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Sample records for genes dual oxidase

  1. Expression of the genes dual oxidase 2, lipocalin 2 and regenerating islet-derived 1 alpha in Crohn's disease

    DEFF Research Database (Denmark)

    Csillag, C.; Nielsen, O.H.; Vainer, Ben

    2007-01-01

    colonoscopically from 33 CD patients and from 17 control subjects. All controls and 10 CD patients were medication-free at the time of colonoscopy. The Human Genome U133 Plus 2.0 GeneChip Array was used for gene profiling. Hybridization data were analysed with dChip software. Results were confirmed by real......, fold change 3.9), codes for a mitogenic protein; this could not be confirmed by RT-PCR. Medication-free patients had no differentially expressed genes as compared with controls. Immunohistochemistry indicated that these proteins were produced by epithelial cells (REG1A, LCN2) and leucocytes (DUOX2...... and LCN2). CONCLUSIONS: As compared with controls, non-inflamed colonic mucosal cells contain two up-regulated genes related to the innate immune system. Up-regulation of these genes, known to be induced by microorganisms, suggests either increased microflora antigenicity or an altered function in mucosal...

  2. Dual functions of transcription factors, transforming growth factor-beta-inducible early gene (TIEG)2 and Sp3, are mediated by CACCC element and Sp1 sites of human monoamine oxidase (MAO) B gene.

    Science.gov (United States)

    Ou, Xiao-Ming; Chen, Kevin; Shih, Jean C

    2004-05-14

    Monoamine oxidases (MAO) A and B catalyze the oxidative deamination of many biogenic and dietary amines. Abnormal expression of MAO has been implicated in several psychiatric and neurodegenerative disorders. Human MAO B core promoter (-246 to -99 region) consists of CACCC element flanked by two clusters of overlapping Sp1 sites. Here, we show that cotransfection with transforming growth factor (TGF)-beta-inducible early gene (TIEG)2 increased MAO B gene expression at promoter, mRNA, protein, and catalytic activity levels in both SH-SY5Y and HepG2 cells. Mutation of the CACCC element increased the MAO B promoter activity, and cotransfection with TIEG2 further increased the promoter activity, suggesting that CACCC was a repressor element. This increase was reduced when the proximal Sp1 overlapping sites was mutated. Similar interactions were found with Sp3. These results showed that TIEG2 and Sp3 were repressors at the CACCC element but were activators at proximal Sp1 overlapping sites of MAO B. Gel-shift and chromatin immunoprecipitation assays showed that TIEG2 and Sp3 bound directly to CACCC element and the proximal Sp1 sites in both synthetic oligonucleotides and natural MAO B core promoter. TIEG2 had a higher affinity to Sp1 sites than CACCC element, whereas Sp3 had an equal affinity to both elements. Thus, TIEG2 was an activator, but Sp3 had no effect on MAO B gene expression. This study provides new insights into MAO B gene expression and illustrates the complexity of gene regulation.

  3. Ovarian dual oxidase (Duox) activity is essential for insect eggshell hardening and waterproofing.

    Science.gov (United States)

    Dias, Felipe A; Gandara, Ana Caroline P; Queiroz-Barros, Fernanda G; Oliveira, Raquel L L; Sorgine, Marcos H F; Braz, Glória R C; Oliveira, Pedro L

    2013-12-06

    In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.

  4. Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

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    Coppée Jean-Yves

    2010-02-01

    Full Text Available Abstract Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III to As(V as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III. To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54 of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III in this microorganism.

  5. Molecular evolution of the polyamine oxidase gene family in Metazoa

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    Polticelli Fabio

    2012-06-01

    Full Text Available Abstract Background Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO and acetylpolyamine oxidase (APAO, specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO, it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. Results We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported

  6. The pea gene NA encodes ent-kaurenoic acid oxidase.

    Science.gov (United States)

    Davidson, Sandra E; Elliott, Robert C; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2003-01-01

    The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

  7. Reactive oxygen species regulate the levels of dual oxidase (Duox1-2 in human neuroblastoma cells.

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    Simona Damiano

    Full Text Available Dual Oxidases (DUOX 1 and 2 are efficiently expressed in thyroid, gut, lung and immune system. The function and the regulation of these enzymes in mammals are still largely unknown. We report here that DUOX 1 and 2 are expressed in human neuroblastoma SK-N-BE cells as well as in a human oligodendrocyte cell line (MO3-13 and in rat brain and they are induced by platelet derived growth factor (PDGF. The levels of DUOX 1 and 2 proteins and mRNAs are induced by reactive oxygen species (ROS produced by the membrane NADPH oxidase. As to the mechanism, we find that PDGF stimulates membrane NADPH oxidase to produce ROS, which stabilize DUOX1 and 2 mRNAs and increases the levels of the proteins. Silencing of gp91(phox (NOX2, or of the other membrane subunit of NADPH oxidase, p22(phox, blocks PDGF induction of DUOX1 and 2. These data unravel a novel mechanism of regulation of DUOX enzymes by ROS and identify a circuitry linking NADPH oxidase activity to DUOX1 and 2 levels in neuroblastoma cells.

  8. Hypoxia-Response Element (HRE)–Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium

    OpenAIRE

    Gao, Song; Zhou, Jing; Zhao, Yinzhi; Toselli, Paul; Li, Wande

    2012-01-01

    Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Compute...

  9. Isolation and analysis of the genes for cytochrome c oxidase in Paracoccus denitrificans

    OpenAIRE

    Raitio, Mirja; Jalli, Tuulikki; Saraste, Matti

    1987-01-01

    Synthetic oligonucleotide probes were used to clone two loci from the chromosomal DNA of Paracoccus denitrificans that contain the genes for cytochrome c oxidase (cytochrome aa3). One locus seems to contain four or five genes probably forming an operon. Two of these code for the oxidase subunits II and III. Three open reading frames are found between the COII and COIII genes. The other locus codes for the subunit I. A short open reading frame is found upstream of this gene. All three subunits...

  10. Hippocampal mitochondrial cytochrome C oxidase activity and gene expression in a rat model of chronic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Qing Zhao; Yingli Zhang; Mingming Zhao; Yu Wang; Ming Ma; Xinquan Gu; Xia Cao

    2011-01-01

    The present study established a rat model of chronic cerebral ischemia using bilateral common carotid artery permanent ligation to analyze cytochrome C oxidase activity and mRNA expression in hippocampal mitochondria.Results showed significantly decreased cytochrome C oxidase activity and cytochrome C oxidase II mRNA expression with prolonged ischemia time.Further analysis revealed five mitochondrial cytochrome C oxidase II gene mutations, two newly generated mutations, and four absent mutational sites at 1 month after cerebral ischemia, as well as three mitochondrial cytochrome C oxidase III gene mutations, including two newly generating mutations, and one disappeared mutational site at 1 month after cerebral ischemia.Results demonstrated that decreased cytochrome C oxidase gene expression and mutations, as well as decreased cytochrome C oxidase activity, resulting in energy dysmetabolism, which has been shown to be involved in the pathological process of ischemic brain injury.

  11. Tetraspanin is required for generation of reactive oxygen species by the dual oxidase system in Caenorhabditis elegans.

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    Hiroki Moribe

    2012-09-01

    Full Text Available Reactive oxygen species (ROS are toxic but essential molecules responsible for host defense and cellular signaling. Conserved NADPH oxidase (NOX family enzymes direct the regulated production of ROS. Hydrogen peroxide (H(2O(2 generated by dual oxidases (DUOXs, a member of the NOX family, is crucial for innate mucosal immunity. In addition, H(2O(2 is required for cellular signaling mediated by protein modifications, such as the thyroid hormone biosynthetic pathway in mammals. In contrast to other NOX isozymes, the regulatory mechanisms of DUOX activity are less understood. Using Caenorhabditis elegans as a model, we demonstrate that the tetraspanin protein is required for induction of the DUOX signaling pathway in conjunction with the dual oxidase maturation factor (DUOXA. In the current study, we show that genetic mutation of DUOX (bli-3, DUOXA (doxa-1, and peroxidase (mlt-7 in C. elegans causes the same defects as a tetraspanin tsp-15 mutant, represented by exoskeletal deficiencies due to the failure of tyrosine cross-linking of collagen. The deficiency in the tsp-15 mutant was restored by co-expression of bli-3 and doxa-1, indicating the involvement of tsp-15 in the generation of ROS. H(2O(2 generation by BLI-3 was completely dependent on TSP-15 when reconstituted in mammalian cells. We also demonstrated that TSP-15, BLI-3, and DOXA-1 form complexes in vitro and in vivo. Cell-fusion-based analysis suggested that association with TSP-15 at the cell surface is crucial for BLI-3 activation to release H(2O(2. This study provides the first evidence for an essential role of tetraspanin in ROS generation.

  12. Molecular cloning and expression analysis of multiple polyphenol oxidase genes in developing wheat (Triticum aestivum) kernels

    Science.gov (United States)

    Polyphenol oxidase (PPO, EC 1.10.31) is a major cause of discoloring in raw dough containing wheat flour. Minimization of PPO activity has proven difficult because bread wheat is genetically complex, composed of the genomes of three grass species. The PPO-A1 and PPO-D1 genes, on chromosomes 2A and...

  13. Monoamine Oxidase a Promoter Gene Associated with Problem Behavior in Adults with Intellectual/Developmental Disabilities

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    May, Michael E.; Srour, Ali; Hedges, Lora K.; Lightfoot, David A.; Phillips, John A., III; Blakely, Randy D.; Kennedy, Craig H.

    2009-01-01

    A functional polymorphism in the promoter of the gene encoding monoamine oxidase A has been associated with problem behavior in various populations. We examined the association of MAOA alleles in adult males with intellectual/developmental disabilities with and without established histories of problem behavior. These data were compared with a…

  14. The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities.

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    Marelja, Zvonimir; Dambowsky, Miriam; Bolis, Marco; Georgiou, Marina L; Garattini, Enrico; Missirlis, Fanis; Leimkühler, Silke

    2014-06-15

    In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po(lpo)) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po(lpo)-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po(lpo) allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.

  15. Biological Effects of Potato Plants Transformation with Glucose Oxidase Gene and their Resistance to Hyperthermia

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    O.I. Grabelnych

    2017-02-01

    Full Text Available It is known that regulation of plant tolerance to adverse environmental factors is connected with short term increase of the concentration of endogenous reactive oxygen species (ROS, which are signalling molecules for the induction of protective mechanisms. Introduction and expression of heterologous gox gene, which encodes glucose oxidase enzyme in plant genome, induce constantly higher content of hydrogen peroxide in plant tissues. It is not known how the introduction of native or modified gox gene affects the plant resistance to high-temperature stress, one of the most commonly used model for the study of stress response and thermal tolerance. In this study, we investigated biological effects of transformation and evaluated the resistance to temperature stress of potato plants with altered levels of glucose oxidase expression. Transformation of potato plants by gox gene led to the more early coming out from tuber dormancy of transformed plants and slower growth rate. Transformants containing the glucose oxidase gene were more sensitive to lethal thermal shock (50 °C, 90 min than the transformant with the empty vector (pBI or untransformed plants (CK. Pre-heating of plants at 37 °C significantly weakened the damaging effect of lethal thermal shock. This attenuation was more significant in the non-transformed plants.

  16. Disruption of the CYTOCHROME C OXIDASE DEFICIENT1 gene leads to cytochrome c oxidase depletion and reorchestrated respiratory metabolism in Arabidopsis.

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    Dahan, Jennifer; Tcherkez, Guillaume; Macherel, David; Benamar, Abdelilah; Belcram, Katia; Quadrado, Martine; Arnal, Nadège; Mireau, Hakim

    2014-12-01

    Cytochrome c oxidase is the last respiratory complex of the electron transfer chain in mitochondria and is responsible for transferring electrons to oxygen, the final acceptor, in the classical respiratory pathway. The essentiality of this step makes it that depletion in complex IV leads to lethality, thereby impeding studies on complex IV assembly and respiration plasticity in plants. Here, we characterized Arabidopsis (Arabidopsis thaliana) embryo-lethal mutant lines impaired in the expression of the CYTOCHROME C OXIDASE DEFICIENT1 (COD1) gene, which encodes a mitochondria-localized PentatricoPeptide Repeat protein. Although unable to germinate under usual conditions, cod1 homozygous embryos could be rescued from immature seeds and developed in vitro into slow-growing bush-like plantlets devoid of a root system. cod1 mutants were defective in C-to-U editing events in cytochrome oxidase subunit2 and NADH dehydrogenase subunit4 transcripts, encoding subunits of respiratory complex IV and I, respectively, and consequently lacked cytochrome c oxidase activity. We further show that respiratory oxygen consumption by cod1 plantlets is exclusively associated with alternative oxidase activity and that alternative NADH dehydrogenases are also up-regulated in these plants. The metabolomics pattern of cod1 mutants was also deeply altered, suggesting that alternative metabolic pathways compensated for the probable resulting restriction in NADH oxidation. Being the first complex IV-deficient mutants described in higher plants, cod1 lines should be instrumental to future studies on respiration homeostasis.

  17. Divergence and adaptive evolution of the gibberellin oxidase genes in plants.

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    Huang, Yuan; Wang, Xi; Ge, Song; Rao, Guang-Yuan

    2015-09-29

    The important phytohormone gibberellins (GAs) play key roles in various developmental processes. GA oxidases (GAoxs) are critical enzymes in GA synthesis pathway, but their classification, evolutionary history and the forces driving the evolution of plant GAox genes remain poorly understood. This study provides the first large-scale evolutionary analysis of GAox genes in plants by using an extensive whole-genome dataset of 41 species, representing green algae, bryophytes, pteridophyte, and seed plants. We defined eight subfamilies under the GAox family, namely C19-GA2ox, C20-GA2ox, GA20ox,GA3ox, GAox-A, GAox-B, GAox-C and GAox-D. Of these, subfamilies GAox-A, GAox-B, GAox-C and GAox-D are described for the first time. On the basis of phylogenetic analyses and characteristic motifs of GAox genes, we demonstrated a rapid expansion and functional divergence of the GAox genes during the diversification of land plants. We also detected the subfamily-specific motifs and potential sites of some GAox genes, which might have evolved under positive selection. GAox genes originated very early-before the divergence of bryophytes and the vascular plants and the diversification of GAox genes is associated with the functional divergence and could be driven by positive selection. Our study not only provides information on the classification of GAox genes, but also facilitates the further functional characterization and analysis of GA oxidases.

  18. Identification and analysis of the Shewanella oneidensis major oxygen-independent coproporphyrinogen III oxidase gene.

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    Al-Sheboul, Suhaila; Saffarini, Daad

    2011-12-01

    Shewanella oneidenesis MR-1 is a facultative anaerobe that can use a large number of electron acceptors including metal oxides. During anaerobic respiration, S. oneidensis MR-1 synthesizes a large number of c cytochromes that give the organism its characteristic orange color. Using a modified mariner transposon, a number of S. oneidensis mutants deficient in anaerobic respiration were generated. One mutant, BG163, exhibited reduced pigmentation and was deficient in c cytochromes normally synthesized under anaerobic condition. The deficiencies in BG163 were due to insertional inactivation of hemN1, which exhibits a high degree of similarity to genes encoding anaerobic coproporphyrinogen III oxidases that are involved in heme biosynthesis. The ability of BG163 to synthesize c cytochromes under anaerobic conditions, and to grow anaerobically with different electron acceptors was restored by the introduction of hemN1 on a plasmid. Complementation of the mutant was also achieved by the addition of hemin to the growth medium. The genome sequence of S. oneidensis contains three putative anaerobic coproporphyrinogen III oxidase genes. The protein encoded by hemN1 appears to be the major enzyme that is involved in anaerobic heme synthesis of S. oneidensis. The other two putative anaerobic coproporphyrinogen III oxidase genes may play a minor role in this process.

  19. Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and radical scavengers.

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    Hofmann, Emily; Webster, Jonathan; Do, Thuy; Kline, Reid; Snider, Lindsey; Hauser, Quintin; Higginbottom, Grace; Campbell, Austin; Ma, Lili; Paula, Stefan

    2016-02-15

    In this study, we evaluated the abilities of a series of chalcones to inhibit the activity of the enzyme xanthine oxidase (XO) and to scavenge radicals. 20 mono- and polyhydroxylated chalcone derivatives were synthesized by Claisen-Schmidt condensation reactions and then tested for inhibitory potency against XO, a known generator of reactive oxygen species (ROS). In parallel, the ability of the synthesized chalcones to scavenge a stable radical was determined. Structure-activity relationship analysis in conjunction with molecular docking indicated that the most active XO inhibitors carried a minimum of three hydroxyl groups. Moreover, the most effective radical scavengers had two neighboring hydroxyl groups on at least one of the two phenyl rings. Since it has been proposed previously that XO inhibition and radical scavenging could be useful properties for reduction of ROS-levels in tissue, we determined the chalcones' effects to rescue neurons subjected to ROS-induced stress created by the addition of β-amyloid peptide. Best protection was provided by chalcones that combined good inhibitory potency with high radical scavenging ability in a single molecule, an observation that points to a potential therapeutic value of this compound class.

  20. Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis.

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    Singtripop, Tippawan; Saeangsakda, Manasawan; Tatun, Nujira; Kaneko, Yu; Sakurai, Sho

    2007-09-01

    The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.

  1. Cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp.

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    Hamed Esmaeil Lashgarian

    2016-10-01

    Full Text Available Cholesterol oxidase (CHO is one of the valuable enzymes that play an important role in: measurement of serum cholesterol, food industry as a biocatalyst and agriculture as a biological larvicide. This enzyme was produced by several bacterial strains. Wild type enzyme produced by Rhodococcus sp. secret two forms of CHO enzyme: extra cellular and membrane bound type which its amount is low and unstable. The goal of the study was cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp. CHO gene was isolated from native bacteria and cloned into pET23a. In the next step, the construct was expressed in E.coli BL21 and induced by different concentration of IPTG ranges from 0.1 - 0.9 mM. This gene contains 1642 bp and encodes a protein consists of 533 amino acids. It has about 96 % homology with CHO gene isolated from Rhodococcus equi. The high expression was obtained in 0.5 mM concentration of IPTG after 4 hour induction. This recombinant enzyme had a molecular weight of 55 kDa, that secretion of intra cellular type is much more than extracellular form. The optimum pH and temperature conditions for the recombinant enzyme were 7.5 and 45°C, respectively. CHO enzyme obtained from Rhodococcus sp. is a cheap enzyme with medical and industrial applications that can be produced easily and purified in large scale with simple methods.

  2. Molecular characterization and expression pattern of tobacco (Nicotiana tabacum ascorbate oxidase gene

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    Zhihua Li

    2016-11-01

    Full Text Available The complete coding sequence of one tobacco (Nicotiana tabacum gene, ascorbate oxidase (AO, was isolated by the application of reverse transcription-polymerase chain reaction. The tobacco AO gene consists of a 1722-bp open reading frame and encodes a protein of 573 amino acids. Sequence comparison analysis revealed that the tobacco AO protein shares high homology with the AO proteins of Lycopersicon esculentum (89%, Populus trichocarpa (75%, soybean (74%, castor bean (73% and peach (73%. The prediction of transmembrane helices showed that tobacco AO might be a transmembrane protein. The expression profile was studied and the results indicated that the tobacco AO gene was diversely expressed in different tobacco tissues, including leaves, stem, roots and flowers. Our experiment laid the grounds for further research on this tobacco gene.

  3. Genetic differentiation of the mitochondrial cytochrome oxidase C subunit I gene in genus Paramecium (Protista, Ciliophora.

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    Yan Zhao

    Full Text Available BACKGROUND: The mitochondrial cytochrome c oxidase subunit I (COI gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure. CONCLUSIONS: Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.

  4. Genetic differentiation of the mitochondrial cytochrome oxidase C subunit I gene in genus Paramecium (Protista, Ciliophora).

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    Zhao, Yan; Gentekaki, Eleni; Yi, Zhenzhen; Lin, Xiaofeng

    2013-01-01

    The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses. We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure. Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.

  5. A Phaseolus vulgaris NADPH oxidase gene is required for root infection by Rhizobia.

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    Montiel, Jesús; Nava, Noreide; Cárdenas, Luis; Sánchez-López, Rosana; Arthikala, Manoj-Kumar; Santana, Olivia; Sánchez, Federico; Quinto, Carmen

    2012-10-01

    Plant NADPH oxidases [respiratory burst oxidase homologs (RBOHs)] have emerged as key players in the regulation of plant-pathogen interactions. Nonetheless, their role in mutualistic associations, such as the rhizobia-legume symbiosis, is poorly understood. In this work, nine members of the Phaseolus vulgaris Rboh gene family were identified. The transcript of one of these, PvRbohB, accumulated abundantly in shoots, roots and nodules. PvRbohB promoter activity was detected in meristematic regions of P. vulgaris roots, as well as during infection thread (IT) progression and nodule development. RNA interference (RNAi)-mediated PvRbohB down-regulation in transgenic roots reduced reactive oxygen species (ROS) production and lateral root density, and greatly impaired nodulation. Microscopy analysis revealed that progression of the ITs was impeded at the base of root hairs in PvRbohB-RNAi roots. Furthermore, the few nodules that formed in PvRbohB-down-regulated roots displayed abnormally wide ITs and reduced nitrogen fixation. These findings indicate that this common bean NADPH oxidase is crucial for successful rhizobial colonization and probably maintains proper IT growth and shape.

  6. Dual oxidase 2 is essential for house dust mite-induced pro-inflammatory cytokine production in human keratinocytes.

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    Ko, Eunbi; Choi, Hyun; Park, Kkot-Nara; Park, Ju-Yearl; Lee, Tae Ryong; Shin, Dong Wook; Bae, Yun Soo

    2015-12-01

    House dust mites (HDMs) are known to trigger chronic inflammation through Toll-like receptors (TLRs) and their signalling cascades. In this study, we found that TLR2 ligation by HDMs induced the activation of dual oxidase 2 (Duox2) and nuclear factor-κB (NF-κB), leading to the production of pro-inflammatory cytokines in human keratinocytes. Stimulation of human keratinocytes with HDMs resulted in increases in interleukin-8 (IL-8) and chemokine (C-C motif) ligand 20 (CCL20) levels. However, pro-inflammatory cytokine production was abolished in keratinocytes transfected with TLR2 siRNA, indicating that HDM-induced cytokine production was mediated via TLR2 signalling. We also examined the function of Duox1/2 isozymes, which are primarily expressed in keratinocytes, in HDM-mediated pro-inflammatory cytokine production. Human keratinocytes transfected with control siRNA or Duox1 siRNA showed no inhibition of IL-8 or CCL20 production in response to HDMs, whereas the silencing of Duox2 expression resulted in a failure to induce cytokine production. Moreover, the phosphorylation and nuclear localization of RelA/p65, a component of NF-κB, were induced by HDMs in human keratinocytes. Transfection of human keratinocytes with TLR2 siRNA or Duox2 siRNA resulted in the complete abolishment of RelA/p65 nuclear localization in response to HDMs. Taken together, these results indicate that the HDM-dependent TLR2-Duox2 signalling axis indeed promotes NF-κB activation, which induces IL-8 and CCL20 production and mediates epidermal keratinocyte inflammation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Cloning and characterization of an up-regulated GA 20-oxidase gene in hybrid maize

    Institute of Scientific and Technical Information of China (English)

    Jinkun Du; Yingyin Yao; Zhongfu Ni; Qixin Sun

    2009-01-01

    Previous studies revealed that GA content and metabolism are positively correlated with a faster shoot growth rate of hybrid, and recently, genes participating in both GA biosynthesis and GA response pathways were also found to be differentially expressed between wheat hybrid and its parental inbreds. In this study, an up-regulated GA 20-oxidase gene in a maize hybrid, designated ZmGA20, was cloned. ZmGA20 contains an open reading frame (ORF) encoding 391 amino acid residues. BLASTX searches in GenBank revealed that the ZmGA20 is homologous to the sequences of GA20ox proteins from different species, and analysis also indicated that ZmGA20 had typical features of GA 20-oxidase proteins, including a "LPWKET" sequence. Semi-quantitative RT-PCR analysis showed that ZmGA20 was expressed in different tissues and/or organs. The expression level of ZmGA20 in the hybrid was higher than that in two parents (in roots, leaves, stems and embryo, and ears). The abundance of ZmGA20 transcript was equal to that of the highly expressed parents, which provided molecular evidence for the observed GA content heterosis in maize hybrids.

