls, NMDA receptor GluRepsilon1 subunit knockout mice displayed larger isoflurane MAC values indicating a resistance to the immobilizing action of isoflurane. Knockout mice were previously shown to have enhanced monoaminergic tone as a result of genetic manipulation, and this increase in MAC could be abolished in our experiments by pretreatment with the serotonin 5-hydroxytryptamine type 2A receptor antagonist ketanserin or with the dopamine D2 receptor antagonist droperidol at doses that did not affect MAC values in wild-type animals. Mutant mice also displayed resistance to the isoflurane MAC-sparing effect of nitrous oxide, but this resistance was similarly abolished by ketanserin and droperidol. Thus, resistance to the immobilizing action of inhaled anesthetics in knockout mice seems to be secondary to increased monoaminergic activation after knockout rather than a direct result of impaired NMDA receptor function. Our results confirm recent findings indicating no critical contribution of NMDA receptors to the immobility induced by isoflurane and nitrous oxide. In addition, they demonstrate the ability of changes secondary to genetic manipulation to affect the results obtained in global knockout

  10. An equine herpesvirus 1 mutant with a lacZ insertion between open reading frames 62 and 63 is replication competent and causes disease in the murine respiratory model.

    PubMed

    Csellner, H; Walker, C; Love, D N; Whalley, J M

    1998-01-01

    An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed that the lacZ insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no modification to late gene expression as monitored by synthesis of EHV-1 glycoproteins C and D. The parental EHV-1 isolate HVS25A used here had almost identical nucleotide sequence to that published for isolate Ab4, in a 1200 bp region surrounding the insert, but lacked a HindIII site corresponding to Ab4 position 109,048. The lacZ62/63-EHV-1 caused respiratory disease in BALB/c mice with clinical signs, histopathology and virus titres in lungs throughout days 1-5 post infection similar to those induced by wt EHV-1. X-gal staining for beta-galactosidase expression in murine lungs clearly demonstrated EHV-1 infection in cells of the bronchiolar epithelium and pulmonary parenchyma, with a peak of infection evident at day 2 post infection, when up to 50% of bronchioles demonstrated blue-staining and thus virus-infected epithelial cells. The construction of this replication competent virus carrying a reporter gene identifies a site for insertion of foreign genes and will facilitate studies on the pathogenesis of EHV-1.

  11. Loss of the Ciliary Kinase Nek8 Causes Left-Right Asymmetry Defects

    PubMed Central

    Manning, Danielle K.; Sergeev, Mikhail; van Heesbeen, Roy G.; Wong, Michael D.; Oh, Jin-Hee; Liu, Yan; Henkelman, R. Mark; Drummond, Iain; Shah, Jagesh V.

    2012-01-01

    A missense mutation in mouse Nek8, which encodes a ciliary kinase, produces the juvenile cystic kidneys (jck) model of polycystic kidney disease, but the functions of Nek8 are incompletely understood. Here, we generated a Nek8-null allele and found that homozygous mutant mice die at birth and exhibit randomization of left-right asymmetry, cardiac anomalies, and glomerular kidney cysts. The requirement for Nek8 in left-right patterning is conserved, as knockdown of the zebrafish ortholog caused randomized heart looping. Ciliogenesis was intact in Nek8-deficient embryos and cells, but we observed misexpression of left-sided marker genes early in development, suggesting that nodal ciliary signaling was perturbed. We also generated jck/Nek8 compound heterozygotes; these mutants developed less severe cystic disease than jck homozygotes and provided genetic evidence that the jck allele may encode a gain-of-function protein. Notably, NEK8 and polycystin-2 (PC2) proteins interact, and we found that Nek8−/− and Pkd2−/− embryonic phenotypes are strikingly similar. Nek8-deficient embryos and cells did express PC2 normally, which localized properly to the cilia. However, similar to cells lacking PC2, NEK8-depleted inner medullary collecting duct cells exhibited a defective response to fluid shear, suggesting that NEK8 may play a role in mediating PC2-dependent signaling. PMID:23274954

  12. The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells

    PubMed Central

    Flowers, Brittany M.; Rusnak, Lauren E.; Wong, Kristen E.; Banks, Dallas A.; Munyikwa, Michelle R.; McFarland, Alexander G.; Hinton, Shantá D.

    2014-01-01

    The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and

  13. Poster session ELIPGRID-PC

    SciTech Connect

    Davidson, J.R.

    1995-02-01

    ELIPGRID-PC, a new personal computer program, has been developed to provide easy access to Singer`s ELIPGRID algorithm for hot-spot detection probabilities. Three features of the program are the ability to determine: (1) the grid size required for specified conditions, (2) the smallest hot spot that can be sampled with a given probability, and (3) the approximate grid size resulting from specified conditions and sampling cost. ELIPGRID-PC also provides probability of detection versus cost data for graphing with spreadsheets or graphics software. The program has been successfully tested using Singer`s published ELIPGRID results. An apparent error in the published ELIPGRID code has been uncovered and an appropriate modification incorporated into the new program.

  14. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    PubMed

    Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  15. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto

    PubMed Central

    Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3’UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3’UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3’UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo. PMID:28253363

  16. CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice.

    PubMed

    Apostol, Barbara L; Simmons, Danielle A; Zuccato, Chiara; Illes, Katalin; Pallos, Judit; Casale, Malcolm; Conforti, Paola; Ramos, Catarina; Roarke, Margaret; Kathuria, Satish; Cattaneo, Elena; Marsh, J Lawrence; Thompson, Leslie Michels

    2008-09-01

    Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the protein Huntingtin (Htt). We previously reported that mutant Htt expression activates the ERK1/2 and JNK pathways [Apostol, B.L., Illes, K., Pallos, J., Bodai, L., Wu, J., Strand, A., Schweitzer, E.S., Olson, J.M., Kazantsev, A., Marsh, J.L., Thompson, L.M., 2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 15, 273-285]. Chemical and genetic modulation of these pathways promotes cell survival and death, respectively. Here we test the ability of two closely related compounds, CEP-11004 and CEP-1347, which inhibit Mixed Lineage Kinases (MLKs) and are neuroprotective, to suppress mutant Htt-mediated pathogenesis in multiple model systems. CEP-11004/CEP-1347 treatment significantly decreased toxicity in mutant Htt-expressing cells that evoke a strong JNK response. However, suppression of cellular dysfunction in cell lines that exhibit only mild Htt-associated toxicity and little JNK activation was associated with activation of ERK1/2. These compounds also reduced neurotoxicity in immortalized striatal neurons from mutant knock-in mice and Drosophila expressing a mutant Htt fragment. Finally, CEP-1347 improved motor performance in R6/2 mice and restored expression of BDNF, a critical neurotrophic factor that is reduced in HD. These studies suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via CEP-1347 up-regulation of BDNF.

  17. PC25{trademark} product and manufacturing experience

    SciTech Connect

    Hall, E.W.; Riley, W.C.; Sandelli, G.J.

    1996-12-31

    Product and manufacturing experience accumulated since the beginning of PC25. A production in 1991 provides a strong base of demonstration and experience for establishing future improvements to the PC25 power plant.

  18. HBV genotypes prevalence, precore and basal core mutants in Morocco.

    PubMed

    Baha, Warda; Ennaji, My Mustapha; Lazar, Fatiha; Melloul, Marouane; El Fahime, Elmostafa; El Malki, Abdelouahad; Bennani, Abdelouaheb

    2012-08-01

    The study of hepatitis B virus (HBV) genomic heterogeneity has become a major issue in investigations aimed at understanding the relationship between HBV mutants and the wide spectrum of clinical and pathological conditions associated with HBV infection. The objective of the current study was to find out the pattern of HBV genotypes circulating in Morocco and to investigate the precore (PC) and basal core promoter (BCP) mutants' status in Moroccan chronic hepatitis B patients. Viral genotypes were determined in 221 chronic carriers using INNO-LiPA HBV assay and hemi-nested PCR. Phylogenetic analysis was performed in 70 samples, and multiplex PCR method was used to confirm some genotyping results. PC and CP mutants were determined using Inno-Lipa. All isolates were successfully genotyped. The genotype distribution was D in 90.45% of cases, A (5.9%), E (1 case), and mixed genotypes (5 A/D and 2 D/F) in 3.17% patients. HBV carried in the HBV/D samples could be assigned to D7 (63.3%), D1 (32.7%) and 2% of strains to each D4 and D5, all HBV/A belonged to A2 subgenotype and HBV/E strain could not be sub-genotyped. In 70 studied strains, HBV mutants were detected in 88.6% of cases; PC mutants were detected in (40%) of patients and 21.5% present a mixture of wild type and G1896A mutation. BCP mutants were observed in 65.7% of cases, 22.9% were found to have the T1762/1764A double mutation, 18.6% had A1762/1764T mutation and 22.9% of patients showed the A1762T/G1764A double mutation with either A1762T/G1764T mutation. Co-infection by PC and BCP mutants was detected in 52.9% of cases. Movement from place to place most likely shapes the observed genotype distribution and consequent prevalence of genotypes other than A2 or D7 in this population. High circulation of PC and BCP mutants is common in chronic hepatitis B infection in Morocco.

  19. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression

    PubMed Central

    Boortz, Kayla A.; Syring, Kristen E.; Pound, Lynley D.; Wang, Yingda; Oeser, James K.; O’Brien, Richard M.

    2016-01-01

    Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans. PMID:27611587

  20. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

    PubMed

    Boortz, Kayla A; Syring, Kristen E; Pound, Lynley D; Wang, Yingda; Oeser, James K; O'Brien, Richard M

    2016-01-01

    Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

  1. Saccharomyces cerevisiae aldolase mutants.

    PubMed Central

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo. PMID:6384192

  2. [Grape seed extract induces morphological changes of prostate cancer PC-3 cells].

    PubMed

    Shang, Xue-Jun; Yin, Hong-Lin; Ge, Jing-Ping; Sun, Yi; Teng, Wen-Hui; Huang, Yu-Feng

    2008-12-01

    To observe the morphological changes of prostate cancer PC-3 cells induced by grape seed extract (GSE). PC-3 cells were incubated with different concentrations of GSE (100, 200 and 300 microg/ml) for 24, 48 and 72 hours, and then observed for morphological changes by invert microscopy, HE staining and transmission electron microscopy. The incubated PC-3 cells appeared round, small, wrinkled and broken under the invert microscope and exhibited the classical morphological characteristics of cell death under the electron microscope, including cell atrophy, increased vacuoles, crumpled nuclear membrane, and chromosome aggregation. GSE can cause morphological changes and induce necrosis and apoptosis of PC-3 cells.

  3. Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe.

    PubMed

    Tu, Wen-Hui; Lv, Ying; Zhang, Yong-Mei; Hou, Wei; Wang, Jin-Yu; Zhang, Yi-Jun; Liu, Hong-Yan; Zhu, Hao-Xiang; Qin, Yan-Li; Mao, Ri-Cheng; Zhang, Ji-Ming

    2015-06-07

    To investigate precore/basal core promoter (PC/BCP) mutants throughout hepatitis B virus (HBV) infection and to determine their relationship to hepatitis B early antigen (HBeAg) titers. We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012. None of the patients received antiviral therapy. HBV DNA from serum, was quantified by real-time PCR. The HBV genotype was determined by direct sequencing of the S gene. We used the Simpleprobe ultrasensitive quantitative method to detect PC/BCP mutants in each patient. We compared the strain number, percentage, and the changes in PC/BCP mutants in different phases, and analyzed the relationship between PC/BCP mutants and HBeAg by multiple linear regression and logistic regression. Patients with HBV infection (n = 191) were assigned to groups by phase: Immune tolerance (IT) = 55, Immune clearance (IC) = 67, Low-replicative (LR) = 49, and HBeAg-negative hepatitis (ENH) = 20. Of the patients (male, 112; female, 79) enrolled, 122 were HBeAg-positive and 69 were HBeAg-negative. The median age was 33 years (range: 18-78 years). PC and BCP mutation detection rates were 84.82% (162/191) and 96.86% (185/191), respectively. In five HBeAg-negative cases, we detected double mutation G1896A/G1899A. The logarithm value of PC mutant quantities (log10 PC) significantly differed in IT, IC, and LR phases, as well as in the ENH phase (F = 49.350, P < 0.001). The logarithm value of BCP mutant quantities (log10 BCP) also differed during the four phases (F = 25.530, P < 0.001). Log10 PC and log10 BCP values were high in the IT and IC phases, decreased in the LR phase, and increased in the ENH phase, although the absolute value at this point remained lower than that in the IT and IC phases. PC mutant quantity per total viral load (PC%) and BCP mutant quantity per total viral load (BCP%) differed between phases (F = 20.040, P < 0.001; F = 10.830, P < 0.001), with PC

  4. Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice.

    PubMed

    Wu, Gengshu; Sher, Roger B; Cox, Gregory A; Vance, Dennis E

    2009-05-01

    Choline kinase in mice is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneously occurring genomic deletion in murine Chkb results in neonatal bone deformity and hindlimb muscular dystrophy. We have investigated the mechanism by which a lack of choline kinase beta, encoded by Chkb, causes hindlimb muscular dystrophy. The biosynthesis of phosphatidylcholine (PC) is impaired in the hindlimbs of Chkb -/- mice, with an accumulation of choline and decreased amount of phosphocholine. The activity of CTP: phosphocholine cytidylyltransferase is also decreased in the hindlimb muscle of mutant mice. Concomitantly, the activities of PC phospholipase C and phospholipase A2 are increased. The mitochondria in Chkb -/- mice are abnormally large and exhibit decreased inner membrane potential. Despite the muscular dystrophy in Chkb -/- mice, we observed increased expression of insulin like growth factor 1 and proliferating cell nuclear antigen. However, regeneration of hindlimb muscles of Chkb -/- mice was impaired when challenged with cardiotoxin. Injection of CDP-choline increased PC content of hindlimb muscle and decreased creatine kinase activity in plasma of Chkb -/- mice. We conclude that the hindlimb muscular dystrophy in Chkb -/- mice is due to attenuated PC biosynthesis and enhanced catabolism of PC.

  5. Role of polycystin-1 in bone remodeling: orthodontic tooth movement study in mutant mice.

    PubMed

    Shalish, Miriam; Will, Leslie A; Fukai, Naomi; Hou, Bo; Olsen, Bjorn R

    2014-09-01

    To test the hypothesis that polycystin-1 (PC1) is involved in orthodontic tooth movement as a mechanical sensor. The response to force application was compared between three mutant and four wild-type 7-week-old mice. The mutant mice were PC1/Wnt1-cre, lacking PC1 in the craniofacial region. An orthodontic closed coil spring was bonded between the incisor and the left first molar, applying 20 g of force for 4 days. Micro-computed tomography, hematoxylin and eosin staining, and tartrate-resistent acid phosphatase (TRAP) staining were used to study the differences in tooth movement among the groups. In the wild-type mice the bonded molar moved mesially, and the periodontal ligament (PDL) was compressed in the compression side. The compression side showed a hyalinized zone, and osteoclasts were identified there using TRAP staining. In the mutant mice, the molar did not move, the incisor tipped palatally, and there was slight widening of the PDL in the tension area. Osteoclasts were not seen on the bone surface or on the compression side. Osteoclasts were only observed on the other side of the bone-in the bone marrow. These results suggest a difference in tooth movement and osteoclast activity between PC1 mutant mice and wild-type mice in response to orthodontic force. The impaired tooth movement and the lack of osteoclasts on the bone surface in the mutant working side may be related to lack of signal from the PDL due to PC1 deficiency.

  6. PC/104 Embedded IOCs at Jefferson Lab

    SciTech Connect

    Jianxun Yan, Trent Allison, Sue Witherspoon, Anthony Cuffe

    2009-10-01

    Jefferson Lab has developed embedded IOCs based on PC/104 single board computers (SBC) for low level control systems. The PC/104 IOCs run EPICS on top of the RTEMS operating system. Two types of control system configurations are used in different applications, PC/104 SBC with commercial PC/104 I/O cards and PC/104 SBC with custom designed FPGA-based boards. RTEMS was built with CEXP shell to run on the PC/104 SBC. CEXP shell provides the function of dynamic object loading, which is similar to the widely used VxWorks operating system. Standard software configurations were setup for PC/104 IOC application development to provide a familiar format for new projects as well as ease the conversion of applications from VME based IOCs to PC/104 IOCs. Many new projects at Jefferson Lab are going to employ PC/104 SBCs as IOCs and some applications have already been running them for accelerator operations. The PC/104 - RTEMS IOC provides a free open source Real-Time Operating System (RTOS), low cost/maintenance, easily installed/ configured, flexible, and reliable solution for accelerator control and 12GeV Upgrade projects.

  7. Different Phenotypes of the Two Chinese Probands with the Same c.889G>A (p.C162Y) Mutation in COCH Gene Verify Different Mechanisms Underlying Autosomal Dominant Nonsyndromic Deafness 9

    PubMed Central

    Wang, Qi; Fei, Peipei; Gu, Hongbo; Zhang, Yanmei; Ke, Xiaomei; Liu, Yuhe

    2017-01-01

    Objectives By analyzing the different phenotypes of two Chinese DFNA9 families with the same mutation located in the intervening region between the LCCL and vWFA domains of cochlin and testing the functional changes in the mutant cochlin, we investigated the different pathogeneses for mutations in LCCL and vWFA domains. Methods Targeted next-generation sequencing for deafness-related genes was used to identify the mutation in the proband in family #208. The probands of family #208 and family #32 with the same p.C162Y mutation were followed for more than 3 years to evaluate the progression of hearing loss and vestibular dysfunction using pure-tone audiometry, caloric testing, electrocochleogram, vestibular-evoked myogenic potential, and video head-impulse test. The disruption of normal cleavage to produce secreted LCCL domain fragments and the tendency to form aggregations of mutant cochlins were tested by in vitro cell experiments. Results The two families showed different clinical symptoms. Family #32 was identified as having early-onset, progressive sensorineural hearing loss, similar to the symptoms in DFNA9 patients with cochlin mutations in the vWFA domain. The proband of family #208 endured late-onset recurrent paroxysmal vertigo attacks and progressively deteriorating hearing, similar to symptoms in those with cochlin mutations in the LCCL domain. We therefore suggest that the disrupted cleavage of the LCCL domain fragment is likely to cause vestibular dysfunction, and aggregation of mutant cochlin caused by mutations in the vWFA domain is responsible for early-onset hearing loss. The p.C162Y mutation causes either disruption of LCCL domain fragment cleavage or aggregation of mutant cochlin, resulting in the different phenotypes in the two families. Conclusion This study demonstrates that DFNA9 families with the same genotype may have significantly different phenotypes. The mutation site in cochlin is related to the pathological mechanism underlying the

  8. Endonuclease IV (nfo) mutant of Escherichia coli.

    PubMed Central

    Cunningham, R P; Saporito, S M; Spitzer, S G; Weiss, B

    1986-01-01

    A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

  9. The mutant phenotype associated with P-element alleles of the vestigial locus in Drosophila melanogaster may be caused by a readthrough transcript initiated at the P-element promoter.

    PubMed Central

    Hodgetts, R B; O'Keefe, S L

    2001-01-01

    We report here the isolation of a new P-element-induced allele of the vestigial locus vg(2a33), the molecular characterization of which allows us to propose a unifying explanation of the phenotypes of the large number of vestigial P-element alleles that now exists. The first P-element allele of vestigial to be isolated was vg(21), which results in a very weak mutant wing phenotype that is suppressed in the P cytotype. By destabilizing vg(2a33) in a dysgenic cross, we isolated the vg(2a33) allele, which exhibits a moderate mutant wing phenotype and is not suppressed by the P cytotype. The new allele is characterized by a 46-bp deletion that removes the 3'-proximal copy of the 11-bp internal repeat from the P element of vg(21). To understand how this subtle difference between the two alleles leads to a rather pronounced difference in their phenotypes, we mapped both the vg and P-element transcription units present in wild type and mutants. Using both 5'-RACE and S1 protection, we found that P-element transcription is initiated 19 bp farther upstream than previously thought. Using primer extension, the start of vg transcription was determined to lie 435 bp upstream of the longest cDNA recovered to date and upstream of the P-element insertion site. Our discovery that the P element is situated within the first vg exon has prompted a reassessment of the large body of genetic data on a series of alleles derived from vg(21). Our current hypothesis to explain the degree of variation in the mutant phenotypes and their response to the P repressor invokes a critical RNA secondary structure in the vg transcript, the formation of which is hindered by a readthrough transcript initiated at the P-element promoter. PMID:11290721

  10. The mutant phenotype associated with P-element alleles of the vestigial locus in Drosophila melanogaster may be caused by a readthrough transcript initiated at the P-element promoter.

    PubMed

    Hodgetts, R B; O'Keefe, S L

    2001-04-01

    We report here the isolation of a new P-element-induced allele of the vestigial locus vg(2a33), the molecular characterization of which allows us to propose a unifying explanation of the phenotypes of the large number of vestigial P-element alleles that now exists. The first P-element allele of vestigial to be isolated was vg(21), which results in a very weak mutant wing phenotype that is suppressed in the P cytotype. By destabilizing vg(2a33) in a dysgenic cross, we isolated the vg(2a33) allele, which exhibits a moderate mutant wing phenotype and is not suppressed by the P cytotype. The new allele is characterized by a 46-bp deletion that removes the 3'-proximal copy of the 11-bp internal repeat from the P element of vg(21). To understand how this subtle difference between the two alleles leads to a rather pronounced difference in their phenotypes, we mapped both the vg and P-element transcription units present in wild type and mutants. Using both 5'-RACE and S1 protection, we found that P-element transcription is initiated 19 bp farther upstream than previously thought. Using primer extension, the start of vg transcription was determined to lie 435 bp upstream of the longest cDNA recovered to date and upstream of the P-element insertion site. Our discovery that the P element is situated within the first vg exon has prompted a reassessment of the large body of genetic data on a series of alleles derived from vg(21). Our current hypothesis to explain the degree of variation in the mutant phenotypes and their response to the P repressor invokes a critical RNA secondary structure in the vg transcript, the formation of which is hindered by a readthrough transcript initiated at the P-element promoter.

  11. Heat shock induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway.

    PubMed

    Kano, Yoshio; Nakagiri, Sachiko; Nohno, Tsutomu; Hiragami, Fukumi; Kawamura, Kenji; Kadota, Michiyo; Numata, Keizo; Koike, Yoshihisa; Furuta, Tomohisa

    2004-11-12

    We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.

  12. Effect of K doping on CuPc: C60 heterojunctions

    NASA Astrophysics Data System (ADS)

    Cheng, Chiu-Ping; Chen, Wen-Yen; Wei, Ching-Hsuan; Pi, Tun-Wen

    2011-12-01

    Here, the electronic properties of K-doped copper phthalocyanine (CuPc): C60 heterojunctions are studied via synchrotron-radiation photoemission. The K-doped heterointerfaces were obtained by means of C60 on K1.5CuPc and CuPc on K3C60. The photoelectron spectra show that the potassium prefers to combine with C60. At the C60/K1.5CuPc interface, the K diffuses and transfers negative charge into the C60 overlayer, while no strong chemical reaction could be found at the CuPc/K3C60 interface. A significant shift of the vacuum level was observed in both cases, which was caused by the charge transfer for the C60/K1.5CuPc and by the induced density of interface states (IDIS) dipole for the CuPc/K3C60. The energy level diagrams show that using C60 adsorption on a K-doped CuPc film is good for the improvement of photovoltaic devices. However, the inverse process, that of CuPc on a K-doped C60, is unfavorable for the photovoltaic effect.

  13. AMPS/PC - AUTOMATIC MANUFACTURING PROGRAMMING SYSTEM

    NASA Technical Reports Server (NTRS)

    Schroer, B. J.

    1994-01-01

    The AMPS/PC system is a simulation tool designed to aid the user in defining the specifications of a manufacturing environment and then automatically writing code for the target simulation language, GPSS/PC. The domain of problems that AMPS/PC can simulate are manufacturing assembly lines with subassembly lines and manufacturing cells. The user defines the problem domain by responding to the questions from the interface program. Based on the responses, the interface program creates an internal problem specification file. This file includes the manufacturing process network flow and the attributes for all stations, cells, and stock points. AMPS then uses the problem specification file as input for the automatic code generator program to produce a simulation program in the target language GPSS. The output of the generator program is the source code of the corresponding GPSS/PC simulation program. The system runs entirely on an IBM PC running PC DOS Version 2.0 or higher and is written in Turbo Pascal Version 4 requiring 640K memory and one 360K disk drive. To execute the GPSS program, the PC must have resident the GPSS/PC System Version 2.0 from Minuteman Software. The AMPS/PC program was developed in 1988.

  14. A novel gain of function mutant in C-kit gene and its tumorigenesis in nude mice

    PubMed Central

    Bai, Chen-Guang; Liu, Xiao-Hong; Xie, Qiang; Feng, Fei; Ma, Da-Lie

    2005-01-01

    AIM: To transfect mutant C-kit cDNA at codon 579 into human embryonic kidney cell line to observe its role in the pathogenesis of gastrointestinal stromal tumor (GIST). METHODS: Eukaryotic expression vectors of pcDNA3-Kit-NW and pcDNA3-Kit-W were constructed. Then pcDNA3-Kit-NW and pcDNA3-Kit-W plasmids were transfected into human embryonic kidney cell line by Lipofectamine. The resistant clone was screened by G418 filtration and identified by sequencing, Western blotting, and immunocytochemical staining. Human embryonic kidney cells were divided into three groups including pcDNA3-Kit-NW, pcDNA3-Kit-W, and vector control groups. Absorbency value with a wavelength of 574 nm was detected by MTT analysis. Mice were injected with three groups of cells. Volume, mass, and histological examinations of the tumors in different groups were measured and compared. RESULTS: The C-kit gene and mutant C-kit gene were successfully cloned into the eukaryotic expression vector pcDNA3. pcDNA3-Kit-NW and pcDNA3-Kit-W were successfully transfected into human embryonic kidney cell line and showed stable expression in this cell line. Cell proliferating activity had significant differences between pcDNA3-Kit-NW and pcDNA3, pcDNA3-Kit-NW and pcDNA3-Kit-W (P<0.05), respectively. Tumors were only observed in nude mice implanted with cells transfected with pcDNA3-Kit-NW. CONCLUSION: Mutation of C-kit gene increases the proliferation activity of human cells and plays an important role in the malignant transformation of GIST. PMID:16437655

  15. Propagation mechanism of daytime Pc 3-4 pulsations observed at synchronous orbit and multiple ground-based stations

    NASA Technical Reports Server (NTRS)

    Yumoto, K.; Saito, T.; Akasofu, S.-I.; Tsurutani, B. T.; Smith, E. J.

    1985-01-01

    Observational data obtained during the last two decades show that the amplitude of daytime Pc 3-4 magnetic pulsations is controlled by the solar wind conditions. The high degree of correlation between the solar wind parameters and Pc 3-4 pulsations in the dayside magnetosphere suggests that the ultimate cause of the daytime Pc 3-4 pulsations must be the interaction of the solar wind with the earth's magnetosphere. The present paper is concerned with details regarding the control of the properties of the Pc 3-4 pulsations by the solar wind parameters, taking into account observations made at multiple ground-based stations. It is attempted to establish the relation between the daytime Pc 3-4 pulsations at the ground stations and the compressional Pc 3-4 waves in the magnetosphere. Attention is given to the most probable propagation mechanism of the daytime Pc 3-4 pulsations in the magnetosphere.

  16. Brassinosteroid Mutants of Crops.

    PubMed

    Bishop, Gerard J.

    2003-12-01

    Plant steroid hormones, brassinosteroids (BRs), were originally isolated from extracts of pollen because of their growth-promoting properties and their potential use for enhancing crop production. Mutants in the biosynthesis, metabolism, and signaling of brassinolide (BL), the most bioactive BR, are important resources in helping to establish BRs' essential role in plant growth and development. The dark green and distinctive dwarf phenotype of BR-related mutants identified in pea, tomato, and rice highlights the importance of BRs in crops. These mutants are helping to elucidate both the conserved and the unique features of BR biosynthesis and signaling. Such insights are providing the key knowledge and understanding that will enable the development of strategies towards the production of crops with enhanced qualities.

  17. Genome sequencing of the Trichoderma reesei QM9136 mutant identifies a truncation of the transcriptional regulator XYR1 as the cause for its cellulase-negative phenotype

    SciTech Connect

    Lichius, Alexander; Bidard, Frédérique; Buchholz, Franziska; Le Crom, Stéphane; Martin, Joel; Schackwitz, Wendy; Austerlitz, Tina; Grigoriev, Igor V; Baker, Scott E; Margeot, Antoine; Seiboth, Bernhard; Kubicek, Christian P

    2015-04-20

    Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a.

  18. Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae.

    PubMed Central

    Letts, V A; Henry, S A

    1985-01-01

    chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. As expected, when chol mutants were starved for ethanolamine, the rates of synthesis of the phospholipids phosphatidylethanolamine and PC declined rapidly. Surprisingly, however, coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. The results obtained suggest that the slowing of PC biosynthesis in ethanolamine-starved chol cells leads to a coordinated decrease in the synthesis of all phospholipids. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed. Images PMID:2991194

  19. PC-based fault finder

    SciTech Connect

    Bengiamin, N.N. ); Jensen, C.A. . Electrical Engineering Dept. Otter Tail Power Co., Fergus Falls, MN . System Protection Group); McMahon, H. )

    1993-07-01

    Electric utilities are continually pressed to stay competitive while meeting the increasing demand of today's sophisticated customer. Advances in electron equipment and the improved array of electric driven devices are setting new standards for improved reliability and quality of service. Besides the specifications on voltage and frequency regulation and the permitted harmonic content, to name a few, the number and duration of service interruptions have a dramatic direct effect on the customer. Accurate fault locating reduces transmission line patrolling and is of particular significance in repairing long lines in rough terrain. Shortened outage times, reduced equipment degrading and stress on the system, fast restored service, and improved revenue are immediate outcomes of fast fault locating which insure minimum loss of system security. This article focuses on a PC-based (DOS) computer program that has unique features for identifying the type of fault and its location on overhead transmission/distribution lines. Balanced and unbalanced faults are identified and located accurately while accounting for changes in conductor sizes and network configuration. The presented concepts and methodologies have been spurred by Otter Tail Power's need for an accurate fault locating scheme to accommodate multiple feeders with mixed lone configurations. A case study based on a section of the Otter Tail network is presented to illustrate the features and capabilities of the developed software.

  20. Isolation and characterization of unusual gin mutants.

    PubMed Central

    Klippel, A; Cloppenborg, K; Kahmann, R

    1988-01-01

    Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation. Images PMID:2974801

  1. Drosophila melanogaster dHCF interacts with both PcG and TrxG epigenetic regulators.

    PubMed

    Rodriguez-Jato, Sara; Busturia, Ana; Herr, Winship

    2011-01-01

    Repression and activation of gene transcription involves multiprotein complexes that modify chromatin structure. The integration of these complexes at regulatory sites can be assisted by co-factors that link them to DNA-bound transcriptional regulators. In humans, one such co-factor is the herpes simplex virus host-cell factor 1 (HCF-1), which is implicated in both activation and repression of transcription. We show here that disruption of the gene encoding the Drosophila melanogaster homolog of HCF-1, dHCF, leads to a pleiotropic phenotype involving lethality, sterility, small size, apoptosis, and morphological defects. In Drosophila, repressed and activated transcriptional states of cell fate-determining genes are maintained throughout development by Polycomb Group (PcG) and Trithorax Group (TrxG) genes, respectively. dHCF mutant flies display morphological phenotypes typical of TrxG mutants and dHCF interacts genetically with both PcG and TrxG genes. Thus, dHCF inactivation enhances the mutant phenotypes of the Pc PcG as well as brm and mor TrxG genes, suggesting that dHCF possesses Enhancer of TrxG and PcG (ETP) properties. Additionally, dHCF interacts with the previously established ETP gene skd. These pleiotropic phenotypes are consistent with broad roles for dHCF in both activation and repression of transcription during fly development.

  2. Polarization of Pc1/EMIC waves and related proton auroras observed at Athabasca

    NASA Astrophysics Data System (ADS)

    Nomura, R.; Shiokawa, K.; Sakaguchi, K.; Otsuka, Y.; Connors, M. G.

    2010-12-01

    Electromagnetic ion cyclotron (EMIC) waves excited in the equatorial region of the magnetosphere by the ion cyclotron instability propagate along magnetic field lines to the ionosphere and are observed as Pc1 geomagnetic pulsations (Pc1) with frequencies at 0.2-5Hz on the ground. These Pc1 waves propagate horizontally through the ionospheric duct. Magnetospheric ions are scattered by the resonance with EMIC waves and precipitate to the ionosphere to cause isolated proton auroras at subauroral latitudes. One-to-one correspondence between isolated proton auroras and Pc1 waves was found by Sakaguchi et al. [JGR, 2008]. The mechanism of ionospheric duct propagation of Pc1 has been studied theoretically for the polarization characteristics [Graifinger, JGR, 1972; Fujita and Tamao, JGR, 1988], and observationally for the possible Pc1 source [e.g., Fraser et al., JATP, 1976] and for the spatial distribution of polarization mode [Hayashi et al., Can J. Phys., 1981] using ground magnetometers. However, comparison between the actual position and size of ionospheric Pc1 sources and the polarization characteristics of Pc1 waves has not been done. In order to investigate this relation, we compare the spectral and polarization parameters of Pc1 waves observed by a 64-Hz sampling induction magnetometer and the position and area of isolated proton auroras observed by an all-sky imager at Athabasca (ATH, 54.7N, 246.7E, magnetic latitude: 61.7N), for 13 one-to-one correspondent events of Pc1 waves and isolated proton auroras reported by Sakaguchi et al. [JGR, 2008]. We found that the major axis direction of Pc1 polarization varies depending on the area of the isolated proton aurora and on the distance from ATH to the aurora. In the presentation, we will discuss these results based on a multi-event study using data from three years of 2005-2008 in the context of the model calculation by Fujita and Tamao [1988].

  3. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  4. Functional roles of PC-PLC and Cdc20 in the cell cycle, proliferation, and apoptosis.

    PubMed

    Chen, Zhiwei; Yu, Yongfeng; Fu, Da; Li, Ziming; Niu, Xiaoming; Liao, Meilin; Lu, Shun

    2010-06-01

    Phosphatidylcholine-specific phospholipase C (PC-PLC) is the major enzyme in the Phosphatidylcholine (PC) cycle and is involved in many long-term cellular responses such as activation, proliferation, and differentiation events. Cell division cycle 20 homolog (Cdc20) is an essential cell-cycle regulator required for the completion of mitosis. Our previous studies identified the interaction between PC-PLC and Cdc20. Through the interaction, Cdc20 could mediate the degradation of PC-PLC by Cdc20-mediated ubiquitin proteasome pathway (UPP). In this study, we found that PC-PLC might not be involved in cancer metastasis. Inhibition of PC-PLC by D609 could cause cell proliferation inhibition and apoptosis inhibition in CBRH-7919 cells. Inhibition of PC-PLC could also influence the cell cycle by arresting the cells in G1 phase, and Cdc20 might be involved in these processes. Taken together, in this report, we provided new evidence for the functional roles of PC-PLC and Cdc20 in the cell cycle, proliferation, and apoptosis in CBRH-7919 cells.

  5. Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

    PubMed Central

    Cesarini, Elisa; Mozzetta, Chiara; Marullo, Fabrizia; Gregoretti, Francesco; Gargiulo, Annagiusi; Columbaro, Marta; Cortesi, Alice; Antonelli, Laura; Di Pelino, Simona; Squarzoni, Stefano; Palacios, Daniela; Zippo, Alessio; Bodega, Beatrice; Oliva, Gennaro

    2015-01-01

    Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors—and how this cross talk influences physiological processes—is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein–mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein–mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors. PMID:26553927

  6. Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes.

    PubMed

    Cesarini, Elisa; Mozzetta, Chiara; Marullo, Fabrizia; Gregoretti, Francesco; Gargiulo, Annagiusi; Columbaro, Marta; Cortesi, Alice; Antonelli, Laura; Di Pelino, Simona; Squarzoni, Stefano; Palacios, Daniela; Zippo, Alessio; Bodega, Beatrice; Oliva, Gennaro; Lanzuolo, Chiara

    2015-11-09

    Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors.

  7. Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system

    PubMed Central

    Ulbrich, Lisa; Favaloro, Flores Lietta; Trobiani, Laura; Marchetti, Valentina; Patel, Vruti; Pascucci, Tiziana; Comoletti, Davide; Marciniak, Stefan J.; De Jaco, Antonella

    2015-01-01

    Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations. PMID:26621873

  8. Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system.

    PubMed

    Ulbrich, Lisa; Favaloro, Flores Lietta; Trobiani, Laura; Marchetti, Valentina; Patel, Vruti; Pascucci, Tiziana; Comoletti, Davide; Marciniak, Stefan J; De Jaco, Antonella

    2016-02-15

    Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations.

  9. Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses.

    PubMed

    Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

    2017-02-15

    HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated.

  10. Do the Pc+ pentaquarks have strange siblings?

    NASA Astrophysics Data System (ADS)

    Lebed, Richard F.

    2015-12-01

    The recent LHCb discovery of states Pc+(4380 ), Pc+(4450 ), believed to be c c ¯u u d pentaquark resonances, begs the question of whether equivalent states with c c ¯→s s ¯ exist, and how they might be produced. The precise analogue to the Pc+ discovery channel Λb→J /ψ K-p , namely, Λc→ϕ π0p , is feasible for this study and indeed is less Cabibbo suppressed, although its limited phase space suggests that evidence of a s s ¯u u d resonance Ps+ would be confined to the kinematic end-point region.

  11. Dispersive Pc1 bursts observed by Freja

    SciTech Connect

    Mursula, K.; Braeysy, T.; Rasinkangas, R.; Tanskanen, P.; Blomberg, L.G.; Lindqvist, P.A.; Marklund, G.T.

    1994-08-15

    The authors report on observation of electromagnetic ion cyclotron waves (Pc1 pulsations) by the Freja satellite on November 18, 1992. These observations are coincident with ground based observation of such pearl like Pc1 pulsations extending over a 12 hour period. This is the first observation by a satellite above the ionosphere of such phenomena. The wave pulsations were observed to come in 10 to 25 second pulses, and to be clearly dispersive in nature. Two spectral bands were observed in all Pc1 pearls. In the longer bursts, the authors observed time differences between the two distinct spectral bands.

  12. Scale-PC shielding analysis sequences

    SciTech Connect

    Bowman, S.M.

    1996-05-01

    The SCALE computational system is a modular code system for analyses of nuclear fuel facility and package designs. With the release of SCALE-PC Version 4.3, the radiation shielding analysis community now has the capability to execute the SCALE shielding analysis sequences contained in the control modules SAS1, SAS2, SAS3, and SAS4 on a MS- DOS personal computer (PC). In addition, SCALE-PC includes two new sequences, QADS and ORIGEN-ARP. The capabilities of each sequence are presented, along with example applications.

  13. The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

    PubMed

    Liang, Zhe; Demko, Viktor; Wilson, Robert C; Johnson, Kenneth A; Ahmad, Rafi; Perroud, Pierre-François; Quatrano, Ralph; Zhao, Sen; Shalchian-Tabrizi, Kamran; Otegui, Marisa S; Olsen, Odd-Arne; Johansen, Wenche

    2013-09-01

    DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.

  14. Magnetospheric filter effect for Pc 3 Alfven mode waves

    NASA Technical Reports Server (NTRS)

    Zhang, X.; Comfort, R. H.; Gallagher, D. L.; Green, J. L.; Musielak, Z. E.; Moore, T. E.

    1995-01-01

    We present a ray-tracing study of the propagation of Pc 3 Alfven mode waves originating at the dayside magnetopause. This study reveals interesting features of magnetospheric filter effect for these waves. Pc 3 Alfven mode waves cannot penetrate to low Earth altitudes unless the wave frequency is below approximately 30 mHz. Configurations of the dispersion curves and the refractive index show that the gyroresonance and pseudo-cutoff introduced by the heavy ion O(+) block the waves. When the O(+) concentration is removed from the plasma composition, the barriers caused by the O(+) no longer exist, and waves with much higher frequencies than 30 mHz can penetrate to low altitudes. The result that the 30 mHz or lower frequency Alfven waves can be guided to low altitudes agrees with ground-based power spectrum observation at high altitudes.

  15. Magnetospheric filter effect for Pc 3 Alfven mode waves

    NASA Technical Reports Server (NTRS)

    Zhang, X.; Comfort, R. H.; Gallagher, D. L.; Green, J. L.; Musielak, Z. E.; Moore, T. E.

    1994-01-01

    We present a ray-tracing study of the propagation of Pc 3 Alfven mode waves originating at the dayside magnetopause. This study reveals interesting features of a magnetospheric filter effect for these waves. Pc 3 Alfven mode waves cannot penetrate to low Earth altitudes unless the wave frequency is below approximately 30 mHz. Configurations of the dispersion curves and the refractive index show that the gyroresonance and pseudo-cutoff introduced by the heavy ion O(+) block the waves. When the O(+) concentration is removed from the plasma composition, the barriers caused by the O(+) no longer exist, and waves with much higher frequencies than 30 mHz can penetrate to low altitudes. The result that the 30 mHz or lower frequency Alfven waves can be guided to low altitudes agrees with ground-based power spectrum observations at high latitudes.

  16. Cytotoxic Effects of the Ethanol Bane Skin Extract in Human Prostate Cancer Pc3 Cells

    PubMed Central

    Amiri, Maryam; Kazerouni, Faranak; Namaki, Saeed; Darbandi Tamijani, Hassan; Rahimipour, Hooman; Boroumand, Nasrin; Barghi, Siyamak; Ebrahimi, Nazanin; Gheibi Hayat, Seyed Mohammad

    2016-01-01

    Background: It is extensively supposed that vegetarian diet could affect cancer progress and increase the influence of formal chemotherapy. Objectives: The present study was designed to determine the effect of the ethanol Bane skin extract against chemo resistant prostate cancer PC3 cells. Materials and Methods: PC3 and L929 cells were cultivated and then incubated in the ethanol Bane skin extract with various concentrations of 0.78, 1.5, 3.13, 6.25, 12.5 mg/mL in 3 times 24, 48, 72 hours. Cytotoxic effect of the ethanol Bane skin extract on PC3 and L929 cells was examined by MTT assay after 24, 48, and 72 hours. Morphology of PC3 cells was evaluated by Gimsa staining. Results: The ethanol Bane skin extract inhibited proliferation and caused cell death with IC50 values of 2.8 mg/mL on PC3 cells and the IC50 was 6.1 mg/mL on l929 cells. Morphological changes and apoptotic bodies were observed in PC3 cells faced with the ethanol Bane skin extract by staining with Gimsa. Conclusions: The ethanol Bane skin extract could repress the growth of PC3 cell line. This inhibitory effect of the Bane extract depended on the dose and the time on PC3. The result of this study shows that the ethanol Bane skin extract includes photochemical and inhibitory function against proliferation and inducer of apoptosis in human prostate cancer PC3 cells and also has less cytotoxic effect on l929 than PC3 cells. The ethanol Bane skin extract might be a good candidate for the new herbal anticancer drug. PMID:27482333

  17. Disruption of the nuclear membrane by perinuclear inclusions of mutant huntingtin causes cell-cycle re-entry and striatal cell death in mouse and cell models of Huntington's disease

    PubMed Central

    Liu, Kuan-Yu; Shyu, Yu-Chiau; Barbaro, Brett A.; Lin, Yuan-Ta; Chern, Yijuang; Thompson, Leslie Michels; James Shen, Che-Kun; Marsh, J. Lawrence

    2015-01-01

    Accumulation of N-terminal fragments of mutant huntingtin (mHTT) in the cytoplasm, nuclei and axons of neurons is a hallmark of Huntington's disease (HD), although how these fragments negatively impact neurons remains unclear. We followed the distribution of mHTT in the striata of transgenic R6/2-J2 HD mice as their motor function declined. The fraction of cells with diffuse, perinuclear or intranuclear mHTT changed in parallel with decreasing motor function. In transgenic mice, medium spiny neurons (MSNs) that exhibited perinuclear inclusions expressed cell-cycle markers typically not seen in the striata of normal mice, and these cells are preferentially lost as disease progresses. Electron microscopy reveals that perinuclear inclusions disrupt the nuclear envelope. The progression of perinuclear inclusions being accompanied by cell-cycle activation and culminating in cell death was also observed in 1° cortical neurons. These observations provide a strong correlation between the subcellular location of mHTT, disruption of the nucleus, re-entry into the cell-cycle and eventual neuronal death. They also highlight the fact that the subcellular distribution of mHTT is highly dynamic such that the distribution of mHTT observed depends greatly on the stage of the disease being examined. PMID:25398943

  18. High Persister Mutants in Mycobacterium tuberculosis

    PubMed Central

    Torrey, Heather L.; Keren, Iris; Via, Laura E.; Lee, Jong Seok; Lewis, Kim

    2016-01-01

    Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

  19. Decay behaviors of the Pc hadronic molecules

    NASA Astrophysics Data System (ADS)

    Lin, Yong-Hui; Shen, Chao-Wei; Guo, Feng-Kun; Zou, Bing-Song

    2017-06-01

    The Pc(4380 ) and Pc(4450 ) states observed recently by the LHCb experiment were proposed to be either D ¯Σc* or D¯*Σc bound states. We analyze the decay behaviors of two such types of hadronic molecules within the effective Lagrangian framework. With branching ratios of ten possible decay channels calculated, it is found that the two types of hadronic molecules have distinguishable decay patterns. While the D ¯Σc* molecule decays dominantly to the D¯*Λc channel with a branching ratio by 2 orders of magnitude larger than to D ¯Λc, the D¯*Σc molecule decays to these two channels with a difference of less than a factor of 2. Our results show that the total decay width of Pc(4380 ) as the spin-parity-3/2- D ¯Σc* molecule is about a factor of 2 larger than the corresponding value for the D¯*Σc molecule. It suggests that the assignment of the D ¯Σc* molecule for Pc(4380 ) is more favorable than the D¯*Σc molecule. In addition, Pc(4450 ) seems to be a D¯*Σc molecule with JP=5/2+ in our scheme. Based on these partial decay widths of the Pc states, we estimate the cross sections for the reactions γ p →J /ψ p and π p →J /ψ p through the s-channel Pc states. The forthcoming γ p experiment at JLAB and the π p experiment at JPARC should be able to pin down the nature of these Pc states.

  20. DIGLIB. PC-DOS Graphics Subroutine Library

    SciTech Connect

    Burleson, R.R.

    1989-02-01

    DIGLIB is a collection of general graphics subroutines. It was designed to be small, reasonably fast, device-independent, and compatible with DEC-supplied operating systems for VAXes, PDP-11s, and LSI-11s, and the DOS operating system for IBM PCs and IBM-compatible machines. DIGLIB/PC runs on IBM PCs under PC-DOS or MS-DOS. The software is readily usable by casual programmers for two-dimensional plotting.