  8. Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

    Science.gov (United States)

    Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

    2015-12-01

    Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene.

  9. Mutations affecting the expression of the MOX gene encoding peroxisomal methanol oxidase in Hansenula polymorpha.

    Science.gov (United States)

    Vallini, V; Berardi, E; Strabbioli, R

    2000-11-01

    In this study, aimed at identifying genetic factors acting positively upon the MOX gene, we report the isolation and characterisation of several methanol utilisation-defective (Mut-) mutants of Hansenula polymorpha. These fall into 12 complementation groups, eight of which show significant reductions in alcohol (methanol) oxidase activity in methanol. Three of these groups, identifying the MUT3, MUT5 and MUT10 loci, exhibit extremely low levels of MOX promoter activity, not only in methanol medium, but also during growth in glycerol or methylamine. We suggest that these loci play a significant role in the derepression of the MOX gene expression. One of these genes (MUT10) also seems to be involved in the utilisation of carbon sources other than methanol, and it is apparent that the same gene plays some role in the biogenesis or in the enlargement of the peroxisome. Three other genes (MUT7, MUT8 and MUT9) appear to be involved in peroxisome biogenesis, whereas most other mutants harbour lesions that leave the peroxisome biogenesis and proliferation unaffected.

  10. The polyphenol oxidase gene family in land plants: Lineage-specific duplication and expansion

    Directory of Open Access Journals (Sweden)

    Tran Lan T

    2012-08-01

    Full Text Available Abstract Background Plant polyphenol oxidases (PPOs are enzymes that typically use molecular oxygen to oxidize ortho-diphenols to ortho-quinones. These commonly cause browning reactions following tissue damage, and may be important in plant defense. Some PPOs function as hydroxylases or in cross-linking reactions, but in most plants their physiological roles are not known. To better understand the importance of PPOs in the plant kingdom, we surveyed PPO gene families in 25 sequenced genomes from chlorophytes, bryophytes, lycophytes, and flowering plants. The PPO genes were then analyzed in silico for gene structure, phylogenetic relationships, and targeting signals. Results Many previously uncharacterized PPO genes were uncovered. The moss, Physcomitrella patens, contained 13 PPO genes and Selaginella moellendorffii (spike moss and Glycine max (soybean each had 11 genes. Populus trichocarpa (poplar contained a highly diversified gene family with 11 PPO genes, but several flowering plants had only a single PPO gene. By contrast, no PPO-like sequences were identified in several chlorophyte (green algae genomes or Arabidopsis (A. lyrata and A. thaliana. We found that many PPOs contained one or two introns often near the 3’ terminus. Furthermore, N-terminal amino acid sequence analysis using ChloroP and TargetP 1.1 predicted that several putative PPOs are synthesized via the secretory pathway, a unique finding as most PPOs are predicted to be chloroplast proteins. Phylogenetic reconstruction of these sequences revealed that large PPO gene repertoires in some species are mostly a consequence of independent bursts of gene duplication, while the lineage leading to Arabidopsis must have lost all PPO genes. Conclusion Our survey identified PPOs in gene families of varying sizes in all land plants except in the genus Arabidopsis. While we found variation in intron numbers and positions, overall PPO gene structure is congruent with the phylogenetic

  11. Gene-Gene-Environment Interactions of Serotonin Transporter, Monoamine Oxidase A and Childhood Maltreatment Predict Aggressive Behavior in Chinese Adolescents

    Science.gov (United States)

    Zhang, Yun; Ming, Qing-sen; Yi, Jin-yao; Wang, Xiang; Chai, Qiao-lian; Yao, Shu-qiao

    2017-01-01

    Gene-environment interactions that moderate aggressive behavior have been identified independently in the serotonin transporter (5-HTT) gene and monoamine oxidase A gene (MAOA). The aim of the present study was to investigate epistasis interactions between MAOA-variable number tandem repeat (VNTR), 5-HTTlinked polymorphism (LPR) and child abuse and the effects of these on aggressive tendencies in a group of otherwise healthy adolescents. A group of 546 Chinese male adolescents completed the Child Trauma Questionnaire and Youth self-report of the Child Behavior Checklist. Buccal cells were collected for DNA analysis. The effects of childhood abuse, MAOA-VNTR, 5-HTTLPR genotypes and their interactive gene-gene-environmental effects on aggressive behavior were analyzed using a linear regression model. The effect of child maltreatment was significant, and a three-way interaction among MAOA-VNTR, 5-HTTLPR and sexual abuse (SA) relating to aggressive behaviors was identified. Chinese male adolescents with high expression of the MAOA-VNTR allele and 5-HTTLPR “SS” genotype exhibited the highest aggression tendencies with an increase in SA during childhood. The findings reported support aggression being a complex behavior involving the synergistic effects of gene-gene-environment interactions. PMID:28203149

  12. Isolation and Characterization of Cucumber ACC Oxidase Gene and Its Upstream

    Institute of Scientific and Technical Information of China (English)

    CHEN Yin-hua; OUYANG Bo; LI Han-xia; ZHANG Jun-hong; YE Zhi-biao

    2005-01-01

    Ethylene has been implicated as a sex-determining hormone in cucumber. Its exogenous application increases femaleness,and gynoecious genotypes were reported to produce more ethylene. 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) is the key enzyme in ethylene biosynthesis. In this study, a 1 200 base pair (bp) candidate fragment was amplified from the cucumber genome with degenerated primers derived from the ACO amino acid consensus sequence among different plant species. The coding region and its upstream (1 155 bp) were obtained by vector-mediated inverse PCR. The novel gene was analyzed by bioinformatics tools. Four exons and three introns were identified in the coding sequence.The spliced length of mRNA was 933 nucleotides (nts) and it encoded 311 amino acids. Phylogenic analysis result of the new gene (CsACO4, GenBank accession number AY450356) was in accordance with the evolution relationship of genetics among various plant species. Northern blotting showed that the gene expressed among female flowers of gynoecious and monoecious genotypes, it could not express in other organs. This implied that the gene might be correlated with the female behavior positively. Further work is on the way to demonstrate the complexity of the relationship between the endogenous ethylene and the sex determination.

  13. Analysis of the cytochrome c oxidase subunit II (COX2) gene in giant panda, Ailuropoda melanoleuca.

    Science.gov (United States)

    Ling, S S; Zhu, Y; Lan, D; Li, D S; Pang, H Z; Wang, Y; Li, D Y; Wei, R P; Zhang, H M; Wang, C D; Hu, Y D

    2017-01-23

    The giant panda, Ailuropoda melanoleuca (Ursidae), has a unique bamboo-based diet; however, this low-energy intake has been sufficient to maintain the metabolic processes of this species since the fourth ice age. As mitochondria are the main sites for energy metabolism in animals, the protein-coding genes involved in mitochondrial respiratory chains, particularly cytochrome c oxidase subunit II (COX2), which is the rate-limiting enzyme in electron transfer, could play an important role in giant panda metabolism. Therefore, the present study aimed to isolate, sequence, and analyze the COX2 DNA from individuals kept at the Giant Panda Protection and Research Center, China, and compare these sequences with those of the other Ursidae family members. Multiple sequence alignment showed that the COX2 gene had three point mutations that defined three haplotypes, with 60% of the sequences corresponding to haplotype I. The neutrality tests revealed that the COX2 gene was conserved throughout evolution, and the maximum likelihood phylogenetic analysis, using homologous sequences from other Ursidae species, showed clustering of the COX2 sequences of giant pandas, suggesting that this gene evolved differently in them.

  14. Expression of ACC Oxidase Gene from Sugarcane Induced by Hormones and Environmental Force

    Institute of Scientific and Technical Information of China (English)

    WANG Ai-qin; YANG Li-tao; WANG Zi-zhang; WEI Yu-tuo; HE Long-fei; LI Yang-rui

    2005-01-01

    In the present study, a full-length cDNA encoding 1-aminocyclopropane-1-carboxylic acid oxidase gene has been cloned from sugarcane (named GZ-ACO). Two primers were designed for coding the ORF in the full-length cDNA of GZ-ACO gene from sugarcane. PCR amplification was performed with sugarcane DNA template, and a fragment of 1 104 bp (GZ34)was obtained. GZ34 was labeled with [α-32p] dCTP as the probe and used for hybridization after cloning and sequencing.Southern blotting analysis indicated that there were at least three other sequences, which weakly hybridized with the GZ34. Northern analysis showed that GZ34 was strongly induced by treatment with IAA, BA, ethephon, LiC1 and cold stress, respectively. As a contrast, the mRNA for ACO gene was at lower levels for both the light-grown and dark-grown plants without additional treatment. There were two transcripts in the dark-grown plants and three transcripts in the treatments with IAA, BA and cold stress, but there was only one transcript in ethephon treatment. It showed that GZ-ACO might be a gene connected with ethylene formation and take part in response to the induction of plant hormone and environmental stress.

  15. The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger

    NARCIS (Netherlands)

    Franken, A.C.W.; Werner, E.R.; Haas, H.; Lokman, B.C.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.; Weert, S. de; Punt, P.J.

    2013-01-01

    Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and

  16. The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger

    NARCIS (Netherlands)

    Franken, A.C.W.; Werner, E.R.; Haas, H.; Lokman, B.C.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.; Weert, S. de; Punt, P.J.

    2013-01-01

    Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and

  17. The role of Coproporphyrinogen III oxidase and Ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger

    NARCIS (Netherlands)

    P.J. Punt; C.A.M.J.J. van den Hondel; H. Haas; A.C.W. Franken; Christien Lokman; E.R. Werner; S. de Weert; A.F.J. Ram

    2013-01-01

    Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and

  18. Collection of mitochondrial cytochrome oxidase I gene sequences from Rhipicephalus ticks from various geographic locations around the world

    Science.gov (United States)

    Determining the origin of the cattle tick, Rhipicephalus microplus, will be helpful to the effort to find biological control agents. Molecular phylogenetics can assist in this determination. Thus, we sequenced and assembled partial gene sequences from the mitochondrial cytochrome oxidase I coding r...

  19. Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development.

    Science.gov (United States)

    García-Martínez, J L; López-Diaz, I; Sánchez-Beltrán, M J; Phillips, A L; Ward, D A; Gaskin, P; Hedden, P

    1997-04-01

    PCR was used with degenerate primers based on conserved amino acid sequences in gibberellin (GA) 20-oxidases to isolate cDNA clones for these enzymes from young seeds of pea (Pisum sativum) and developing embryos of French bean (Phaseolus vulgaris). One GA 20-oxidase cDNA (Ps27-12) was obtained from pea and three (Pv 15-11, Pv73-1 and Pv85-26) from bean. Their identities were confirmed by demonstrating that fusion proteins expressed in Escherichia coli exhibited GA 20-oxidase activity, converting [14C]GA12 to [14C]GA9. The intermediates in this three-step reaction, GA15 and GA24, were also identified as products. The expression proteins from three of the clones (Ps27-12, Pv15-11 and Pv73-1) were also shown to convert GA53 to GA20, as effectively as they did GA12. On the basis of transcript levels measured by northern blot analysis, the pea GA 20-oxidase gene is most highly expressed in young leaves, fully expanded internodes, very young seeds (until 4 days after anthesis) and expanding pods (from 3 days after anthesis at least until day 6). Expression in pods from 3-day-old unpollinated ovaries is higher than in those from pollinated ovaries. Treatment of unpollinated ovaries with GA3 to induce parthenocarpic fruit-set severely reduced the amount of GA 20-oxidase mRNA, whereas treatment with 2,4-D, although inducing fruit-set, did not reduce the levels of these transcripts. Plant decapitation above an unpollinated ovary resulted in very high levels of GA 20-oxidase mRNA in the pod. The three GA 20-oxidase genes from French bean showed very different patterns of expression: Pv 15-1 was expressed in the roots, young leaves, and developing seeds, but most highly in immature cotyledons, while Pv73-1 has a similar expression pattern to Ps27-12, with transcripts found only in young seeds and young leaves, where it was particularly abundant. Transcripts corresponding to Pv85-26 were detected in developing seeds, and just traces in the young leaves. Southern blot analysis

  20. Global Transcriptomic Analysis of Targeted Silencing of Two Paralogous ACC Oxidase Genes in Banana

    Science.gov (United States)

    Xia, Yan; Kuan, Chi; Chiu, Chien-Hsiang; Chen, Xiao-Jing; Do, Yi-Yin; Huang, Pung-Ling

    2016-01-01

    Among 18 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologous genes existing in the banana genome there are two genes, Mh-ACO1 and Mh-ACO2, that participate in banana fruit ripening. To better understand the physiological functions of Mh-ACO1 and Mh-ACO2, two hairpin-type siRNA expression vectors targeting both the Mh-ACO1 and Mh-ACO2 were constructed and incorporated into the banana genome by Agrobacterium-mediated transformation. The generation of Mh-ACO1 and Mh-ACO2 RNAi transgenic banana plants was confirmed by Southern blot analysis. To gain insights into the functional diversity and complexity between Mh-ACO1 and Mh-ACO2, transcriptome sequencing of banana fruits using the Illumina next-generation sequencer was performed. A total of 32,093,976 reads, assembled into 88,031 unigenes for 123,617 transcripts were obtained. Significantly enriched Gene Oncology (GO) terms and the number of differentially expressed genes (DEGs) with GO annotation were ‘catalytic activity’ (1327, 56.4%), ‘heme binding’ (65, 2.76%), ‘tetrapyrrole binding’ (66, 2.81%), and ‘oxidoreductase activity’ (287, 12.21%). Real-time RT-PCR was further performed with mRNAs from both peel and pulp of banana fruits in Mh-ACO1 and Mh-ACO2 RNAi transgenic plants. The results showed that expression levels of genes related to ethylene signaling in ripening banana fruits were strongly influenced by the expression of genes associated with ethylene biosynthesis. PMID:27681726

  1. Global Transcriptomic Analysis of Targeted Silencing of Two Paralogous ACC Oxidase Genes in Banana

    Directory of Open Access Journals (Sweden)

    Yan Xia

    2016-09-01

    Full Text Available Among 18 1-aminocyclopropane-1-carboxylic acid (ACC oxidase homologous genes existing in the banana genome there are two genes, Mh-ACO1 and Mh-ACO2, that participate in banana fruit ripening. To better understand the physiological functions of Mh-ACO1 and Mh-ACO2, two hairpin-type siRNA expression vectors targeting both the Mh-ACO1 and Mh-ACO2 were constructed and incorporated into the banana genome by Agrobacterium-mediated transformation. The generation of Mh-ACO1 and Mh-ACO2 RNAi transgenic banana plants was confirmed by Southern blot analysis. To gain insights into the functional diversity and complexity between Mh-ACO1 and Mh-ACO2, transcriptome sequencing of banana fruits using the Illumina next-generation sequencer was performed. A total of 32,093,976 reads, assembled into 88,031 unigenes for 123,617 transcripts were obtained. Significantly enriched Gene Oncology (GO terms and the number of differentially expressed genes (DEGs with GO annotation were ‘catalytic activity’ (1327, 56.4%, ‘heme binding’ (65, 2.76%, ‘tetrapyrrole binding’ (66, 2.81%, and ‘oxidoreductase activity’ (287, 12.21%. Real-time RT-PCR was further performed with mRNAs from both peel and pulp of banana fruits in Mh-ACO1 and Mh-ACO2 RNAi transgenic plants. The results showed that expression levels of genes related to ethylene signaling in ripening banana fruits were strongly influenced by the expression of genes associated with ethylene biosynthesis.

  2. Global Transcriptomic Analysis of Targeted Silencing of Two Paralogous ACC Oxidase Genes in Banana.

    Science.gov (United States)

    Xia, Yan; Kuan, Chi; Chiu, Chien-Hsiang; Chen, Xiao-Jing; Do, Yi-Yin; Huang, Pung-Ling

    2016-09-26

    Among 18 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologous genes existing in the banana genome there are two genes, Mh-ACO1 and Mh-ACO2, that participate in banana fruit ripening. To better understand the physiological functions of Mh-ACO1 and Mh-ACO2, two hairpin-type siRNA expression vectors targeting both the Mh-ACO1 and Mh-ACO2 were constructed and incorporated into the banana genome by Agrobacterium-mediated transformation. The generation of Mh-ACO1 and Mh-ACO2 RNAi transgenic banana plants was confirmed by Southern blot analysis. To gain insights into the functional diversity and complexity between Mh-ACO1 and Mh-ACO2, transcriptome sequencing of banana fruits using the Illumina next-generation sequencer was performed. A total of 32,093,976 reads, assembled into 88,031 unigenes for 123,617 transcripts were obtained. Significantly enriched Gene Oncology (GO) terms and the number of differentially expressed genes (DEGs) with GO annotation were 'catalytic activity' (1327, 56.4%), 'heme binding' (65, 2.76%), 'tetrapyrrole binding' (66, 2.81%), and 'oxidoreductase activity' (287, 12.21%). Real-time RT-PCR was further performed with mRNAs from both peel and pulp of banana fruits in Mh-ACO1 and Mh-ACO2 RNAi transgenic plants. The results showed that expression levels of genes related to ethylene signaling in ripening banana fruits were strongly influenced by the expression of genes associated with ethylene biosynthesis.

  3. Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

    Directory of Open Access Journals (Sweden)

    Nuria Troyano-Suárez

    2015-01-01

    Full Text Available Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK, a scaffold protein at cell-extracellular matrix (ECM adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

  4. Monoamine oxidase A gene DNA hypomethylation - a risk factor for panic disorder?

    Science.gov (United States)

    Domschke, Katharina; Tidow, Nicola; Kuithan, Henriette; Schwarte, Kathrin; Klauke, Benedikt; Ambrée, Oliver; Reif, Andreas; Schmidt, Hartmut; Arolt, Volker; Kersting, Anette; Zwanzger, Peter; Deckert, Jürgen

    2012-10-01

    The monoamine oxidase A (MAOA) gene has been suggested as a prime candidate in the pathogenesis of panic disorder. In the present study, DNA methylation patterns in the MAOA regulatory and exon 1/intron 1 region were investigated for association with panic disorder with particular attention to possible effects of gender and environmental factors. Sixty-five patients with panic disorder (44 females, 21 males) and 65 healthy controls were analysed for DNA methylation status at 42 MAOA CpG sites via direct sequencing of sodium bisulfate treated DNA extracted from blood cells. The occurrence of recent positive and negative life events was ascertained. Male subjects showed no or only very minor methylation with some evidence for relative hypomethylation at one CpG site in intron 1 in patients compared to controls. Female patients exhibited significantly lower methylation than healthy controls at 10 MAOA CpG sites in the promoter as well as in exon/intron 1, with significance surviving correction for multiple testing at four CpG sites (p≤0.001). Furthermore, in female subjects the occurrence of negative life events was associated with relatively decreased methylation, while positive life events were associated with increased methylation. The present pilot data suggest a potential role of MAOA gene hypomethylation in the pathogenesis of panic disorder particularly in female patients, possibly mediating a detrimental influence of negative life events. Future studies are warranted to replicate the present finding in independent samples, preferably in a longitudinal design.

  5. Life without putrescine: disruption of the gene-encoding polyamine oxidase in Ustilago maydis odc mutants.

    Science.gov (United States)

    Valdés-Santiago, Laura; Guzmán-de-Peña, Doralinda; Ruiz-Herrera, José

    2010-11-01

    In previous communications the essential role of spermidine in Ustilago maydis was demonstrated by means of the disruption of the genes encoding ornithine decarboxylase (ODC) and spermidine synthase (SPE). However, the assignation of specific roles to each polyamine in different cellular functions was not possible because the spermidine added to satisfy the auxotrophic requirement of odc/spe double mutants is partly back converted into putrescine. In this study, we have approached this problem through the disruption of the gene-encoding polyamine oxidase (PAO), required for the conversion of spermidine into putrescine, and the construction of odc/pao double mutants that were unable to synthesize putrescine by either ornithine decarboxylation or retroconversion from spermidine. Phenotypic analysis of the mutants provided evidence that putrescine is only an intermediary in spermidine biosynthesis, and has no direct role in cell growth, dimorphic transition, or any other vital function of U. maydis. Nevertheless, our results show that putrescine may play a role in the protection of U. maydis against salt and osmotic stress, and possibly virulence. Evidence was also obtained that the retroconversion of spermidine into putrescine is not essential for U. maydis growth but may be important for its survival under natural conditions.

  6. Molecular cloning and expression analysis of duplicated polyphenol oxidase genes reveal their functional differentiations in sorghum.