  1. Modulation of PLAGL2 transactivation by positive cofactor 2 (PC2), a component of the ARC/Mediator complex.

    PubMed

    Wezensky, Sara J; Hanks, Tracey S; Wilkison, Michelle J; Ammons, Mary Cloud; Siemsen, Daniel W; Gauss, Katherine A

    2010-02-15

    The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter (NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-alpha-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.

  2. MODULATION OF PLAGL2 TRANSACTIVATION BY POSITIVE COFACTOR 2 (PC2), A COMPONENT OF THE ARC/MEDIATOR COMPLEX

    PubMed Central

    Wezensky, Sara J.; Hanks, Tracey S.; Wilkison, Michelle J.; Ammons, Mary Cloud; Siemsen, Daniel W.; Gauss, Katherine A.

    2009-01-01

    The pleomorphic adenoma gene (PLAG) family of transcription factors regulate a wide-range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter (NCF2). The PLAGL2 binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-α-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation. PMID:20025940

  3. Mutations in GANAB, Encoding the Glucosidase IIα Subunit, Cause Autosomal-Dominant Polycystic Kidney and Liver Disease.

    PubMed

    Porath, Binu; Gainullin, Vladimir G; Cornec-Le Gall, Emilie; Dillinger, Elizabeth K; Heyer, Christina M; Hopp, Katharina; Edwards, Marie E; Madsen, Charles D; Mauritz, Sarah R; Banks, Carly J; Baheti, Saurabh; Reddy, Bharathi; Herrero, José Ignacio; Bañales, Jesús M; Hogan, Marie C; Tasic, Velibor; Watnick, Terry J; Chapman, Arlene B; Vigneau, Cécile; Lavainne, Frédéric; Audrézet, Marie-Pierre; Ferec, Claude; Le Meur, Yannick; Torres, Vicente E; Harris, Peter C

    2016-06-02

    Autosomal-dominant polycystic kidney disease (ADPKD) is a common, progressive, adult-onset disease that is an important cause of end-stage renal disease (ESRD), which requires transplantation or dialysis. Mutations in PKD1 or PKD2 (∼85% and ∼15% of resolved cases, respectively) are the known causes of ADPKD. Extrarenal manifestations include an increased level of intracranial aneurysms and polycystic liver disease (PLD), which can be severe and associated with significant morbidity. Autosomal-dominant PLD (ADPLD) with no or very few renal cysts is a separate disorder caused by PRKCSH, SEC63, or LRP5 mutations. After screening, 7%-10% of ADPKD-affected and ∼50% of ADPLD-affected families were genetically unresolved (GUR), suggesting further genetic heterogeneity of both disorders. Whole-exome sequencing of six GUR ADPKD-affected families identified one with a missense mutation in GANAB, encoding glucosidase II subunit α (GIIα). Because PRKCSH encodes GIIβ, GANAB is a strong ADPKD and ADPLD candidate gene. Sanger screening of 321 additional GUR families identified eight further likely mutations (six truncating), and a total of 20 affected individuals were identified in seven ADPKD- and two ADPLD-affected families. The phenotype was mild PKD and variable, including severe, PLD. Analysis of GANAB-null cells showed an absolute requirement of GIIα for maturation and surface and ciliary localization of the ADPKD proteins (PC1 and PC2), and reduced mature PC1 was seen in GANAB(+/-) cells. PC1 surface localization in GANAB(-/-) cells was rescued by wild-type, but not mutant, GIIα. Overall, we show that GANAB mutations cause ADPKD and ADPLD and that the cystogenesis is most likely driven by defects in PC1 maturation.

  4. The Alexander Disease–Causing Glial Fibrillary Acidic Protein Mutant, R416W, Accumulates into Rosenthal Fibers by a Pathway That Involves Filament Aggregation and the Association of αB-Crystallin and HSP27

    PubMed Central

    Perng, Ming Der; Su, Mu; Wen, Shu Fang; Li, Rong; Gibbon, Terry; Prescott, Alan R.; Brenner, Michael; Quinlan, Roy A.

    2006-01-01

    Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed “Rosenthal fibers,” contain the protein chaperones αB-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of αB-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease. PMID:16826512

  5. CARES/PC - CERAMICS ANALYSIS AND RELIABILITY EVALUATION OF STRUCTURES

    NASA Technical Reports Server (NTRS)

    Szatmary, S. A.

    1994-01-01

    The beneficial properties of structural ceramics include their high-temperature strength, light weight, hardness, and corrosion and oxidation resistance. For advanced heat engines, ceramics have demonstrated functional abilities at temperatures well beyond the operational limits of metals. This is offset by the fact that ceramic materials tend to be brittle. When a load is applied, their lack of significant plastic deformation causes the material to crack at microscopic flaws, destroying the component. CARES/PC performs statistical analysis of data obtained from the fracture of simple, uniaxial tensile or flexural specimens and estimates the Weibull and Batdorf material parameters from this data. CARES/PC is a subset of the program CARES (COSMIC program number LEW-15168) which calculates the fast-fracture reliability or failure probability of ceramic components utilizing the Batdorf and Weibull models to describe the effects of multi-axial stress states on material strength. CARES additionally requires that the ceramic structure be modeled by a finite element program such as MSC/NASTRAN or ANSYS. The more limited CARES/PC does not perform fast-fracture reliability estimation of components. CARES/PC estimates ceramic material properties from uniaxial tensile or from three- and four-point bend bar data. In general, the parameters are obtained from the fracture stresses of many specimens (30 or more are recommended) whose geometry and loading configurations are held constant. Parameter estimation can be performed for single or multiple failure modes by using the least-squares analysis or the maximum likelihood method. Kolmogorov-Smirnov and Anderson-Darling goodness-of-fit tests measure the accuracy of the hypothesis that the fracture data comes from a population with a distribution specified by the estimated Weibull parameters. Ninety-percent confidence intervals on the Weibull parameters and the unbiased value of the shape parameter for complete samples are provided

  6. CARES/PC - CERAMICS ANALYSIS AND RELIABILITY EVALUATION OF STRUCTURES

    NASA Technical Reports Server (NTRS)

    Szatmary, S. A.

    1994-01-01

    The beneficial properties of structural ceramics include their high-temperature strength, light weight, hardness, and corrosion and oxidation resistance. For advanced heat engines, ceramics have demonstrated functional abilities at temperatures well beyond the operational limits of metals. This is offset by the fact that ceramic materials tend to be brittle. When a load is applied, their lack of significant plastic deformation causes the material to crack at microscopic flaws, destroying the component. CARES/PC performs statistical analysis of data obtained from the fracture of simple, uniaxial tensile or flexural specimens and estimates the Weibull and Batdorf material parameters from this data. CARES/PC is a subset of the program CARES (COSMIC program number LEW-15168) which calculates the fast-fracture reliability or failure probability of ceramic components utilizing the Batdorf and Weibull models to describe the effects of multi-axial stress states on material strength. CARES additionally requires that the ceramic structure be modeled by a finite element program such as MSC/NASTRAN or ANSYS. The more limited CARES/PC does not perform fast-fracture reliability estimation of components. CARES/PC estimates ceramic material properties from uniaxial tensile or from three- and four-point bend bar data. In general, the parameters are obtained from the fracture stresses of many specimens (30 or more are recommended) whose geometry and loading configurations are held constant. Parameter estimation can be performed for single or multiple failure modes by using the least-squares analysis or the maximum likelihood method. Kolmogorov-Smirnov and Anderson-Darling goodness-of-fit tests measure the accuracy of the hypothesis that the fracture data comes from a population with a distribution specified by the estimated Weibull parameters. Ninety-percent confidence intervals on the Weibull parameters and the unbiased value of the shape parameter for complete samples are provided

  7. Misfolded opsin mutants display elevated β -sheet structure

    DOE PAGES

    Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; ...

    2015-09-07

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

  8. Misfolded Opsin Mutants Display Elevated β-Sheet Structure

    PubMed Central

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

    2015-01-01

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself. PMID:26358292

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

    PubMed

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

    2015-10-07

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

  10. APC-PC Combined Scheme in Gilbert Two State Model: Proposal and Study

    NASA Astrophysics Data System (ADS)

    Bulo, Yaka; Saring, Yang; Bhunia, Chandan Tilak

    2016-08-01

    In an automatic repeat request (ARQ) scheme, a packet is retransmitted if it gets corrupted due to transmission errors caused by the channel. However, an erroneous packet may contain both erroneous bits and correct bits and hence it may still contain useful information. The receiver may be able to combine this information from multiple erroneous copies to recover the correct packet. Packet combining (PC) is a simple and elegant scheme of error correction in transmitted packet, in which two received copies are XORed to obtain the bit location of erroneous bits. Thereafter, the packet is corrected by bit inversion of bit located as erroneous. Aggressive packet combining (APC) is a logic extension of PC primarily designed for wireless communication with objective of correcting error with low latency. PC offers higher throughput than APC, but PC does not correct double bit errors if occur in same bit location of erroneous copies of the packet. A hybrid technique is proposed to utilize the advantages of both APC and PC while attempting to remove the limitation of both. In the proposed technique, applications of APC-PC on Gilbert two state model has been studied. The simulation results show that the proposed technique offers better throughput than the conventional APC and lesser packet error rate than PC scheme.

  11. APC-PC Combined Scheme in Gilbert Two State Model: Proposal and Study

    NASA Astrophysics Data System (ADS)

    Bulo, Yaka; Saring, Yang; Bhunia, Chandan Tilak

    2017-04-01

    In an automatic repeat request (ARQ) scheme, a packet is retransmitted if it gets corrupted due to transmission errors caused by the channel. However, an erroneous packet may contain both erroneous bits and correct bits and hence it may still contain useful information. The receiver may be able to combine this information from multiple erroneous copies to recover the correct packet. Packet combining (PC) is a simple and elegant scheme of error correction in transmitted packet, in which two received copies are XORed to obtain the bit location of erroneous bits. Thereafter, the packet is corrected by bit inversion of bit located as erroneous. Aggressive packet combining (APC) is a logic extension of PC primarily designed for wireless communication with objective of correcting error with low latency. PC offers higher throughput than APC, but PC does not correct double bit errors if occur in same bit location of erroneous copies of the packet. A hybrid technique is proposed to utilize the advantages of both APC and PC while attempting to remove the limitation of both. In the proposed technique, applications of APC-PC on Gilbert two state model has been studied. The simulation results show that the proposed technique offers better throughput than the conventional APC and lesser packet error rate than PC scheme.

  12. PcchiB1, encoding a class V chitinase, is affected by PcVelA and PcLaeA, and is responsible for cell wall integrity in Penicillium chrysogenum.

    PubMed

    Kamerewerd, Jens; Zadra, Ivo; Kürnsteiner, Hubert; Kück, Ulrich

    2011-11-01

    Penicillin production in Penicillium chrysogenum is controlled by PcVelA and PcLaeA, two components of the regulatory velvet-like complex. Comparative microarray analysis with mutants lacking PcVelA or PcLaeA revealed a set of 62 common genes affected by the loss of both components. A downregulated gene in both knockout strains is PcchiB1, potentially encoding a class V chitinase. Under nutrient-depleted conditions, transcript levels of PcchiB1 are strongly upregulated, and the gene product contributes to more than 50 % of extracellular chitinase activity. Functional characterization by generating PcchiB1-disruption strains revealed that PcChiB1 is responsible for cell wall integrity and pellet formation in P. chrysogenum. Further, fluorescence microscopy with a DsRed-labelled chitinase suggests a cell wall association of the protein. An unexpected phenotype occurred when knockout strains were grown on media containing N-acetylglucosamine as the sole C and N source, where, in contrast to the recipient, a penicillin producer strain, the mutants and an ancestral strain show distinct mycelial growth. We discuss the relevance of this class V chitinase for morphology in an industrially important fungus.

  13. Antioxidation activity of tetrahydrobiopterin in pheochromocytoma PC 12 cells.

    PubMed

    Shen, R S; Zhang, Y X

    1991-01-01

    Rat pheochromocytoma PC 12 cells are susceptible to the oxidative toxicity caused by H2O2, nitrofurantoin, dopamine, and xanthine/xanthine oxidase reaction. The cytotoxicities of these agents are greatly reduced by the simultaneous presence of 0.1 mM tetrahydrobiopterin (BH4), 3 units/ml horseradish peroxidase, 0.2 mM NADH, and 0.1 units/ml sheep liver dihydropteridine reductase (DHPR). Individually, BH4, NADH and DHPR have no protection against H2O2 toxicity in PC 12 cells. Peroxidase alone offers 58% of protection if cells are incubated in the medium but only 3% in Dulbecco's phosphate buffered saline. The efficiency of the BH4-mediated antioxidation system in PC 12 cells is equal to or better than ascorbic acid and catalase, depending on the source of the reactive O2 species (ROS). The reactions responsible for the BH4-antioxidation system may consist of the non-enzymatic and the peroxidase-catalyzed reduction of H2O2 to H2O by BH4 and the regeneration of BH4 by DHPR using NADH as the cofactor. The components of this defence mechanism against ROS are all normal cellular constituents and are ubiquitous in nature. This DHPR-catalyzed redox cycling of BH4 may constitute an as yet little-known antioxidation system in mammalian cells.

  14. Characterization of shrunken endosperm mutants in barley.

    PubMed

    Ma, Jian; Jiang, Qian-Tao; Wei, Long; Wang, Ji-Rui; Chen, Guo-Yue; Liu, Ya-Xi; Li, Wei; Wei, Yu-Ming; Liu, Chunji; Zheng, You-Liang

    2014-04-10

    Despite numerous studies on shrunken endosperm mutants caused by either maternal tissues (seg) or kernel per se (sex) in barley, the molecular mechanism for all of the eight seg mutants (seg1-seg8) and some sex mutants is yet to be uncovered. In this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm (seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9% (seg4a) to 25.8% (seg1). SDS-PAGE analysis revealed that 87 kDa proteins corresponding to the starch branching enzyme II (SBEII) and starch synthase II (SSII) were not present in seg1, seg3, seg6, and seg7 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of seg1, seg3, seg6, and seg7 mutants decreased in varying degrees to lower levels until 27 days after anthesis (DAA) after reaching the peak at 15-21 DAA, which differed from the pattern of normal barley accessions. Further characterization of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sex1 had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg7 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.

  15. Incomplete processing of proinsulin to insulin accompanied by elevation of Des-31,32 proinsulin intermediates in islets of mice lacking active PC2.

    PubMed

    Furuta, M; Carroll, R; Martin, S; Swift, H H; Ravazzola, M; Orci, L; Steiner, D F

    1998-02-06

    The prohormone convertases PC2 (SPC2) and PC3/PC1 (SPC3) are the major precursor processing endoproteases in a wide variety of neural and endocrine tissues. Both enzymes are normally expressed in the islet beta cells and participate in proinsulin processing. Recently we generated mice lacking active PC2 due to a disruption of the PC2 gene (Furuta, M., Yano, H., Zhou, A., Rouillé, Y., Holst, J. J., Carroll, R. J., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). Here we report that these PC2 mutant mice have elevated circulating proinsulin, comprising 60% of immunoreactive insulin-like components. Acid ethanol extractable proinsulin from pancreas is also significantly elevated, representing about 35% of total immunoreactive insulin-like components. These increased amounts of proinsulin are mainly stored in secretory granules, giving rise to an altered appearance on electron microscopy. In pulse-chase experiments, the mutant islets incorporate lesser amounts of isotopic amino acids into insulin-related components than normal islets. In both wild-type and mutant islets, proinsulin I was processed more rapidly to insulin, reflecting the preference of both PC2 and PC3 for substrates having a basic amino acid positioned four residues upstream of the cleavage site. The overall half-time for the conversion of proinsulin to insulin is increased approximately 3-fold in the mutant islets and is associated with a 4-5-fold greater elevation of des-31,32 proinsulin, an intermediate that is formed by the preferential cleavage of proinsulin at the B chain-C-peptide junction by PC3 and is C-terminally processed to remove Arg31 and Arg32 by carboxypeptidase E. The constitutive release of newly synthesized proinsulin from both mutant and wild-type islets during the first 1-2 h of chase was normal (<2% of total). These results demonstrate that PC2 plays an essential role in proinsulin processing in vivo, but is

  16. Adapting PC104Plus for Space

    NASA Technical Reports Server (NTRS)

    Abbott, Larry; Cox, Gary; Nguyen, Hai

    2000-01-01

    This article addresses the issues associated with adapting the commercial PC104Plus standard and its associated architecture to the requirements of space applications. In general, space applications exhibit extreme constraints on power, weight, and volume. EMI and EMC are also issues of significant concern. Additionally, space applications have to survive high radiation environment. Finally, NASA is always concerned about achieving cost effective solutions that are compatible with safety and launch constraints. Weight and volume constraints are directly related to high launch cost. Power on the other hand is not only related to the high launch costs, but are related to the problem of dissipating the resulting heat once in space. The article addresses why PC104Plus is an appropriate solution for the weight and volume issues. The article also addresses what NASA did electrically to reduce power consumption and mechanically dissipate the associated heat in a microgravity and vacuum environment, and how these solutions allow NASA to integrate various sizes of ruggedized custom PC104 boards with COTS, PC104 complaint boards for space applications. In addition to the mechanical changes to deal with thermal dissipation NASA also made changes to minimize EMI. Finally, radiation issues are addressed as well as the architectural and testing solutions and the implications for use of COTS PC104Plus boards.

  17. The Cellular Prion Protein (PrPC): Its Physiological Function and Role in Disease

    PubMed Central

    Westergard, Laura; Christensen, Heather M.; Harris, David A.

    2007-01-01

    Prion diseases are caused by conversion of a normal cell-surface glycoprotein (PrPC) into a conformationally altered isoform (PrPSc) that is infectious in the absence of nucleic acid. Although a great deal has been learned about PrPSc and its role in prion propagation, much less is known about the physiological function of PrPC. In this review, we will summarize some of the major proposed functions for PrPC, including protection against apoptotic and oxidative stress, cellular uptake or binding of copper ions, transmembrane signaling, formation and maintenance of synapses, and adhesion to the extracellular matrix. We will also outline how loss or subversion of the cytoprotective or neuronal survival activities of PrPC might contribute to the pathogenesis of prion diseases, and how similar mechanisms are probably operative in other neurodegenerative disorders. PMID:17451912

  18. Protective effects of ginsenoside Rg1 against colistin sulfate-induced neurotoxicity in PC12 cells.

    PubMed

    Jiang, Guo-Zheng; Li, Ji-Chang

    2014-03-01

    The present study aimed to examine the protective effect of ginsenoside Rg1 against colistin-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Ginsenoside Rg1 was shown to elevate cell viability, decrease levels of malondialdehyde and intracellular reactive oxygen species, enhance activity of superoxide dismutase and glutathione, and decrease the release of cytochrome-c, formation of DNA fragmentation in colistin-treated PC12 cells. Ginsenoside Rg1 also reversed the increased caspase-9 and -3 mRNA levels caused by colistin in PC12 cells. These results suggest that ginsenoside Rg1 exerts a neuroprotective effect on colistin-induced neurotoxicity in PC12 cells, at least in part, via the inhibition of oxidative stress, prevention of apoptosis mediated via mitochondria pathway. Co-administration of ginsenoside Rg1 highlights the potential to increase the therapeutic index of colistin.

  19. Broad spectral sensitivity and improved efficiency in CuPc/Sub-Pc organic photovoltaic devices

    NASA Astrophysics Data System (ADS)

    Kumar, Hemant; Kumar, Pankaj; Bhardwaj, Ramil; Sharma, G. D.; Chand, Suresh; Jain, S. C.; Kumar, Vikram

    2009-01-01

    We demonstrate organic photovoltaic devices incorporating two donors, namely, copper phthalocyanine (CuPc) and boron sub-phthalocyanine chloride (Sub-Pc) in association with single acceptor fullerene (C60) with sensitivity extending across the visible solar spectrum. It has been found that the absorption in different spectral regions in CuPc and Sub-Pc results in efficient harvesting of incident light photons which leads to enhanced power conversion efficiency (η). An enhancement in η from 0.64%, in the device architecture indium-tin-oxide (ITO)/CuPc(20 nm)/C60(40 nm)/bathophenanthroline (BPhen) (8 nm)/Al(150 nm), to ~1.3% in the optimized device having a 2 nm layer of Sub-Pc in the geometry ITO/CuPc(18 nm)/Sub-Pc(2 nm)/C60 (40 nm)/BPhen (8 nm)/Al(150 nm) has been observed. This enhancement in η is dominantly attributed to the increment in short circuit current density (Jsc) due to efficient photon harvesting by incorporation of dual donors.

  20. Native Mutant Huntingtin in Human Brain

    PubMed Central

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

    2012-01-01

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

  1. Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig.

    PubMed

    Coleman, Stewart; Choi, K Yeon; McGregor, Alistair

    2017-09-01

    Guinea pig cytomegalovirus (GPCMV) encodes a homolog pentameric complex (PC) for specific cell tropism and congenital infection. In human cytomegalovirus, the PC is an important antibody neutralizing target and GPCMV studies will aid in the development of intervention strategies. Deletion mutants of the C-terminal domains of unique PC proteins (UL128, UL130 and UL131 homologs) were unable to form a PC in separate transient expression assays. Minor modifications to the UL128 homolog (GP129) C-terminal domain enabled PC formation but viruses encoding these mutants had altered tropism to renal and placental trophoblast cells. Mutation of the presumptive CC chemokine motif encoded by GP129 was investigated by alanine substitution of the CC motif (codons 26-27) and cysteines (codons 47 and 62). GP129 chemokine mutants formed PC but GP129 chemokine mutant viruses had reduced epitropism. A GP129 chemokine mutant virus pathogenicity study demonstrated reduced viral load to target organs but highly extended viremia. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Mutant presenilin2 promotes apoptosis through the p53/miR-34a axis in neuronal cells.

    PubMed

    Li, Liu-Hong; Tu, Qiu-Yun; Deng, Xiao-Hua; Xia, Jian; Hou, De-Ren; Guo, Ke; Zi, Xiao-Hong

    2017-05-01

    Neurodegenerative disorders have attracted attention in last decades due to their high incidence in the world. The p53/miR-34a axis triggers apoptosis and suppresses viability in multiple types of cells, but little is known about its role in neurodegenerative diseases. In this study, we showed that presenilin (PS)-2, a major gene associated with familial Alzheimer's disease (AD) could trigger the apoptosis through the p53/miR-34a axis in PC12 cells. First we found that PC12 cell viability was downregulated by PS-2 and mutant PS-2 overexpression, especially by mutant PS-2 overexpression. Then, we established a mutant PS-2-overexpressing PC12 cell line and confirmed that mutant PS-2 induced not only p53 but also miR-34a expression. The transfection of miR-34a inhibitor reversed PS-2-induced effects on cellular viability and apoptosis. Mutant PS-2 overexpression promoted caspase-3 expression, reduced Sirt1 and Bcl-2 expression, all of which were miR-34a downstream genes related with cell apoptosis. Moreover, mutant PS-2 also activated the p53/miR-34a axis and induced apoptosis in AD transgenic mice brain. These results implied that mutant PS-2 might promote the apoptosis of neuronal cells through triggering the p53/miR-34a axis. Altogether our results provide a novel insight into neurodegenerative disease and deepen our understandings of AD pathogenic processes.