    Science.gov (United States)

    Yan, Song; Li, Sujuan; Zhai, Guowei; Lu, Ping; Deng, Hui; Zhu, Shan; Huang, Renliang; Shao, Jianfeng; Tao, Yuezhi; Zou, Guihua

    2017-10-01

    Polyphenol oxidase (PPO) is believed to play a role in plant growth, reproduction, and resistance to pathogens and pests. PPO causes browning of grains in cereals. In this study, genetic mapping of sorghum grain for phenol color reaction (PHR) was performed using a recombinant inbred line population. Only one locus was detected between SSR markers SM06072 and Xtxp176 on chromosome 6. Two linked orthologous genes (Sb06PPO1 and Sb06PPO2) within the mapped region were discovered and cloned. Transformation experiments using Nipponbare (a PHR negative rice cultivar) showed that Sb06PPO1 from LTR108 and two Sb06PPO2 alleles from both varieties could complement Nipponbare, whereas Sb06PPO1 from 654 could not. Subsequent quantitative real-time PCR (qPCR) experiments showed that Sb06PPO1 and Sb06PPO2 functioned diversely, Sb06PPO1 was mainly expressed in young panicles before flowering. Sb06PPO2 was strongly expressed in flowering panicles, especially in hulls and branches at filling stage. Moreover, the expression of Sb06PPO1 was found to be significantly up-regulated by exogenous ABA and salt, whereas Sb06PPO2 was not changed significantly, further demonstrating functional differentiation between the two genes. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Molecular evolution of the cytochrome c oxidase subunit 5A gene in primates

    Directory of Open Access Journals (Sweden)

    Wildman Derek E

    2008-01-01

    Full Text Available Abstract Background Many electron transport chain (ETC genes show accelerated rates of nonsynonymous nucleotide substitutions in anthropoid primate lineages, yet in non-anthropoid lineages the ETC proteins are typically highly conserved. Here, we test the hypothesis that COX5A, the ETC gene that encodes cytochrome c oxidase subunit 5A, shows a pattern of anthropoid-specific adaptive evolution, and investigate the distribution of this protein in catarrhine brains. Results In a dataset comprising 29 vertebrate taxa, including representatives from all major groups of primates, there is nearly 100% conservation of the COX5A amino acid sequence among extant, non-anthropoid placental mammals. The most recent common ancestor of these species lived about 100 million years (MY ago. In contrast, anthropoid primates show markedly elevated rates of nonsynonymous evolution. In particular, branch site tests identify five positively selected codons in anthropoids, and ancestral reconstructions infer that substitutions in these codons occurred predominantly on stem lineages (anthropoid, ape and New World monkey and on the human terminal branch. Examination of catarrhine brain samples by immunohistochemistry characterizes for the first time COX5A protein distribution in the primate neocortex, and suggests that the protein is most abundant in the mitochondria of large-size projection neurons. Real time quantitative PCR supports previous microarray results showing COX5A is expressed in cerebral cortical tissue at a higher level in human than in chimpanzee or gorilla. Conclusion Taken together, these results suggest that both protein structural and gene regulatory changes contributed to COX5A evolution during humankind's ancestry. Furthermore, these findings are consistent with the hypothesis that adaptations in ETC genes contributed to the emergence of the energetically expensive anthropoid neocortex.

  8. Sudden infant death syndrome (SIDS) and polymorphisms in Monoamine oxidase A gene (MAOA): a revisit.

    Science.gov (United States)

    Groß, Maximilian; Bajanowski, Thomas; Vennemann, Mechtild; Poetsch, Micaela

    2014-01-01

    Literature describes multiple possible links between genetic variations in the neuroadrenergic system and the occurrence of sudden infant death syndrome. The X-chromosomal Monoamine oxidase A (MAOA) is one of the genes with regulatory activity in the noradrenergic and serotonergic neuronal systems and a polymorphism of the promoter which affects the activity of this gene has been proclaimed to contribute significantly to the prevalence of sudden infant death syndrome (SIDS) in three studies from 2009, 2012 and 2013. However, these studies described different significant correlations regarding gender or age of children. Since several studies, suggesting associations between genetic variations and SIDS, were disproved by follow-up analysis, this study was conducted to take a closer look at the MAOA gene and its polymorphisms. The functional MAOA promoter length polymorphism was investigated in 261 SIDS cases and 93 control subjects. Moreover, the allele distribution of 12 coding and non-coding single nucleotide polymorphisms (SNPs) of the MAOA gene was examined in 285 SIDS cases and 93 controls by a minisequencing technique. In contrast to prior studies with fewer individuals, no significant correlations between the occurrence of SIDS and the frequency of allele variants of the promoter polymorphism could be demonstrated, even including the results from the abovementioned previous studies. Regarding the SNPs, three statistically significant associations were observed which had not been described before. This study clearly disproves interactions between MAOA promoter polymorphisms and SIDS, even if variations in single nucleotide polymorphisms of MAOA should be subjected to further analysis to clarify their impact on SIDS.

  9. ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation.

    Science.gov (United States)

    de Pater, Sylvia; Pinas, Johan E; Hooykaas, Paul J J; van der Zaal, Bert J

    2013-05-01

    Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5' coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10⁻³ per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10⁻³ per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5' and the 3' end of the gene. One plant line contained a PPO gene repaired only at the 5' end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.

  10. Direct electron transfer of glucose oxidase and dual hydrogen peroxide and glucose detection based on water-dispersible carbon nanotubes derivative

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hsiao-Chien [Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, 250, Wuxing St., Taipei 11031, Taiwan (China); Tu, Yi-Ming; Hou, Chung-Che [Department of Chemical and Materials Engineering, Chang Gung University, 259 Wen-Hwa 1st Rd., Tao-Yuan 33302, Taiwan (China); Lin, Yu-Chen [Wah Hong industrial Co. Ltd., 6 Lixing St., Guantian Dist., Tainan City 72046,Taiwan (China); Chen, Ching-Hsiang [Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, 43 Keelung Rd., Sec. 4, Taipei 10607, Taiwan (China); Yang, Kuang-Hsuan, E-mail: khy@mail.vnu.edu.tw [Department of Food and Beverage Management, Vanung University, 1, Van Nung Rd., Shuei-Wei Li, Chung-Li City 32061, Taiwan (China)

    2015-03-31

    Highlights: • Dual hydrogen peroxide and glucose sensor. • Direct electrochemistry of glucose oxidase used MWCNT-Py/GC electrode. • Change sensing function by adjusting pH value. - Abstract: A water-dispersible multi-walled carbon nanotubes (MWCNTs) derivative, MWCNTs-1-one-dihydroxypyridine (MWCNTs-Py) was synthesis via Friedel–Crafts chemical acylation. Raman spectra demonstrated the conjugated level of MWCNTs-Py was retained after this chemical modification. MWCNTs-Py showed dual hydrogen peroxide (H{sub 2}O{sub 2}) and glucose detections without mutual interference by adjusting pH value. It was sensitive to H{sub 2}O{sub 2} in acidic solution and displayed the high performances of sensitivity, linear range, response time and stability; meanwhile it did not respond to H{sub 2}O{sub 2} in neutral solution. In addition, this positively charged MWCNTs-Py could adsorb glucose oxidase (GOD) by electrostatic attraction. MWCNTs-Py-GOD/GC electrode showed the direct electron transfer (DET) of GOD with a pair of well-defined redox peaks, attesting the bioactivity of GOD was retained due to the non-destroyed immobilization. The high surface coverage of active GOD (3.5 × 10{sup −9} mol cm{sup −2}) resulted in exhibiting a good electrocatalytic activity toward glucose. This glucose sensor showed high sensitivity (68.1 μA mM{sup −1} cm{sup −2}) in a linear range from 3 μM to 7 mM in neutral buffer solution. The proposed sensor could distinguish H{sub 2}O{sub 2} and glucose, thus owning high selectivity and reliability.

  11. Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

    Science.gov (United States)

    Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-08-01

    Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks.

  12. [Prolonging the vase life of carnation "Mabel" through integrating repeated ACC oxidase genes into its genome].

    Science.gov (United States)

    Yu, Yi-Xun; Bao, Man-Zhu

    2004-10-01

    Carnation (Dianthus caryophyllus L.) is one of the most important cut flowers. The cultivar "Mabel" of carnation was transformed with direct repeat gene of ACC oxidase, the key enzyme in ethylene synthesis, driven by the CaMV35S promoter mediated by Agrobacterium tumefacien. Hygromycin phosphotransferase (HPT) gene was used as selection marker. Leaf explants were pre-cultured on shoot-inducing medium for 2 d, then immersed in Agrobacterium suspension for 8-12 min. Co-cultivation was carried out on the medium (MS+BA 1.0 mg/L+NAA 0.3 mg/L +Acetosyringone 100 micromol/L, pH 5.8-6.0) for 3 d. After that transformants were obtained by transferring explants to selection medium supplemented with 5 mg/L hygromycin (Hyg) and 400 mg/L cefotaxime (Cef). Southern blotting detection showed that a foreign gene was integrated into the carnation genome and 3 transgenic lines (T257, T299 and T273 line) obtained. Addition of acetosyringone and the time of co-culture were the main factors that influenced transformation frequency. After being transplanted to soil, transgenic plants were grew normally in greenhouse. Ethylene production of cut flower of transgenic T257 line was 95% lower than that of the control, and that of T299 line was reduced by 90% than that of the control, while that of transgenic T273 line has no of significantly different from control. Vase life of transgenic T257 line was 5 d longer than that of the control line at 25 degrees C.

  13. The ccoNOQP gene cluster codes for a cb-type cytochrome oxidase that functions in aerobic respiration of Rhodobacter capsulatus.

    Science.gov (United States)

    Thöny-Meyer, L; Beck, C; Preisig, O; Hennecke, H

    1994-11-01

    The genes for a new type of a haem-copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe. Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb-type oxidase. CcoN is a b-type cytochrome, CcoO and CcoP are membrane-bound mono- and dihaem c-type cytochromes and CcoQ is a small membrane protein of unknown function. Genes for a similar oxidase are also present in other non-rhizobial bacterial species such as Azotobacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis. A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R. capsulatus and which is probably identical to the cbb3-type oxidase that was recently purified from a different strain of the same species. Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen-fixation activity.

  14. Stress-induced co-expression of two alternative oxidase (VuAox1 and 2b) genes in Vigna unguiculata.

    Science.gov (United States)

    Costa, José Hélio; Mota, Erika Freitas; Cambursano, Mariana Virginia; Lauxmann, Martin Alexander; de Oliveira, Luciana Maia Nogueira; Silva Lima, Maria da Guia; Orellano, Elena Graciela; Fernandes de Melo, Dirce

    2010-05-01

    Cowpea (Vigna unguiculata) alternative oxidase is encoded by a small multigene family (Aox1, 2a and 2b) that is orthologous to the soybean Aox family. Like most of the identified Aox genes in plants, VuAox1 and VuAox2 consist of 4 exons interrupted by 3 introns. Alignment of the orthologous Aox genes revealed high identity of exons and intron variability, which is more prevalent in Aox1. In order to determine Aox gene expression in V. unguiculata, a steady-state analysis of transcripts involved in seed development (flowers, pods and dry seeds) and germination (soaked seeds) was performed and systemic co-expression of VuAox1 and VuAox2b was observed during germination. The analysis of Aox transcripts in leaves from seedlings under different stress conditions (cold, PEG, salicylate and H2O2 revealed stress-induced co-expression of both VuAox genes. Transcripts of VuAox2a and 2b were detected in all control seedlings, which was not the case for VuAox1 mRNA. Estimation of the primary transcript lengths of V. unguiculata and soybean Aox genes showed an intron length reduction for VuAox1 and 2b, suggesting that the two genes have converged in transcribed sequence length. Indeed, a bioinformatics analysis of VuAox1 and 2b promoters revealed a conserved region related to a cis-element that is responsive to oxidative stress. Taken together, the data provide evidence for co-expression of Aox1 and Aox2b in response to stress and also during the early phase of seed germination. The dual nature of VuAox2b expression (constitutive and induced) suggests that the constitutive Aox2b gene of V. unguiculata has acquired inducible regulatory elements.

  15. Mutations in the SURF1 gene associated with Leigh syndrome and cytochrome C oxidase deficiency.

    Science.gov (United States)

    Péquignot, M O; Dey, R; Zeviani, M; Tiranti, V; Godinot, C; Poyau, A; Sue, C; Di Mauro, S; Abitbol, M; Marsac, C

    2001-05-01

    Cytochrome c oxidase (COX) deficiency is one of the major causes of Leigh Syndrome (LS), a fatal encephalopathy of infancy or childhood, characterized by symmetrical lesions in the basal ganglia and brainstem. Mutations in the nuclear genes encoding COX subunits have not been found in patients with LS and COX deficiency, but mutations have been identified in SURF1. SURF1 encodes a factor involved in COX biogenesis. To date, 30 different mutations have been reported in 40 unrelated patients. We aim to provide an overview of all known mutations in SURF1, and to propose a common nomenclature. Twelve of the mutations were insertion/deletion mutations in exons 1, 4, 6, 8, and 9; 10 were missense/nonsense mutations in exons 2, 4, 5, 7, and 8; and eight were detected at splicing sites in introns 3 to 7. The most frequent mutation was 312_321del 311_312insAT which was found in 12 patients out of 40. Twenty mutations have been described only once. We also list all polymorphisms discovered to date.

  16. Molecular Phylogeny of Giant Clams Based on Mitochondrial DNA Cytochrome C Oxidase I Gene

    Directory of Open Access Journals (Sweden)

    AGUS NURYANTO

    2007-12-01

    Full Text Available There is an uncertainty for the relationships among giant clam species of Tridacninae, in particular among species belongs to subgenus Chametrachea i.e. Tridacna crocea, T. maxima, and T. squamosa based on different genetic markers. This study examined the relationships among three species within subgenus Chametrachea compared to the previous studies. Neighbour Joining, Maximum Parsimony and Maximum Likelihood tree were constructed based on 455 bp of the mitochondrial DNA cytochrome c oxidase I gene from T. crocea, T. squamosa, T. maxima, T. gigas, and several sequences derived from Genbank for the outgroups. The results showed that giant clams formed a monophyletic group. Within Tridacna group, T. crocea was more closely related to T. squamosa than to T. maxima and they formed a monophyletic group. T. crocea and T. squamosa were sister taxa and sister group to T. maxima and T. gigas. Close affinity between T. crocea and T. squamosa was also supported by high similarity on nucleotide level (94.30% and concordant with the results of the previous studies using mitochondrial 16S rRNA and nuclear 18S rRNA.

  17. PHYLOGENY OF ANGIOSTRONGYLUS CANTONENSIS IN THAILAND BASED ON CYTOCHROME C OXIDASE SUBUNIT I GENE SEQUENCE.

    Science.gov (United States)

    Apichat, Vitta; Narongrit, Srisongcram; Jittranuch, Thiproaj; Anucha, Wongma; Wilaiwan, Polsut; Chamaiporn, Fukruksa; Thatcha, Yimthin; Bandid, Mangkit; Aunchalee, Thanwisai; Paron, Dekumyoy

    2016-05-01

    Angiostrongylus cantonensis is an emerging infectious agent causing eosinophilic meningitis or meningoencephalitis in humans with clinical manifestation of severe headache. Molecular genetic studies on classification and phylogeny of A. cantonensis in Thailand are limited. This study surveyed A. cantonensis larvae prevalence in natural intermediate hosts across Thailand and analyzed their phylogenetic relationships. A total of 14,032 freshwater and land snails were collected from 19 provinces of Thailand. None of Filopaludina sp, Pomacea sp, and Cyclophorus sp were infected with Angiostrongylus larvae, whereas Achatina fulica, Cryptozona siamensis, and Megaustenia siamensis collected from Kalasin, Kamphaeng Phet, Phetchabun, Phitsanulok, and Tak Provinces were infected, with C. siamensis being the common intermediate host. Based on morphology, larvae isolated from 11 samples of these naturally infected snails preliminarily were identified as A. cantonensis. Comparison of partial nucleotide sequences of cytochrome c oxidase subunit I gene revealed that four sequences are identical to A. cantonensis haplotype ac4 from Bangkok and the other seven to that of A. cantonensis isolate AC Thai, indicating two independent lineages of A. cantonensis in Thailand.

  18. Molecular evolution at the cytochrome oxidase subunit 2 gene among divergent populations of the intertidal copepod, Tigriopus californicus.

    Science.gov (United States)

    Rawson, Paul D; Burton, Ronald S

    2006-06-01

    The cytochrome c oxidase subunit 2 gene (COII) encodes a highly conserved protein that is directly responsible for the initial transfer of electrons from cytochrome c to cytochrome c oxidase (COX) crucial to the production of ATP during cellular respiration. Despite its integral role in electron transport, we have observed extensive intraspecific nucleotide and amino acid variation among 26 full-length COII sequences sampled from seven populations of the marine copepod, Tigriopus californicus. Although intrapopulation divergence was virtually nonexistent, interpopulation divergence at the COII locus was nearly 20% at the nucleotide level, including 38 nonsynonymous substitutions. Given the high degree of interaction between the cytochrome c oxidase subunit 2 protein (COX2) and the nuclear-encoded subunits of COX and cytochrome c (CYC), we hypothesized that some codons in the COII gene are likely to be under positive selection in order to compensate for amino acid substitutions in other subunits. Estimates of the ratio of nonsynonymous to synonymous substitution (omega), obtained using a series of maximum likelihood models of codon substitution, indicated that the majority of codons in T. californicus COII are under strong purifying selection (omega < 1), while approximately 4% of the sites in this gene appear to evolve under relaxed selective constraint (omega = 1). A branch-site maximum likelihood model identified three sites that may have experienced positive selection within the central California sequence clade in our COII phylogeny; these results are consistent with previous studies showing functional and fitness consequences among interpopulation hybrids between central and northern California populations.

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

    Directory of Open Access Journals (Sweden)

    Xiumei Zhen

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

  20. Shape-Controlled Generation of Gold Nanoparticles Assisted by Dual-Molecules: The Development of Hydrogen Peroxide and Oxidase-Based Biosensors

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    Chifang Peng

    2014-01-01

    Full Text Available With the assist of dual-molecules, 2-(N-morpholinoethanesulfonic acid (MES and sodium citrate, gold nanoparticles (GNPs with different shapes can be generated in the H2O2-mediated reduction of chloroauric acid. This one-pot reaction can be employed to sensitively detect H2O2, probe substrates or enzymes in oxidase-based reactions as well as prepare branched GNPs controllably. By the “naked eye,” 20 μM H2O2, 0.1 μM glucose, and 0.26 U/mL catalase could be differentiated, respectively. By spectrophotometer, the detected limits of H2O2, glucose, and catalase were 1.0 μM, 0.01 μM, and 0.03 U/mL, respectively, and the detection linear ranges for them were 5.0–400 μM, 0.01–0.3 mM, and 0.03–0.78 U/mL, respectively. The proposed “dual-molecules assist” strategy probably paves a new way for the fabrication of nanosensors based on the growth of anisotropic metal nanoparticles, and the developed catalase sensor can probably be utilized to fabricate ultrasensitive ELISA methods for various analytes.

  1. [Association between the canine monoamine oxidase B (MAOB) gene polymorphisms and behavior of puppies in open-field test].

    Science.gov (United States)

    Li, Xiao-Hui; Xu, Han-Kun; Mao, Da-Gan; Ma, Da-Jun; Chen, Peng; Yang, Li-Guo

    2006-11-01

    Excitability, activity and exploration behavior of puppies in a novel open-field were tested in a total of 204 two-month-old German shepherd dog, labrador retriever or English springer spaniel puppies. The polymorphisms of monoamine oxidase B gene (MAOB) were detected by PCR-RFLP. Statistics analysis indicated that genotype and allele frequencies of the polymorphisms were significantly different among three breeds (P open-field test. The results showed that MAOB gene polymorphisms had a significant effect on walking time, squares crossed, lying time, the times of standing up against walls(P open-field test and TT genotype has favorable effects in these behavior traits.

  2. The role of the monoamine oxidase A gene in moderating the response to adversity and associated antisocial behavior: a review

    Directory of Open Access Journals (Sweden)

    Buades-Rotger M

    2014-07-01

    Full Text Available Macià Buades-Rotger,1,2 David Gallardo-Pujol1,3 1Department of Personality, Faculty of Psychology, University of Barcelona, Barcelona, Spain; 2Department of Neurology, University of Lübeck, Lübeck, Germany; 3Institute for Brain, Cognition and Behavior (IR3C, University of Barcelona, Barcelona, Spain Abstract: Hereditary factors are increasingly attracting the interest of behavioral scientists and practitioners. Our aim in the present article is to introduce some state-of-the-art topics in behavioral genetics, as well as selected findings in the field, in order to illustrate how genetic makeup can modulate the impact of environmental factors. We focus on the most-studied polymorphism to date for antisocial responses to adversity: the monoamine oxidase A gene. Advances, caveats, and promises of current research are reviewed. We also discuss implications for the use of genetic information in applied settings. Keywords: behavioral genetics, antisocial behaviors, monoamine oxidase A

  3. Hypoxia-response element (HRE)-directed transcriptional regulation of the rat lysyl oxidase gene in response to cobalt and cadmium.

    Science.gov (United States)

    Gao, Song; Zhou, Jing; Zhao, Yinzhi; Toselli, Paul; Li, Wande

    2013-04-01

    Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5'-ACGTG-3') exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10-100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)-treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at -387/-383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.

  4. Correlation between single nucleotide polymorphisms of NADPH oxidase p22phox gene and ischemic stroke in Shanghai Han population

    Directory of Open Access Journals (Sweden)

    Wei XU

    2015-09-01

    Full Text Available Objective This paper aims to investigate the distribution of genotypes and alleles of nicotinamide adenine dinucleotide phosphate (NADPH oxidase p22phox -930A/G, 242C/T and -675A/T, so as to evaluate the association between three single-nucleotide polymorphisms (SNPs and risk of atherosclerotic ischemic stroke in permanent resident population of Han nationality living in Shanghai area. Methods The genotypes and allele frequencies of NADPH oxidase p22phox subunit -930A/G, 242C/T and -675A/T were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis in 205 patients with ischemic stroke and 136 healthy controls. Results In patients with ischemic stroke, the results of PCR-RFLP in variant genetic loci were different. For -930A/G, one band appeared at 268 bp of genotype AA; 2 bands appeared at 197 and 71 bp of genotype GG; 3 bands appeared at 268, 197 and 71 bp of genotype AG. For 242C/T, one band appeared at 348 bp of genotype CC; 2 bands appeared at 188 and 160 bp of genotype TT; 3 bands appeared at 348, 188 and 160 bp of genotype CT. For -675A/T, 2 bands appeared at 158 and 54 bp of genotype TT; 3 bands appeared at 212, 158 and 54 bp of genotype AT. The genotypes and allele frequency of all three SNPs of NADPH oxidase p22phox gene had no significant difference between ischemic stroke patients and healthy controls (P > 0.05. Conclusions The genetic polymorphism of NADPH oxidase p22phox gene -930A/G, 242C/T and -675A/T might have no association with ischemic stroke. DOI: 10.3969/j.issn.1672-6731.2015.09.011

  5. Expressional studies of the aldehyde oxidase (AOX1) gene during myogenic differentiation in C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kamli, Majid Rasool; Kim, Jihoe; Pokharel, Smritee; Jan, Arif Tasleem [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Lee, Eun Ju [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Bovine Genome Resources Bank, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Choi, Inho, E-mail: inhochoi@ynu.ac.kr [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Bovine Genome Resources Bank, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of)

    2014-08-08

    Highlights: • AOX1 contributes to the formation of myotube. • Silencing of AOX1 reduces myotube formation. • AOX1 regulates MyoG gene expression. • AOX1 contributes to myogenesis via H{sub 2}O{sub 2}. - Abstract: Aldehyde oxidases (AOXs), which catalyze the hydroxylation of heterocycles and oxidation of a wide variety of aldehydic compounds, have been present throughout evolution from bacteria to humans. While humans have only a single functional aldehyde oxidase (AOX1) gene, rodents are endowed with four AOXs; AOX1 and three aldehyde oxidase homologs (AOH1, AOH2 and AOH3). In continuation of our previous study conducted to identify genes differentially expressed during myogenesis using a microarray approach, we investigated AOX1 with respect to its role in myogenesis to conceptualize how it is regulated in C2C12 cells. The results obtained were validated by silencing of the AOX1 gene. Analysis of their fusion index revealed that formation of myotubes showed a marked reduction of up to 40% in AOX1{sub kd} cells. Expression of myogenin (MYOG), one of the marker genes used to study myogenesis, was also found to be reduced in AOX1{sub kd} cells. AOX1 is an enzyme of pharmacological and toxicological importance that metabolizes numerous xenobiotics to their respective carboxylic acids. Hydrogen peroxide (H{sub 2}O{sub 2}) produced as a by-product in this reaction is considered to be involved as a part of the signaling mechanism during differentiation. An observed reduction in the level of H{sub 2}O{sub 2} among AOX1{sub kd} cells confirmed production of H{sub 2}O{sub 2} in the reaction catalyzed by AOX1. Taken together, these findings suggest that AOX1 acts as a contributor to the process of myogenesis by influencing the level of H{sub 2}O{sub 2}.

  6. MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors.

    Science.gov (United States)

    Roth, Justin C; Alberti, Michael O; Ismail, Mourad; Lingas, Karen T; Reese, Jane S; Gerson, Stanton L

    2015-01-22

    The DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin(-)Sca-1(+)c-kit(+) (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT(+)GFP(+)) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect.

  7. NUCLEOTIDE-SEQUENCE OF THE LAST EXON OF THE GENE FOR HUMAN CYTOCHROME-C-OXIDASE SUBUNIT-VIB AND ITS FLANKING REGIONS

    NARCIS (Netherlands)

    TAANMAN, JW; SCHRAGE, C; BOKMA, E; REUVEKAMP, P; AGSTERIBBE, E; DEVRIES, H

    1991-01-01

    A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit VIb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncoding re

  8. Spermine oxidase maintains basal skeletal muscle gene expression and fiber size and is strongly repressed by conditions that cause skeletal muscle atrophy.

    Science.gov (United States)

    Bongers, Kale S; Fox, Daniel K; Kunkel, Steven D; Stebounova, Larissa V; Murry, Daryl J; Pufall, Miles A; Ebert, Scott M; Dyle, Michael C; Bullard, Steven A; Dierdorff, Jason M; Adams, Christopher M

    2015-01-15

    Skeletal muscle atrophy is a common and debilitating condition that remains poorly understood at the molecular level. To better understand the mechanisms of muscle atrophy, we used mouse models to search for a skeletal muscle protein that helps to maintain muscle mass and is specifically lost during muscle atrophy. We discovered that diverse causes of muscle atrophy (limb immobilization, fasting, muscle denervation, and aging) strongly reduced expression of the enzyme spermine oxidase. Importantly, a reduction in spermine oxidase was sufficient to induce muscle fiber atrophy. Conversely, forced expression of spermine oxidase increased muscle fiber size in multiple models of muscle atrophy (immobilization, fasting, and denervation). Interestingly, the reduction of spermine oxidase during muscle atrophy was mediated by p21, a protein that is highly induced during muscle atrophy and actively promotes muscle atrophy. In addition, we found that spermine oxidase decreased skeletal muscle mRNAs that promote muscle atrophy (e.g., myogenin) and increased mRNAs that help to maintain muscle mass (e.g., mitofusin-2). Thus, in healthy skeletal muscle, a relatively low level of p21 permits expression of spermine oxidase, which helps to maintain basal muscle gene expression and fiber size; conversely, during conditions that cause muscle atrophy, p21 expression rises, leading to reduced spermine oxidase expression, disruption of basal muscle gene expression, and muscle fiber atrophy. Collectively, these results identify spermine oxidase as an important positive regulator of muscle gene expression and fiber size, and elucidate p21-mediated repression of spermine oxidase as a key step in the pathogenesis of skeletal muscle atrophy.

  9. Spermine oxidase maintains basal skeletal muscle gene expression and fiber size and is strongly repressed by conditions that cause skeletal muscle atrophy

    Science.gov (United States)

    Bongers, Kale S.; Fox, Daniel K.; Kunkel, Steven D.; Stebounova, Larissa V.; Murry, Daryl J.; Pufall, Miles A.; Ebert, Scott M.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.

    2014-01-01

    Skeletal muscle atrophy is a common and debilitating condition that remains poorly understood at the molecular level. To better understand the mechanisms of muscle atrophy, we used mouse models to search for a skeletal muscle protein that helps to maintain muscle mass and is specifically lost during muscle atrophy. We discovered that diverse causes of muscle atrophy (limb immobilization, fasting, muscle denervation, and aging) strongly reduced expression of the enzyme spermine oxidase. Importantly, a reduction in spermine oxidase was sufficient to induce muscle fiber atrophy. Conversely, forced expression of spermine oxidase increased muscle fiber size in multiple models of muscle atrophy (immobilization, fasting, and denervation). Interestingly, the reduction of spermine oxidase during muscle atrophy was mediated by p21, a protein that is highly induced during muscle atrophy and actively promotes muscle atrophy. In addition, we found that spermine oxidase decreased skeletal muscle mRNAs that promote muscle atrophy (e.g., myogenin) and increased mRNAs that help to maintain muscle mass (e.g., mitofusin-2). Thus, in healthy skeletal muscle, a relatively low level of p21 permits expression of spermine oxidase, which helps to maintain basal muscle gene expression and fiber size; conversely, during conditions that cause muscle atrophy, p21 expression rises, leading to reduced spermine oxidase expression, disruption of basal muscle gene expression, and muscle fiber atrophy. Collectively, these results identify spermine oxidase as an important positive regulator of muscle gene expression and fiber size, and elucidate p21-mediated repression of spermine oxidase as a key step in the pathogenesis of skeletal muscle atrophy. PMID:25406264

  10. Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments.

    Science.gov (United States)

    Hamamura, N; Macur, R E; Korf, S; Ackerman, G; Taylor, W P; Kozubal, M; Reysenbach, A-L; Inskeep, W P

    2009-02-01

    The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroA-like) from pure-culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite-oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite-oxidizing environments. Geochemical analyses, including measurement of arsenite-oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA-like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6-3.6) Fe-oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near-neutral (pH 6.2-8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and beta-Proteobacteria. Modified primers designed around previously characterized and newly identified aroA-like genes successfully amplified new lineages of aroA-like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA-like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences

  11. Alternative Splicing and Differential Expression of Two Transcripts of Nicotine Adenine Dinucleotide Phosphate Oxidase B Gene from Zea mays

    Institute of Scientific and Technical Information of China (English)

    Fan Lin; Yun Zhang; Ming-Yi Jiang

    2009-01-01

    With the exception of rice, little is known about the existence of respiratory burst oxidase homolog (rboh) gene in cereals. The present study reports the cloning and analysis of a novel rboh gene, termed ZmrbohB, from maize (Zea mays L.). The full-length cDNA of ZmrbohB encodes a 942 amino acid protein containing all of the respiratory burst oxidase homolog catalytically critical motifs.Altemative splicing of ZmrbohB has generated two transcript isoforms, ZmrbohB-α and -β. Spliced transcript ZmrbohB-β retains an unspliced intron 11 that carries a premature termination codon and probably leads to nonsense-mediated mRNA decay. Expression analysis showed that two splice isoforms were differentially expressed in various tissues and at different developmental stages, and the major product was ZmrbohB-α. The transcripts of ZmrbohB-α accumulated markedly when the maize seedlings were subjected to various abiotic stimuli, such as wounding, cold (4℃), heat (40℃), UV and salinity stress. In addition, several abiotic stimuli also affected the alternative splicing pattern of ZmrbohB except wounding. These results provide new insight into roles in the expression regulation of plant rboh genes and suggest that ZmrbohB gene may play a role in response to environmental stresses.

  12. Tet1 oxidase regulates neuronal gene transcription, active DNA hydroxymethylation, object location memory, and threat recognition memory

    Directory of Open Access Journals (Sweden)

    Dinesh Kumar

    2015-10-01

    Full Text Available A dynamic equilibrium between DNA methylation and demethylation of neuronal activity-regulated genes is crucial for memory processes. However, the mechanisms underlying this equilibrium remain elusive. Tet1 oxidase has been shown to play a key role in the active DNA demethylation in the central nervous system. In this study, we used Tet1 gene knockout (Tet1KO mice to examine the involvement of Tet1 in memory consolidation and storage in the adult brain. We found that Tet1 ablation leads to altered expression of numerous neuronal activity-regulated genes, compensatory upregulation of active demethylation pathway genes, and upregulation of various epigenetic modifiers. Moreover, Tet1KO mice showed an enhancement in the consolidation and storage of threat recognition (cued and contextual fear conditioning and object location memories. We conclude that Tet1 plays a critical role in regulating neuronal transcription and in maintaining the epigenetic state of the brain associated with memory consolidation and storage.

  13. hNIS-IRES-eGFP Dual Reporter Gene Imaging

    Directory of Open Access Journals (Sweden)

    Jiantu Che

    2005-04-01

    Full Text Available The human and rodent sodium iodide symporters (NIS have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES-linked human NIS (hNIS-enhanced green fluorescent protein (eGFP hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4– to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.

  14. Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

    Science.gov (United States)

    Sekar, T V; Foygel, K; Willmann, J K; Paulmurugan, R

    2013-05-01

    Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

  15. Over-expression of polyphenol oxidase gene in strawberry fruit delays the fungus infection process

    Science.gov (United States)

    Polyphenols are secondary metabolites widely present in plants and beneficial to human health. In this study, the changes of polyphenol contents during strawberry fruit development as well as changes of polyphenol oxidase (PPO) was analyzed. The polyphenol content showed declining trend during fruit...

  16. Structural analysis of tissues affected by cytochrome C oxidase deficiency due to mutations in the SCO2 gene.

    Science.gov (United States)

    Vesela, Katerina; Hulkova, Helena; Hansikova, Hana; Zeman, Jiri; Elleder, Milan

    2008-01-01

    Structural and histochemical studies carried out in a series of seven cases (from five families) with isolated cytochrome c oxidase (COX) deficiency caused by mutations in the SCO2 gene (1, 2) disclosed changes concentrated in the nervous system, skeletal muscle and myocardium. In five patients homozygous for the E140K mutation, the phenotype was predominantly neuromuscular and the average life span ranged between 9 and 15 months. In two cases, the course was more rapid (death at 7 and 11 weeks of life) and featured marked cardiac hypertrophy (3- and 4-fold increase in heart weight). This predominantly cardiomyopathic phenotype was associated with compound heterozygosity (E140K with another nonsense mutation) in the SCO2 gene. Polioencephalopathy with neurodegeneration and neuronal drop out was present in all cases with evidence that retinal neurons might be seriously affected too. Involvement of spinal motoneurons together with cytochrome c oxidase deficiency in muscle represents a "double hit" for the skeletal muscle. The mitochondrial population was not found to be significantly increased or structurally altered, with the exception of two compound heterozygotes in which the cardiac mitochondria were increased in number and size. Our report extends knowledge of the pathology of COX deficiency caused by mutations in the SCO2 gene.

  17. Dysbindin and d-amino-acid-oxidase gene polymorphisms associated with positive and negative symptoms in schizophrenia

    DEFF Research Database (Denmark)

    Wirgenes, Katrine V; Djurovic, Srdjan; Agartz, Ingrid;

    2009-01-01

    BACKGROUND: Schizophrenia is a genetically complex disorder with an unknown pathophysiology. Several genes implicated in glutamate metabolism have been associated with the disorder. Recent studies of polymorphisms in the dystrobrevin-binding protein 1 gene (DTNBP1; dysbindin) and D......-amino-acid-oxidase (DAO) gene, both involved in glutamate receptor function, reported associations with negative symptoms and with anxiety and depression, respectively, when measured with the Positive and Negative Syndrome Scale (PANSS). METHODS: In the present study, the suggested association between dysbindin and DAO...... single nucleotide polymorphisms (SNPs) and PANSS scores was analyzed in 155 Norwegian schizophrenia patients. RESULTS: There was a significant association between the dysbindin SNP rs3213207 and severity of both negative symptoms and total symptom load, as well as between the DAO SNP rs2070587 and total...

  18. ASSIGNMENT OF THE GENE CODING FOR HUMAN CYTOCHROME-C-OXIDASE SUBUNIT-VIB TO CHROMOSOME-19, BAND-Q13.1, BY FLUORESCENCE INSITU HYBRIDIZATION

    NARCIS (Netherlands)

    TAANMAN, JW; VANDERVEEN, AY; SCHRAGE, C; DEVRIES, H; BUYS, CHCM

    1991-01-01

    A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence

  19. Elimination of manganese(II,III) oxidation in Pseudomonas putida GB-1 by a double knockout of two putative multicopper oxidase genes.

    Science.gov (United States)

    Geszvain, Kati; McCarthy, James K; Tebo, Bradley M

    2013-01-01

    Bacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase in Pseudomonas putida GB-1 has long remained unknown. To identify the P. putida GB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG, mofA, moxA, and mopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11 Pseudomonas species. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes.

  20. Analysis of Taxonomic Relationships and Species Divergence Of Libellulidae (Odonata: Anisoptera Members Using Cytochrome Oxidase I Gene

    Directory of Open Access Journals (Sweden)

    Jisha Krishnan, E. K.,

    2016-04-01

    Full Text Available Libellulidae are commonly called ‘skimmers’ or ‘perchers’ representing the largest dragonfly family in the world. They are cosmopolitan in distribution and consist of 142 genera and 871 species. This family displays remarkable diversity in behaviour and morphology and consequently focused on studies of comparative population ecology, sexual selection, phylogeography and the evolution of mating behaviour. In the present study we deciphered the phylogenetic relationships of nine Libellulidae members by Neighbour-joining (NJ and Maximum likelihood methods using partial cytochrome oxidase I gene as the marker. The phylogenetic tree inferred the sister clade relationship of the representing libellulidae members and confirmed the evolutionary divergence in relation with branch length. The averages A+T content of all these species are 62.03 % while G+C content is 37.97 % showing strong A+T bias. The transition/ transversion ratio are found to be 0.858 for purines and 2.533 for pyramidines indicating higher mutations are exhibited by the transition of Thymine, Uracil and Cytosine. The present study thus concluded that the mitochondrial cytochrome oxidase subunit I (COI gene sequence of Libellulidae members demonstrated substantial variation; therefore it can be used for molecular taxonomy and for the phylogenetic studies.

  1. Exploring regulation genes involved in the expression of L-amino acid oxidase in Pseudoalteromonas sp. Rf-1.

    Directory of Open Access Journals (Sweden)

    Zhiliang Yu

    Full Text Available Bacterial L-amino acid oxidase (LAAO is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO.

  2. Colorimetric biosensing of targeted gene sequence using dual nanoparticle platforms

    Directory of Open Access Journals (Sweden)

    Thavanathan J

    2015-04-01

    Full Text Available Jeevan Thavanathan,1 Nay Ming Huang,1 Kwai Lin Thong2 1Low Dimension Material Research Center, Department of Physics, 2Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia Abstract: We have developed a colorimetric biosensor using a dual platform of gold nanoparticles and graphene oxide sheets for the detection of Salmonella enterica. The presence of the invA gene in S. enterica causes a change in color of the biosensor from its original pinkish-red to a light purplish solution. This occurs through the aggregation of the primary gold nanoparticles–conjugated DNA probe onto the surface of the secondary graphene oxide–conjugated DNA probe through DNA hybridization with the targeted DNA sequence. Spectrophotometry analysis showed a shift in wavelength from 525 nm to 600 nm with 1 µM of DNA target. Specificity testing revealed that the biosensor was able to detect various serovars of the S. enterica while no color change was observed with the other bacterial species. Sensitivity testing revealed the limit of detection was at 1 nM of DNA target. This proves the effectiveness of the biosensor in the detection of S. enterica through DNA hybridization. Keywords: biosensor, DNA hybridization, DNA probe, gold nanoparticles, graphene oxide, Salmonella enterica

  3. High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization.

    Science.gov (United States)

    Gao, Zhaowei; Li, Zhuofu; Zhang, Yuhong; Huang, Huoqing; Li, Mu; Zhou, Liwei; Tang, Yunming; Yao, Bin; Zhang, Wei

    2012-03-01

    The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35-40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml(-1) (2.5 g protein l(-1)) in a 3 l fermentor--410% higher than GOD-w (148 U ml(-1)), and thus is a low-cost alternative for the bread baking industry.

  4. A novel phylogeny and morphological reconstruction of the PIN genes and first phylogeny of the ACC-oxidases (ACOs

    Directory of Open Access Journals (Sweden)

    Ronald Matthew Clouse

    2014-06-01

    Full Text Available The PIN and ACO gene families present interesting questions about the evolution of plant physiology, including testing hypotheses about the ecological drivers of their diversification and whether unrelated genes have been recruited for similar functions. The PIN-formed proteins contribute to the polar transport of auxin, a hormone which regulates plant growth and development. PIN loci are categorized into groups according to their protein length and structure, as well as subcellular localization. An interesting question with PIN genes is the nature of the ancestral form and location. ACOs are members of a superfamily of oxygenases and oxidases that catalyze the last step of ethylene synthesis, which regulates many aspects of the plant life cycle. We used publicly available PIN and ACO sequences to conduct phylogenetic analyses. Third codon positions of these genes in monocots have a high GC content, which could be historical but is more likely due to a mutational bias. Thus we developed methods to extract phylogenetic information from nucleotide sequences while avoiding this convergent feature. One method consisted in using only A-T transformations, and another used only the first and second codon positions for serine, which can only take A or T and G or C, respectively. We also conducted tree-searches for both gene families using unaligned amino acid sequences and dynamic homology. PIN genes appear to have diversified earlier than ACOs, with monocot and dicot copies more mixed in the phylogeny. However, gymnosperm PINs appear to be derived and not closely related to those from primitive plants. We find strong support for a long PIN gene ancestor with short forms subsequently evolving one or more times. ACO genes appear to have diversified mostly since the dicot-monocot split, as most genes cluster into a small number of monocot and dicot clades when the tree is rooted by genes from mosses. Gymnosperm ACOs were recovered as closely related and

  5. A novel phylogeny and morphological reconstruction of the PIN genes and first phylogeny of the ACC-oxidases (ACOs).

    Science.gov (United States)

    Clouse, Ronald M; Carraro, Nicola

    2014-01-01

    The PIN and ACO gene families present interesting questions about the evolution of plant physiology, including testing hypotheses about the ecological drivers of their diversification and whether unrelated genes have been recruited for similar functions. The PIN-formed proteins contribute to the polar transport of auxin, a hormone which regulates plant growth and development. PIN loci are categorized into groups according to their protein length and structure, as well as subcellular localization. An interesting question with PIN genes is the nature of the ancestral form and location. ACOs are members of a superfamily of oxygenases and oxidases that catalyze the last step of ethylene synthesis, which regulates many aspects of the plant life cycle. We used publicly available PIN and ACO sequences to conduct phylogenetic analyses. Third codon positions of these genes in monocots have a high GC content, which could be historical but is more likely due to a mutational bias. Thus, we developed methods to extract phylogenetic information from nucleotide sequences while avoiding this convergent feature. One method consisted in using only A-T transformations, and another used only the first and second codon positions for serine, which can only take A or T and G or C, respectively. We also conducted tree-searches for both gene families using unaligned amino acid sequences and dynamic homology. PIN genes appear to have diversified earlier than ACOs, with monocot and dicot copies more mixed in the phylogeny. However, gymnosperm PINs appear to be derived and not closely related to those from primitive plants. We find strong support for a long PIN gene ancestor with short forms subsequently evolving one or more times. ACO genes appear to have diversified mostly since the dicot-monocot split, as most genes cluster into a small number of monocot and dicot clades when the tree is rooted by genes from mosses. Gymnosperm ACOs were recovered as closely related and derived.

  6. Identification of forensically important blow fly species (Diptera: Calliphoridae) in China by mitochondrial cytochrome oxidase I gene differentiation

    Institute of Scientific and Technical Information of China (English)

    Qin-Lai Liu; Li Yang; Kun-Lu Wu; Ling-mei Lan; Jiang-Feng Wang; Yao-Qing Chen; Ji-Feng Cai; Yun-Feng Chang; Yan Gu; Ya-Dong Guo; Xing-Hua Wang; Ji-FangWeng; Ming Zhong; Xiang Wang

    2011-01-01

    Unambiguous and rapid sarcosaphagous insect species identification is an essential requirement for forensic investigations. Although some insect species are difficult to classify morphologically, they can be effectively identified using molecular methods based on similarity with abundant authenticated reference DNA sequences in local databases.However, local databases are still relatively incomplete in China because of the large land area with distinct regional conditions. In this study, 75 forensically important blow flies were collected from 23 locations in 16 Chinese provinces, and a 278-bp segment of the cytochrome oxidase subunit Ⅰ gene of all specimens was successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all Calliphorid specimens were properly assigned into nine species with relatively strong supporting values, thus indicating that the 278-bp cytochrome oxidase subunit one region is suitable for identification of Calliphorid species. The clear difference between intraspecific threshold and interspecific divergence confirmed the potential of this region for Calliphorid species identification,especially for distinguishing between morphologically similar species. Intraspecific geographic variations were observed in Lucilia sericata (Meigen, 1826) and Lucilia caesar (Linnaeus, 1758).

  7. Knockdown of the Rhipicephalus microplus cytochrome c oxidase subunit III gene is associated with a failure of Anaplasma marginale transmission.

    Directory of Open Access Journals (Sweden)

    Thais D Bifano

    Full Text Available Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH were constructed, and five up-regulated genes {glutathione S-transferase (GST, cytochrome c oxidase sub III (COXIII, dynein (DYN, synaptobrevin (SYN and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission.