  3. Toll-like receptor 9 agonist IMO cooperates with cetuximab in K-ras mutant colorectal and pancreatic cancers.

    PubMed

    Rosa, Roberta; Melisi, Davide; Damiano, Vincenzo; Bianco, Roberto; Garofalo, Sonia; Gelardi, Teresa; Agrawal, Sudhir; Di Nicolantonio, Federica; Scarpa, Aldo; Bardelli, Alberto; Tortora, Giampaolo

    2011-10-15

    K-Ras somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong in vivo activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab. In the present study, we investigated, both in vitro and in vivo, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring K-Ras mutations and resistance to EGFR inhibitors. We showed that IMO was able to significantly restore the sensitivity of K-Ras mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen-activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. In vivo, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using K-Ras mutant colorectal cancer cell models obtained through homologous recombination technology. We showed that IMO markedly inhibits growth of K-Ras mutant colon and pancreatic cancers in vitro and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for K-Ras wild-type as well for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancers. ©2011 AACR.

  4. Emissions tracking system (ETS-PC) software

    SciTech Connect

    Weatherbee, J. Jr.; Kress, T.

    1997-12-31

    The U.S. EPA Acid Rain Division developed and is maintaining the Emissions Tracking System (ETS) to receive, store and analyze data from continuous emissions monitors (CEMs) submitted by utilities affected by the 1990 Clean Air Act. This paper will describe ETS-PC, a PC application developed by EPA to assist utilities in analyzing and submitting emission data files each quarter. ETS-PC includes quality assurance software which helps utilities identify possible errors in their quarterly data files (QDFs) prior to submission. It also includes communications software which allows utilities to transfer QDFs via modem directly to the EPA mainframe computer located in Research Triangle Park, NC. After a file is transferred, users are provided with immediate feedback from the mainframe in the form of a file transfer receipt and summary.

  5. The IBM PC at NASA Ames

    NASA Technical Reports Server (NTRS)

    Peredo, James P.

    1988-01-01

    Like many large companies, Ames relies very much on its computing power to get work done. And, like many other large companies, finding the IBM PC a reliable tool, Ames uses it for many of the same types of functions as other companies. Presentation and clarification needs demand much of graphics packages. Programming and text editing needs require simpler, more-powerful packages. The storage space needed by NASA's scientists and users for the monumental amounts of data that Ames needs to keep demand the best database packages that are large and easy to use. Availability to the Micom Switching Network combines the powers of the IBM PC with the capabilities of other computers and mainframes and allows users to communicate electronically. These four primary capabilities of the PC are vital to the needs of NASA's users and help to continue and support the vast amounts of work done by the NASA employees.

  6. ECB deacylase mutants

    DOEpatents

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  7. Aluminum electrolytic capacitors for tablet PC

    NASA Astrophysics Data System (ADS)

    Liu, Longchun; Dong, Liangwei; Li, Qinglong; Xu, Xiangyang

    2017-07-01

    Based on the operating conditions of tablet PC, this paper presents the design of a long load life aluminum electrolytic capacitor. Due to the key technology breakthrough of electrolyte with low resistance and excellent temperature stability, the capacitor boasts low leakage current, low impedance, high frequency, high ripple resistance and high temperature resistance. In the meantime, it can pass 5000 h of durability test with load at 105∘C. The aluminum electrolytic capacitor can be used in tablet PC with long load life.

  8. Neurosecretory Habituation in PC12 Cells: Modulation During Parallel Habituation

    NASA Astrophysics Data System (ADS)

    Martin, Paul T.; Koshland, Daniel E., Jr.

    1995-05-01

    PC12 cells habituate during repetitive stimulation with acetylcholine, bradykinin, or high potassium. Interspersing these stimulants did not affect the rate of habituation of the others, but it could modulate the amplitude of the norepinephrine secretion each could achieve. Stimulation with acetylcholine inhibited norepinephrine secretion caused by high potassium and bradykinin stimulation, while high potassium had no effect on acetylcholine or bradykinin, and bradykinin increased secretion caused by acetylcholine. Changes in norepinephrine secretion resulting from any of these stimulants correlated with changes in internal calcium levels. Cyclic AMP-, protein kinase C-, and calmodulin-dependent second messenger pathways all modulated norepinephrine secretion caused by acetylcholine and high potassium and showed a distinct hierarchy in their effectiveness. These data demonstrate that different receptor pathways can change the norepinephrine response of one another while not changing the levels of the molecules responsible for habituation.

  9. [Breeding and characterization of laccase-producing Phanerochaete chrysosporium mutant resistant to nutritional repression].

    PubMed

    Qiu, Ailian; Li, Wenyan; Zheng, Yaotong; Fan, Xiaojing; Ye, Youxian; Meng, Yan

    2011-03-01

    To screen Phanerochaete chrysosporium mutants resisting nutritional repression and to characterize laccase produced by the mutants. We used repeated UV mutagenesis and screened the mutant strains by using the guaiacol nitrogen sufficient differential medium. We characterized enzymes production mechanism of the nutritional regulation through comparing the differences of cell growth and enzyme-production kinetics under different nutritional conditions; We validated production of laccase by Phanerochaete chrysosporium through measurements of the heat treatment, removal of manganese ion and addition of the catalase. Three different methods were validated that both strains of pcR5305 and pcR5324 can produce laccase under the nitrogen limitation (N-L) and nitrogen sufficient (N-S) conditions. Under the N-L conditions, pcR5305 can produce 203.5 U/L laccase and pcR5324 can produce 187.6 U/L laccase; Under the N-S conditions, pcR5305 can produce 220.6 U/L laccase and pcR5324 can produce 183.9 U/L laccase. The original strain pc530 only can produce very little laccase under either conditions. The laccase-production regulation mechanisms of the two strains are different: Production of laccase and the cell growth by pcR5305 are in synchronism. However production of the laccase by pcR5324 is repressed by nutrition. Both strains have the capacity of resisting nutritional repression and produce lignin peroxidase and manganese peroxidase with high yield. (LiP 1343.2, MnP 252.2 U/L and LiP 1169.5, MnP 172.4 U/L respectively). The mutants of Phanerochaete chrysosporium can produce laccase. At same time they showed the capacity of resisting nutritional repression and production of laccase, lignin peroxidase and manganese peroxidase. Our results possess high value for production, application and fundamental research. We provided new strains and established a very good foundation for the further research of metabolic regulation of ligninolytic enzymes production.

  10. The antiandrogen bicalutamide activates the androgen receptor (AR) with a mutation in codon 741 through the mitogen activated protein kinase (MARK) pathway in human prostate cancer PC3 cells.

    PubMed

    Terakawa, Tomoaki; Miyake, Hideaki; Kumano, Masafumi; Sakai, Iori; Fujisawa, Masato

    2010-11-01

    The objective of this study was to assess the effect of antiandrogen on the activation of mutated androgen receptor (AR) and its signaling pathway in prostate cancer. We transfected the AR gene with a point mutation at codon 741 (tryptophan to leucine; W741L) into human androgen-independent prostate cancer PC3 cells lacking the expression of AR, and established PC3 cells overexpressing mutant type AR (PC3/W741L). Changes in the phenotype in these cells were compared to those in PC3 cells transfected with wild- type AR (PC3/Wild) and control vector alone (PC3/Co). There was no significant differences in the growth among PC3/Co, PC3/Wild and PC3/W741L cells. A transactivation assay using these cells showed that bicalutamide activated W741L mutant type AR, but not wild-type AR, while hydroxyflutamide failed to activate either type of ARs. Treatment with specific inhibitors of the MAPK or STST3 pathway (UO126 or AG490, respectively), in contrast to treatment with the Akt pathway inhibitor LY294002, significantly inhibited the dihydrotestosterone-induced activation of both wild-type and mutant ARs; however, activation of W741L mutant AR by bicalutamide was significantly inhibited by treatment with UO126, in contrast to treatment with AG490 or LY294002. Furthermore, treatment of PC3/W741L with bicalutamide, in contrast to treatment with hydroxyflutamide, resulted in significant upregulation of phosphorylated p44/42 MAPK. These findings suggest that the MAPK pathway might be involved in the activation of the AR with a point mutation at codon 741 induced by treatment with the antiandrogen bicalutamide.

  11. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    NASA Astrophysics Data System (ADS)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  12. Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin

    PubMed Central

    Tan, Z; Dai, W; van Erp, T G M; Overman, J; Demuro, A; Digman, M A; Hatami, A; Albay, R; Sontag, E M; Potkin, K T; Ling, S; Macciardi, F; Bunney, W E; Long, J D; Paulsen, J S; Ringman, J M; Parker, I; Glabe, C; Thompson, L M; Chiu, W; Potkin, S G

    2015-01-01

    Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT. PMID:26100538

  13. Stretch Your PC Dollars--Buy Clones.

    ERIC Educational Resources Information Center

    True, John

    1986-01-01

    Relates how the story of how San Francisco State University evaluated IBM PC look-alikes, considered some of the risks involved, and decided to purchase over 100 of them. Questions of compatibility, vendor longevity, support, and other risk management issues are discussed. (Author/MLW)

  14. PC Based Video on Demand Trials.

    ERIC Educational Resources Information Center

    Branch, Philip; Durran, Jennifer

    Many educational institutions have a substantial personal computer (PC) network that can be adapted to provide digital video on demand, as well as PCs that can be used as video on demand clients. To gain insight into the issues involved in using this technology in an educational environment that relies heavily on video, a simple, low cost video on…

  15. Multitasking Operating Systems for the IBM PC.

    ERIC Educational Resources Information Center

    Owen, G. Scott

    1985-01-01

    The ability of a microcomputer to execute several programs at the same time is called "multitasking." The nature and use of one multitasking operating system Concurrent PC-DOS from Digital Research (the developers of the CP/M operating system) are discussed. (JN)

  16. Stretch Your PC Dollars--Buy Clones.

    ERIC Educational Resources Information Center

    True, John

    1986-01-01

    Relates how the story of how San Francisco State University evaluated IBM PC look-alikes, considered some of the risks involved, and decided to purchase over 100 of them. Questions of compatibility, vendor longevity, support, and other risk management issues are discussed. (Author/MLW)

  17. An Introduction To PC-TRIM.

    Treesearch

    John R. Mills

    1989-01-01

    The timber resource inventory model (TRIM) has been adapted to run on person al computers. The personal computer version of TRIM (PC-TRIM) is more widely used than its mainframe parent. Errors that existed in previous versions of TRIM have been corrected. Information is presented to help users with program input and output management in the DOS environment, to...

  18. Combating adverse selection in secondary PC markets.

    PubMed

    Hickey, Stewart W; Fitzpatrick, Colin

    2008-04-15

    Adverse selection is a significant contributor to market failure in secondary personal computer (PC) markets. Signaling can act as a potential solution to adverse selection and facilitate superior remarketing of second-hand PCs. Signaling is a means whereby usage information can be utilized to enhance consumer perception of both value and utility of used PCs and, therefore, promote lifetime extension for these systems. This can help mitigate a large portion of the environmental impact associated with PC system manufacture. In this paper, the computer buying and selling behavior of consumers is characterized via a survey of 270 Irish residential users. Results confirm the existence of adverse selection in the Irish market with 76% of potential buyers being unwilling to purchase and 45% of potential vendors being unwilling to sell a used PC. The so-called "closet affect" is also apparent with 78% of users storing their PC after use has ceased. Results also indicate that consumers place a higher emphasis on specifications when considering a second-hand purchase. This contradicts their application needs which are predominantly Internet and word-processing/spreadsheet/presentation applications, 88% and 60% respectively. Finally, a market solution utilizing self monitoring and reporting technology (SMART) sensors for the purpose of real time usage monitoring is proposed, that can change consumer attitudes with regard to second-hand computer equipment.

  19. Multitasking Operating Systems for the IBM PC.

    ERIC Educational Resources Information Center

    Owen, G. Scott

    1985-01-01

    The ability of a microcomputer to execute several programs at the same time is called "multitasking." The nature and use of one multitasking operating system Concurrent PC-DOS from Digital Research (the developers of the CP/M operating system) are discussed. (JN)

  20. PC Kiosk Trends in Rural India

    ERIC Educational Resources Information Center

    Toyama, Kentaro; Kiri, Karishma; Menon, Deepak; Sethi, Suneet; Pal, Joyojeet; Srinivasan, Janaki

    2006-01-01

    This article presents a series of preliminary, quantitative results on rural PC kiosks in India. An analysis of the data confirms many expected trends and correlations and shows that kiosks still face the challenge of sustainability as a business. This study is based on questionnaires presented to kiosk operators and customers of kiosks operated…

  1. Mathematics Instruction and the Tablet PC

    ERIC Educational Resources Information Center

    Fister, K. Renee; McCarthy, Maeve L.

    2008-01-01

    The use of tablet PCs in teaching is a relatively new phenomenon. A cross between a notebook computer and a personal digital assistant (PDA), the tablet PC has all of the features of a notebook with the additional capability that the screen can also be used for input. Tablet PCs are usually equipped with a stylus that allows the user to write on…

  2. PC Kiosk Trends in Rural India

    ERIC Educational Resources Information Center

    Toyama, Kentaro; Kiri, Karishma; Menon, Deepak; Sethi, Suneet; Pal, Joyojeet; Srinivasan, Janaki

    2006-01-01

    This article presents a series of preliminary, quantitative results on rural PC kiosks in India. An analysis of the data confirms many expected trends and correlations and shows that kiosks still face the challenge of sustainability as a business. This study is based on questionnaires presented to kiosk operators and customers of kiosks operated…

  3. Experience using EPICS on PC platforms

    SciTech Connect

    Hill, J.O.; Kasemire, K.U.

    1998-03-01

    The Experimental Physics and Industrial Control System (EPICS) has been widely adopted in the accelerator community. Although EPICS is available on many platforms, the majority of implementations have used UNIX workstations as clients, and VME- or VXI-based processors for distributed input output controllers. Recently, a significant portion of EPICS has been ported to personal computer (PC) hardware platforms running Microsoft`s operating systems, and also Wind River System`s real time vxWorks operating system. This development should significantly reduce the cost of deploying EPICS systems, and the prospect of using EPICS together with the many high quality commercial components available for PC platforms is also encouraging. A hybrid system using both PC and traditional platforms is currently being implemented at LANL for LEDA, the low energy demonstration accelerator under construction as part of the Accelerator Production of Tritium (APT) project. To illustrate these developments the authors compare their recent experience deploying a PC-based EPICS system with experience deploying similar systems based on traditional (UNIX-hosted) EPICS hardware and software platforms.

  4. Jargon that Computes: Today's PC Terminology.

    ERIC Educational Resources Information Center

    Crawford, Walt

    1997-01-01

    Discusses PC (personal computer) and telecommunications terminology in context: Integrated Services Digital Network (ISDN); Asymmetric Digital Subscriber Line (ADSL); cable modems; satellite downloads; T1 and T3 lines; magnitudes ("giga-,""nano-"); Central Processing Unit (CPU); Random Access Memory (RAM); Universal Serial Bus…

  5. Jargon that Computes: Today's PC Terminology.

    ERIC Educational Resources Information Center

    Crawford, Walt

    1997-01-01

    Discusses PC (personal computer) and telecommunications terminology in context: Integrated Services Digital Network (ISDN); Asymmetric Digital Subscriber Line (ADSL); cable modems; satellite downloads; T1 and T3 lines; magnitudes ("giga-,""nano-"); Central Processing Unit (CPU); Random Access Memory (RAM); Universal Serial Bus…

  6. Experience in setting up a PC cluster

    NASA Astrophysics Data System (ADS)

    Lin, Ganghua; Zhang, Mei

    2004-09-01

    In this paper we summary and present our thinking and experience in setting up a PC cluster, with a consideration that the described thinking and experience may be relevant to or useful for those who intend to buy a similar cluster in the near future.

  7. Wf/pc Cycle 1 Cal: Non-Sv PC Flats 1

    NASA Astrophysics Data System (ADS)

    MacKenty, John

    1990-12-01

    This program takes "uniform illuminated" pictures of the earth to obtain the "instrument signature" for flat field corrections. This proposal contains observations for all PC filters used by GOs and GTOs during Cycle 1 which were not included in the SV program. The camera - filter combinations done in this proposal are: PC: F368M,F588N,F631N,F648M,F875M,F1042M These are the PC flats which are not expected to saturate on the bright earth.

  8. Prion Propagation in Cells Expressing PrP Glycosylation Mutants

    PubMed Central

    Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-01-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

  9. Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells.

    PubMed Central

    Gutacker, C; Klock, G; Diel, P; Koch-Brandt, C

    1999-01-01

    Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression. PMID:10215617

  10. Binding of bovine T194A PrPC by PrPSc-specific antibodies

    PubMed Central

    Madampage, Claudia A; Määttänen, Pekka; Marciniuk, Kristen; Brownlie, Robert; Andrievskaia, Olga; Potter, Andrew; Cashman, Neil R; Lee, Jeremy S; Napper, Scott

    2013-01-01

    Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrPC) into an infectious, pathological conformation (PrPSc). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrPSc-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrPC associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrPC that is associated with early onset of familial dementia. With nanopore analysis, under non-denaturing conditions, we observed binding of the SN6b antibody to bPrP(T194A). This interaction was confirmed through ELISAs as well as immunoprecipitation of the recombinant and cellularly expressed forms of bPrP(T194A). This interaction did not promote formation of a protease resistant conformation of PrP in vitro. Collectively, these findings support the disease-specific approach for immunotherapy of prion diseases but also suggest that the concept of conformation-specific immunotherapy may be complicated in individuals who are genetically predisposed to PrPC misfolding. PMID:23787697

  11. Arabidopsis MET1 cytosine methyltransferase mutants.

    PubMed Central

    Kankel, Mark W; Ramsey, Douglas E; Stokes, Trevor L; Flowers, Susan K; Haag, Jeremy R; Jeddeloh, Jeffrey A; Riddle, Nicole C; Verbsky, Michelle L; Richards, Eric J

    2003-01-01

    We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis. PMID:12663548

  12. Isolation of a Defective Prion Mutant from Natural Scrapie

    PubMed Central

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

    2016-01-01

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

  13. Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

    PubMed Central

    Manos, M M; Gluzman, Y

    1985-01-01

    To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants. Images PMID:2981330

  14. Methods of producing protoporphyrin IX and bacterial mutants therefor

    DOEpatents

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  15. Nerve Growth Factor Receptor TrkA, a New Receptor in Insulin Signaling Pathway in PC12 Cells*

    PubMed Central

    Geetha, Thangiah; Rege, Shraddha D.; Mathews, Salome E.; Meakin, Susan O.; White, Morris F.; Babu, Jeganathan Ramesh

    2013-01-01

    TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells. PMID:23749991

  16. Nerve growth factor receptor TrkA, a new receptor in insulin signaling pathway in PC12 cells.

    PubMed

    Geetha, Thangiah; Rege, Shraddha D; Mathews, Salome E; Meakin, Susan O; White, Morris F; Babu, Jeganathan Ramesh

    2013-08-16

    TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.

  17. alpha Pix enhances mutant huntingtin aggregation.

    PubMed

    Eriguchi, Makoto; Mizuta, Haruo; Luo, Shouqing; Kuroda, Yasuo; Hara, Hideo; Rubinsztein, David C

    2010-03-15

    Huntington's disease is caused by polyglutamine-expanded mutant huntingtin (muhtt), an aggregation-prone protein. We identified the Pak-interacting exchange factor (alpha Pix/Cool2) as a novel huntingtin (htt) interacting protein, after screening actin-cytoskeleton organization-related factors. Using immunoprecipitation experiments, we show that alpha Pix binds to both the N-terminal of wild-type htt (wthtt) and mutant htt (muthtt). Colocalization studies revealed that alpha Pix accumulates in muthtt aggregates. Deletion analysis suggested that the dbl homology (DH) and pleckstrin homology (PH) domains of alpha Pix are required for its interaction with htt. Overexpression of alpha Pix enhanced muthtt aggregation by inducing SDS-soluble muthtt-muthtt interactions. Conversely, knocking down alpha Pix attenuated muhtt aggregation. These findings suggest that alpha Pix plays an important role in muthtt aggregation.

  18. Adenosine-dependent activation of tyrosine hydroxylase is defective in adenosine kinase-deficient PC12 cells.

    PubMed Central

    Erny, R; Wagner, J A

    1984-01-01

    (R)-N6-Phenylisopropyladenosine (PIA) stimulates dopa production 3- to 5-fold in PC12 cells, with a half-maximal effective concentration (EC50) of 50 nM. This increase can be explained by a stable activation of tyrosine hydroxylase [TyrOHase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] when it is phosphorylated by a cAMP-dependent protein kinase. The activation of TyrOHase is mediated by the adenosine-dependent activation of adenylate cyclase (EC50 = 600 nM). PIA (10 microM) is as effective as cholera toxin or dibutyryl cAMP in activating TyrOHase in wild-type cells. Adenosine kinase-deficient mutants of PC12 were found to be resistant to PIA-dependent activation of TyrOHase (EC50 = 100-1000 nM). This phenomenon was explored in detail in one adenosine kinase-deficient mutant and was shown to occur because the mutant was resistant to the adenosine-dependent activation of adenylate cyclase. In this mutant, TyrOHase was activated 14-fold by cholera toxin, suggesting that activated TyrOHase is about 14 times as active as unactivated TyrOHase. These studies with kinase-deficient PC12 cells provide genetic evidence that adenosine-dependent activation of TyrOHase is mediated by acute increases in cAMP. When the adenosine receptor found on PC12 cells is expressed in vivo, it might function as either a presynaptic (i.e., localized on the nerve terminal) or a postsynaptic (i.e., localized on the cell body or dendrite) receptor that regulates rates of transmitter synthesis in response to cell activity. PMID:6146982

  19. Cell-cycle-dependent PC-PLC regulation by APC/C(Cdc20)-mediated ubiquitin-proteasome pathway.

    PubMed

    Fu, Da; Ma, Yushui; Wu, Wei; Zhu, Xuchao; Jia, Chengyou; Zhao, Qianlei; Zhang, Chunyi; Wu, Xing Zhong

    2009-07-01

    Phosphatidylcholine-specific phospholipase C (PC-PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC-PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC-PLC were co-immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH-7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC-PLC and Cdc20 were co-localized in the perinuclear endoplasmic reticulum region (the "juxtanuclear quality control" compartment, JUNQ). The expression level and activities of PC-PLC changed in a cell-cycle-dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC-PLC, and caused PC-PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC-PLC regulation in cell cycles is controlled by APC/C(Cdc20)-mediated UPP. 2009 Wiley-Liss, Inc.

  20. PC, a Novel Oral Insecticidal Toxin from Bacillus bombysepticus Involved in Host Lethality via APN and BtR-175.

    PubMed

    Lin, Ping; Cheng, Tingcai; Jin, Shengkai; Wu, Yuqian; Fu, Bohua; Long, Renwen; Zhao, Ping; Xia, Qingyou

    2015-06-09

    Insect pests have developed resistance to chemical insecticides, insecticidal toxins as bioinsecticides or genetic protection built into crops. Consequently, novel, orally active insecticidal toxins would be valuable biological alternatives for pest control. Here, we identified a novel insecticidal toxin, parasporal crystal toxin (PC), from Bacillus bombysepticus (Bb). PC shows oral pathogenic activity and lethality towards silkworms and Cry1Ac-resistant Helicoverpa armigera strains. In vitro assays, PC after activated by trypsin binds to BmAPN4 and BtR-175 by interacting with CR7 and CR12 fragments. Additionally, trypsin-activated PC demonstrates cytotoxicity against Sf9 cells expressing BmAPN4, revealing that BmAPN4 serves as a functional receptor that participates in Bb and PC pathogenicity. In vivo assay, knocking out BtR-175 increased the resistance of silkworms to PC. These data suggest that PC is the first protein with insecticidal activity identified in Bb that is capable of causing silkworm death via receptor interactions, representing an important advance in our understanding of the toxicity of Bb and the contributions of interactions between microbial pathogens and insects to disease pathology. Furthermore, the potency of PC as an insecticidal protein makes it a good candidate for inclusion in integrated agricultural pest management systems.