  8. Analysis of the cytochrome c oxidase subunit 1 (COX1) gene reveals the unique evolution of the giant panda.

    Science.gov (United States)

    Hu, Yao-Dong; Pang, Hui-Zhong; Li, De-Sheng; Ling, Shan-Shan; Lan, Dan; Wang, Ye; Zhu, Yun; Li, Di-Yan; Wei, Rong-Ping; Zhang, He-Min; Wang, Cheng-Dong

    2016-11-05

    As the rate-limiting enzyme of the mitochondrial respiratory chain, cytochrome c oxidase (COX) plays a crucial role in biological metabolism. "Living fossil" giant panda (Ailuropoda melanoleuca) is well-known for its special bamboo diet. In an effort to explore functional variation of COX1 in the energy metabolism behind giant panda's low-energy bamboo diet, we looked at genetic variation of COX1 gene in giant panda, and tested for its selection effect. In 1545 base pairs of the gene from 15 samples, 9 positions were variable and 1 mutation leaded to an amino acid sequence change. COX1 gene produces six haplotypes, nucleotide (pi), haplotype diversity (Hd). In addition, the average number of nucleotide differences (k) is 0.001629±0.001036, 0.8083±0.0694 and 2.517, respectively. Also, dN/dS ratio is significantly below 1. These results indicated that giant panda had a low population genetic diversity, and an obvious purifying selection of the COX1 gene which reduces synthesis of ATP determines giant panda's low-energy bamboo diet. Phylogenetic trees based on the COX1 gene were constructed to demonstrate that giant panda is the sister group of other Ursidae. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Transcript Profiling Reveals the Presence of Abiotic Stress and Developmental Stage Specific Ascorbate Oxidase Genes in Plants

    Science.gov (United States)

    Batth, Rituraj; Singh, Kapil; Kumari, Sumita; Mustafiz, Ananda

    2017-01-01

    Abiotic stress and climate change is the major concern for plant growth and crop yield. Abiotic stresses lead to enhanced accumulation of reactive oxygen species (ROS) consequently resulting in cellular damage and major losses in crop yield. One of the major scavengers of ROS is ascorbate (AA) which acts as first line of defense against external oxidants. An enzyme named ascorbate oxidase (AAO) is known to oxidize AA and deleteriously affect the plant system in response to stress. Genome-wide analysis of AAO gene family has led to the identification of five, three, seven, four, and six AAO genes in Oryza sativa, Arabidopsis, Glycine max, Zea mays, and Sorghum bicolor genomes, respectively. Expression profiling of these genes was carried out in response to various abiotic stresses and during various stages of vegetative and reproductive development using publicly available microarray database. Expression analysis in Oryza sativa revealed tissue specific expression of AAO genes wherein few members were exclusively expressed in either root or shoot. These genes were found to be regulated by both developmental cues as well as diverse stress conditions. The qRT-PCR analysis in response to salinity and drought stress in rice shoots revealed OsAAO2 to be the most stress responsive gene. On the other hand, OsAAO3 and OsAAO4 genes showed enhanced expression in roots under salinity/drought stresses. This study provides lead about important stress responsive AAO genes in various crop plants, which could be used to engineer climate resilient crop plants. PMID:28261251

  10. Genome-wide analysis and identification of cytokinin oxidase/dehydrogenase (CKX gene family in foxtail millet (Setaria italica

    Directory of Open Access Journals (Sweden)

    Yuange Wang

    2014-08-01

    Full Text Available Cytokinin oxidase/dehydrogenase (CKX; EC.1.5.99.12 regulates cytokinin (CK level in plants and plays an essential role in CK regulatory processes. CKX proteins are encoded by a small gene family with a varying number of members in different plants. In spite of their physiological importance, systematic analyses of SiCKX genes in foxtail millet have not yet been examined. In this paper, we report the genome wide isolation and characterization of SiCKXs using bioinformatic methods. A total of 11 members of the family were identified in the foxtail millet genome. SiCKX genes were distributed in seven chromosomes (chromosome 1, 3, 4, 5, 6, 7, and 11. The coding sequences of all the SiCKX genes were disrupted by introns, with numbers varying from one to four. These genes expanded in the genome mainly due to segmental duplication events. Multiple alignment and motif display results showed that all SiCKX proteins share FAD- and CK-binding domains. Putative cis-elements involved in Ca2 +-response, abiotic stress response, light and circadian rhythm regulation, disease resistance and seed development were present in the promoters of SiCKX genes. Expression data mining suggested that SiCKX genes have diverse expression patterns. Real-time PCR analysis indicated that all 11 SiCKX genes were up-regulated in embryos under 6-BA treatment, and some were NaCl or PEG inducible. Collectively, these results provide molecular insights into CKX research in plants.

  11. A first look at ARFome: dual-coding genes in mammalian genomes.

    Directory of Open Access Journals (Sweden)

    Wen-Yu Chung

    2007-05-01

    Full Text Available Coding of multiple proteins by overlapping reading frames is not a feature one would associate with eukaryotic genes. Indeed, codependency between codons of overlapping protein-coding regions imposes a unique set of evolutionary constraints, making it a costly arrangement. Yet in cases of tightly coexpressed interacting proteins, dual coding may be advantageous. Here we show that although dual coding is nearly impossible by chance, a number of human transcripts contain overlapping coding regions. Using newly developed statistical techniques, we identified 40 candidate genes with evolutionarily conserved overlapping coding regions. Because our approach is conservative, we expect mammals to possess more dual-coding genes. Our results emphasize that the skepticism surrounding eukaryotic dual coding is unwarranted: rather than being artifacts, overlapping reading frames are often hallmarks of fascinating biology.

  12. Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

    Indian Academy of Sciences (India)

    B. Mahendran; S. K. Ghosh; S. C. Kundu

    2006-04-01

    We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkwormspecies that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA and coxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) for coxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.

  13. Molecular detection of field isolates of Turkey Eimeria by polymerase chain reaction amplification of the cytochrome c oxidase I gene.

    Science.gov (United States)

    Rathinam, T; Gadde, U; Chapman, H D

    2015-07-01

    Oocysts of Eimeria spp. were isolated from litter samples obtained from 30 commercial turkey farms. Genomic DNA was extracted from clean oocysts, and polymerase chain amplification of the species-specific cytochrome c oxidase subunit I (COI) gene was performed for five species of turkey Eimeria. The species tested were Eimeria adenoeides, Eimeria meleagrimitis, Eimeria meleagridis, Eimeria dispersa, and Eimeria gallopavonis. All DNA samples were positive for E. meleagrimitis, nine were positive for E. adenoeides, two were positive for E. dispersa, and none for E. meleagridis and E. gallopavonis. E. meleagrimitis occurred as a single species in 21 (70 %) of the farms while 9 (30 %) farms had a mixed species with E. meleagrimitis and E. adenoeides and 2 (7 %) were triple positive with E. meleagrimitis, E. adenoeides, and E. dispersa. This is the first account of the field prevalence of turkey Eimeria species using molecular methods.

  14. Genome-wide identification and expression analysis of the polyamine oxidase gene family in sweet orange (Citrus sinensis).

    Science.gov (United States)

    Wang, Wei; Liu, Ji-Hong

    2015-01-25

    Polyamine oxidases (PAOs) are FAD-dependent enzymes associated with polyamine catabolism. In plants, increasing evidences support that PAO genes play essential roles in abiotic and biotic stresses response. In this study, six putative PAO genes (CsPAO1-CsPAO6) were unraveled in sweet orange (Citrus sinensis) using the released citrus genome sequences. A total of 203 putative cis-regulatory elements involved in hormone and stress response were predicted in 1.5-kb promoter regions at the upstream of CsPAOs. The CsPAOs can be divided into four major groups, with similar organizations with their counterparts of Arabidopsis thaliana. Transcripts of CsPAOs were detected in leaf, stem, cotyledon, and root, with the highest levels detected in the roots. The CsPAOs displayed various responses to exogenous treatments with polyamines and ABA and were differentially altered by abiotic stresses, including cold, salt, and mannitol. Overexpression of CsPAO3 in tobacco demonstrated that spermidine and spermine were decreased in the transgenic line, while putrescine was significantly enhanced, implying a potential role of this gene in polyamine back conversion. These data provide valuable knowledge for understanding the roles of the PAO genes in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Allelic variations in the CYBA gene of NADPH oxidase and risk of kidney complications in patients with type 1 diabetes.

    Science.gov (United States)

    Patente, Thiago A; Mohammedi, Kamel; Bellili-Muñoz, Naïma; Driss, Fathi; Sanchez, Manuel; Fumeron, Frédéric; Roussel, Ronan; Hadjadj, Samy; Corrêa-Giannella, Maria Lúcia; Marre, Michel; Velho, Gilberto

    2015-09-01

    Oxidative stress plays a pivotal role in the pathophysiology of diabetic nephropathy, and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system is an important source of reactive oxygen species in hyperglycemic conditions in the kidney. Plasma concentration of advanced oxidation protein products (AOPP), a marker of oxidative stress, is increased in patients with diabetic nephropathy. We investigated associations of variants in the CYBA gene, encoding the regulatory subunit p22(phox) of NADPH oxidase, with diabetic nephropathy and plasma AOPP and myeloperoxidase (MPO) concentrations in type 1 diabetic patients. Seven SNPs in the CYBA region were analyzed in 1357 Caucasian subjects with type 1 diabetes from the SURGENE (n=340), GENEDIAB (n=444), and GENESIS (n=573) cohorts. Duration of follow-up was 10, 9, and 6 years, respectively. Cox proportional hazards and logistic regression analyses were used to estimate hazard ratios (HR) or odds ratios (OR) for incidence and prevalence of diabetic nephropathy. The major G-allele of rs9932581 was associated with the incidence of renal events defined as new cases of microalbuminuria or the progression to a more severe stage of nephropathy during follow-up (HR 1.59, 95% CI 1.17-2.18, P=0.003) in SURGENE. The same allele was associated with established/advanced nephropathy (OR 1.52, 95% CI 1.22-1.92, P=0.0001) and with the incidence of end-stage renal disease (ESRD) (HR 2.01, 95% CI 1.30-3.24, P=0.001) in GENEDIAB/GENESIS pooled studies. The risk allele was also associated with higher plasma AOPP concentration in subsets of SURGENE and GENEDIAB, with higher plasma MPO concentration in a subset of GENEDIAB, and with lower estimated glomerular filtration rate (eGFR) in the three cohorts. In conclusion, a functional variant in the promoter of the CYBA gene was associated with lower eGFR and with prevalence and incidence of diabetic nephropathy and ESRD in type 1 diabetic patients. These results are consistent with

  16. Dual localized mitochondrial and nuclear proteins as gene expression regulators in plants?

    Directory of Open Access Journals (Sweden)

    Philippe eGiegé

    2012-09-01

    Full Text Available Mitochondria heavily depend on the coordinated expression of both mitochondrial and nuclear genomes because some of their most significant activities are held by multi-subunit complexes composed of both mitochondrial and nuclear encoded proteins. Thus, precise communication and signaling pathways are believed to exist between the two compartments. Proteins dual localized to both mitochondria and the nucleus make excellent candidates for a potential involvement in the envisaged communication. Here, we review the identified instances of dual localized nucleo-mitochondrial proteins with an emphasis on plant proteins and discuss their functions, which are seemingly mostly related to gene expression regulation. We discuss whether dual localization could be achieved by dual targeting and / or by re-localization and try to apprehend the signals required for the respective processes. Finally, we propose that in some instances, dual localized mitochondrial and nuclear proteins might act as retrograde signaling molecules for mitochondrial biogenesis.

  17. Molecular, phylogenetic and comparative genomic analysis of the cytokinin oxidase/dehydrogenase gene family in the Poaceae.

    Science.gov (United States)

    Mameaux, Sabine; Cockram, James; Thiel, Thomas; Steuernagel, Burkhard; Stein, Nils; Taudien, Stefan; Jack, Peter; Werner, Peter; Gray, John C; Greenland, Andy J; Powell, Wayne

    2012-01-01

    The genomes of cereals such as wheat (Triticum aestivum) and barley (Hordeum vulgare) are large and therefore problematic for the map-based cloning of agronomicaly important traits. However, comparative approaches within the Poaceae permit transfer of molecular knowledge between species, despite their divergence from a common ancestor sixty million years ago. The finding that null variants of the rice gene cytokinin oxidase/dehydrogenase 2 (OsCKX2) result in large yield increases provides an opportunity to explore whether similar gains could be achieved in other Poaceae members. Here, phylogenetic, molecular and comparative analyses of CKX families in the sequenced grass species rice, brachypodium, sorghum, maize and foxtail millet, as well as members identified from the transcriptomes/genomes of wheat and barley, are presented. Phylogenetic analyses define four Poaceae CKX clades. Comparative analyses showed that CKX phylogenetic groupings can largely be explained by a combination of local gene duplication, and the whole-genome duplication event that predates their speciation. Full-length OsCKX2 homologues in barley (HvCKX2.1, HvCKX2.2) and wheat (TaCKX2.3, TaCKX2.4, TaCKX2.5) are characterized, with comparative analysis at the DNA, protein and genetic/physical map levels suggesting that true CKX2 orthologs have been identified. Furthermore, our analysis shows CKX2 genes in barley and wheat have undergone a Triticeae-specific gene-duplication event. Finally, by identifying ten of the eleven CKX genes predicted to be present in barley by comparative analyses, we show that next-generation sequencing approaches can efficiently determine the gene space of large-genome crops. Together, this work provides the foundation for future functional investigation of CKX family members within the Poaceae. © 2011 National Institute of Agricultural Botany (NIAB). Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell

  18. Study of a possible role of the monoamine oxidase A (MAOA) gene in paranoid schizophrenia among a Chinese population.

    Science.gov (United States)

    Sun, Yuhui; Zhang, Jiexu; Yuan, Yanbo; Yu, Xin; Shen, Yan; Xu, Qi

    2012-01-01

    Monoamine oxidase A (MAOA) is the enzyme responsible for degradation of several monoamines, such as dopamine and serotonin that are considered as being two of the most important neurotransmitters involved in the pathophysiology of schizophrenia. To study a possible role of the MAOA gene in conferring susceptibility to schizophrenia, the present study genotyped the variable number of tandem repeat (VNTR) polymorphism and 41 SNPs across this gene among 555 unrelated patients with paranoid schizophrenia and 567 unrelated healthy controls. Quantitative real-time PCR analysis was employed to quantify expression of MAOA mRNA in 73 drug-free patients. While none of these genotyped DNA markers showed allelic association with paranoid schizophrenia, haplotypic association was found for the VNTR-rs6323, VNTR-rs1137070, and VNTR-rs6323-rs1137070 haplotypes in female subjects. Nevertheless, no significant change of the expression of MAOA mRNA was detected in either female or male patients with paranoid schizophrenia. Our study suggests that the interaction between genetic variants within the MAOA gene may contribute to an increased risk of paranoid schizophrenia, but the precise mechanism needs further investigation.

  19. Breadfruit (Artocarpus altilis) gibberellin 2-oxidase genes in stem elongation and abiotic stress response.

    Science.gov (United States)

    Zhou, Yuchan; Underhill, Steven J R

    2016-01-01

    Breadfruit (Artocarpus altilis) is a traditional staple tree crop in the Oceania. Susceptibility to windstorm damage is a primary constraint on breadfruit cultivation. Significant tree loss due to intense tropical windstorm in the past decades has driven a widespread interest in developing breadfruit with dwarf stature. Gibberellin (GA) is one of the most important determinants of plant height. GA 2-oxidase is a key enzyme regulating the flux of GA through deactivating biologically active GAs in plants. As a first step toward understanding the molecular mechanism of growth regulation in the species, we isolated a cohort of four full-length GA2-oxidase cDNAs, AaGA2ox1- AaGA2ox4 from breadfruit. Sequence analysis indicated the deduced proteins encoded by these AaGA2oxs clustered together under the C19 GA2ox group. Transcripts of AaGA2ox1, AaGA2ox2 and AaGA2ox3 were detected in all plant organs, but exhibited highest level in source leaves and stems. In contrast, transcript of AaGA2ox4 was predominantly expressed in roots and flowers, and displayed very low expression in leaves and stems. AaGA2ox1, AaGA2ox2 and AaGA2ox3, but not AaGA2ox4 were subjected to GA feedback regulation where application of exogenous GA3 or gibberellin biosynthesis inhibitor, paclobutrazol was shown to manipulate the first internode elongation of breadfruit. Treatments of drought or high salinity increased the expression of AaGA2ox1, AaGA2ox2 and AaGA2ox4. But AaGA2ox3 was down-regulated under salt stress. The function of AaGA2oxs is discussed with particular reference to their role in stem elongation and involvement in abiotic stress response in breadfruit. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  20. Estradiol plays a role in regulating the expression of lysyl oxidase family genes in mouse urogenital tissues and human Ishikawa cells.

    Science.gov (United States)

    Zong, Wen; Jiang, Yan; Zhao, Jing; Zhang, Jian; Gao, Jian-gang

    2015-10-01

    The lysyl oxidase (LOX) family encodes the copper-dependent amine oxidases that play a key role in determining the tensile strength and structural integrity of connective tissues by catalyzing the crosslinking of elastin or collagen. Estrogen may upregulate the expression of LOX and lysyl oxidase-like 1 (LOXL1) in the vagina. The objective of this study was to determine the effect of estrogen on the expression of all LOX family genes in the urogenital tissues of accelerated ovarian aging mice and human Ishikawa cells. Mice and Ishikawa cells treated with estradiol (E2) showed increased expression of LOX family genes and transforming growth factor β1 (TGF-β1). Ishikawa cells treated with TGF-β1 also showed increased expression of LOX family genes. The Ishikawa cells were then treated with either E2 plus the TGF-β receptor (TGFBR) inhibitor SB431542 or E2 alone. The expression of LOX family genes induced by E2 was reduced in the Ishikawa cells treated with TGFBR inhibitor. Our results showed that E2 increased the expression of the LOX family genes, and suggest that this induction may be mediated by the TGF-β signal pathway. E2 may play a role in regulating the expression of LOX family genes.

  1. Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

    Science.gov (United States)

    De Meyer, Sofie E; Briscoe, Leah; Martínez-Hidalgo, Pilar; Agapakis, Christina M; de-Los Santos, Paulina Estrada; Seshadri, Rekha; Reeve, Wayne; Weinstock, George; O'Hara, Graham; Howieson, John G; Hirsch, Ann M

    2016-08-01

    Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

  2. Involvement of the NADH oxidase-encoding noxA gene in oxidative stress responses in Corynebacterium glutamicum.

    Science.gov (United States)

    Park, Jung Chul; Kim, Younhee; Lee, Heung-Shick

    2015-02-01

    Corynebacterium glutamicum ORF NCgl0328, designated noxA, encodes an NADH oxidase enzyme. The noxA gene, which was preferentially expressed in the log growth phase, was found to be under the control of the whcA, whcB, and whcE genes, which play regulatory roles in cells under oxidative stress. While noxA transcription was minimal in whcE-deleted mutant cells (ΔwhcE) during growth, its transcription was maximal even in the stationary phase in ΔwhcA cells. The transcription levels of noxA in ΔwhcB and whcB-overexpressing cells were comparable to the levels only in the log growth phase in ΔwhcA and whcA-overexpressing cells, respectively. Direct binding of purified WhcA to the promoter region of noxA was observed in vitro. The DNA-protein interaction was only possible in the presence of the reducing agent dithiothreitol. A noxA-deleted mutant strain and a strain overexpressing the noxA gene (P180-noxA) were established, and these strains were found to exhibit defective cell growth. The ΔnoxA and P180-noxA strains were sensitive to the redox-cycling oxidant menadione, suggesting a role of noxA in redox balancing. Accordingly, the purified NoxA enzyme exhibited NADH-oxidizing activity. Taken together, these data show that noxA plays a role in oxidative stress responses and also that the gene is under direct control of the WhcA protein, which was shown to be a regulatory DNA-binding protein. Furthermore, the involvement and roles of the whcA, whcB, and whcE genes in regulating the expression of noxA were demonstrated.

  3. A versatile and efficient markerless gene disruption system for Acidithiobacillus thiooxidans: application for characterizing a copper tolerance related multicopper oxidase gene.

    Science.gov (United States)

    Wen, Qing; Liu, Xiangmei; Wang, Huiyan; Lin, Jianqun

    2014-11-01

    The acidophilic bioleaching bacteria can usually survive in high concentrations of copper ions because of their special living environment. However, little is known about the copper homeostatic mechanisms of Acidithiobacillus thiooxidans, an important member of bioleaching bacteria. Here, a putative multicopper oxidase gene (cueO) was detected from the draft genome of A. thiooxidans ATCC 19377. The transcriptional level of cueO in response to 10 mM CuSO₄was upregulated 25.01 ± 2.59 folds. The response of P(cueO) to copper was also detected and might be stimulated by a putative CueR protein. Then, by using the counter-selectable marker lacZ and enhancing the expression of endonuclease I-SceI with tac promoter, a modified markerless gene disruption system was developed and the cueO gene disruption mutant (ΔcueO) of A. thiooxidans was successfully constructed with a markedly improved second homologous recombination frequency of 0.28 ± 0.048. The ΔcueO mutant was more sensitive to external copper and nearly completely lost the phenoloxidase activity; however, the activity could be restored after complementing the cueO gene. All results suggest the close relation of cueO gene to copper tolerance in A. thiooxidans. In addition, the developed efficient markerless gene knockout method can also be introduced into other Acidithiobacillus strains.

  4. The Influence of Co-Suppressing Tomato 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Ⅰ on the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

    Institute of Scientific and Technical Information of China (English)

    HU Zong-li; CHEN Xu-qing; CHEN Guo-ping; L(U) Li-juan; Grierson Donald

    2007-01-01

    The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase I on the expression of fruit ripening-related and pathogenesis-related protein genes, and on the biosynthesis of endogenous ethylene and storage ability of fruits. Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato (Lycopersicon esculentum) were cloned, such as the 1-aminocyclopropane-1-carboxylic acid oxidase 1 gene (LeACO1), 1-aminocyclopropane-1-carboxylic acid oxidase 3 gene (LeAC03), EIN3-binding F-box 1 gene (LeEBF1), pathogenesisrelated protein 1 gene (LePR1), pathogenesis-related protein 5 gene (LePR5), and pathogenesis-related protein osmotin precursor gene (LeNP24) by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato Ailsa Craig (AC++) and the LeACO1 co-suppression tomatoes (V1187 and T4B), respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carried out. The hybridization results indicated that the expression of fruit ripening-related genes such as LeACO3 and LeEBF1, and pathogenesis-related protein genes such as LePR1, LePR5, and LeNP24, were reduced sharply, and the ethylene production in the fruits, wounded leaves decreased and the storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.

  5. Genetic characterization of Bagarius species using cytochrome c oxidase I and cytochrome b genes.

    Science.gov (United States)

    Nagarajan, Muniyandi; Raja, Manikam; Vikram, Potnuru

    2016-09-01

    In this study, we first inferred the genetic variability of two Bagarius bagarius populations collected from Ganges and Brahmaputra rivers of India using two mtDNA markers. Sequence analysis of COI gene did not show significant differences between two populations whereas cytochrome b gene showed significant differences between two populations. Followed by, genetic relationship of B. bagarius and B. yarrielli was analyzed using COI and cytochrome b gene and the results showed a higher level genetic variation between two species. The present study provides support for the suitability of COI and cytochrome b genes for the identification of B. bagarius and B. yarrielli.

  6. Risk assessment for Helicoverpa zea (Lepidoptera: Noctuidae) resistance on dual-gene versus single-gene corn.

    Science.gov (United States)

    Edwards, Kristine T; Caprio, Michael A; Allen, K Clint; Musser, Fred R

    2013-02-01

    Recent Environmental Protection Agency (EPA) decisions regarding resistance management in Bt-cropping systems have prompted concern in some experts that dual-gene Bt-corn (CrylA.105 and Cry2Ab2 toxins) may result in more rapid selection for resistance in Helicoverpa zea (Boddie) than single-gene Bacillus thuringiensis (Bt)-corn (CrylAb toxin). The concern is that Bt-toxin longevity could be significantly reduced with recent adoption of a natural refuge for dual-gene Bt-cotton (CrylAc and Cry2Ab2 toxins) and concurrent reduction in dual-gene corn refuge from 50 to 20%. A population genetics framework that simulates complex landscapes was applied to risk assessment. Expert opinions on effectiveness of several transgenic corn and cotton varieties were captured and used to assign probabilities to different scenarios in the assessment. At least 350 replicate simulations with randomly drawn parameters were completed for each of four risk assessments. Resistance evolved within 30 yr in 22.5% of simulations with single-gene corn and cotton with no volunteer corn. When volunteer corn was added to this assessment, risk of resistance evolving within 30 yr declined to 13.8%. When dual-gene Bt-cotton planted with a natural refuge and single-gene corn planted with a 50% structured refuge was simulated, simultaneous resistance to both toxins never occurred within 30 yr, but in 38.5% of simulations, resistance evolved to toxin present in single-gene Bt-corn (CrylAb). When both corn and cotton were simulated as dual-gene products, cotton with a natural refuge and corn with a 20% refuge, 3% of simulations evolved resistance to both toxins simultaneously within 30 yr, while 10.4% of simulations evolved resistance to CrylAb/c toxin.

  7. Structural, phylogenetic and docking studies of D-amino acid oxidase activator (DAOA, a candidate schizophrenia gene

    Directory of Open Access Journals (Sweden)

    Sehgal Sheikh

    2013-01-01

    Full Text Available Abstract Background Schizophrenia is a neurodegenerative disorder that occurs worldwide and can be difficult to diagnose. It is the foremost neurological disorder leading to suicide among patients in both developed and underdeveloped countries. D-amino acid oxidase activator (DAOA, also known as G72, is directly implicated in the glutamateric hypothesis of schizophrenia. It activates D-amino acid oxidase, which oxidizes D-serine, leading to modulation of the N-methyl-D-aspartate receptor. Methods MODELLER (9v10 was utilized to generate three dimensional structures of the DAOA candidate gene. The HOPE server was used for mutational analysis. The Molecular Evolutionary Genetics Analysis (MEGA5 tool was utilized to reconstruct the evolutionary history of the candidate gene DAOA. AutoDock was used for protein-ligand docking and Gramm-X and PatchDock for protein-protein docking. Results A suitable template (1ZCA was selected by employing BLASTp on the basis of 33% query coverage, 27% identity and E-value 4.9. The Rampage evaluation tool showed 91.1% favored region, 4.9% allowed region and 4.1% outlier region in DAOA. ERRAT demonstrated that the predicted model had a 50.909% quality factor. Mutational analysis of DAOA revealed significant effects on hydrogen bonding and correct folding of the DAOA protein, which in turn affect protein conformation. Ciona was inferred as the outgroup. Tetrapods were in their appropriate clusters with bifurcations. Human amino acid sequences are conserved, with chimpanzee and gorilla showing more than 80% homology and bootstrap value based on 1000 replications. Molecular docking analysis was employed to elucidate the binding mode of the reported ligand complex for DAOA. The docking experiment demonstrated that DAOA is involved in major amino acid interactions: the residues that interact most strongly with the ligand C28H28N3O5PS2 are polar but uncharged (Gln36, Asn38, Thr 122 and non-polar hydrophobic (Ile119, Ser171

  8. Identification of a gene causing human cytochrome c oxidase deficiency by integrative genomics

    DEFF Research Database (Denmark)

    Mootha, Vamsi K; Lepage, Pierre; Miller, Kathleen;

    2003-01-01

    Identifying the genes responsible for human diseases requires combining information about gene position with clues about biological function. The recent availability of whole-genome data sets of RNA and protein expression provides powerful new sources of functional insight. Here we illustrate how...

  9. Characterization of Fasciola hepatica genotypes from cattle and sheep in Iran using cytochrome C oxidase gene (CO1).

    Science.gov (United States)

    Moazeni, Mohammad; Sharifiyazdi, Hassan; Izadpanah, Afshin

    2012-06-01

    The present study compared the genetic variation among 19 different isolates of Fasciola hepatica from cattle and sheep in different areas of Iran using sequence data for mitochondrial DNA gene, the subunit 1 of cytochrome C oxidase gene (CO1). Four different CO1 genotypes were detected among F. hepatica isolates that showed five variable nucleotide positions (accession nos.; GQ398051, GQ398052, GQ398053, GQ398054). Nucleotide sequence variation among 19 isolates for CO1 analyzed in this study ranged from 0% to 0.98% in Iran. Among the five polymorphism sites identified in this study, only one (T to G at position 51 in 5'end of GQ175362) resulted in putative amino acid alteration of phenylalanine (TTT) to leucine (TTG) in CO1. A phylogenetic analysis of the sequence data revealed that host associations and geographic location are likely not useful markers for Fasciola genotype classification. In addition, morphological analysis showed that the ratios of body length and body width of some (n = 5) of the 19 examined F. hepatica isolates were intermediate between F. hepatica and Fasciola gigantica, representing the substantial polymorphism of the F. hepatica species and the difficulty in the accurate recognition based on morphological features. In conclusion, Iranian F. hepatica exhibited the presence of considerable genetic diversity at CO1.

  10. Apparent selection intensity for the cytochrome oxidase subunit I gene varies with mode of reproduction in echinoderms.

    Science.gov (United States)

    Foltz, David W; Hrincevich, Adam W; Rocha-Olivares, Axayácatl

    2004-10-01

    When most amino acid substitutions in protein-coding genes are slightly deleterious rather than selectively neutral, life history differences can potentially modify the effective population size or the selective regime, resulting in altered ratios of non-synonymous to synonymous substitutions among taxa. We studied substitution patterns for the mitochondrial cytochrome oxidase subunit I (COI) gene in a sea star genus (Leptasterias spp.) with an obligate brood-protecting mode of reproduction and small-scale population genetic subdivision, and compared the results to available COI sequences in nine other genera of echinoderms with pelagic larvae: three sea stars, five sea urchins and one brittle star. We predicted that this life history difference would be associated with differences in the ratio of non-synonymous (dN) to synonymous (dS) substitution rates. Leptasterias had a significantly greater dN/dS ratio (both between species and within species), a significantly smaller transition/transversion rate ratio, and a significantly lower average nucleotide diversity within species, than did the non-brooding genera. Other explanations for the results, such as altered mutation rates or selective sweeps, were not supported by the data analysis. These findings highlight the potential influence of reproductive traits and other life history factors on patterns of nucleotide substitution within and between species.

  11. Novel Homozygous Missense Mutation in SPG20 Gene Results in Troyer Syndrome Associated with Mitochondrial Cytochrome c Oxidase Deficiency.

    Science.gov (United States)

    Spiegel, Ronen; Soiferman, Devorah; Shaag, Avraham; Shalev, Stavit; Elpeleg, Orly; Saada, Ann

    2016-08-19

    Troyer syndrome is an autosomal recessive form of hereditary spastic paraplegia (HSP) caused by deleterious mutations in the SPG20 gene. Although the disease is associated with a loss of function mechanism of spartin, the protein encoded by SPG20, the precise pathogenesis is yet to be elucidated. Recent data indicated an important role for spartin in both mitochondrial maintenance and function. Here we report a child presenting with progressive spastic paraparesis, generalized muscle weakness, dysarthria, impaired growth, and severe isolated decrease in muscle cytochrome c oxidase (COX) activity. Whole exome sequencing identified the homozygous c.988A>G variant in SPG20 gene (p.Met330Val) resulting in almost complete loss of spartin in skeletal muscle. Further analyses demonstrated significant tissue specific reduction of COX 4, a nuclear encoded subunit of COX, in muscle suggesting a role for spartin in proper mitochondrial respiratory chain function mediated by COX activity. Our findings need to be verified in other Troyer syndrome patients in order to classify it as a form of HSP caused by mitochondrial dysfunction.

  12. Diversity and abundance of the arsenite oxidase gene aioA in geothermal areas of Tengchong, Yunnan, China.

    Science.gov (United States)

    Jiang, Zhou; Li, Ping; Jiang, Dawei; Wu, Geng; Dong, Hailiang; Wang, Yanhong; Li, Bing; Wang, Yanxin; Guo, Qinghai

    2014-01-01

    A total of 12 samples were collected from the Tengchong geothermal areas of Yunnan, China, with the goal to assess the arsenite (AsIII) oxidation potential of the extant microbial communities as inferred by the abundance and diversity of the AsIII oxidase large subunit gene aioA relative to geochemical context. Arsenic concentrations were higher (on average 251.68 μg/L) in neutral or alkaline springs than in acidic springs (on average 30.88 μg/L). aioA abundance ranged from 1.63 × 10(1) to 7.08 × 10(3) per ng of DNA and positively correlated with sulfide and the ratios of arsenate (AsV):total dissolved arsenic (AsTot). Based on qPCR estimates of bacterial and archaeal 16S rRNA gene abundance, aioA-harboring organisms comprised as much as ~15% of the total community. Phylogenetically, the major aioA sequences (270 total) in the acidic hot springs (pH 3.3-4.4) were affiliated with Aquificales and Rhizobiales, while those in neutral or alkaline springs (pH 6.6-9.1) were inferred to be primarily bacteria related to Thermales and Burkholderiales. Interestingly, aioA abundance at one site greatly exceeded bacterial 16S rRNA gene abundance, suggesting these aioA genes were archaeal even though phylogenetically these aioA sequences were most similar to the Aquificales. In summary, this study described novel aioA sequences in geothermal features geographically far removed from those in the heavily studied Yellowstone geothermal complex.

  13. Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits.

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    Yu-Li Liu

    Full Text Available D-amino acid oxidase (DAO has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016. This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001 and replicated samples (corrected p = 0.0003. The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia.

  14. Association of nicotinamide adenine dinucleotide phosphate oxidase p22phox gene 549C>T polymorphism with coronary artery disease

    Institute of Scientific and Technical Information of China (English)

    LIU Tong-tao; WANG Li-li; FANG Sheng-xia; JIA Chong-qi

    2012-01-01

    Background The p22phox is a critical component of the superoxide-generating vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.Several polymorphisms in p22phox gene are studied for their association with cardiovascular diseases.However,no publication is available to assess the relation of 549C>T polymorphism in p22phox gene to coronary artery disease (CAD) risk.This study was to investigate the effect of the p22phox gene 549C>T polymorphism on CAD risk.Methods Hospital-based case-control study was conducted with 297 CAD patients and 343 healthy persons as the control group.Polymerase chain reaction and pyrosequencing using PSQ 96 MA Pyrosequencer (Biotage AB) were used to detect the polymorphisms.Multiple Logistic regression model was used to adjust the potential confounders and to estimate odds ratio (OR) with 95% confidence intervals (Cls).Results The observed genotype frequencies of this polymorphism obeyed the Hardy-Weinberg equilibrium in both cases (P=0.439) and controls (P=0.668).The frequency of mutant genotypes (TT+CT) in cases (41.08%) was higher than that in controls (36.73%) with an OR=-1.20 (95% CI=0.87-1.65).After the adjustment of the potential confounders,there was a significant association of the mutant genotypes with increased risk of CAD (OR=1.57,95% CI=1.01-2.46,P=0.047).Conclusions The mutant genotypes of the p22phox gene 549C>T polymorphism had a significant effect on the increased risk of CAD in this studied population.

  15. Expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and mitochondrial oxidative phosphorylation in septic patients

    Science.gov (United States)

    Nucci, Laura A.; Santos, Sidnéia S.; Brunialti, Milena K. C.; Sharma, Narendra Kumar; Machado, Flavia R.; Assunção, Murillo; de Azevedo, Luciano C. P.

    2017-01-01

    Background and objectives Sepsis is a complex disease that is characterized by activation and inhibition of different cell signaling pathways according to the disease stage. Here, we evaluated genes involved in the TLR signaling pathway, oxidative phosphorylation and oxidative metabolism, aiming to assess their interactions and resulting cell functions and pathways that are disturbed in septic patients. Materials and methods Blood samples were obtained from 16 patients with sepsis secondary to community acquired pneumonia at admission (D0), and after 7 days (D7, N = 10) of therapy. Samples were also collected from 8 healthy volunteers who were matched according to age and gender. Gene expression of 84 genes was performed by real-time polymerase chain reactions. Their expression was considered up- or down-regulated when the fold change was greater than 1.5 compared to the healthy volunteers. A p-value of ≤ 0.05 was considered significant. Results Twenty-two genes were differently expressed in D0 samples; most of them were down-regulated. When gene expression was analyzed according to the outcomes, higher number of altered genes and a higher intensity in the disturbance was observed in non-survivor than in survivor patients. The canonical pathways altered in D0 samples included interferon and iNOS signaling; the role of JAK1, JAK2 and TYK2 in interferon signaling; mitochondrial dysfunction; and superoxide radical degradation pathways. When analyzed according to outcomes, different pathways were disturbed in surviving and non-surviving patients. Mitochondrial dysfunction, oxidative phosphorylation and superoxide radical degradation pathway were among the most altered in non-surviving patients. Conclusion Our data show changes in the expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and oxidative phosphorylation. Importantly, distinct patterns are clearly observed in surviving and non-surviving patients. Interferon signaling, marked by

  16. Hodgkin-Reed-Sternberg cells in classical Hodgkin lymphoma show alterations of genes encoding the NADPH oxidase complex and impaired reactive oxygen species synthesis capacity.

    Science.gov (United States)

    Giefing, Maciej; Winoto-Morbach, Supandi; Sosna, Justyna; Döring, Claudia; Klapper, Wolfram; Küppers, Ralf; Böttcher, Sebastian; Adam, Dieter; Siebert, Reiner; Schütze, Stefan

    2013-01-01

    The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL.

  17. Uric acid stimulates endothelin-1 gene expression associated with NADPH oxidase in human aortic smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Hung-hsing CHAO; Ju-chi LIU; Jia-wei LIN; Cheng-hsien CHEN; Chieh-hsi WU; Tzu-hurng CHENG

    2008-01-01

    Aim: Recent experimental and human studies have shown that hyperuricemia is associated with hypertension and cardiovascular diseases. Elevated levels of endotheliu-1 (ET-1) has been regarded as one of the most powerful indepen-dent predictors of cardiovascular diseases. For investigating whether uric acidinduced vascular diseases are related to ET-1, the uric acid-induced ET-1 expression in human aortic smooth muscle cells (HASMC) was examined. Methods: Cultured HASMC treated with uric acid, cell proliferation and ET-1 expression were examined. Antioxidant pretreatments on uric acid-induced extracellular signal-regulated kinases (ERK) phosphorylation were carried out to elucidate the redox-sensitive pathway in proliferation and ET-1 gene expression. Results: Uric acid was found to increase HASMC proliferation, ET-1 expression and reactive oxygen species production. The ability of both N-acetylcysteine and apocynin (1-[4-hydroxy-3-methoxyphenyl]ethanone, a NADPH oxidase inhibitor) to inhibit uric acid-induced ET-1 secretion and cell proliferation suggested the involvement of intracellular redox pathways. Furthermore, apocynin, and p47phox small interfering RNA knockdown inhibited ET-1 secretion and cell proliferation induced by uric acid. Inhibition of ERK by U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) significantly suppressed uric acid-induced ET-I expression, implicating this pathway in the response to uric acid. In addition, uric acid increased the transcription factor activator protein-1 (AP-1) medi-ated reporter activity, as well as the ERK phosphorylation. Mutational analysis of the ET-1 gene promoter showed that the AP-1 binding site was an important cis-element in uric acid-induced ET-1 gene expression. Conclusion: This is the first observation of ET-1 regulation by uric acid in HASMC, which implicates the important role of uric acid in the vascular changes associated with hypertension and vascular diseases.

  18. Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression.

    Science.gov (United States)

    Macková, Hana; Hronková, Marie; Dobrá, Jana; Turečková, Veronika; Novák, Ondřej; Lubovská, Zuzana; Motyka, Václav; Haisel, Daniel; Hájek, Tomáš; Prášil, Ilja Tom; Gaudinová, Alena; Štorchová, Helena; Ge, Eva; Werner, Tomáš; Schmülling, Thomas; Vanková, Radomíra

    2013-07-01

    Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

  19. D-amino acid oxidase gene therapy sensitizes glioma cells to the antiglycolytic effect of 3-bromopyruvate.

    Science.gov (United States)

    El Sayed, S M; Abou El-Magd, R M; Shishido, Y; Chung, S P; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

    2012-01-01

    Glioma tumors are refractory to conventional treatment. Glioblastoma multiforme is the most aggressive type of primary brain tumors in humans. In this study, we introduce oxidative stress-energy depletion (OSED) therapy as a new suggested treatment for glioblastoma. OSED utilizes D-amino acid oxidase (DAO), which is a promising therapeutic protein that induces oxidative stress and apoptosis through generating hydrogen peroxide (H2O2). OSED combines DAO with 3-bromopyruvate (3BP), a hexokinase II (HK II) inhibitor that interferes with Warburg effect, a metabolic alteration of most tumor cells that is characterized by enhanced aerobic glycolysis. Our data revealed that 3BP induced depletion of energetic capabilities of glioma cells. 3BP induced H2O2 production as a novel mechanism of its action. C6 glioma transfected with DAO and treated with D-serine together with 3BP-sensitized glioma cells to 3BP and decreased markedly proliferation, clonogenic power and viability in a three-dimensional tumor model with lesser effect on normal astrocytes. DAO gene therapy using atelocollagen as an in vivo transfection agent proved effective in a glioma tumor model in Sprague-Dawley (SD) rats, especially after combination with 3BP. OSED treatment was safe and tolerable in SD rats. OSED therapy may be a promising therapeutic modality for glioma.

  20. Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene.

    Science.gov (United States)

    Sánchez, Elizabeth; Cáceres, Omar; Náquira, César; Garcia, David; Patiño, Gladys; Silvia, Herrera; Volotão, Aline C; Fernandes, Octavio

    2010-09-01

    Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  1. Molecular characterization of Echinococcus granulosusfrom Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

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    Elizabeth Sánchez

    2010-09-01

    Full Text Available Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10, have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44, sheep (41 and humans (14 from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4. The G1 genotype is considered the most widespread and infectious form of E. granulosusworldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  2. ASSESSMENT OF GENETIC VARIATION OF PEARL OYSTER, Pinctada maxima, BASED ON THE ANALYSIS OF MITOCHONDRIAL CYTOCHROME OXIDASE SUBUNIT I GENE

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    Achmad Sudradjat

    2009-06-01

    Full Text Available Pearl oyster, Pinctada maxima is one of economical ly important species in aquaculture, particularly in pearl industry. Information on genetic variation of pearl oyster is required in order to be able to make a sound management of it’s natural populations and to utilize it to improve the quality of pearl culture. Five populations from different geographic locations of pearl oyster, Pinctada maxima, (Sumbawa, Bali, Selat Sunda, Belitung, and South Sulawesi were analyzed for genetic variation within a 750-base pair region of the Mitochondrial Cytochrome Oxidase subunit I (MtCOI gene using Restriction Fragment Length Polymorphism (RFLP technique. The analysis of 25 pearl oyster samples, their haplotype diversity ranged from 0.0970 to 0.1939 and the number of haplotype in each population ranged from three to five haplotypes. Clustering of populations based on Nei’s genetic distances and constructed using unweighted pair-group method with Arithmetic mean (UPGMA showed that the populations were clustered into two groups: Belitung, Selat Sunda, Bali and Sumbawa in one group, while South Sulawesi in the second group.

  3. Population genetic structure of Gasterophilus pecorum in the Kalamaili Nature Reserve, Xinjiang, based on mitochondrial cytochrome oxidase (COI) gene sequence.

    Science.gov (United States)

    Wang, W; Zhang, D; Hu, D; Chu, H; Cao, J; Ente, M; Jiang, G; Li, K

    2014-08-01

    Gasterophilosis is a significant threat to equids in the desert steppe of Xinjiang, China, where Gasterophilus pecorum (Fabricius) (Diptera: Gasterophilidae) is the dominant botfly species. A population analysis was conducted on 195 individual G. pecorum larvae from three host species, Przewalski's horse, the domestic horse and the Asiatic wild ass. The distribution of haplotypes of the maternally inherited mitochondrial cytochrome oxidase subunit I (COI) gene was analysed to assess the population differentiation of G. pecorum. High haplotype diversity was observed among G. pecorum populations from all host species, indicating that the G. pecorum infecting one host had multiple maternal ancestors. A phylogenetic tree showed six clades, suggesting a high degree of genetic differentiation. A constructed haplotype network described both the origin of the haplotypes and the population structure. The findings indicated that G. pecorum infections within Przewalski's horses were mainly transmitted from Asiatic wild asses. Clade 1 was found to be the most primitive group and to have evolved to be highly adaptable to the desert steppe. Clade 2 originated from Clade 1, potentially as a result of the annual migration of domestic horses. Revealing the differentiation of the G. pecorum population is important for elucidating the aetiology of Gasterophilus infection in Xinjiang and for planning appropriate control measures. © 2014 The Royal Entomological Society.

  4. Combination of polymorphic variants in serotonin transporter and monoamine oxidase-A genes may influence the risk for early-onset alcoholism.

    Science.gov (United States)

    Bordukalo-Niksic, Tatjana; Stefulj, Jasminka; Matosic, Ana; Mokrovic, Gordana; Cicin-Sain, Lipa

    2012-12-30

    The combinatory effect of polymorphisms in serotonin transporter and monoamine oxidase-A genes on the aetiopathogenesis of alcoholism was investigated in a sample of 714 individuals. Increased frequency of subjects having three 'suspected' genotypes (5-HTTLPR-LL, STin2-1010 and MAO-A 3-repeat allele) was found among type-2 alcoholic patients (P=0.0189). Results highlight serotonergic/genetic contribution to early-onset alcoholism.

  5. Alternative Oxidase Gene Family in Hypericum perforatum L.: Characterization and Expression at the Post-germinative Phase

    Science.gov (United States)

    Velada, Isabel; Cardoso, Hélia G.; Ragonezi, Carla; Nogales, Amaia; Ferreira, Alexandre; Valadas, Vera; Arnholdt-Schmitt, Birgit

    2016-01-01

    Alternative oxidase (AOX) protein is located in the inner mitochondrial membrane and is encoded in the nuclear genome being involved in plant response upon a diversity of environmental stresses and also in normal plant growth and development. Here we report the characterization of the AOX gene family of Hypericum perforatum L. Two AOX genes were identified, both with a structure of four exons (HpAOX1, acc. KU674355 and HpAOX2, acc. KU674356). High variability was found at the N-terminal region of the protein coincident with the high variability identified at the mitochondrial transit peptide. In silico analysis of regulatory elements located at intronic regions identified putative sequences coding for miRNA precursors and trace elements of a transposon. Simple sequence repeats were also identified. Additionally, the mRNA levels for the HpAOX1 and HpAOX2, along with the ones for the HpGAPA (glyceraldehyde-3-phosphate dehydrogenase A subunit) and the HpCAT1 (catalase 1), were evaluated during the post-germinative development. Gene expression analysis was performed by RT-qPCR with accurate data normalization, pointing out HpHYP1 (chamba phenolic oxidative coupling protein 1) and HpH2A (histone 2A) as the most suitable reference genes (RGs) according to GeNorm algorithm. The HpAOX2 transcript demonstrated larger stability during the process with a slight down-regulation in its expression. Contrarily, HpAOX1 and HpGAPA (the corresponding protein is homolog to the chloroplast isoform involved in the photosynthetic carbon assimilation in other plant species) transcripts showed a marked increase, with a similar expression pattern between them, during the post-germinative development. On the other hand, the HpCAT1 (the corresponding protein is homolog to the major H2O2-scavenging enzyme in other plant species) transcripts showed an opposite behavior with a down-regulation during the process. In summary, our findings, although preliminary, highlight the importance to

  6. Alternative Oxidase Gene Family in Hypericum perforatum L.: Characterization and Expression at the Post-germinative Phase.