  1. PC, a Novel Oral Insecticidal Toxin from Bacillus bombysepticus Involved in Host Lethality via APN and BtR-175

    PubMed Central

    Lin, Ping; Cheng, Tingcai; Jin, Shengkai; Wu, Yuqian; Fu, Bohua; Long, Renwen; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Insect pests have developed resistance to chemical insecticides, insecticidal toxins as bioinsecticides or genetic protection built into crops. Consequently, novel, orally active insecticidal toxins would be valuable biological alternatives for pest control. Here, we identified a novel insecticidal toxin, parasporal crystal toxin (PC), from Bacillus bombysepticus (Bb). PC shows oral pathogenic activity and lethality towards silkworms and Cry1Ac-resistant Helicoverpa armigera strains. In vitro assays, PC after activated by trypsin binds to BmAPN4 and BtR-175 by interacting with CR7 and CR12 fragments. Additionally, trypsin-activated PC demonstrates cytotoxicity against Sf9 cells expressing BmAPN4, revealing that BmAPN4 serves as a functional receptor that participates in Bb and PC pathogenicity. In vivo assay, knocking out BtR-175 increased the resistance of silkworms to PC. These data suggest that PC is the first protein with insecticidal activity identified in Bb that is capable of causing silkworm death via receptor interactions, representing an important advance in our understanding of the toxicity of Bb and the contributions of interactions between microbial pathogens and insects to disease pathology. Furthermore, the potency of PC as an insecticidal protein makes it a good candidate for inclusion in integrated agricultural pest management systems. PMID:26057951

  2. Impaired Terminal Differentiation of Hippocampal Granule Neurons and Defective Contextual Memory in PC3/Tis21 Knockout Mice

    PubMed Central

    Costanzi, Marco; Leonardi, Luca; Cinà, Irene; Micheli, Laura; Nutini, Michele; Longone, Patrizia; Oh, S. Paul; Cestari, Vincenzo; Tirone, Felice

    2009-01-01

    Neurogenesis in the dentate gyrus of the adult hippocampus has been implicated in neural plasticity and memory, but the molecular mechanisms controlling the proliferation and differentiation of newborn neurons and their integration into the synaptic circuitry are still largely unknown. To investigate this issue, we have analyzed the adult hippocampal neurogenesis in a PC3/Tis21-null mouse model. PC3/Tis21 is a transcriptional co-factor endowed with antiproliferative and prodifferentiative properties; indeed, its upregulation in neural progenitors has been shown to induce exit from cell cycle and differentiation. We demonstrate here that the deletion of PC3/Tis21 causes an increased proliferation of progenitor cells in the adult dentate gyrus and an arrest of their terminal differentiation. In fact, in the PC3/Tis21-null hippocampus postmitotic undifferentiated neurons accumulated, while the number of terminally differentiated neurons decreased of 40%. As a result, PC3/Tis21-null mice displayed a deficit of contextual memory. Notably, we observed that PC3/Tis21 can associate to the promoter of Id3, an inhibitor of proneural gene activity, and negatively regulates its expression, indicating that PC3/Tis21 acts upstream of Id3. Our results identify PC3/Tis21 as a gene required in the control of proliferation and terminal differentiation of newborn neurons during adult hippocampal neurogenesis and suggest its involvement in the formation of contextual memories. PMID:20020054

  3. A PC based fault diagnosis expert system

    NASA Technical Reports Server (NTRS)

    Marsh, Christopher A.

    1990-01-01

    The Integrated Status Assessment (ISA) prototype expert system performs system level fault diagnosis using rules and models created by the user. The ISA evolved from concepts to a stand-alone demonstration prototype using OPS5 on a LISP Machine. The LISP based prototype was rewritten in C and the C Language Integrated Production System (CLIPS) to run on a Personal Computer (PC) and a graphics workstation. The ISA prototype has been used to demonstrate fault diagnosis functions of Space Station Freedom's Operation Management System (OMS). This paper describes the development of the ISA prototype from early concepts to the current PC/workstation version used today and describes future areas of development for the prototype.

  4. IBM PC enhances the world's future

    NASA Technical Reports Server (NTRS)

    Cox, Jozelle

    1988-01-01

    Although the purpose of this research is to illustrate the importance of computers to the public, particularly the IBM PC, present examinations will include computers developed before the IBM PC was brought into use. IBM, as well as other computing facilities, began serving the public years ago, and is continuing to find ways to enhance the existence of man. With new developments in supercomputers like the Cray-2, and the recent advances in artificial intelligence programming, the human race is gaining knowledge at a rapid pace. All have benefited from the development of computers in the world; not only have they brought new assets to life, but have made life more and more of a challenge everyday.

  5. PC12 differentiation on biopolymer nanostructures

    NASA Astrophysics Data System (ADS)

    Cecchini, Marco; Bumma, Giorgia; Serresi, Michela; Beltram, Fabio

    2007-12-01

    The study of nervous system regeneration and axonal outgrowth control are relevant in several research areas, like neurophysiology or biomedical engineering. Among the elements that control neuron dynamics, the host substrate topography is a key parameter in determining cell differentiation. We present time-lapse experiments to analyze the differentiation dynamics of PC12 cells on nanopatterned biocompatible substrates. 200 nm depth gratings were fabricated on tissue-culture polystyrene substrates by nanoimprint lithography; different linewidths and pitches were compared down to 500 nm and 1000 nm, respectively. PC12 cells were cultured on these substrates and, following NGF administration to the medium, body morphology, cell movement and neuritogenesis were monitored at different time periods. In addition to demonstrating guided differentiation, our studies show complex time variations in body morphology and axon length, and guided cell movement. We show unstable synaptic connections and cell-body polarization, and the competition between topographical guidance and cell-cell interactions.

  6. Identification of Two Nickel Ion-Induced Genes, NCI16 and PcGST1, in Paramecium caudatum

    PubMed Central

    Haga, Nobuyuki; Nakano, Takanari; Ikeda, Masaaki; Katayama, Shigehiro; Awata, Takuya

    2014-01-01

    Here, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (∼16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; ∼25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H2O2 concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H2O2, implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tert-butylhydroquinone (tBHQ) but not by H2O2, suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an ∼0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control. PMID:25001407

  7. Virtual Reality at the PC Level

    NASA Technical Reports Server (NTRS)

    Dean, John

    1998-01-01

    The main objective of my research has been to incorporate virtual reality at the desktop level; i.e., create virtual reality software that can be run fairly inexpensively on standard PC's. The standard language used for virtual reality on PC's is VRML (Virtual Reality Modeling Language). It is a new language so it is still undergoing a lot of changes. VRML 1.0 came out only a couple years ago and VRML 2.0 came out around last September. VRML is an interpreted language that is run by a web browser plug-in. It is fairly flexible in terms of allowing you to create different shapes and animations. Before this summer, I knew very little about virtual reality and I did not know VRML at all. I learned the VRML language by reading two books and experimenting on a PC. The following topics are presented: CAD to VRML, VRML 1.0 to VRML 2.0, VRML authoring tools, VRML browsers, finding virtual reality applications, the AXAF project, the VRML generator program, web communities and future plans.

  8. Spent fuel pin temperature PC code

    SciTech Connect

    Fischer, L.E.

    1985-03-01

    During an annual outage, a Pressurized Water Reactor (PWR) may discharge 60 or more spent fuel bundles into its storage pool. Most early PWRs were built to store 3 to 5 years of spent fuel in their pools and are beginning to exceed their capacities. One method currently being developed and licensed for expanding spent fuel storage capabilities is the dry storage of spent fuel in large casks. To reduce the probability of gross failures of fuel cladding during dry storage in casks, the fuel pin temperatures must be shown to remain within acceptable limits. LLNL has developed, for the Nuclear Regulatory Commission, a personal computer (PC) code for calculating fuel pin temperatures on the IBM PC. The code uses the Wooton-Epstein Correlation to calculate the pin temperatures and has been benchmarked against test data. An iterative type of solution is used to calculate the fuel pin temperatures for specified heat fluxes and pin configurations. The PC code is useful in performing confirmatory analyses and comparing the results with those submitted by applicants applying for storage licenses. 5 references, 2 tables.

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

    PubMed

    Yuan, Fei; Snapp, Erik L; Novikoff, Phyllis M; Suadicani, Sylvia O; Spray, David C; Potvin, Barry; Wolkoff, Allan W; Stanley, Pamela

    2014-01-01

    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.

  10. Human Liver Cell Trafficking Mutants: Characterization and Whole Exome Sequencing

    PubMed Central

    Yuan, Fei; Snapp, Erik L.; Novikoff, Phyllis M.; Suadicani, Sylvia O.; Spray, David C.; Potvin, Barry; Wolkoff, Allan W.; Stanley, Pamela

    2014-01-01

    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. PMID:24466322

  11. Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms.

    PubMed

    Tang, Qiong-Yao; Zhang, Fei-Fei; Xu, Jie; Wang, Ran; Chen, Jian; Logothetis, Diomedes E; Zhang, Zhe

    2016-01-05

    Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects.

  12. Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells.

    PubMed

    Han, Jung Min; Kim, Jae Ho; Lee, Byoung Dae; Lee, Sang Do; Kim, Yong; Jung, Yon Woo; Lee, Sukmook; Cho, Wonhwa; Ohba, Motoi; Kuroki, Toshio; Suh, Pann-Ghill; Ryu, Sung Ho

    2002-03-08

    Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.

  13. Familial dementia caused by polymerization of mutant neuroserpin.

    PubMed

    Davis, R L; Shrimpton, A E; Holohan, P D; Bradshaw, C; Feiglin, D; Collins, G H; Sonderegger, P; Kinter, J; Becker, L M; Lacbawan, F; Krasnewich, D; Muenke, M; Lawrence, D A; Yerby, M S; Shaw, C M; Gooptu, B; Elliott, P R; Finch, J T; Carrell, R W; Lomas, D A

    1999-09-23

    Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutations, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

  14. Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein

    PubMed Central

    Lawson, Victoria A.; Lumicisi, Brooke; Welton, Jeremy; Machalek, Dorothy; Gouramanis, Katrina; Klemm, Helen M.; Stewart, James D.; Masters, Colin L.; Hoke, David E.; Collins, Steven J.; Hill, Andrew F.

    2010-01-01

    Background The accumulation of protease resistant conformers of the prion protein (PrPres) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. Methodology/Principal Finding In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrPres formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrPC substrate was found to specifically prevent PrPres formation seeded by mouse derived PrPSc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrPres formation, while having no effect on PrPres formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. Conclusions/Significance Cofactor requirements for PrPres formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains. PMID:20808809

  15. The USL NASA PC R and D development environment standards

    NASA Technical Reports Server (NTRS)

    Dominick, Wayne D. (Editor); Moreau, Dennis R.

    1984-01-01

    The development environment standards which have been established in order to control usage of the IBM PC/XT development systems and to prevent interference between projects being currently developed on the PC's are discussed. The standards address the following areas: scheduling PC resources; login/logout procedures; training; file naming conventions; hard disk organization; diskette care; backup procedures; and copying policies.

  16. Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

    PubMed Central

    Hauben, K J; Bartlett, D H; Soontjens, C C; Cornelis, K; Wuytack, E Y; Michiels, C W

    1997-01-01

    Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing. PMID:9055412

  17. IBM PC/IX operating system evaluation plan

    NASA Technical Reports Server (NTRS)

    Dominick, Wayne D. (Editor); Granier, Martin; Hall, Philip P.; Triantafyllopoulos, Spiros

    1984-01-01

    An evaluation plan for the IBM PC/IX Operating System designed for IBM PC/XT computers is discussed. The evaluation plan covers the areas of performance measurement and evaluation, software facilities available, man-machine interface considerations, networking, and the suitability of PC/IX as a development environment within the University of Southwestern Louisiana NASA PC Research and Development project. In order to compare and evaluate the PC/IX system, comparisons with other available UNIX-based systems are also included.

  18. PcFKH1, a novel regulatory factor from the forkhead family, controls the biosynthesis of penicillin in Penicillium chrysogenum.

    PubMed

    Domínguez-Santos, Rebeca; García-Estrada, Carlos; Kosalková, Katarina; Prieto, Carlos; Santamarta, Irene; Martín, Juan-Francisco

    2015-08-01

    Penicillin biosynthesis in Penicillium chrysogenum (re-identified as Penicillium rubens) is a good example of a biological process subjected to complex global regulatory networks and serves as a model to study fungal secondary metabolism. The winged-helix family of transcription factors recently described, which includes the forkhead type of proteins, is a key type of regulatory proteins involved in this process. In yeasts and humans, forkhead transcription factors are involved in different processes (cell cycle regulation, cell death control, pre-mRNA processing and morphogenesis); one member of this family of proteins has been identified in the P. chrysogenum genome (Pc18g00430). In this work, we have characterized this novel transcription factor (named PcFKH1) by generating knock-down mutants and overexpression strains. Results clearly indicate that PcFKH1 positively controls antibiotic biosynthesis through the specific interaction with the promoter region of the penDE gene, thus regulating penDE mRNA levels. PcFKH1 also binds to the pcbC promoter, but with low affinity. In addition, it also controls other ancillary genes of the penicillin biosynthetic process, such as phlA (encoding phenylacetyl CoA ligase) and ppt (encoding phosphopantetheinyl transferase). PcFKH1 also plays a role in conidiation and spore pigmentation, but it does not seem to be involved in hyphal morphology or cell division in the improved laboratory reference strain Wisconsin 54-1255. A genome-wide analysis of processes putatively coregulated by PcFKH1 and PcRFX1 (another winged-helix transcription factor) in P. chrysogenum provided evidence of the global effect of these transcription factors in P. chrysogenum metabolism. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  19. Wf/pc Cycle 1 Calib: Non-Sv PC Flats 2

    NASA Astrophysics Data System (ADS)

    MacKenty, John

    1990-12-01

    This program takes "uniform illuminated" pictures of the earth to obtain the "instrument signature" for flat field corrections. This proposal series contains observations for all PC filters used by GOs and GTOs during Cycle 1 which were not included in the SV program. The camera - filter combinations done in this proposal are: PC: F569W, F606W, F675W, F725LP, F791W, F814W, F850LP (these are done in combination with F122M) These are the PC flats which are expected to saturate on the bright earth without a neutral density filter. The following observations are also included to permit correction for the F122M filter: F517N+F122M, F517N, F664N+F122M, F664N, F718M+F122M, F718M, F875M+F122M, F875M

  20. Ibrutinib selectively and irreversibly targets EGFR (L858R, Del19) mutant but is moderately resistant to EGFR (T790M) mutant NSCLC Cells

    PubMed Central

    Wang, Wenchao; Hu, Chen; Ye, Zi; Zhao, Zheng; Wang, Li; Li, Xixiang; Yu, Kailin; Liu, Juan; Wu, Jiaxin; Yan, Xiao-E; Zhao, Peng; Wang, Jinhua; Wang, Chu; Weisberg, Ellen L.; Gray, Nathanael S.; Yun, Cai-Hong; Liu, Jing; Chen, Liang; Liu, Qingsong

    2015-01-01

    Through comprehensive comparison study, we found that ibrutinib, a clinically approved covalent BTK kinase inhibitor, was highly active against EGFR (L858R, del19) mutant driven NSCLC cells, but moderately active to the T790M ‘gatekeeper’ mutant cells and not active to wild-type EGFR NSCLC cells. Ibrutinib strongly affected EGFR mediated signaling pathways and induced apoptosis and cell cycle arrest (G0/G1) in mutant EGFR but not wt EGFR cells. However, ibrutinib only slowed down tumor progression in PC-9 and H1975 xenograft models. MEK kinase inhibitor, GSK1120212, could potentiate ibrutinib's effect against the EGFR (L858R/T790M) mutation in vitro but not in vivo. These results suggest that special drug administration might be required to achieve best clinical response in the ongoing phase I/II clinical trial with ibrutinib for NSCLC. PMID:26375053

  1. Sim2 Mutants Have Developmental Defects Not Overlapping with Those of Sim1 Mutants

    PubMed Central

    Goshu, Eleni; Jin, Hui; Fasnacht, Rachel; Sepenski, Mike; Michaud, Jacques L.; Fan, Chen-Ming

    2002-01-01

    The mouse genome contains two Sim genes, Sim1 and Sim2. They are presumed to be important for central nervous system (CNS) development because they are homologous to the Drosophila single-minded (sim) gene, mutations in which cause a complete loss of CNS midline cells. In the mammalian CNS, Sim2 and Sim1 are coexpressed in the paraventricular nucleus (PVN). While Sim1 is essential for the development of the PVN (J. L. Michaud, T. Rosenquist, N. R. May, and C.-M. Fan, Genes Dev. 12:3264-3275, 1998), we report here that Sim2 mutant has a normal PVN. Analyses of the Sim1 and Sim2 compound mutants did not reveal obvious genetic interaction between them in PVN histogenesis. However, Sim2 mutant mice die within 3 days of birth due to lung atelectasis and breathing failure. We attribute the diminished efficacy of lung inflation to the compromised structural components surrounding the pleural cavity, which include rib protrusions, abnormal intercostal muscle attachments, diaphragm hypoplasia, and pleural mesothelium tearing. Although each of these structures is minimally affected, we propose that their combined effects lead to the mechanical failure of lung inflation and death. Sim2 mutants also develop congenital scoliosis, reflected by the unequal sizes of the left and right vertebrae and ribs. The temporal and spatial expression patterns of Sim2 in these skeletal elements suggest that Sim2 regulates their growth and/or integrity. PMID:12024028

  2. Genetic analysis of sex chromosomal meiotic mutants in Drosophilia melanogaster.

    PubMed

    Baker, B S; Carpenter, A T

    1972-06-01

    A total of 209 ethyl methanesulfonate-treated X chromosomes were screened for meiotic mutants that either (1) increased sex or fourth chromosome nondisjunction at either meiotic division in males; (2) allowed recombination in such males; (3) increased nondisjunction of the X chromosome at either meiotic division in females; or (4) caused such females, when mated to males heterozygous for Segregation-Distorter (SD) and a sensitive homolog to alter the strength of meiotic drive in males.-Twenty male-specific meiotic mutants were found. Though the rates of nondisjunction differed, all twenty mutants were qualitatively similar in that (1) they alter the disjunction of the X chromosome from the Y chromosome; (2) among the recovered sex-chromosome exceptional progeny, there is a large excess of those derived from nullo-XY as compared to XY gametes; (3) there is a negative correlation between the frequency of sex-chromosome exceptional progeny and the frequency of males among the regular progeny. In their effects on meiosis these mutants are similar to In(1)sc(4L)sc(8R), which is deleted for the basal heterochromatin. These mutants, however, have normal phenotypes and viabilities when examined as X/0 males, and furthermore, a mapping of two of the mutants places them in the euchromatin of the X chromosome. It is suggested that these mutants are in genes whose products are involved in insuring the proper functioning of the basal pairing sites which are deleted in In(1)sc(4L)sc(8R), and in addition that there is a close connection, perhaps causal, between the disruption of normal X-Y pairing (and, therefore, disjunction) and the occurrence of meiotic drive in the male.-Eleven mutants were found which increased nondisjunction in females. These mutants were characterized as to (1) the division at which they acted; (2) their effect on recombination; (3) their dominance; (4) their effects on disjunction of all four chromosome pairs. Five female mutants caused a nonuniform

  3. Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells.

    PubMed

    Korhonen, J M; Saïd, F A; Wong, A J; Kaplan, D R

    1999-12-24

    The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.

  4. Silymarin protects against acrylamide-induced neurotoxicity via Nrf2 signalling in PC12 cells.

    PubMed

    Li, Liang; Sun, Hong-Yang; Liu, Wei; Zhao, Hong-Yu; Shao, Mei-Li

    2017-04-01

    Silymarin (SM) is a well-known antioxidant, anti-inflammatory and anti-cancer compound extracted from the milk thistle. Here, we investigated the protective effect of SM against acrylamide (AA)-induced neurotoxicity, mainly caused by oxidative stress, via activation of the nuclear transcription factor E2-related factor 2 (Nrf2) signalling pathway in PC12 cells. The MTT reduction assay was used to measure cell viability in various drug-treated groups and demonstrated that SM could increase cell viability in AA-treated PC12 cells. We then measured the reactive oxygen species (ROS) levels by the peroxide-sensitive fluorescent probe DCFH-DA and intracellular glutathione (GSH) and malondialdehyde (MDA) levels by absorption spectrophotometry. Our data revealed that SM could reduce ROS and MDA levels and increase GSH levels in AA-induced PC12 cells. To identify a potential mechanism for SM-induced protection, we measured the mRNA and protein expression levels of Nrf2 and its downstream target antioxidants glutathione peroxidase (Gpx), glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM) by quantitative real-time PCR and Western blot, respectively. The results suggested that SM could activate Nrf2 signalling and increase the expression of Nrf2, Gpx, GCLC and GCLM in AA-treated PC12 cells. In conclusion, SM can effectively alleviate AA-induced neurotoxicity in PC12 cells.

  5. The Local Population of White Dwarfs within 25 pc

    NASA Astrophysics Data System (ADS)

    Holberg, Jay B.; Oswalt, Terry D.; Sion, Edward M.

    2015-01-01

    We have extended the detailed survey of the local white dwarf population from 20 pc to 25 pc, effectively doubling the sample volume to now include 231 stars. The present 25 pc has an estimated completeness of 70% (the corresponding 20 pc sample is now 85% complete). The space density of white dwarfs remains at 4.8 ± 0.5 x 10-3 pc-3. There exists a curious excess of single stars in the sample 70% vs 30% in systems with one or more companions. A pronounced apparent deficiency remains between the eleven known Sirius-like systems present in the 20 pc sample and only a single such system presently known in the extended 25 pc sample. Also demonstrated, using explicit individual white dwarf cooling ages, is the feasibility of estimating the white dwarf birth rates over the last ~ 5 Gyr.This work is supported by NSF grant AST-1413537

  6. Securing your PC and protecting your privacy.

    PubMed

    Schloman, Barbara F

    2004-10-22

    Working in a networked information environment brings new opportunities for getting and sharing information. Regrettably, these benefits of the Internet are challenged by forces that would interfere to satisfy their own profit or malevolent motives. Your networked computer can be infected by viruses, worms, or Trojan horses or infiltrated by spyware, adware, or pop-ups. Without being aware of the dangers and taking precautionary steps, your PC is susceptible to being compromised and your privacy invaded. This column will highlight some of the dangers and offer basic steps for securing your computer and protecting your privacy.

  7. Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity

    PubMed Central

    Sun, Xin; Marque, Leonard O.; Cordner, Zachary; Pruitt, Jennifer L.; Bhat, Manik; Li, Pan P.; Kannan, Geetha; Ladenheim, Ellen E.; Moran, Timothy H.; Margolis, Russell L.; Rudnicki, Dobrila D.

    2014-01-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient–derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of HdhQ7/Q150 knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT. PMID:25035419

  8. Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

    PubMed Central

    Noel, K D; Sanchez, A; Fernandez, L; Leemans, J; Cevallos, M A

    1984-01-01

    Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained. Images PMID:6325385

  9. Novel modular domain PB1 recognizes PC motif to mediate functional protein–protein interactions

    PubMed Central

    Ito, Takashi; Matsui, Yasushi; Ago, Tetsuro; Ota, Kazuhisa; Sumimoto, Hideki

    2001-01-01

    Modular domains mediating specific protein–protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67phox, which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40phox (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67phox, which abolishes its interaction with Cdc24p, confers on cells temperature- sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40phox, which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules. PMID:11483497

  10. Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse.