    Science.gov (United States)

    Velada, Isabel; Cardoso, Hélia G; Ragonezi, Carla; Nogales, Amaia; Ferreira, Alexandre; Valadas, Vera; Arnholdt-Schmitt, Birgit

    2016-01-01

    Alternative oxidase (AOX) protein is located in the inner mitochondrial membrane and is encoded in the nuclear genome being involved in plant response upon a diversity of environmental stresses and also in normal plant growth and development. Here we report the characterization of the AOX gene family of Hypericum perforatum L. Two AOX genes were identified, both with a structure of four exons (HpAOX1, acc. KU674355 and HpAOX2, acc. KU674356). High variability was found at the N-terminal region of the protein coincident with the high variability identified at the mitochondrial transit peptide. In silico analysis of regulatory elements located at intronic regions identified putative sequences coding for miRNA precursors and trace elements of a transposon. Simple sequence repeats were also identified. Additionally, the mRNA levels for the HpAOX1 and HpAOX2, along with the ones for the HpGAPA (glyceraldehyde-3-phosphate dehydrogenase A subunit) and the HpCAT1 (catalase 1), were evaluated during the post-germinative development. Gene expression analysis was performed by RT-qPCR with accurate data normalization, pointing out HpHYP1 (chamba phenolic oxidative coupling protein 1) and HpH2A (histone 2A) as the most suitable reference genes (RGs) according to GeNorm algorithm. The HpAOX2 transcript demonstrated larger stability during the process with a slight down-regulation in its expression. Contrarily, HpAOX1 and HpGAPA (the corresponding protein is homolog to the chloroplast isoform involved in the photosynthetic carbon assimilation in other plant species) transcripts showed a marked increase, with a similar expression pattern between them, during the post-germinative development. On the other hand, the HpCAT1 (the corresponding protein is homolog to the major H2O2-scavenging enzyme in other plant species) transcripts showed an opposite behavior with a down-regulation during the process. In summary, our findings, although preliminary, highlight the importance to

  7. Coptotermes gestroi (Isoptera: Rhinotermitidae) in Brazil: possible origins inferred by mitochondrial cytochrome oxidase II gene sequences.

    Science.gov (United States)

    Martins, C; Fontes, L R; Bueno, O C; Martins, V G

    2010-09-01

    The Asian subterranean termite, Coptotermes gestroi, originally from northeast India through Burma, Thailand, Malaysia, and the Indonesian archipelago, is a major termite pest introduced in several countries around the world, including Brazil. We sequenced the mitochondrial COII gene from individuals representing 23 populations. Phylogenetic analysis of COII gene sequences from this and other studies resulted in two main groups: (1) populations of Cleveland (USA) and four populations of Malaysia and (2) populations of Brazil, four populations of Malaysia, and one population from each of Thailand, Puerto Rico, and Key West (USA). Three new localities are reported here, considerably enlarging the distribution of C. gestroi in Brazil: Campo Grande (state of Mato Grosso do Sul), Itajaí (state of Santa Catarina), and Porto Alegre (state of Rio Grande do Sul).

  8. Utility of the cytochrome c oxidase subunit I gene for the diagnosis of toxoplasmosis using PCR.

    Science.gov (United States)

    Feng, Xue; Norose, Kazumi; Li, Kexin; Hikosaka, Kenji

    2017-10-01

    Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, which belongs to the phylum Apicomplexa. Since this parasite causes severe clinical symptoms in immunocompromised patients, early diagnosis of toxoplasmosis is essential. PCR is currently used for early diagnosis, but there is no consensus regarding the most effective method for amplifying Toxoplasma DNA. In this study, we considered the utility of the cytochrome c subunit I (cox1) gene, which is encoded in the mitochondrial DNA of this parasite, as a novel target of PCR for the diagnosis of toxoplasmosis. To do this, we compared its copy number per haploid nuclear genome and the detection sensitivity of cox1-PCR with the previously reported target genes B1 and 18S rRNA and the AF146527 repeat element. We found that the copy number of cox1 was high and that the PCR using cox1 primers was more efficient at amplifying Toxoplasma DNA than the other PCR targets examined. In addition, PCR using clinical samples indicated that the cox1 gene would be useful for the diagnosis of toxoplasmosis. These findings suggest that use of cox1-PCR would facilitate the diagnosis of toxoplasmosis in clinical laboratories. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences.

    Science.gov (United States)

    Azevedo, M I; Botton, S A; Pereira, D I B; Robe, L J; Jesus, F P K; Mahl, C D; Costa, M M; Alves, S H; Santurio, J M

    2012-09-14

    Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum. Copyright © 2012 Elsevier

  10. Exploring laccase-like multicopper oxidase genes from the ascomycete Trichoderma reesei: a functional, phylogenetic and evolutionary study

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    Levasseur Anthony

    2010-08-01

    Full Text Available Abstract Background The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO genes. The purpose of this work was i to validate the function of a candidate LMCO gene from T. reesei, and ii to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection. Results After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detected when ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followed by biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimal temperature and pH were respectively 40-45°C and 4, by using ABTS as substrate. TrLAC1 showed broad pH stability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was also confirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed light on the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO and classical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and the results showed that positive selection drove the evolution of sequences leading to well-known laccases involved in ligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functional studies between classical laccases and LMCO from T. reesei. Conclusions Homologous production and evolutionary studies of the first

  11. Alternative oxidase gene family in Hypericum perforatum L.: characterization and expression at the post-germinative phase

    Directory of Open Access Journals (Sweden)

    Isabel Velada

    2016-08-01

    Full Text Available Alternative oxidase (AOX protein is located in the inner mitochondrial membrane and is encoded in the nuclear genome being involved in plant response upon diversity of environmental stresses and also in normal plant growth and development. Here we report the characterization of the AOX gene family of Hypericum perforatum L. Two AOX genes were identified, both with a structure of four exons (HpAOX1, acc. KU674355 and HpAOX2, acc. KU674356. High variability was found at the N-terminal region of the protein coincident with the high variability identified at the mitochondrial transit peptide. In silico analysis of regulatory elements located at intronic regions identified putative sequences coding for miRNA precursors and trace elements of a transposon. Simple sequence repeats were also identified. Additionally, the mRNA levels for the HpAOX1 and HpAOX2, along with the ones for the HpGAPA and the HpCAT1, were evaluated during the post-germinative development. The gene expression analysis was performed by RT-qPCR with accurate data normalization, pointing out HpHYP1 and HpH2A as the most suitable RGs according to GeNorm algorithm. The HpAOX2 transcript demonstrated larger stability during the process with a slight down-regulation in its expression. Contrarily, HpAOX1 and HpGAPA (the corresponding protein is homolog to the chloroplast isoform involved in the photosynthetic carbon assimilation in other species transcripts showed a marked increase, with a similar expression pattern between them, during the post-germinative development. On the other hand, the HpCAT1 (the corresponding protein is homolog to the major H2O2-scavenging enzyme in other species transcripts showed an opposite behavior with a down-regulation during the process. In summary, our findings, although preliminary, highlight the importance to investigate in more detail the participation of AOX genes during the post-germinative development in Hypericum, in order to explore their

  12. Expression of alternative oxidase in tomato

    Energy Technology Data Exchange (ETDEWEB)

    Kakefuda, M.; McIntosh, L. (Michigan State Univ., East Lansing (USA))

    1990-05-01

    Tomato fruit ripening is characterized by an increase in ethylene biosynthesis, a burst in respiration (i.e. the climacteric), fruit softening and pigmentation. As whole tomatoes ripened from mature green to red, there was an increase in the alternative oxidase capacity. Aging pink tomato slices for 24 and 48 hrs also showed an increase of alternative oxidase and cytochrome oxidase capacities. Monoclonal antibodies prepared to the Sauromatum guttatum alternative oxidase were used to follow the appearance of alternative oxidase in tomato fruits. There is a corresponding increase in a 36kDa protein with an increase in alternative oxidase capacity. Effects of ethylene and norbornadiene on alternative oxidase capacity were also studied. We are using an alternative oxidase cDNA clone from potato to study the expression of mRNA in ripening and wounded tomatoes to determine if the gene is transcriptionally regulated.

  13. CYP99A3: Functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice

    Science.gov (United States)

    Wang, Qiang; Hillwig, Matthew L.; Peters, Reuben J.

    2013-01-01

    SUMMARY Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochromes P450 mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNAi double knock-down of this pair of closely related CYP reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which ultimately was achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that, while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis. PMID:21175892

  14. Heterologous expression of the glucose oxidase gene in Trichoderma atroviride leads enhanced ability to attack phytopathogenic fungi and induction of plant systemic disease resistance

    Institute of Scientific and Technical Information of China (English)

    Robert L Mach; Brunner Kurt; Matteo Lorito; Susanne Zeilinger; Rosalia Ciliento; Sheridan Woo

    2004-01-01

    @@ A transgenic strain of Trichoderma atroviride that expresses the Aspergillus niger glucose oxidase gene goxA under a homologous pathogen-inducible promoter (nag1) has been constructed, with the aim of increasing the ability of this biocontrol agent (BCA) to attack phytopathogenic fungi and enhance plant systemic disease resistance. The sporulation and growth rate of the transgenic progenies were similar to the wild-type strain Pl. goxA expression occurred immediately after contact with the plant pathogen,and the glucose oxidase formed was secreted extracellularly. The transformed strain SJ3 4, containing 12-14 copies of the transgene, produced significantly less N-acetyl-glucosaminidase and endochitinase then wild type. However, the ability of its culture filtrate to inhibit the germination of Botrytis cinerea spores was increased by about 3-fold. In comparison to P1, the transgenic strain more quickly overgrew and lysed in vitro the pathogens Rhizoctonia solani and Pythium ultimum.

  15. Functional expression of the Acanthamoeba castellanii alternative oxidase in Escherichia coli; regulation of the activity and evidence for Acaox gene function.

    Science.gov (United States)

    Antos-Krzeminska, Nina; Jarmuszkiewicz, Wieslawa

    2014-06-01

    To evidence Acanthamoeba castellanii alternative oxidase (AcAOX) gene product function, we studied alterations in the levels of mRNA and protein and AcAOX activity during growth in amoeba batch culture. Moreover, heterologous expression of AcAOX in AOX-deficient Escherichia coli confirmed by the protein immunodetection and functional studies was performed. Despite the presence of native bo and bd quinol oxidases in E. coli membrane, from which the latter is known to be cyanide-resistant, functional expression of AcAOX in E. coli conferred cyanide-resistant benzohydroxamate-sensitive respiration on the bacteria. Moreover, AcAOX activity in transformed bacteria was stimulated by GMP and inhibited by ATP, indicating that AcAOX is regulated by mutual exclusion of purine nucleotides, which was previously demonstrated in the mitochondria of A. castellanii.

  16. Association between monoamine oxidase A gene promoter 30 bp repeat polymorphism and tardive dyskinesia in Chinese schizophrenics

    Institute of Scientific and Technical Information of China (English)

    Changhe Fan; Lihua Li; Yan Fu; Hehuang Deng; Xiangjiao Liao; Youcai Zhou

    2006-01-01

    BACKGROUND: The pathophysiology of tardive dyskinesia (TD) is not yet fully understood. With the hypothesis of altered dopaminergic neurotransmission, altered activities of dopamine degrading enzymes such as monoamine oxidase A (MAOA) and their coding genes are supposed to be related to the pathophysiology of TD.OBJECTIVE: To investigate possible association between 30 bp variable number tandem repeat (VNTR) polymorphism in the promoter of MAOA gene and susceptibility, severity of neuroleptic induced TD in Chinese Han people in Guandong Province.DESIGN: Non-randomization-synchronization controlled study. SETTING: Guangdong Mental Health Institute, Guangdong Provincial People's Hospital; Guangzhou Psychiatric Hospital; Affiliated Psychiatric Hospital of Guangzhou Municipal Bureau of Civil Administration. PARTICIPANTS: A total of 179 subjects were enrolled in the study. All subjects were sporadic and genetically unrelated Chinese schizophrenic patients who were hospitalizing in Guangzhou Psychiatric Hospital or Affiliated Psychiatric Hospital of Guangzhou Municipal Bureau of Civil Administration during January to April 2005. The diagnosis of schizophrenia was made according to the criteria of Diagnostic and Statistic Manual of Mental Disorder-the third edition-revised (DSM-Ⅲ-R). Among all patients, 88 were diagnosed as with TD and 91 without TD according to the research diagnostic criteria described by Schooler-Kane. Informed consent was obtained from all subjects or their relatives.METHODS: ① TD severity was assessed with the AIMS which was a 5-degree rating scale from 0 to 4 (corresponding to none, minimal, mild, moderate and severe, respectively). The study was approved by the Ethics Committees of the two hospitals and informed consent was obtained from all subjects or their relatives. ② The polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) techniques were used to detect MAOA gene 30 bp VNTR polymorphism in schizophrenic patients

  17. Scale Alpha and Beta of Quantitative Convergence and Chemical Reactivity Analysis in Dual Cholinesterase/Monoamine Oxidase Inhibitors for the Alzheimer Disease Treatment Using Density Functional Theory (DFT

    Directory of Open Access Journals (Sweden)

    Alejandro Morales-Bayuelo

    2013-01-01

    Full Text Available Molecular quantum similarity descriptors and Density Functional Theory (DFT based reactivity descriptors were studied for a series of cholinesterase/monoamine oxidase inhibitors used for the Alzheimer's disease treatment (AD. This theoretical study is expected to shed some light onto some molecular aspects that could contribute to the knowledge of the molecular mechanics behind interactions of these molecules with acetylcholinesterase (AChE and butyrylcholinesterase (BuChE, as well as with monoamine oxidase (MAO A and B. The Topogeometrical Superposition Algorithm to handle flexible molecules (TGSA-Flex alignment method was used to solve the problem of the relative orientation in the quantum similarity (QS field. Using the molecular quantum similarity (MQS field and reactivity descriptors supported in the DFT was possible the quantification of the steric and electrostatic effects through of the Coulomb and Overlap quantitative convergence scales (alpha and beta. In addition, an analysis of reactivity indexes is development, using global and local descriptors, identifying the binding sites and selectivity in the (cholinesterase/monoamine oxidase inhibitors, understanding the retrodonor process, and showing new insight for drugs design in a disease of difficult control as Alzheimer.

  18. Severity of ulcerative colitis is associated with a polymorphism at diamine oxidase gene but not at histamine N-methyltransferase gene

    Institute of Scientific and Technical Information of China (English)

    Elena García-Martín; Juan L Mendoza; Carmen Martínez; Carlos Taxonera; Elena Urcelay; José M Ladero; Emilio G de la Concha; Manuel Díaz-Rubio; José AG Agúndez

    2006-01-01

    AIM: To analyse the role of two common polymorphisms in genes coding for histamine metabolising enzymes as it relates to the risk to develop ulcerative colitis (UC) and the clinical course of these patients.METHODS: A cohort of 229 unrelated patients with UC recruited from a single centre and 261 healthy volunteers were analysed for the presence of Thr105Ile and His645Asp amino acid substitutions at histamine N-methyltransferase (HNMT) and diamine oxidase (ABP1) enzymes, respectively, by amplification-restriction procedures. All patients were phenotyped and followed up for at least 2 years (mean time 11 years).RESULTS: There were no significant differences in the distribution of ABP1 alleles between ulcerative colitis patients and healthy individuals [OR (95% CI) for variant alleles= 1.22 (0.91-1.61)]. However, mutated ABP1 alleles were present with higher frequency among the 58 patients that required immunosuppresive drugs [OR (95 % CI) for carriers of mutated alleles 2.41 (1.21-4.83;P=0.006)], with a significant gene-dose effect (P= 0.0038). In agreement with the predominant role of ABP1 versus HNMT on local histamine metabolism in human bowel, the frequencies for carriers ofHNMT genotypes or mutated alleles were similar among patients,regardless clinical evolution, and control individuals.CONCLUSION: The His645Asp polymorphism of the histamine metabolising enzyme ABP1 is related to severity of ulcerative colitis.

  19. RNAi Plasmid Construction of PttGA 20-oxidase Gene%PttGA20-氧化酶基因dsRNA抑止载体的构建

    Institute of Scientific and Technical Information of China (English)

    周冰彬; 陈晓阳

    2008-01-01

    [Objective] Genetic Engineering technology was used to regulate the expression of PttGA20-oxidase gene thus restrained plant height growth and internode elongation for cultivating dwarfed plant. [Method] Based on the RNAi principle, the gene specific sequences of PttGA20-oxidase in the antisense and sense orientations interrupted by a gene sequence from GUS were cloned into a binary vector pBI121. The selection marker gene npt Ⅱ was replaced with bar gene to RNAi plasmid. [Result] After undergone different endonuclease restrictions, the constructed constraint vector released different segments whose sizes were similar to that of target segment, which demonstrated that the RNAi plasmid of PttGA20-oxidase gene was successfully constructed. [Conclusion] The experiment provided a new way for culturing dwarfed plant.

  20. Religion, fertility and genes: a dual inheritance model

    Science.gov (United States)

    Rowthorn, Robert

    2011-01-01

    Religious people nowadays have more children on average than their secular counterparts. This paper uses a simple model to explore the evolutionary implications of this difference. It assumes that fertility is determined entirely by culture, whereas subjective predisposition towards religion is influenced by genetic endowment. People who carry a certain ‘religiosity’ gene are more likely than average to become or remain religious. The paper considers the effect of religious defections and exogamy on the religious and genetic composition of society. Defections reduce the ultimate share of the population with religious allegiance and slow down the spread of the religiosity gene. However, provided the fertility differential persists, and people with a religious allegiance mate mainly with people like themselves, the religiosity gene will eventually predominate despite a high rate of defection. This is an example of ‘cultural hitch-hiking’, whereby a gene spreads because it is able to hitch a ride with a high-fitness cultural practice. The theoretical arguments are supported by numerical simulations. PMID:21227968

  1. Human retina-specific amine oxidase: genomic structure of the gene (AOC2), alternatively spliced variant, and mRNA expression in retina.

    Science.gov (United States)

    Imamura, Y; Noda, S; Mashima, Y; Kudoh, J; Oguchi, Y; Shimizu, N

    1998-07-15

    Previously, we reported the isolation of cDNA for human retina-specific amine oxidase (RAO) and the expression of RAO exclusively in retina. Bacterial artificial chromosome clones containing the human RAO gene (AOC2) were mapped to human chromosome 17q21 (Imamura et al., 1997, Genomics 40: 277-283). Here, we report the complete genomic structure of the RAO gene, including 5' flanking sequence, and mRNA expression in retina. The human RAO gene spans 6 kb and is composed of four exons corresponding to the amino acid sequence 1-530, 530-598, 598-641, and 642-729 separated by three introns of 3000, 310, and 351 bp. Screening of a human retina cDNA library revealed the existence of an alternatively spliced cDNA variant with an additional 81 bp at the end of exon 2. The sizes of exons and the locations of exon/intron boundaries in the human RAO gene showed remarkable similarity to those of the human kidney diamine oxidase gene (AOC1). In situ hybridization revealed that mRNA coding for RAO is expressed preferentially in the ganglion cell layer of the mouse retina. We designed four sets of PCR primers to amplify four exons, which will be valuable for analyzing mutations in patients with ocular diseases affecting the retinal ganglion cell layer.

  2. Variation in the Lysyl Oxidase (LOX) Gene Is Associated with Keratoconus in Family-Based and Case-Control Studies

    Science.gov (United States)

    Bykhovskaya, Yelena; Li, Xiaohui; Epifantseva, Irina; Haritunians, Talin; Siscovick, David; Aldave, Anthony; Szczotka-Flynn, Loretta; Iyengar, Sudha K.; Taylor, Kent D.; Rotter, Jerome I.; Rabinowitz, Yaron S

    2012-01-01

    Purpose. Keratoconus is a bilateral noninflammatory progressive corneal disorder with complex genetic inheritance and a common cause for cornea transplantation in young adults. A genomewide linkage scan in keratoconus families identified a locus at 5q23.2, overlapping the gene coding for the lysyl oxidase (LOX). LOX encodes an enzyme responsible for collagen cross-linking in a variety of tissues including the cornea. Corneal collagen cross-linking with long-wave ultraviolet light and riboflavin is a promising new treatment for keratoconus. To determine whether LOX is a genetic determinant of the pathogenesis of keratoconus, we analyzed association results of LOX polymorphisms in two independent case-control samples and in keratoconus families. Methods. Association results were analyzed of single-nucleotide polymorphisms (SNPs) in the LOX gene from a Genome-Wide Association Study (GWAS) investigation in two independent panels of patients with keratoconus and controls and in keratoconus families. Results. Evidence of association was found at SNPs rs10519694 and rs2956540 located in intron 4 of LOX in the GWAS discovery case-control panel with P values of 2.3 × 10−3 and 7 × 10−3, respectively. The same two SNPs were found to be associated with keratoconus by family-based association testing with P values of 2.7 × 10−3 and 7.7 × 10−4, respectively. Meta P values of 4.0 × 10−5 and 4.0 × 10−7 were calculated for SNPs rs10519694 and rs2956540 by analyzing case-control and family samples simultaneously. Sequencing of LOX exons in a subset of keratoconus patients identified two polymorphisms, rs1800449 and rs2288393, located in LOX transcripts I and II, associated with keratoconus in case-control and family samples with a meta P value of 0.02. Conclusions. Results provided strong genetic evidence that LOX variants lead to increased susceptibility to developing of keratoconus. PMID:22661479

  3. Arsenic and phosphate rock impacted the abundance and diversity of bacterial arsenic oxidase and reductase genes in rhizosphere of As-hyperaccumulator Pteris vittata.

    Science.gov (United States)

    Han, Yong-He; Fu, Jing-Wei; Xiang, Ping; Cao, Yue; Rathinasabapathi, Bala; Chen, Yanshan; Ma, Lena Q

    2017-01-05

    Microbially-mediated arsenic (As) transformation in soils affects As speciation and plant uptake. However, little is known about the impacts of As on bacterial communities and their functional genes in the rhizosphere of As-hyperaccumulator Pteris vittata. In this study, arsenite (AsIII) oxidase genes (aroA-like) and arsenate (AsV) reductase genes (arsC) were amplified from three soils, which were amended with 50mgkg(-1) As and/or 1.5% phosphate rock (PR) and grew P. vittata for 90 d. The aroA-like genes in the rhizosphere were 50 times more abundant than arsC genes, consistent with the dominance of AsV in soils. According to functional gene alignment, most bacteria belonged to α-, β- and γ-Proteobacteria. Moreover, aroA-like genes showed a higher biodiversity than arsC genes based on clone library analysis and could be grouped into nine clusters based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Besides, AsV amendment elevated aroA-like gene diversity, but decreased arsC gene diversity. Redundancy analysis indicated that soil pH, available Ca and P, and AsV concentration were key factors driving diverse compositions in aroA-like gene community. This work identified new opportunities to screen for As-oxidizing and/or -reducing bacteria to aid phytoremediation of As-contaminated soils.