    PubMed

    Rahimi Balaei, Maryam; Jiao, Xiaodan; Ashtari, Niloufar; Afsharinezhad, Pegah; Ghavami, Saeid; Marzban, Hassan

    2016-01-15

    Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2) mouse (nax--naked-ataxia mutant mouse) correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc) degeneration. Loss of Pcs in the nax cerebellum is compartmentalized and harmonized to the classic pattern of gene expression of the cerebellum in the wild type mouse. Usually, degeneration starts in the anterior and posterior zones and continues to the central and nodular zones of cerebellum. Studies have suggested that the p75 neurotrophin receptor (NTR) plays a role in Pc degeneration; thus, in this study, we investigated the p75NTR pattern and protein expression in the cerebellum of the nax mutant mouse. Despite massive Pc degeneration that was observed in the nax mouse cerebellum, p75NTR pattern expression was similar to the HSP25 pattern in nax mice and comparable with wild type sibling cerebellum. In addition, immunoblot analysis of p75NTR protein expression did not show any significant difference between nax and wild type sibling (p > 0.5). In comparison with wild type counterparts, p75NTR pattern expression is aligned with the fundamental cytoarchitecture organization of the cerebellum and is unchanged in the nax mouse cerebellum despite the severe neurodevelopmental disorder accompanied with Pc degeneration.

  11. Photodynamic therapy with the phthalocyanine photosensitizer Pc 4 of SW480 human colon cancer xenografts in athymic mice.

    PubMed

    Whitacre, C M; Feyes, D K; Satoh, T; Grossmann, J; Mulvihill, J W; Mukhtar, H; Oleinick, N L

    2000-05-01

    Photodynamic therapy (PDT) using the silicon phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N-(CH3)2] is an oxidative stress associated with induction of apoptosis in various cell types. We assessed the effectiveness of Pc 4-PDT on SW480 colon cancer xenografts grown in athymic nude mice. Animals bearing xenografts were treated with 1 mg/kg body weight Pc 4 and 48 h later were irradiated with 150 J/cm2 672-nm light from a diode laser delivered at 150 mW/cm2. Biochemical studies were performed in xenografts resected at various time points up to 26 h after Pc 4-PDT treatment, whereas tumor size was evaluated over a 4-week period in parallel experiments. In the tumors resected for biochemical studies, apoptosis was visualized by activation of caspase-9 and caspase-3 and a gradual increase in the cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to a maximum of approximately 60% of the total PARP present at approximately 26 h. At that time all Pc 4-PDT-treated tumors had regressed significantly. Two signaling responses that have previously been shown to be associated with Pc 4-PDT-induced apoptosis in cultured cells, p38 mitogen-activated protein kinase and p21/WAF1/Cip1, were examined. A marked increase in phosphorylation of p38 was observed within 1 h after Pc 4-PDT without changes in levels of the p38 protein. Levels of p21 were not altered in the xenografts in correspondence with the presence of mutant p53 in SW480 cells. Evaluation of tumor size showed that tumor growth resumed after a delay of 9-15 days. Our results suggest that: (a) Pc 4-PDT is effective in the treatment of SW480 human colon cancer xenografts independent of p53 status; (b) PARP cleavage may be mediated by caspase-9 and caspase-3 activation in the Pc 4-PDT-treated tumors; and (c) p38 phosphorylation may be a trigger of apoptosis in response to PDT in vivo in this tumor model.

  12. Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae

    SciTech Connect

    Letts, V.A.; Henry, S.A.

    1985-08-01

    Saccharomyces cerevisiae mutants, chol, are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. These mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). The authors exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. Coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.

  13. Retinal degeneration mutants in the mouse.

    PubMed

    Chang, B; Hawes, N L; Hurd, R E; Davisson, M T; Nusinowitz, S; Heckenlively, J R

    2002-02-01

    The Jackson Laboratory, having the world's largest collection of mouse mutant stocks and genetically diverse inbred strains, is an ideal place to look for genetically determined eye variations and disorders. Through ophthalmoscopy, electroretinography and histology, we have discovered disorders affecting all aspects of the eye including the lid, cornea, iris, lens and retina, resulting in corneal disorders, cataracts, glaucoma and retinal degenerations. Mouse models of retinal degeneration have been investigated for many years in the hope of understanding the causes of photoreceptor cell death. Sixteen naturally occurring mouse mutants that manifest degeneration of photoreceptors in the retina with preservation of all other retinal cell types have been found: retinal degeneration (formerly rd, identical with rodless retina, r, now Pde6b(rd1)); Purkinje cell degeneration (pcd); nervous (nr); retinal degeneration slow (rds, now Prph(Rd2)); retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinal degeneration 4 (Rd4); retinal degeneration 5 (rd5, now tub); vitiligo (vit, now Mitf(mi-vit)); retinal degeneration 6 (rd6); retinal degeneration 7 (rd7, now Nr2e3(rd7)); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8 (rd8); retinal degeneration 9 (Rd9); retinal degeneration 10 (rd10, now Pde6b(rd10)); and cone photoreceptor function loss (cpfl1). In this report, we first review the genotypes and phenotypes of these mutants and second, list the mouse strains that carry each mutation. We will also provide detailed information about the cpfl1 mutation. The phenotypic characteristics of cpfl1 mice are similar to those observed in patients with complete achromatopsia (ACHM2, OMIM 216900) and the cpfl1 mutation is the first naturally-arising mutation in mice to cause cone-specific photoreceptor function loss. cpfl1 mice may provide a model for congenital achromatopsia in humans.

  14. ICAN - INTEGRATED COMPOSITE ANALYZER (IBM PC VERSION)

    NASA Technical Reports Server (NTRS)

    Murthy, P. L.

    1994-01-01

    , macromechanics, and laminate analysis including the hygrothermal response of fiber composites. ICAN output includes the various ply and composite properties, composite structural response, and composite stress analysis results with details of failure. Output can be tailored to specific needs by choosing the appropriate options. Two machine versions of ICAN are available. The IBM 370 series version (LEW-14468) is written in FORTRAN IV for the IBM 370 series computers running OS/TSS. The IBM PC version (LEW-15592) is written in FORTRAN 77 for use on the IBM PC series computers running MS-DOS and Microsoft FORTRAN 5.1. The IBM 370 version requires 3.5Mb of memory for execution. No sample executable is provided. For the IBM PC version, a sample executable, along with sample input and output data, is included on the distribution medium. Although the included executable requires a math coprocessor, the ICAN source can be recompiled into an executable which does not require a math coprocessor. The standard distribution medium for the IBM 370 version of ICAN is a 9-track 1600 BPI magnetic tape in EBCDIC CARD IMAGE format. The standard distribution medium for the IBM PC version is one 5.25 inch 360K MS-DOS format diskette. The contents of the diskette are compressed using the PKWARE archiving tools. The utility to unarchive the files, PKUNZIP.EXE, is included. ICAN was developed in 1986 and the IBM PC version was released in 1992.

  15. Assessment of the PrPc Amino-Terminal Domain in Prion Species Barriers

    PubMed Central

    Davenport, Kristen A.; Henderson, Davin M.; Mathiason, Candace K.

    2016-01-01

    ABSTRACT Chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle are prion diseases that are caused by the same protein-misfolding mechanism, but they appear to pose different risks to humans. We are interested in understanding the differences between the species barriers of CWD and BSE. We used real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, the templated misfolding of the normal prion protein, PrPc, to a pathogenic, amyloid isoform, scrapie prion protein, PrPSc. We examined the role of the PrPc amino-terminal domain (N-terminal domain [NTD], amino acids [aa] 23 to 90) in cross-species conversion by comparing the conversion efficiency of various prion seeds in either full-length (aa 23 to 231) or truncated (aa 90 to 231) PrPc. We demonstrate that the presence of white-tailed deer and bovine NTDs hindered seeded conversion of PrPc, but human and bank vole NTDs did the opposite. Additionally, full-length human and bank vole PrPcs were more likely to be converted to amyloid by CWD prions than were their truncated forms. A chimera with replacement of the human NTD by the bovine NTD resembled human PrPc. The requirement for an NTD, but not for the specific human sequence, suggests that the NTD interacts with other regions of the human PrPc to increase promiscuity. These data contribute to the evidence that, in addition to primary sequence, prion species barriers are controlled by interactions of the substrate NTD with the rest of the substrate PrPc molecule. IMPORTANCE We demonstrate that the amino-terminal domain of the normal prion protein, PrPc, hinders seeded conversion of bovine and white-tailed deer PrPcs to the prion forms, but it facilitates conversion of the human and bank vole PrPcs to the prion forms. Additionally, we demonstrate that the amino-terminal domain of human and bank vole PrPcs requires interaction with the rest of the molecule to facilitate conversion by

  16. THE ORIGIN OF OB CLUSTERS: FROM 10 pc TO 0.1 pc

    SciTech Connect

    Liu Hauyu Baobab; Wang Ke; Ho, Paul T. P.; Zhang Qizhou; Quintana-Lacaci, Guillermo; Li Zhiyun; Zhang Zhiyu E-mail: kwang@cfa.harvard.edu E-mail: quintana@iram.es E-mail: zl4h@virginia.edu

    2012-01-20

    We observe the 1.2 mm continuum emission around the OB cluster-forming region G10.6-0.4, using the MAMBO-2 bolometer array of the IRAM 30 m telescope and the Submillimeter Array (SMA). Comparison of the Spitzer 24 {mu}m and 8 {mu}m images with our 1.2 mm continuum maps reveal an ionization front of an H II region, the photon-dominated layer, and several 5 pc scale filaments that follow the outer edge of the photon-dominated layer. The filaments, which are resolved in the MAMBO-2 observations, show regularly spaced parsec-scale molecular clumps, embedded with a cluster of dense molecular cores as shown in the SMA 0.87 mm observations. Toward the center of the G10.6-0.4 region, the combined SMA+IRAM 30 m continuum image reveals several parsec-scale protrusions. They may continue down to within 0.1 pc of the geometric center of a dense 3 pc scale structure, where a 200 M{sub Sun} OB cluster resides. The observed filaments may facilitate mass accretion onto the central cluster-forming region in the presence of strong radiative and mechanical stellar feedback. Their filamentary geometry may also facilitate fragmentation. We did not detect any significant polarized emission at 0.87 mm in the inner 1 pc region with SMA.

  17. PC-based Multiple Information System Interface (PC/MISI) detailed design and implementation plan

    NASA Technical Reports Server (NTRS)

    Dominick, Wayne D. (Editor); Hall, Philip P.

    1985-01-01

    The design plan for the personal computer multiple information system interface (PC/MISI) project is discussed. The document is intended to be used as a blueprint for the implementation of the system. Each component is described in the detail necessary to allow programmers to implement the system. A description of the system data flow and system file structures is given.

  18. (TFPP)Eu[Pc(OPh)8]Eu[Pc(OPh)8]/CuPc two-component bilayer heterojunction-based organic transistors with high ambipolar performance.

    PubMed

    Gao, Dameng; Zhang, Xia; Kong, Xia; Chen, Yanli; Jiang, Jianzhuang

    2015-02-04

    Organic thin film transistor (OTFT) devices fabricated by the solution-based QLS technique from a mixed (phthalocyaninato)(porphyrinato) europium complex (TFPP)Eu[Pc(OPh)8]Eu[Pc(OPh)8] exhibit air-stable ambipolar performance with mobilities of 6.0 × 10(-5) cm(2) V(-1) s(-)1 for holes and 1.4 × 10(-4) cm(2) V(-1) s(-1) for electrons, respectively. In good contrast, the two-component bilayer heterojunction thin film devices constructed by directly growing (TFPP)Eu[Pc(OPh)8]Eu[Pc(OPh)8] on vacuum deposited (VCD) CuPc film using solution based QLS method were revealed to show unprecedented ambipolar performance with carrier mobilities of 0.16 cm(2) V(-1) s(-1) for holes and 0.30 cm(2) V(-1) s(-1) for electrons. In addition to the intrinsic role of p-type organic semiconductor, the VCD CuPc film on the substrate also acts as a good template that induces significant improvement over the molecular ordering of triple-decker compound in the film. In particular, it results in the change in the aggregation mode of (TFPP)Eu[Pc(OPh)8]Eu[Pc(OPh)8] from J-type in the single-layer film to H-type in the bilayer film according to the UV-vis, XRD, and AFM observations.

  19. [Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis].

    PubMed

    Kurbatova, E M; Dutova, T A; Serkova, N N; Rabinovich, Ia M; Trotsenko, Iu A

    2004-05-01

    After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation

  20. Ypr140wp, 'the yeast tafazzin', displays a mitochondrial lysophosphatidylcholine (lyso-PC) acyltransferase activity related to triacylglycerol and mitochondrial lipid synthesis.

    PubMed

    Testet, Eric; Laroche-Traineau, Jeanny; Noubhani, Abdelmajid; Coulon, Denis; Bunoust, Odile; Camougrand, Nadine; Manon, Stephen; Lessire, René; Bessoule, Jean-Jacques

    2005-05-01

    When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC [lysophosphatidylcholine (1-acylglycerophosphorylcholine)] acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein capable of catalysing the acylation of lyso-PC molecules to form PC. Fluorescence microscopy analysis of living yeasts revealed that the fusion protein Ypr140w-green fluorescent protein is targeted to the mitochondria. Moreover, in contrast with wild-type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction. When yeast cells were grown in the presence of lactate, the mutant synthesized 2-fold more triacylglycerols when compared with the wild-type. Moreover, its mitochondrial membranes contained a lesser amount of PC and cardiolipin, and the fatty acid composition of these latter was greatly changed. These modifications were accompanied by a 2-fold increase in the respiration rates (states 3 and 4) of the mitochondria. The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wDelta cells is discussed.

  1. Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells.

    PubMed Central

    Szeberényi, J; Cai, H; Cooper, G M

    1990-01-01

    A dominant inhibitory mutation of Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21 (Asn-17)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The growth of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-17)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve growth factor (NGF) or fibroblast growth factor (FGF). These cells, however, were still able to respond with neurite outgrowth to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-17)Ha-ras. However, lower levels of p21(Asn-17) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-17) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-17)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells. Images PMID:2118994

  2. METCAN-PC - METAL MATRIX COMPOSITE ANALYZER

    NASA Technical Reports Server (NTRS)

    Murthy, P. L.

    1994-01-01

    High temperature metal matrix composites offer great potential for use in advanced aerospace structural applications. The realization of this potential however, requires concurrent developments in (1) a technology base for fabricating high temperature metal matrix composite structural components, (2) experimental techniques for measuring their thermal and mechanical characteristics, and (3) computational methods to predict their behavior. METCAN (METal matrix Composite ANalyzer) is a computer program developed to predict this behavior. METCAN can be used to computationally simulate the non-linear behavior of high temperature metal matrix composites (HT-MMC), thus allowing the potential payoff for the specific application to be assessed. It provides a comprehensive analysis of composite thermal and mechanical performance. METCAN treats material nonlinearity at the constituent (fiber, matrix, and interphase) level, where the behavior of each constituent is modeled accounting for time-temperature-stress dependence. The composite properties are synthesized from the constituent instantaneous properties by making use of composite micromechanics and macromechanics. Factors which affect the behavior of the composite properties include the fabrication process variables, the fiber and matrix properties, the bonding between the fiber and matrix and/or the properties of the interphase between the fiber and matrix. The METCAN simulation is performed as point-wise analysis and produces composite properties which are readily incorporated into a finite element code to perform a global structural analysis. After the global structural analysis is performed, METCAN decomposes the composite properties back into the localized response at the various levels of the simulation. At this point the constituent properties are updated and the next iteration in the analysis is initiated. This cyclic procedure is referred to as the integrated approach to metal matrix composite analysis. METCAN-PC

  3. METCAN-PC - METAL MATRIX COMPOSITE ANALYZER

    NASA Technical Reports Server (NTRS)

    Murthy, P. L.

    1994-01-01

    High temperature metal matrix composites offer great potential for use in advanced aerospace structural applications. The realization of this potential however, requires concurrent developments in (1) a technology base for fabricating high temperature metal matrix composite structural components, (2) experimental techniques for measuring their thermal and mechanical characteristics, and (3) computational methods to predict their behavior. METCAN (METal matrix Composite ANalyzer) is a computer program developed to predict this behavior. METCAN can be used to computationally simulate the non-linear behavior of high temperature metal matrix composites (HT-MMC), thus allowing the potential payoff for the specific application to be assessed. It provides a comprehensive analysis of composite thermal and mechanical performance. METCAN treats material nonlinearity at the constituent (fiber, matrix, and interphase) level, where the behavior of each constituent is modeled accounting for time-temperature-stress dependence. The composite properties are synthesized from the constituent instantaneous properties by making use of composite micromechanics and macromechanics. Factors which affect the behavior of the composite properties include the fabrication process variables, the fiber and matrix properties, the bonding between the fiber and matrix and/or the properties of the interphase between the fiber and matrix. The METCAN simulation is performed as point-wise analysis and produces composite properties which are readily incorporated into a finite element code to perform a global structural analysis. After the global structural analysis is performed, METCAN decomposes the composite properties back into the localized response at the various levels of the simulation. At this point the constituent properties are updated and the next iteration in the analysis is initiated. This cyclic procedure is referred to as the integrated approach to metal matrix composite analysis. METCAN-PC

  4. ERK5 Activity Is Required for Nerve Growth Factor-induced Neurite Outgrowth and Stabilization of Tyrosine Hydroxylase in PC12 Cells*

    PubMed Central

    Obara, Yutaro; Yamauchi, Arata; Takehara, Shin; Nemoto, Wataru; Takahashi, Maho; Stork, Philip J. S.; Nakahata, Norimichi

    2009-01-01

    Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt2cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 μm, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt2cAMP, but BIX02188 and BIX02189, at 30 μm, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt2cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt2cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process. PMID:19581298

  5. Leptin gene promoter DNA methylation in WNIN obese mutant rats.

    PubMed

    Kalashikam, Rajender Rao; Inagadapa, Padmavathi J N; Thomas, Anju Elizabeth; Jeyapal, Sugeetha; Giridharan, Nappan Veettil; Raghunath, Manchala

    2014-02-05

    Obesity has become an epidemic in worldwide population. Leptin gene defect could be one of the causes for obesity. Two mutant obese rats WNIN/Ob and WNIN/GROb, isolated at National Centre for Laboratory Animal Sciences (NCLAS), Hyderabad, India, were found to be leptin resistant. The present study aims to understand the regulatory mechanisms underlying the resistance by promoter DNA methylation of leptin gene in these mutant obese rats. Male obese mutant homozygous, carrier and heterozygous rats of WNIN/Ob and WNIN/GROb strain of 6 months old were studied to check the leptin gene expression (RT-PCR) and promoter DNA methylation (MassARRAY Compact system, SEQUENOM) of leptin gene by invivo and insilico approach. Homozygous WNIN/Ob and WNIN/GROb showed significantly higher leptin gene expression compared to carrier and lean counterparts. Leptin gene promoter DNA sequence region was analyzed ranging from transcription start site (TSS) to-550 bp length and found four CpGs in this sequence among them only three CpG loci (-309, -481, -502) were methylated in these WNIN mutant rat phenotypes. The increased percentage of methylation in WNIN mutant lean and carrier phenotypes is positively correlated with transcription levels. Thus genetic variation may have effect on methylation percentages and subsequently on the regulation of leptin gene expression which may lead to obesity in these obese mutant rat strains.

  6. Induction of petite yeast mutants by membrane-active agents.

    PubMed

    Jiménez, J; Longo, E; Benítez, T

    1988-12-01

    Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.

  7. Induction of petite yeast mutants by membrane-active agents.

    PubMed Central

    Jiménez, J; Longo, E; Benítez, T

    1988-01-01

    Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents. PMID:3066293

  8. Plasma mutant α-galactosidase A protein and globotriaosylsphingosine level in Fabry disease.

    PubMed

    Tsukimura, Takahiro; Nakano, Sachie; Togawa, Tadayasu; Tanaka, Toshie; Saito, Seiji; Ohno, Kazuki; Shibasaki, Futoshi; Sakuraba, Hitoshi

    2014-01-01

    Fabry disease is an X-linked genetic disorder characterized by deficient activity of α-galactosidase A (GLA) and accumulation of glycolipids, and various GLA gene mutations lead to a wide range of clinical phenotypes from the classic form to the later-onset one. To investigate the biochemical heterogeneity and elucidate the basis of the disease using available clinical samples, we measured GLA activity, GLA protein and accumulated globotriaosylsphingosine (Lyso-Gb3), a biomarker of this disease, in plasma samples from Fabry patients. The analysis revealed that both the enzyme activity and the protein level were apparently decreased, and the enzyme activity was well correlated with the protein level in many Fabry patients. In these cases, a defect of biosynthesis or excessive degradation of mutant GLAs should be involved in the pathogenesis, and the residual protein level would determine the accumulation of Lyso-Gb3 and the severity of the disease. However, there are some exceptional cases, i.e., ones harboring p.C142Y, p.R112H and p.M296I, who exhibit a considerable amount of GLA protein. Especially, a subset of Fabry patients with p.R112H or p.M296I has been attracted interest because the patients exhibit almost normal plasma Lyso-Gb3 concentration. Structural analysis revealed that C142Y causes a structural change at the entrance of the active site. It will lead to a complete enzyme activity deficiency, resulting in a high level of plasma Lyso-Gb3 and the classic Fabry disease. On the other hand, it is thought that R112H causes a relatively large structural change on the molecular surface, and M296I a small one in a restricted region from the core to the surface, both the structural changes being far from the active site. These changes will cause not only partial degradation but also degeneration of the mutant GLA proteins, and the degenerated enzymes exhibiting small and residual activity remain and probably facilitate degradation of Lyso-Gb3 in plasma, leading

  9. Nighttime Pc3 pulsations: MM100 and MAGDAS observations

    NASA Astrophysics Data System (ADS)

    Yagova, Nadezda V.; Heilig, Balazs; Pilipenko, Vyacheslav A.; Yoshikawa, Akimasa; Nosikova, Nataliya S.; Yumoto, Kiyohumi; Reda, Jan

    2017-05-01

    In this paper, we present a statistical and case analysis of nighttime Pc3 pulsations observed from middle to equatorial latitudes during the year 2003. We found two groups of nighttime Pc3 pulsations. Pc3s of the first group are in fact the nightside counterpart of morning Pc3 pulsations with large azimuthal scales slowly attenuating toward midnight. Such night signatures of morning Pc3 waves are observed during the periods of fast solar wind (V>500 km/s). The second type is the locally generated night Pc3 pulsations. They can be observed under moderate solar wind velocities. Maximal occurrence rates and amplitudes for these pulsations are recorded at middle geomagnetic latitudes near the local magnetic midnight. Probably, they are associated with auroral activations or local non-substorm bursty processes.[Figure not available: see fulltext.][Figure not available: see fulltext.

  10. Interaction of PC4 with melted DNA inhibits transcription.

    PubMed

    Werten, S; Stelzer, G; Goppelt, A; Langen, F M; Gros, P; Timmers, H T; Van der Vliet, P C; Meisterernst, M

    1998-09-01

    PC4 is a nuclear DNA-binding protein that stimulates activator-dependent class II gene transcription in vitro. Recent biochemical and X-ray analyses have revealed a unique structure within the C-terminal domain of PC4 that binds tightly to unpaired double-stranded (ds)DNA. The cellular function of this evolutionarily conserved dimeric DNA-binding fold is unknown. Here we demonstrate that PC4 represses transcription through this motif. Interaction with melted promoters is not required for activator-dependent transcription in vitro. The inhibitory activity is attenuated on bona fide promoters by (i) transcription factor TFIIH and (ii) phosphorylation of PC4. PC4 remains a potent inhibitor of transcription in regions containing unpaired ds DNA, in single-stranded DNA that can fold into two antiparallel strands, and on DNA ends. Our observations are consistent with a novel inhibitory function of PC4.

  11. Acquired Resistance to the Mutant-Selective EGFR Inhibitor AZD9291 Is Associated with Increased Dependence on RAS Signaling in Preclinical Models.

    PubMed

    Eberlein, Catherine A; Stetson, Daniel; Markovets, Aleksandra A; Al-Kadhimi, Katherine J; Lai, Zhongwu; Fisher, Paul R; Meador, Catherine B; Spitzler, Paula; Ichihara, Eiki; Ross, Sarah J; Ahdesmaki, Miika J; Ahmed, Ambar; Ratcliffe, Laura E; O'Brien, Elizabeth L Christey; Barnes, Claire H; Brown, Henry; Smith, Paul D; Dry, Jonathan R; Beran, Garry; Thress, Kenneth S; Dougherty, Brian; Pao, William; Cross, Darren A E

    2015-06-15

    Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. ©2015 American Association for Cancer Research.

  12. Acquired resistance to mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling in preclinical models

    PubMed Central

    Eberlein, Catherine A.; Stetson, Daniel; Markovets, Aleksandra A.; Al-Kadhimi, Katherine J.; Lai, Zhongwu; Fisher, Paul R.; Meador, Catherine B.; Spitzler, Paula; Ichihara, Eiki; Ross, Sarah J.; Ahdesmaki, Miika J.; Ahmed, Ambar; Ratcliffe, Laura E.; Christey O’Brien, Elizabeth L.; Barnes, Claire H.; Brown, Henry; Smith, Paul D.; Dry, Jonathan R.; Beran, Garry; Thress, Kenneth S.; Dougherty, Brian; Pao, William; Cross, Darren A. E.

    2015-01-01

    Resistance to targeted EGFR inhibitors is likely to develop in EGFR mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR TKIs including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002 or AZD9291. Compared to parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumours in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumours. Further, these findings suggest that NRAS modifications in tumour samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. PMID:25870145

  13. Features of Pc5 pulsations in the geomagnetic field, auroral luminosity, and Riometer absorption

    NASA Astrophysics Data System (ADS)

    Belakhovsky, V. B.; Pilipenko, V. A.; Samsonov, S. N.; Lorentsen, D.

    2016-01-01

    Simultaneous morning Pc5 pulsations ( f ~ 3-5 mHz) in the geomagnetic field, aurora intensities (in the 557.7 and 630.0 nm oxygen emissions and the 471.0 nm nitrogen emission), and riometer absorption, were studied based on the CARISMA, CANMOS, and NORSTAR network data for the event of January 1, 2000. According to the GOES-8 satellite observations, these Pc5 geomagnetic pulsations are observed as incompressible Alfvén waves with toroidal polarization in the magnetosphere. Although the Pc5 pulsation frequencies in auroras, the geomagnetic field, and riometer absorption are close to one another, stable phase relationships are not observed between them. Far from all trains of geomagnetic Pc5 pulsations are accompanied by corresponding auroral pulsations; consequently, geomagnetic pulsations are primary with respect to auroral pulsations. Both geomagnetic and auroral pulsations propagate poleward, and the frequency decreases with increasing geomagnetic latitude. When auroral Pc5 pulsations appear, the ratio of the 557.7/630.0 nm emission intensity sharply increases, which indicates that auroral pulsations result from not simply modulated particle precipitation but also an additional periodic acceleration of auroral electrons by the wave field. A high correlation is not observed between Pc5 pulsations in auroras and the riometer absorption, which indicates that these pulsations have a common source but different generation mechanisms. Auroral luminosity modulation is supposedly related to the interaction between Alfvén waves and the region with the field-aligned potential drop above the auroral ionosphere, and riometer absorption modulation is caused by the scattering of energetic electrons by VLF noise pulsations.

  14. [Report on the VDÄPC Fellowship].

    PubMed

    Sarantopoulos, E

    2016-04-01

    "Orandum est ut sit mens sana in corpore sano" ("You should pray for a healthy mind in a healthy body"). This phrase is a shortened citation from the satirical work of the Roman poet Juvenal. It highlights the significant role of physical as well as mental health. Aesthetic ideals have existed since the Archaic age, but they are not necessarily the same in different continents. Being familiar with aesthetic ideals in different cultures might help to accommodate patients' needs and wishes in today's globalised world. Therefore, fellowship programs such as the program organised by the Association of German Aesthetic Plastic Surgeons ("VDÄPC") are very important for young plastic surgeons who are interested in improving their surgical skills and experience. After all, aesthetic surgery is a dynamic specialty, which requires aesthetic plastic surgeons to undergo continued medical education.

  15. On the Internet with a PC

    NASA Astrophysics Data System (ADS)

    Woronow, Alex; Dare, Scott

    1995-07-01

    Using a PC to access the Internet and all its services is not difficult—once basic computer hardware, software, and network connections have been installed and configured. Many Internet networking problems can be avoided by contacting network experts before selecting the hardware and network software. This article will introduce some of the hardware, software, and Internet networking options available to the would-be Internet Surfer, and some of the pitfalls that one might encounter. Once the network hardware and software are installed successfully, one can explore the Internet through a number of different software packages that allow the sharing of computer files and printers, as well as logging onto remote host-computers, searching World Wide Web, posting and reading notices on virtually any topic, and sending and receiving electronic mail. This article will describe the functions of several programs that can access these Internet services and how to obtain them.

  16. Pathway Controlled Penetration (PcP)

    SciTech Connect

    Knight, Earl E.; Rougier, Esteban; Zubelewicz, Aleksander

    2012-08-29

    The technical approach employs advanced computational simulation tools to demonstrate how current assets can destroy RWK-RFI-12-0001's HDBT, a tunnel complex with two portals built into the base of a granite mountain. The granite over layer is assumed to be 60 meters thick over both portals and 80 meters over the facility's mission space. Key S&T is the completed development of a highly innovative viscoplastic fracture material model, 3D parallel gas-fracture capabilities into FDEM, and a stochastic handling of the material properties. Phase I - Develop and validate code simulation tools: (1) develop, incorporate and validate AZ-Frac material model for granite; and (2) Develop and incorporate gas-driven-fracture modeling into LANL's FDEM MUNROU code; (3) Develop and incorporate stochastic features into FDEM modeling. Phase II - Conduct PcP analysis on above HDBT: (1) Acquire HDBT design data, develop simulation model; and (2) Evaluate and select most promising defeat alternative. Phase III - Deliver code, train Service target analysts, and conduct simulations against real world HDBTs. PcP uses advanced computer simulations to enhance HDBT functional defeat efforts. Newly developed material models that account for fractural energy coupled with the finite discrete element methodology (FDEM) will provide targeting packages that will create penetration avenues for current or future lethality options. This novel computational approach requires full 3D geologic and structure characterization as well as significant high performance computing capabilities. The goal is to distinctively alter the targeting paradigm by leveraging critical DoD assets along with insitu geologic strata. In other words, assets will utilize underground rock structure to their benefit by creating rubbilization zones that will allow pathway controlled penetration.

  17. Microcomputer Applications with PC LAN (Local Area Network) in Battleships.

    DTIC Science & Technology

    1988-12-01

    9 G. INTEL 80386 MICROPROCESSOR ARCHITECTURE 9 1. Cache Strategy 10 2. Memory Management 10 3. Multitasking 11 4. Software...shrinks, it becomes more clear that the traditional modes of battle planning and management are no longer practical. Many of these problems, like...Card in it. Each PC must run the PC Network program to manage its participation in the network. Each PC must have a cable to connect it to the

  18. Bacteriorhodopsin mutants of Halobacterium sp. GRB. II. Characterization of mutants.

    PubMed

    Soppa, J; Otomo, J; Straub, J; Tittor, J; Meessen, S; Oesterhelt, D

    1989-08-05

    The bacterioopsin genes of Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F. (1984) Mol. & Gen. Genet. 194, 91-97) wild type and 10 independent mutants of different phenotypes have been cloned and sequenced. The wild type gene has two conservative changes compared to the gene of Halobacterium halobium, so that the proteins of the two species are identical. Six different mutations at five different codons have been found, leading to the following amino acid changes compared to the wild type: Trp10----Cys (three cases), Tyr57----Asn, Asp85----Glu, Asp06----Asn (three cases), Asp96----Gly, Trp138----Arg. A first characterization of the mutant proteins is given, and their implications for models of bacteriorhodopsin structure and function are discussed.

  19. Pearl structures of Pc1 geomagnetic pulsations observed at multipoint ground stations at Russia, Japan and Canada

    NASA Astrophysics Data System (ADS)

    Jun, C.; Shiokawa, K.; Connors, M. G.; Schofield, I.; Poddelsky, I.; Shevtsov, B.

    2013-12-01

    Pc1 geomagnetic pulsations propagate from high to low latitudes through the ionospheric wave duct. A few papers had shown longitudinal propagation of Pc1 pulsations [e.g., Kawamura et al. 1981; Sakaguchi et al.2012]. Despite these previous researches, diurnal variations of longitudinally-distributed Pc1 pulsations and the pearl structures at different stations have not been investigated yet. In order to understand generation and propagation processes of Pc1 pulsations in the magnetosphere and the ionosphere, it is necessary to investigate spatial distribution of Pc1 pulsations using magnetometers at longitudinally and latitudinally separated ground stations. We have investigated spatial distributions of the Pc1 pulsations observed by induction magnetometers at three ground stations at Moshiri (MOS) in Japan, Magadan (MGD) in far-eastern Russia and Athabasca (ATH) in central Canada from January 2009 to December 2011. Simultaneous Pc1 events observed at MGD and ATH occurred in the morning and afternoon sectors. This result is consistent with the global distribution of EMIC waves observed in space [Min et al. 2012]. The simultaneous Pc1 events with high coherence (> 0.5) observed at ATH and MGD concentrates in the afternoon to pre-midnight sector. The Pc1 frequencies of the simultaneous Pc1 events at ATH and MGD in the afternoon to pre-midnight sector were higher than those in the post-midnight to morning sector. Most of the simultaneous Pc1 events with high coherence observed at ATH and MGD have different pearl structures. This result indicates that the pearl structures should be not caused in the magnetosphere, and rather made during the propagation in the ionospheric duct. Simultaneous Pc1 events observed at MGD and MOS at subauroral and middle latitudes, respectively, were most frequently observed at night suggesting that propagation in the ionospheric duct suffers less attenuation at night. In the presentation we discuss these results in combination with the EMIC

  20. Newcastle disease virus-induced apoptosis in PC12 pheochromocytoma cells.

    PubMed

    Szeberényi, József; Fábián, Zsolt; Töröcsik, Beáta; Kiss, Katalin; Csatary, Laszlo K

    2003-01-01

    The avian paramyxovirus Newcastle disease virus (NDV) causes severe infections in birds. It is essentially nonpathogenic in rodents and human beings but was found to have an oncolytic potential against certain types of human malignancies. An attenuated NDV vaccine (designated MTH-68/H) was found to cause regression of various human tumors, but the mechanism of its oncolytic action and its selectivity toward malignant cells remain poorly understood. NDV was reported to cause apoptotic death in several avian cultured cell types. Programmed cell death may thus be the basis for the oncolytic effect of NDV vaccines. To test this possibility, we chose the PC12 rat pheochromocytoma cell line, a widely used model system for apoptosis. The MTH-68/H vaccine was found to cause apoptotic death of PC12 cells in a dose-dependent manner. A brief exposure of cells to the virus was found to trigger the apoptotic response. Cell death induced by the vaccine was not accompanied by significant alterations in the major mitogen-activated protein kinase pathways of these cells. Apoptotic DNA fragmentation was not affected by stimulating growth factor pathways or signaling mechanisms mediated by protein kinase C or the second messenger, calcium. In contrast, stimulation of protein kinase A by cyclic adenosine monophosphate analogs gave partial protection against the virus. PC12 cells thus provide a useful model system to study the effects of NDV on cell survival at the molecular level.

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

    PubMed Central

    Norman, Kaitlyn L.

    2016-01-01

    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. PMID:26453908

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

    PubMed

    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.

  3. ELIPGRID-PC: A PC program for calculating hot spot probabilities

    SciTech Connect

    Davidson, J.R.

    1994-10-01

    ELIPGRID-PC, a new personal computer program has been developed to provide easy access to Singer`s 1972 ELIPGRID algorithm for hot-spot detection probabilities. Three features of the program are the ability to determine: (1) the grid size required for specified conditions, (2) the smallest hot spot that can be sampled with a given probability, and (3) the approximate grid size resulting from specified conditions and sampling cost. ELIPGRID-PC also provides probability of hit versus cost data for graphing with spread-sheets or graphics software. The program has been successfully tested using Singer`s published ELIPGRID results. An apparent error in the original ELIPGRID code has been uncovered and an appropriate modification incorporated into the new program.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

    PubMed Central

    Vivoli, Mirella; Caulfield, Thomas R.; Martínez-Mayorga, Karina; Johnson, Alan T.; Jiao, Guan-Sheng

    2012-01-01

    The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub- and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes. PMID:22169851

  6. Docosahexaenoic acid phospholipid differentially modulates the conformation of G90V and N55K rhodopsin mutants associated with retinitis pigmentosa.

    PubMed

    Dong, Xiaoyun; Herrera-Hernández, María Guadalupe; Ramon, Eva; Garriga, Pere

    2017-05-01

    Rhodopsin is the visual photoreceptor of the retinal rod cells that mediates dim light vision and a prototypical member of the G protein-coupled receptor superfamily. The structural stability and functional performance of rhodopsin are modulated by membrane lipids. Docosahexaenoic acid has been shown to interact with native rhodopsin but no direct evidence has been established on the effect of such lipid on the stability and regeneration of rhodopsin mutants associated with retinal diseases. The stability and regeneration of two thermosensitive mutants G90V and N55K, associated with the retinal degenerative disease retinitis pigmentosa, have been analyzed in docosohexaenoic phospholipid (1,2-didocosa-hexaenoyl-sn-glycero-3-phosphocholine; DDHA-PC) liposomes. G90V mutant reconstituted in DDHA-PC liposomes significantly increased its thermal stability, but N55K mutant showed similar thermal sensitivity both in dodecyl maltoside detergent solution and in DDHA-PC liposomes. The retinal release process, measured by fluorescence spectroscopy, became faster in the lipid system for the two mutants. The opsin conformation was stabilized for the G90V mutant allowing improved retinal uptake whereas no chromophore binding could be detected for N55K opsin after photoactivation. The results emphasize the distinct role of DHA on different phenotypic rhodopsin mutations associated with classical (G90V) and sector (N55K) retinitis pigmentosa. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Electronic properties of CuPc and H2Pc: an experimental and theoretical study.

    PubMed

    Nardi, Marco Vittorio; Detto, Francesca; Aversa, Lucrezia; Verucchi, Roberto; Salviati, Giancarlo; Iannotta, Salvatore; Casarin, Maurizio

    2013-08-21

    Phthalocyanine (H2Pc) and its open-shell copper complex (CuPc) deposited on amorphous gold films have been studied by combining the outcomes of several synchrotron based spectroscopic tools (X-ray photoelectron spectroscopy, UV photoelectron spectroscopy and near-edge X-ray absorption fine structure, NEXAFS, spectroscopy) with those of density functional theory (DFT) calculations. The assignment of experimental evidence has been guided by the results of DFT numerical experiments carried out on isolated molecules. With specific reference to CuPc NEXAFS data collected at the N K-edge, they have been assigned by using the open-shell time-dependent DFT (TDDFT) in the framework of the zeroth order regular approximation (ZORA) scalar relativistic approach. The agreement between theory and experiment has been found to be satisfactory, thus indicating that the open-shell TDDFT (F. Wang and T. Ziegler, Mol. Phys., 2004, 102, 2585) may be used with some confidence to look into the X-ray absorption spectroscopy results pertinent to transition metal complexes. As far as the metal-ligand interaction is concerned, the combined use of NEXAFS spectroscopy and DFT outcomes ultimately testified the significant ionic contribution characterizing the bonding between the metal centre and the nitrogen atoms of the phthalocyanine coordinative pocket.

  8. Quantification of PrPC in bovine peripheral tissues: Analysis in wild-type and PrPC-deficient cattle.

    PubMed

    Kobayashi, Shin-Ichi; Ano, Yasuhisa; Sakudo, Akikazu; Yukawa, Masayoshi; Sigiura, Katsuaki; Manabe, Noboru; Nakayama, Hiroyuki; Onodera, Takashi

    2009-01-01

    Cellular PrP (PrPC) is necessary for bovine spongiform encephalopathy (BSE) infection. The purpose of the present experiment was the quantification of PrPC in peripheral tissues to assess the risk of BSE infection from these tissues. The tissue distribution of PrPC was examined by a sandwich enzyme-linked immunosorbent assay (sELISA) and histochemical analysis. PrPC-deficient cows were used as a negative control. The sELISA revealed that the brain contained the highest PrPC content (10.7 µg/g tissue), while other organs/tissues harbored lower amounts, in decreasing order as follows: longissimus capitis muscle, iliocostalis thoracis muscle, splenius muscle, biceps femoris muscle, triceps brachii muscle, longissimus thoracis muscle, ileum, jejunum, duodenum, colon, cecum, apex linguae, omotransversarius muscle, posterior part of the corpus linguae, anterior part of the corpus linguae and radix linguae (5.2- to 31-fold less PrPC than the brain). In the tissue/organs of PrP-deficient cows, PrPC levels were under the limit of detection. Histochemical analysis showed that PrPC was expressed in nerve cells in intestinal tissues. The presence of PrPC in the bovine tongue, skeletal muscles and intestines raises the possibility of PrPSc accumulation in these tissues, indicating that these organs/tissues may serve as potential sources of BSE infection.

  9. Zebrafish Genomic Instability Mutants and Cancer Susceptibility

    PubMed Central

    Moore, Jessica L.; Rush, Lindsay M.; Breneman, Carol; Mohideen, Manzoor-Ali P. K.; Cheng, Keith C.

    2006-01-01

    Somatic loss of tumor suppressor gene function comprising the second hit of Knudson's two-hit hypothesis is important in human cancer. A genetic screen was performed in zebrafish (Danio rerio) to find mutations that cause genomic instability (gin), as scored by Streisinger's mosaic-eye assay that models this second hit. The assay, based on a visible test for loss of wild-type gene function at a single locus, golden, is representative of genomewide events. Twelve ENU-induced genomic instability (gin) mutations were isolated. Most mutations showed weak dominance in heterozygotes and all showed a stronger phenotype in homozygotes. Trans-heterozygosity for 7 of these mutations showed greatly enhanced instability. A variety of spontaneous tumors were found in heterozygous adults from all gin lines, consistent with the expectation that genomic instability (mutator) mutations can accelerate carcinogenesis. The incidence of spontaneous cancer at 30–34 months was increased 9.6-fold in heterozygotes for the mutant with the strongest phenotype, gin-10. Tumors were seen in skin, colon, kidney, liver, pancreas, ovary, testis, and neuronal tissues, with multiple tumors in some fish. The study of these mutants will add to our understanding of the mechanisms of somatic loss of gene function and how those mechanisms contribute to cancer susceptibility. PMID:16888336

  10. Sigma-1 receptor agonist PRE084 is protective against mutant huntingtin-induced cell degeneration: involvement of calpastatin and the NF-κB pathway

    PubMed Central

    Hyrskyluoto, A; Pulli, I; Törnqvist, K; Huu Ho, T; Korhonen, L; Lindholm, D

    2013-01-01

    Alterations in mitochondria and increased oxidative stress are associated with the disease progression in Huntington's disease (HD). Endoplasmic reticulum (ER) stress and oxidative damage are linked through the close communication between the ER and mitochondria. Sigma-1 receptor (Sig-1R) is a chaperone protein in the ER that is involved in ER stress regulation, but little is known about its role in HD or the mechanisms for cell protection. Here we show that the Sig-1R agonist, PRE084 increases cell survival and counteracts the deleterious effects caused by N-terminal mutant huntingtin proteins in neuronal PC6.3 cells. Particularly, PRE084 increased the levels of cellular antioxidants by activating the NF-κB pathway that is compromised by the expression of mutant huntingtin proteins. These results show that the Sig-1R agonist has beneficial effects in models of HD and that compounds affecting the Sig-1R may be promising targets for future drug development in HD. PMID:23703391

  11. Anticancer effects of ethanolic neem leaf extract on prostate cancer cell line (PC-3).

    PubMed

    Kumar, Suresh; Suresh, P K; Vijayababu, M R; Arunkumar, A; Arunakaran, J

    2006-04-21

    Prostate cancer (PC) is the most prevalent cancer and the leading cause of male cancer death. Azadirachta indica (neem tree) has been used successfully centuries to reduce tumors by herbalists throughout Southeast Asia. Here the present study indicated that an ethanolic extract of neem has been shown to cause cell death of prostate cancer cells (PC-3) by inducing apoptosis as evidenced by a dose-dependent increase in DNA fragmentation and a decrease in cell viability. Western blot studies indicated that treatment with neem extract showed decreased level of Bcl-2, which is anti-apoptotic protein and increased the level of Bax protein. So the neem extract could be potentially effective against prostate cancer treatment.

  12. Identical substitutions in magnesium chelatase paralogs result in chlorophyll deficient soybean mutants

    USDA-ARS?s Scientific Manuscript database

    The soybean (Glycine max (L.) Merr.) chlorophyll deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a non-synonymous nucleotide substitution in the third exon of a Mg-chelat...

  13. PC7 and the related proteases Furin and Pace4 regulate E-cadherin function during blastocyst formation.

    PubMed

    Bessonnard, Sylvain; Mesnard, Daniel; Constam, Daniel B

    2015-09-28

    The first cell differentiation in mammalian embryos segregates polarized trophectoderm cells from an apolar inner cell mass (ICM). This lineage decision is specified in compacted morulae by cell polarization and adhesion acting on the Yes-associated protein in the Hippo signaling pathway, but the regulatory mechanisms are unclear. We show that morula compaction and ICM formation depend on PC7 and the related proprotein convertases (PCs) Furin and Pace4 and that these proteases jointly regulate cell-cell adhesion mediated by E-cadherin processing. We also mapped the spatiotemporal activity profiles of these proteases by live imaging of a transgenic reporter substrate in wild-type and PC mutant embryos. Differential inhibition by a common inhibitor revealed that all three PCs are active in inner and outer cells, but in partially nonoverlapping compartments. E-cadherin processing by multiple PCs emerges as a novel mechanism to modulate cell-cell adhesion and fate allocation.

  14. PC7 and the related proteases Furin and Pace4 regulate E-cadherin function during blastocyst formation

    PubMed Central

    Mesnard, Daniel

    2015-01-01

    The first cell differentiation in mammalian embryos segregates polarized trophectoderm cells from an apolar inner cell mass (ICM). This lineage decision is specified in compacted morulae by cell polarization and adhesion acting on the Yes-associated protein in the Hippo signaling pathway, but the regulatory mechanisms are unclear. We show that morula compaction and ICM formation depend on PC7 and the related proprotein convertases (PCs) Furin and Pace4 and that these proteases jointly regulate cell–cell adhesion mediated by E-cadherin processing. We also mapped the spatiotemporal activity profiles of these proteases by live imaging of a transgenic reporter substrate in wild-type and PC mutant embryos. Differential inhibition by a common inhibitor revealed that all three PCs are active in inner and outer cells, but in partially nonoverlapping compartments. E-cadherin processing by multiple PCs emerges as a novel mechanism to modulate cell–cell adhesion and fate allocation. PMID:26416966

  15. Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

    PubMed Central

    Hamam, Ahmed

    2012-01-01

    We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters—a mechanosensitive channel homolog (MscS), a Ca2+/H+ exchange protein (cax), and Ca2+-ATPases (nca-1, nca-2, nca-3)—as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H+-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca2+ levels, indicative of lesions in Ca2+ homeostasis. However, the net Ca2+ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca2+-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca2+ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca2+] was elevated. Thus, although Ca2+ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654–661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H+-ATPase activity. PMID:22408225

  16. Cytotoxic, Genotoxic, and Neurotoxic Effects of Mg, Pb, and Fe on Pheochromocytoma (PC-12) Cells

    PubMed Central

    Sanders, Talia; Liu, Yi-Ming; Tchounwou, Paul B.

    2014-01-01

    Metals such as lead (Pb), magnesium (Mg), and iron (Fe) are ubiquitous in the environment as a result of natural occurrence and anthropogenic activities. Although Mg, Fe and others are considered essential elements, high level of exposure has been associated with severe adverse health effects including cardiovascular, hematological, nephrotoxic, hepatotoxic, and neurologic abnormalities in humans. In the present study we hypothesized that Mg, Pb, and Fe are cytotoxic, genotoxic and neurotoxic, and their toxicity is mediated through oxidative stress and alteration in protein expression. To test the hypothesis, we used the pheochromocytoma (PC-12) cell line as a neuro cell model and performed the LDH assay for cell viability, Comet assay for DNA damage, Western blot for oxidative stress, and HPLC-MS to assess the concentration levels of neurological biomarkers such as glutamate, dopamine (DA), and 3-methoxytyramine (3-MT). The results of this study clearly show that Mg, Pb, and Fe, respectively in the form of MgSO4, Pb(NO3)2, FeCl2, and FeCl3 induce cytotoxicity, oxidative stress, and genotoxicity in PC-12 cells. In addition, exposure to these metallic compounds caused significant changes in the concentration levels of glutamate, dopamine, and 3-MT in PC-12 cells. Taken together the findings suggest that MgSO4, Pb(NO3)2, FeCl2, and FeCl3 have the potential to induce substantial toxicity to PC-12 cells. PMID:24942330

  17. [Nimotuzumab significantly enhances chemosensitivity of
PC9 human lung adenocarcinoma cells to paclitaxel in vitro].

    PubMed

    Xiao, Yu; Cao, Baoshan; Liang, Li

    2015-02-01

    Nimotuzumab is a humanized IgG1 type monoclonal antibody targeting epidermal growth factor receptor, and can enhance chemosensitivity and radiosensitivity of certain cancers. The aim of this study is to investigate the effects of nimotuzumab on the chemosensitivities of PC9 human lung adenocarcinoma cells to common chemtherapeutic drugs including ciaplatin, gemcitabine, paclitaxel, pemetrexed and vinorelbine, and to elucidate possible mechanisms. PC9 human lung adenocarcinoma cell line was used in the study. Cell proliferation was determined by WST-1 assay and cell apoptosis was detected by TUNEL assay. Cell cycle distribution was analyzed by DNA analysis with FACS. Tublin and microfilaments were observed by immunofluorescence staining. Nimotuzumab significantly enhanced the chemosensitivity of PC9 cells to paclitaxel. Cell proliferation was inhibited significantly (P<0.05) and cell apoptosis rate was higher in nimotuzumab combined with low dose paclitaxel (0.05 μg/mL) group (P=0.013). G2/M arrest was increased significantly by nimotuzumab combined with paclitaxel group (P<0.05). Nimotuzumab caused aggregation of tublin and microfilaments into well organized microtubules. Nimotuzumab enhanced the chemosensitivity of PC9 cell to paclitaxel by enhancing G2/M arrest and aggregation of tublin and microfilaments. Therefore, Nimotuzumab combined with taxane drugs could be a potential effective regimen in non-small cell lung cancer.

  18. Color Shift Investigations for LED Secondary Optical Designs: Comparison between BPA-PC and PMMA

    NASA Astrophysics Data System (ADS)

    Lu, Guangjun; Yazdan Mehr, M.; van Driel, W. D.; Fan, Xuejun; Fan, Jiajie; Jansen, K. M. B.; Zhang, G. Q.

    2015-07-01

    Recently, color shift of LED-based lighting products has attracted much attention due to its increasing impact on the field application. However, significant research investigations on the color shift mechanisms are not publically available especially for important transmission materials used for secondary optical design. In this paper, broadly used such commercial materials (BPA-PC and PMMA) are experimentally investigated on the color shift effects during aging. Besides this, color shift mechanisms of degradation of transmittance are also studied. Results revealed: (1) Inconsistent degradation of wavelength-dependent transmittance induces the decrease of the blue/yellow light intensity ratio and thus gives rise to the color shift toward the yellow field, which is the color shift mechanism of BPA-PC; (2) Even for the non-aged BPA-PC, the transmittance varies with wavelength in the visible light field due to the chemistry of the material, which caused the change of intensify ratio of blue light to yellow light in the SPD, leading to color change in perception; (3) Oxidation plays a key role in the degradation of transmittance at around the peak wavelength of the blue light field, which is in correlation with the discoloration of thermally-aged BPA-PC materials. By contrast, for the PMMA specimen aged up to 3000 h, oxidation was neither occurred at 85 °C, nor with additional exposure to blue light, nor even with additional humidity of 85%RH.

  19. MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features

    PubMed Central

    Li, Xiaoran; Zhong, Yali; Lu, Jie; Axcrona, Karol; Eide, Lars; Syljuåsen, Randi G.; Peng, Qian; Wang, Junbai; Zhang, Hongquan; Goscinski, Mariusz Adam; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

    2016-01-01

    Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features. PMID:27248169

  20. Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome

    PubMed Central

    Burnett, Lisa C.; LeDuc, Charles A.; Sulsona, Carlos R.; Paull, Daniel; Rausch, Richard; Eddiry, Sanaa; Carli, Jayne F. Martin; Morabito, Michael V.; Skowronski, Alicja A.; Hubner, Gabriela; Zimmer, Matthew; Wang, Liheng; Day, Robert; Levy, Brynn; Dubern, Beatrice; Poitou, Christine; Clement, Karine; Rosenbaum, Michael; Salles, Jean Pierre; Tauber, Maithe; Egli, Dieter

    2016-01-01

    Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell–derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p–/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p–/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p–/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH–releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency. PMID:27941249

  1. Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2).

    PubMed

    Grieben, Mariana; Pike, Ashley C W; Shintre, Chitra A; Venturi, Elisa; El-Ajouz, Sam; Tessitore, Annamaria; Shrestha, Leela; Mukhopadhyay, Shubhashish; Mahajan, Pravin; Chalk, Rod; Burgess-Brown, Nicola A; Sitsapesan, Rebecca; Huiskonen, Juha T; Carpenter, Elisabeth P

    2017-02-01

    Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.

  2. Habituation in the Single Cell: Diminished Secretion of Norepinephrine with Repetitive Depolarization of PC12 Cells

    NASA Astrophysics Data System (ADS)

    McFadden, Philip N.; Koshland, Daniel E., Jr.

    1990-03-01

    Neuronally differentiated PC12 cells secrete decreasing amounts of [^3H]norepinephrine when repetitively stimulated by depolarizing concentrations of potassium ion. The decreasing response shows attributes that have been classically ascribed to response habituation, a behavior commonly observed in nervous systems but found here in a homogeneous cell type. Alteration of the habituation pattern was caused by activators of the protein kinase C pathway and of voltage-gated calcium channels.

  3. Hydrogen bonds in PC{sub 61}BM solids

    SciTech Connect

    Sheng, Chun-Qi; Li, Wen-Jie; Du, Ying-Ying; Chen, Guang-Hua; Chen, Zheng; Li, Hai-Yang; Li, Hong-Nian

    2015-09-15

    We have studied the hydrogen bonds in PC{sub 61}BM solids. Inter-molecular interaction is analyzed theoretically for the well-defined monoclinic (P2{sub 1}/n) structure. The results indicate that PC{sub 61}BM combines into C–H⋯O{sub d} bonded molecular chains, where O{sub d} denotes the doubly-bonded O atom of PC{sub 61}BM. The molecular chains are linked together by C–H⋯O{sub s} bonds, where O{sub s} denotes the singly-bonded O atom of PC{sub 61}BM. To reveal the consequences of hydrogen bond formation on the structural properties of PC{sub 61}BM solids (not limited to the monoclinic structure), we design and perform some experiments for annealed samples with the monoclinic (P2{sub 1}/n) PC{sub 61}BM as starting material. The experiments include differential scanning calorimetry, X-ray diffraction and infrared absorption measurements. Structural phase transitions are observed below the melting point. The C–H⋯O{sub d} bonds seem persisting in the altered structures. The inter-molecular hydrogen bonds can help to understand the phase separation in polymer/PC{sub 61}BM blends and may be responsible for the existence of liquid PC{sub 61}BM.

  4. PC2 Ovotransferrin: Characterization and Alternative Immunotherapeutic Activity

    PubMed Central

    Chiurciu, Constantin; Chiurciu, Viorica; Oporanu, Mariana; Pătrașcu, Ionel Victor; Mihai, Iuliana; Tablică, Mădălina

    2017-01-01

    Characterization and evaluation of immunotherapeutic potential of ovotransferrin PC2 (OTf PC2) were performed in this study. The ovoprotein was obtained from egg white from hens immunized with bacterial antigens, pathogenic for humans. For the negative control samples, OTf was extracted from eggs collected from Specific Pathogen-Free (SPF) hens and purified by affinity chromatography on Protein G-agarose column with two eluting peaks: I, representing ovalbumin, and II, ovotransferrin. The final apo-OTf form was reached by successive precipitation with ammonium sulfate and citric acid and the holo-OTf form by saturating the apo-form with FeCl3. Multiple OTf PC2 samples were analyzed through Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and, based on the molecular marker migration model, the ovotransferrin (76.5 kDa) and ovalbumin (45 kDa) were detected. The agglutination reaction exhibited statistically significant high specificity of the multiple OTf PC2, by reacting with the antigens used for hens' immunization. Following ELISA, it was established that OTf PC2 from hyperimmune eggs has specificity for all antigens; the antibody titer was high, indicating that OTf PC2 possesses immunological properties similar to immunoglobulin Y (Ig Y). This study suggests that OTf PC2 immunological activity may play a crucial role in the prevention and treatment of infections resistant to antibiotics and OTf PC2 can also act as a valuable nutraceutical. PMID:28408944

  5. Transforming PC Power Supplies into Smart Car Battery Conditioners

    ERIC Educational Resources Information Center

    Rodriguez-Ascariz, J. M.; Boquete-Vazquez, L.

    2011-01-01

    This paper describes a laboratory project consisting of a PC power supply modification into an intelligent car-battery conditioner with both wireless and wired networking capabilities. Adding a microcontroller to an average PC power supply transforms it into a flexible, intelligent device that can be configured and that is suitable to keep car…

  6. Chromosomal Distribution of PcG Proteins during Drosophila Development

    PubMed Central

    Nègre, Nicolas; Hennetin, Jérôme; Sun, Ling V; Lavrov, Sergey; Bellis, Michel; White, Kevin P

    2006-01-01

    Polycomb group (PcG) proteins are able to maintain the memory of silent transcriptional states of homeotic genes throughout development. In Drosophila, they form multimeric complexes that bind to specific DNA regulatory elements named PcG response elements (PREs). To date, few PREs have been identified and the chromosomal distribution of PcG proteins during development is unknown. We used chromatin immunoprecipitation (ChIP) with genomic tiling path microarrays to analyze the binding profile of the PcG proteins Polycomb (PC) and Polyhomeotic (PH) across 10 Mb of euchromatin. We also analyzed the distribution of GAGA factor (GAF), a sequence-specific DNA binding protein that is found at most previously identified PREs. Our data show that PC and PH often bind to clustered regions within large loci that encode transcription factors which play multiple roles in developmental patterning and in the regulation of cell proliferation. GAF co-localizes with PC and PH to a limited extent, suggesting that GAF is not a necessary component of chromatin at PREs. Finally, the chromosome-association profile of PC and PH changes during development, suggesting that the function of these proteins in the regulation of some of their target genes might be more dynamic than previously anticipated. PMID:16613483

  7. Transforming PC Power Supplies into Smart Car Battery Conditioners

    ERIC Educational Resources Information Center

    Rodriguez-Ascariz, J. M.; Boquete-Vazquez, L.

    2011-01-01

    This paper describes a laboratory project consisting of a PC power supply modification into an intelligent car-battery conditioner with both wireless and wired networking capabilities. Adding a microcontroller to an average PC power supply transforms it into a flexible, intelligent device that can be configured and that is suitable to keep car…

  8. Attention selection, distractor suppression and N2pc.

    PubMed

    Mazza, Veronica; Turatto, Massimo; Caramazza, Alfonso

    2009-01-01

    N2pc is generally interpreted as the electrocortical correlate of the distractor-suppression mechanisms through which attention selection takes place in humans. Here, we present data that challenge this common N2pc interpretation. In Experiment 1, multiple distractors induced greater N2pc amplitudes even when they facilitated target identification, despite the suppression account of the N2pc predicted the contrary; in Experiment 2, spatial proximity between target and distractors did not affect the N2pc amplitude, despite resulting in more interference in response times; in Experiment 3, heterogeneous distractors delayed response times but did not elicit a greater N2pc relative to homogeneous distractors again in contrast with what would have predicted the suppression hypothesis. These results do not support the notion that the N2pc unequivocally mirrors distractor-suppression processes. We propose that the N2pc indexes mechanisms involved in identifying and localizing relevant stimuli in the scene through enhancement of their features and not suppression of distractors.

  9. PrPC from stem cells to cancer

    PubMed Central

    Martin-Lannerée, Séverine; Hirsch, Théo Z.; Hernandez-Rapp, Julia; Halliez, Sophie; Vilotte, Jean-Luc; Launay, Jean-Marie; Mouillet-Richard, Sophie

    2014-01-01

    The cellular prion protein PrPC was initially discovered as the normal counterpart of the pathological scrapie prion protein PrPSc, the main component of the infectious agent of Transmissible Spongiform Encephalopathies. While clues as to the physiological function of this ubiquitous protein were greatly anticipated from the development of knockout animals, PrP-null mice turned out to be viable and to develop without major phenotypic abnormalities. Notwithstanding, the discovery that hematopoietic stem cells from PrP-null mice have impaired long-term repopulating potential has set the stage for investigating into the role of PrPC in stem cell biology. A wealth of data have now exemplified that PrPC is expressed in distinct types of stem cells and regulates their self-renewal as well as their differentiation potential. A role for PrPC in the fate restriction of embryonic stem cells has further been proposed. Paralleling these observations, an overexpression of PrPC has been documented in various types of tumors. In line with the contribution of PrPC to stemness and to the proliferation of cancer cells, PrPC was recently found to be enriched in subpopulations of tumor-initiating cells. In the present review, we summarize the current knowledge of the role played by PrPC in stem cell biology and discuss how the subversion of its function may contribute to cancer progression. PMID:25364760

  10. A PC based computerized maintenance system

    SciTech Connect

    Pruett, D.P.; Walker, G.D.; Imel, G.R.

    1990-03-01

    The present regulatory climate in the research reactor community has made an easily manageable and auditable maintenance system a necessity. We at NRAD have developed a computer-based system that is easy to implement and use, meets all our regulatory and reporting requirements, and is extremely useful to us in our daily operations. The system, developed at the NRAD reactor facility at Argonne National Laboratory in Idaho Falls, Idaho, uses DBASE-III coupled with C language routines, written for specific purposes. It is a menu-driven system that can be mastered in a short period of time and maintained with only a few hours of computer operation per month. It uses three computer processes: job scheduling, file updating, and report preparation, to produce schedules, work orders, and miscellaneous report forms. The heart of the system is an IBM PC with a 10 MB hard disk, providing adequate data storage capacity for a facility the size of NRAD. The computer is totally dedicated to the maintenance system, thus guarding against inadvertent loss of, or damage to, data files. Computer operator training time is minimized by the menu driven program. Multiple operators can share the computer operation responsibilities, and maintain the system with only 12 to 16 hours of computer operation per month. The system is adaptable to almost any facility, and can be altered and expanded to satisfy changing requirements. 7 figs.

  11. Pc-Based Floating Point Imaging Workstation

    NASA Astrophysics Data System (ADS)

    Guzak, Chris J.; Pier, Richard M.; Chinn, Patty; Kim, Yongmin

    1989-07-01

    The medical, military, scientific and industrial communities have come to rely on imaging and computer graphics for solutions to many types of problems. Systems based on imaging technology are used to acquire and process images, and analyze and extract data from images that would otherwise be of little use. Images can be transformed and enhanced to reveal detail and meaning that would go undetected without imaging techniques. The success of imaging has increased the demand for faster and less expensive imaging systems and as these systems become available, more and more applications are discovered and more demands are made. From the designer's perspective the challenge to meet these demands forces him to attack the problem of imaging from a different perspective. The computing demands of imaging algorithms must be balanced against the desire for affordability and flexibility. Systems must be flexible and easy to use, ready for current applications but at the same time anticipating new, unthought of uses. Here at the University of Washington Image Processing Systems Lab (IPSL) we are focusing our attention on imaging and graphics systems that implement imaging algorithms for use in an interactive environment. We have developed a PC-based imaging workstation with the goal to provide powerful and flexible, floating point processing capabilities, along with graphics functions in an affordable package suitable for diverse environments and many applications.

  12. Physical Properties of PC-PMMA Multilayers

    NASA Astrophysics Data System (ADS)

    Rahman, Arifur; Baer, Eric; Chipara, Alin Cristian; Vajtai, Robert; Ajayan, Pullickel M.; Hinthorne, James; Elamin, Ibrahim; Chipara, Mircea; Eric Baer Collaboration; Pullickel Ajayan Collaboration; Mircea Chipara Collaboration

    2015-03-01

    Multilayers of polycarbonate (PC) and polymethylmethacrylate (PMMA) have been obtained by the layer multiplying coextrusion method. Each sample (1024 layers, of equal thickness, with individual thickness between 10 and 200 nm) has been investigated at room temperature by Wide Angle X-Ray Scattering (WAXS) using a Bruker Discovery 8 spectrometer (Cu K α radiation), Raman spectroscopy (Bruker Senterra confocal Raman spectrometer operating at 785 nm), FTIR spectroscopy (Tensor 27 Bruker), and UV-Vis spectroscopy. Further details about the glass transition temperature in these samples have been obtained by Dynamical Mechanical Analysis, DMA, (TA Instruments Q800) at various frequencies in the range 1 to 100 Hz. Isothermal Differential Scanning Calorimetry, DSC, (TA Instruments Q200) was used to investigate the effect of the thickness of the polymeric film on the crystallization processes. Non-isothermal DSC measurements aimed at the identification and location of the main phase transitions (glass, crystallization, and melting) occurring in these multilayers. The effects of confinement on the phase transitions occurring in these multilayers are discussed in detail.

  13. Monte Carlo tests of the ELIPGRID-PC algorithm

    SciTech Connect

    Davidson, J.R.

    1995-04-01

    The standard tool for calculating the probability of detecting pockets of contamination called hot spots has been the ELIPGRID computer code of Singer and Wickman. The ELIPGRID-PC program has recently made this algorithm available for an IBM{reg_sign} PC. However, no known independent validation of the ELIPGRID algorithm exists. This document describes a Monte Carlo simulation-based validation of a modified version of the ELIPGRID-PC code. The modified ELIPGRID-PC code is shown to match Monte Carlo-calculated hot-spot detection probabilities to within {plus_minus}0.5% for 319 out of 320 test cases. The one exception, a very thin elliptical hot spot located within a rectangular sampling grid, differed from the Monte Carlo-calculated probability by about 1%. These results provide confidence in the ability of the modified ELIPGRID-PC code to accurately predict hot-spot detection probabilities within an acceptable range of error.

  14. Changes in temperature preferences and energy homeostasis in dystroglycan mutants.

    PubMed

    Takeuchi, Ken-Ichi; Nakano, Yoshiro; Kato, Utako; Kaneda, Mizuho; Aizu, Masako; Awano, Wakae; Yonemura, Shigenobu; Kiyonaka, Shigeki; Mori, Yasuo; Yamamoto, Daisuke; Umeda, Masato

    2009-03-27

    Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.

  15. Auditory development in progressive motor neuronopathy mouse mutants.

    PubMed

    Volkenstein, Stefan; Brors, Dominik; Hansen, Stefan; Berend, Achim; Mlynski, Robert; Aletsee, Christoph; Dazert, Stefan

    2009-11-06

    The present study was performed to elucidate the hearing development in the progressive motor neuronopathy (pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance.

  16. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    PubMed Central

    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

  17. Possible generation mechanisms for Pc1 pearl structures in the ionosphere based on 6 years of ground observations in Canada, Russia, and Japan

    NASA Astrophysics Data System (ADS)

    Jun, Chae-Woo; Shiokawa, Kazuo; Connors, Martin; Schofield, Ian; Poddelsky, Igor; Shevtsov, Boris

    2016-05-01

    We investigate pearl structures (amplitude modulations) of Pc1 pulsations simultaneously observed at Athabasca (ATH, 54.7°N, 246.7°E, L = 4.3) in Canada, Magadan (MGD, 60.1°N, 150.7°E, L = 2.6) in Russia, and Moshiri (MOS, 44.4°N, 142.3°E, L = 1.5) in Japan. From 6 years of ground observations, from 2008 to 2013, we selected 84 Pc1 events observed simultaneously at the longitudinally separated stations (ATH and MGD) and 370 events observed at the latitudinally separated stations (MGD and MOS), all with high coherence (>0.7) of Pc1 waveforms. We calculated the cross-correlation coefficient (similarity: r) for the Pc1 pearl structures and found that more than half of the events in both pairs had low similarity (r < 0.7), indicating that most Pc1 waves exhibit different pearl structures at different stations. We found that high-similarity Pc1 pearl structures (r > 0.7) at the longitudinally separated stations are concentrated from 6 to 15 UT when both stations are in the nighttime. The similarity of Pc1 pearl structures tends to show a negative correlation with the standard deviation of the polarization angle in both pairs. The observed repetition period of Pc1 pearl structures has a clear positive correlation with the repetition period estimated from Pc1 bandwidth by assuming beating of different frequencies. From these results, we suggest that ionospheric beating effect could be a dominant process for the generation of Pc1 pearl structures. Beating processes in the ionosphere with a spatially distributed ionospheric source can cause the different shapes of Pc1 pearl structures at different observation points during ionospheric duct propagation.

  18. PC4/Tis7/IFRD1 Stimulates Skeletal Muscle Regeneration and Is Involved in Myoblast Differentiation as a Regulator of MyoD and NF-κB*

    PubMed Central

    Micheli, Laura; Leonardi, Luca; Conti, Filippo; Maresca, Giovanna; Colazingari, Sandra; Mattei, Elisabetta; Lira, Sergio A.; Farioli-Vecchioli, Stefano; Caruso, Maurizia; Tirone, Felice

    2011-01-01

    In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis. PMID:21127072

  19. Proposed Nomenclature for Mutants of Adenoviruses

    PubMed Central

    Ginsberg, Harold S.; Williams, James F.; Doerfler, Walter H.; Shimojo, Hiroto

    1973-01-01

    In accord with the nomenclature proposed for mutants of simian virus 40 the same rules, with minor modifications, are recommended for naming mutants of adenoviruses. It is further suggested that these rules, which pertain to a system of classification based primarily upon complementation analysis, also be applied to mutants of other DNA-containing ani