  4. Cloning and expression analysis of the Ccrboh gene encoding respiratory burst oxidase in Citrullus colocynthis and grafting onto Citrullus lanatus (watermelon).

    Science.gov (United States)

    Si, Ying; Dane, Fenny; Rashotte, Aaron; Kang, Kwonkyoo; Singh, Narendra K

    2010-06-01

    A full-length drought-responsive gene Ccrboh, encoding the respiratory burst oxidase homologue (rboh), was cloned in Citrullus colocynthis, a very drought-tolerant cucurbit species. The robh protein, also named NADPH oxidase, is conserved in plants and animals, and functions in the production of reactive oxygen species (ROS). The Ccrboh gene accumulated in a tissue-specific pattern when C. colocynthis was treated with PEG, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), or NaCl, while the homologous rboh gene did not show any change in C. lanatus var. lanatus, cultivated watermelon, during drought. Grafting experiments were conducted using C. colocynthis or C. lanatus as the rootstock or scion. Results showed that the rootstock significantly affects gene expression in the scion, and some signals might be transported from the root to the shoot. Ccrboh in C. colocynthis was found to function early during plant development, reaching high mRNA transcript levels 3 d after germination. The subcellular location of Ccrboh was investigated by transient expression of the 35S::Ccrboh::GFP fusion construct in protoplasts. The result confirmed that Ccrboh is a transmembrane protein. Our data suggest that Ccrboh might be functionally important during the acclimation of plants to stress and also in plant development. It holds great promise for improving drought tolerance of other cucurbit species.

  5. RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit.

    Science.gov (United States)

    Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom; Yeong, Wee Chien; Pillai, Vilasini

    2014-06-19

    The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

  6. RNA Interference of 1-Aminocyclopropane-1-carboxylic Acid Oxidase (ACO1 and ACO2 Genes Expression Prolongs the Shelf Life of Eksotika (Carica papaya L. Papaya Fruit

    Directory of Open Access Journals (Sweden)

    Rogayah Sekeli

    2014-06-01

    Full Text Available The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6. Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

  7. Protein trans-splicing based dual-vector delivery of the coagulation factor Ⅷ gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.

  8. Phenolic profiles and polyphenol oxidase (PPO) gene expression of red clover (Trifolium pratense) selected for decreased postharvest browning

    Science.gov (United States)

    Red clover (Trifolium pratense L.) is a legume forage abundant in phenolic compounds. It tends to brown when cut for hay, due to oxidation of phenolic compounds catalyzed by polyphenol oxidase (PPO), and subsequent binding to proteins. Selecting for a greener hay may provide information about the re...

  9. IDENTIFICATION OF 3 HUMAN PSEUDOGENES FOR SUBUNIT-VIB OF CYTOCHROME-C-OXIDASE - A MOLECULAR RECORD OF GENE EVOLUTION

    NARCIS (Netherlands)

    TAANMAN, JW; SCHRAGE, C; REUVEKAMP, P; BIJL, J; HARTOG, M; DEVRIES, H; AGSTERIBBE, E

    1991-01-01

    Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned human cDNA coding for COX subunit VIb. The nucleotide sequences of the pseudogenes, designated PSI-COX6b-1, PSI-COX6b-2 and PSI-COX6b-3, were determined.

  10. IDENTIFICATION OF 3 HUMAN PSEUDOGENES FOR SUBUNIT-VIB OF CYTOCHROME-C-OXIDASE - A MOLECULAR RECORD OF GENE EVOLUTION

    NARCIS (Netherlands)

    TAANMAN, JW; SCHRAGE, C; REUVEKAMP, P; BIJL, J; HARTOG, M; DEVRIES, H; AGSTERIBBE, E

    1991-01-01

    Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned human cDNA coding for COX subunit VIb. The nucleotide sequences of the pseudogenes, designated PSI-COX6b-1, PSI-COX6b-2 and PSI-COX6b-3, were determined.

  11. A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors

    Directory of Open Access Journals (Sweden)

    Uhde-Stone Claudia

    2012-08-01

    Full Text Available Abstract Background Transcription activator-like effectors (TALEs are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking. Results We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP applications. Conclusion We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.

  12. The terminal oxidases of Paracoccus denitrificans

    OpenAIRE

    de Gier, Jan-Willem L.; Lübben, Mathias; Reijnders, Willem N.M.; Tipker, Corinne A.; Slotboom, Dirk-Jan; Van Spanning, Rob J. M.; Stouthamer, Adriaan H.; van der Oost, John

    1994-01-01

    Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ΔctaDI, ΔctaDII), indicating that they are isoforms of subunit I of the aa3-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. This protohaem...

  13. Relationship between reduced nicotinamide adenine dinucleotide phosphate oxidase subunit p22phox gene polymorphism and obstructive sleep apnea-hypopnea syndrome in the Chinese Han population

    Institute of Scientific and Technical Information of China (English)

    LIU Hui-guo; LIU Kui; ZHOU Yan-ning; XU Yong-jian

    2009-01-01

    Background Increased production of reactive oxygen species (ROS) is thought to play a major role in the pathogenesis of obstructive sleep apnea-hypopnea syndrome (OSAHS). The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is an important source of ROS. The p22phox subunit is polymorphic with a C242T variant that changes histidine-72 for a tyrosine in the potential heme binding site. This study aimed to investigate the relationship between NADPH oxidase subunit p22phox gene polymorphism and OSAHS. Methods The genotypes of p22phox polymorphism were determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay in 176 unrelated subjects of the Han population in southern region of China (including 107 OSAHS subjects and 69 non-OSAHS subjects), while the plasma concentration of superoxide dismutase (SOD) was detected in the two groups, and p22phox mRNA expression in peripheral blood mononuclear cell (PBMC) was determined with reverse transcription polymerase chain reaction (RT-PCR).Results The phagocyte NADPH oxidase subunit p22phox mRNA expression was significantly increased in the OSAHS group than that in the non-OSAHS group (P<0.01). Compared with the non-OSAHS control group ((85.31±9.23) U/ml), the levels of SOD were lower in patients with OSAHS ((59.65±11.61) U/ml (P<0.01). There were significant differences in genotypes distribution in p22phox polymorphism between the two groups (P=0.02). Compared with the non-OSAHS control group, the OSAHS group had a significantly higher T allele frequency in p22phox polymorphism (P=0.03). There were independent effects of p22phox polymorphism on body mass index (BMI), neck circumference (NC), waist-to-hip ratio (WHR) in the OSAHS group, and the carriers of the T allele of p22phox polymorphism had greater NC, WHR, systolic blood pressure (SBP), diastolic blood pressure (DBP) and apnea-hypopnea index (AHI) (P <0.05), but the carriers of the T allele had lower SOD

  14. [Post-translational ligation and function of dual-vector transferred split CFTR gene].

    Science.gov (United States)

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2010-01-01

    The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

  15. Neural stem cell-based dual suicide gene delivery for metastatic brain tumors.

    Science.gov (United States)

    Wang, C; Natsume, A; Lee, H J; Motomura, K; Nishimira, Y; Ohno, M; Ito, M; Kinjo, S; Momota, H; Iwami, K; Ohka, F; Wakabayashi, T; Kim, S U

    2012-11-01

    In our previous works, we demonstrated that human neural stem cells (NSCs) transduced with the cytosine deaminase (CD) gene showed remarkable 'bystander killer effect' on glioma and medulloblastoma cells after administration of the prodrug 5-fluorocytosine (5-FC). In addition, herpes simplex virus thymidine kinase (TK) is a widely studied enzyme used for suicide gene strategies, for which the prodrug is ganciclovir (GCV). To apply this strategy to brain metastasis treatment, we established here a human NSC line (F3.CD-TK) expressing the dual suicide genes CD and TK. We examined whether F3.CD-TK cells intensified the antitumor effect on lung cancer brain metastases. In vitro studies showed that F3.CD-TK cells exerted a marked bystander effect on human lung cancer cells after treatment with 5-FC and GCV. In a novel experimental brain metastases model, intravenously administered F3 cells migrated near lung cancer metastatic lesions, which were induced by the injection of lung cancer cells via the intracarotid artery. More importantly, F3.CD-TK cells in the presence of prodrugs 5-FC and GCV decreased tumor size and considerably prolonged animal survival. The results of the present study indicate that the dual suicide gene-engineered, NSC-based treatment strategy might offer a new promising therapeutic modality for brain metastases.

  16. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    Science.gov (United States)

    Loera-Castañeda, Verónica; Sandoval-Ramírez, Lucila; Pacheco Moisés, Fermín Paul; Macías-Islas, Miguel Ángel; Alatorre Jiménez, Moisés Alejandro; González-Renovato, Erika Daniela; Cortés-Enríquez, Fernando; Célis de la Rosa, Alfredo; Velázquez-Brizuela, Irma E.

    2014-01-01

    Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD. PMID:24701363

  17. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Verónica Loera-Castañeda

    2014-01-01

    Full Text Available Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS. Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12% harbored the A8027G polymorphism and three of them were early onset (EO AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn’t been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

  18. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease.

    Science.gov (United States)

    Loera-Castañeda, Verónica; Sandoval-Ramírez, Lucila; Pacheco Moisés, Fermín Paul; Macías-Islas, Miguel Ángel; Alatorre Jiménez, Moisés Alejandro; González-Renovato, Erika Daniela; Cortés-Enríquez, Fernando; Célis de la Rosa, Alfredo; Velázquez-Brizuela, Irma E; Ortiz, Genaro Gabriel

    2014-01-01

    Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

  19. Overexpression of a Maize Sulfite Oxidase Gene in Tobacco Enhances Tolerance to Sulfite Stress via Sulfite Oxidation and CAT-Mediated H2O2 Scavenging

    Science.gov (United States)

    Xia, Zongliang; Sun, Kaile; Wang, Meiping; Wu, Ke; Zhang, Hua; Wu, Jianyu

    2012-01-01

    Sulfite oxidase (SO) plays an important role in sulfite metabolism. To date, the molecular mechanisms of sulfite metabolism in plants are largely unknown. Previously, a full-length cDNA of the putative sulfite oxidase gene from maize (ZmSO) was cloned, and its response to SO2/sulfite stress at the transcriptional level was characterized. In this study, the recombinant ZmSO protein was purified from E.coli. It exhibited sulfite-dependent activity and had strong affinity for the substrate sulfite. Over-expression (OE) of ZmSO in tobacco plants enhanced their tolerance to sulfite stress. The plants showed much less damage, less sulfite accumulation, but greater amounts of sulfate. This suggests that tolerance of transgenic plants to sulfite was enhanced by increasing SO expression levels. Interestingly, H2O2 accumulation levels by histochemical detection and quantitative determination in the OE plants were much less than those in the wild-type upon sulfite stress. Furthermore, reductions of catalase levels detected in the OE lines were considerably less than in the wild-type plants. This indicates that SO may play an important role in protecting CAT from inhibition by excess sulfite. Collectively, these data demonstrate that transgenic tobacco plants over-expressing ZmSO enhance tolerance to excess sulfite through sulfite oxidation and catalase-mediated hydrogen peroxide scavenging. This is the first SO gene from monocots to be functionally characterized. PMID:22693572

  20. Overexpression of a maize sulfite oxidase gene in tobacco enhances tolerance to sulfite stress via sulfite oxidation and CAT-mediated H2O2 scavenging.

    Directory of Open Access Journals (Sweden)

    Zongliang Xia

    Full Text Available Sulfite oxidase (SO plays an important role in sulfite metabolism. To date, the molecular mechanisms of sulfite metabolism in plants are largely unknown. Previously, a full-length cDNA of the putative sulfite oxidase gene from maize (ZmSO was cloned, and its response to SO(2/sulfite stress at the transcriptional level was characterized. In this study, the recombinant ZmSO protein was purified from E. coli. It exhibited sulfite-dependent activity and had strong affinity for the substrate sulfite. Over-expression (OE of ZmSO in tobacco plants enhanced their tolerance to sulfite stress. The plants showed much less damage, less sulfite accumulation, but greater amounts of sulfate. This suggests that tolerance of transgenic plants to sulfite was enhanced by increasing SO expression levels. Interestingly, H(2O(2 accumulation levels by histochemical detection and quantitative determination in the OE plants were much less than those in the wild-type upon sulfite stress. Furthermore, reductions of catalase levels detected in the OE lines were considerably less than in the wild-type plants. This indicates that SO may play an important role in protecting CAT from inhibition by excess sulfite. Collectively, these data demonstrate that transgenic tobacco plants over-expressing ZmSO enhance tolerance to excess sulfite through sulfite oxidation and catalase-mediated hydrogen peroxide scavenging. This is the first SO gene from monocots to be functionally characterized.

  1. Evidence for Lateral Transfer of Genes Encoding Ferredoxins, Nitroreductases, NADH Oxidase, and Alcohol Dehydrogenase 3 from Anaerobic Prokaryotes to Giardia lamblia and Entamoeba histolytica

    Science.gov (United States)

    Nixon, Julie E. J.; Wang, Amy; Field, Jessica; Morrison, Hilary G.; McArthur, Andrew G.; Sogin, Mitchell L.; Loftus, Brendan J.; Samuelson, John

    2002-01-01

    Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica. PMID

  2. [Integration of different T-DNA structures of ACC oxidase gene into carnation genome extended cut flower vase-life differently].

    Science.gov (United States)

    Yu, Yi-Xun; Bao, Man-Zhu

    2004-09-01

    The cultivar 'Master' of carnation (Dianthus caryophyllus L.) was transformed with four T-DNA structures containing sense, antisense, sense direct repeat and antisense direct repeat gene of ACC oxidase mediated by Agrobacterium tumefaciens. Southern blotting detection showed that foreign gene was integrated into the carnation genome and 14 transgenic lines were obtained. The transgenic plants were transplanted to soil and grew normally in greenhouse. Of the 12 transgenic lines screened, the cut flower vase life of 8 transgenic lines is up to 11 days and the longest one is 12.8 days while the vase life of the control is 5.8 days under 25 degrees C. The vase life of 2 lines out of 3 with single sense ACO gene is same as that of the control, while the vase life of 3 lines out of 4 with single antisense ACO gene is prolonged. The vase life of cut flowers of 5 lines with direct repeat ACO genes is all prolonged by about 6 days, while the vase life of 3 out of 7 lines with single ACO gene is same as that of the control. During the senescence of cut flowers, the ethylene production of the most of the transgenic lines decreased significantly, and the production of ethylene is not detectable in lines T456, T556 and T575. The results of the research demonstrate that antisense foreign gene inhibits expression of endogenesis gene more significantly than sense one. Both sense direct repeat and antisense direct repeat foreign genes can suppress endogenous gene expression more significantly comparing to single foreign genes. The transgenic lines obtained from this research are useful to minimize carnation cut flower transportation and storage expenses.

  3. Single-cell analysis reveals gene-expression heterogeneity in syntrophic dual-culture of Desulfovibrio vulgaris with Methanosarcina barkeri

    Science.gov (United States)

    Qi, Zhenhua; Pei, Guangsheng; Chen, Lei; Zhang, Weiwen

    2014-12-01

    Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

  4. Acute kidney injury observed during phase 1 clinical trials of a novel xanthine oxidase/URAT1 dual inhibitor PF-06743649.

    Science.gov (United States)

    Dua, Pinky; Gurrell, Rachel; Kirby, Simon; Sudworth, Maria; Loudon, Peter T

    2016-08-01

    The objective of these clinical studies was to assess the safety and urate lowering activity of a novel urate transporter 1 (URAT1)/ xanthine oxidase (XO) inhibitor PF-06743649 in healthy subjects and gout patients. Escalating doses of PF-06743649 or placebo were given to healthy young subjects, healthy elderly subjects and gout patients. Serum uric acid (sUA) and urinary pharmacodynamic markers were assayed, and safety was assessed by collection of adverse events and assessment of safety labs, ECGs and vital signs. Administration of PF-06743649 led to rapid decrease in sUA in all cohorts; in gout patients, a change from baseline of 69 % was observed for the 40 mg dose. Urinary and serum biomarkers were consistent with inhibition of both URAT1 and XO. Although dosing was otherwise well tolerated, two subjects experienced serious adverse events of acute kidney injury. Both subjects exhibited increased serum creatinine and blood urea nitrogen in the first 3 days post first dose and were hospitalised. One subject exhibited oliguria for the first 24 h. Both subjects made a complete recovery with minimal intervention. PF-06743649 was effective at rapidly lowering sUA, but further development was terminated for an identified renal safety risk.

  5. Status and Advances of Researches on GA 20-oxidases

    Institute of Scientific and Technical Information of China (English)

    Li Wei; Chen Xiaoyang; Li Hui; Guo Hai

    2003-01-01

    GA 20-oxidase, the most important limiting enzyme, can catalyze a series of oxidization of GA biosynthesis pathwayfrom GA12 to GA9 and from GA53 to GA20 in the higher plants. This paper reviews the studies on the characters of GA 20-oxidase,the gene and the protein of GA 20-oxidase and the regulation of GA 20-oxidase gene expression in recent years. At the same time,the prospects for the gene transformation of GA 20-oxidase in agriculture, forestry and horticulture are also discussed.

  6. Molecular cloning and sequence analysis of a PVGOX gene encoding glucose oxidase in Penicillium viticola F1 strain and it's expression quantitation.

    Science.gov (United States)

    Khan, Ibrar; Qayyum, Sadia; Ahmed, Shehzad; Niaz, Zeeshan; Fatima, Nighat; Chi, Zhen-Ming

    2016-11-05

    The PVGOX gene (accession number: KT452630) was isolated from genomic DNA of the marine fungi Penicillium viticola F1 by Genome Walking and their expression analysis was done by Fluorescent RT-PCR. An open reading frame of 1806bp encoding a 601 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 65,535.4 was characterized. The deduced protein showed 75%, 71%, 69% and 64% identity to those deduced from the glucose oxidase (GOX) genes from different fungal strains including; Talaromyces variabilis, Beauveria bassiana, Aspergillus terreus, and Aspergillus niger, respectively. The promoter of the gene (intronless) had two TATA boxes around the base pair number -88 and -94 and as well as a CAAT box at -100. However, the terminator of the PVGOX gene does not contain any polyadenylation site (AATAAA). The protein deduced from the PVGOX gene had a signal peptide containing 17 amino acids, three cysteine residues and six potential N-linked glycosylation sites, among them, -N-K-T-Y- at 41 amino acid, -N-R-S-L- at 113 amino acid, -N-G-T-I- at 192 amino acid, -N-T-T-A at 215 amino acid, -N-F-T-E at 373 amino acid and -N-V-T-A- at 408 amino acid were the most possible N-glycosylation sites. Furthermore, the relative transcription level of the PVGOX gene was also stimulated in the presence of 4% (w/v) of calcium carbonate and 0.5 % (v/v) of CSL in the production medium compared with that of the PVGOX gene when the fungal strain F1 was grown in the absence of calcium carbonate and CSL in the production medium, suggesting that under the optimal conditions, the expression of the PVGOX gene responsible for gluconic acid biosynthesis was enhanced, leading to increased gluconic acid production. Therefore, the highly glycosylated oxidase enzyme produced by P. viticola F1 strain might be a good producer in the fermentation process for the industrial level production of gluconic acid.

  7. C242T polymorphism of the NADPH oxidase p22PHOX gene and its association with endothelial dysfunction in asymptomatic individuals with essential systemic hypertension.

    Science.gov (United States)

    Rafiq, Adnan; Aslam, Khursheed; Malik, Rouf; Afroze, Dil

    2014-05-01

    Vascular oxidative stress is an important factor in hypertension-associated vascular damage and is mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. The C242T polymorphism at the p22PHOX gene affects binding of p22PHOX to heme, leading to variants of NADPH oxidase that produce different levels of reactive oxygen species (ROS). Specific variations in ROS are associated with an altered risk of developing cardiovascular disease. In the present study, 140 permanent Kashmiri-resident individuals were recruited (75 with essential systemic hypertension and 65 normotensive controls). Endothelial function was assessed non-invasively using high-resolution ultrasonography of the brachial artery. Endothelium-dependent vasoreactivity was expressed in terms of flow-mediated dilation. The TT genotype was identified in 2% of hypertensive and 7% of normotensive individuals. Frequency of the T-allele was not observed as significantly different between hypertensive and normotensive individuals (P=0.24; OR=0.4; 95% CI, 0.07-2.2). Blood pressure or the prevalence of hypertension did not vary between C242T p22PHOX genotypes or in the presence or absence of the T-allele.

  8. Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation

    DEFF Research Database (Denmark)

    Kulp, M. S.; Frickel, E. M.; Ellgaard, Lars

    2006-01-01

    Native disulfide bond formation in eukaryotes is dependent on protein-disulfide isomerase (PDI) and its homologs, which contain varying combinations of catalytically active and inactive thioredoxin domains. However, the specific contribution of PDI to the formation of new disulfides versus...... reduction/rearrangement of non-native disulfides is poorly understood. We analyzed the role of individual PDI domains in disulfide bond formation in a reaction driven by their natural oxidant, Ero1p. We found that Ero1p oxidizes the isolated PDI catalytic thioredoxin domains, A and A' at the same rate....... In contrast, we found that in the context of full-length PDI, there is an asymmetry in the rate of oxidation of the two active sites. This asymmetry is the result of a dual effect: an enhanced rate of oxidation of the second catalytic (A') domain and the substrate-mediated inhibition of oxidation of the first...

  9. The sense and antisense expression of gibberellin 20-oxidase gene (rga5) in rice and its effects on GA1 level and agronomic traits

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A gibberellin 20-oxidase gene rga5 was isolated by PCR from genomic DNA of rice (Oryza sativa ssp indica) cultivars 'Aizizhan' and 'Nante'. Compared with the reported OsGA20ox, the rga5 was partial-frame-shifted with 11 different amino acids. Then the rga5 with CaMV 35S promotor and NOS terminator was inserted into the polylinker site of pCambia1301 to construct sense and antisense gene expressing vectors pSrga5 and pArga5. The transgenic plants were obtained by biolistic bombardment with pSrga5 or pArga5. The transgenic rice plants showed that the over- expression and antisense-expression of rga5 have remarkable effects on the biological characters of rice. The sense transgenic plants showed heightening with longer spike, more seed-bearing and unaffected flowering, whereas antisense transgenic plants showed dwarfing, early-flowering with slender stem, dark leaf color, shorter leaf and shorter spike. The PCR amplification and Southern blot hybridization showed that the rga5 has been integrated into the transgenic rice genome and the transcription of rga5 was identified by Northern blot hybridization. In the sense transgenic plants the GA1 content increased of about 50%, however, the antisense transgenic rice decreased of 90% approximately compared with control plant 'Zhonghua 8'. These results demonstrated that the rga5 is a functional gene encoding gibberellin 20-oxidase in rice. Over-expressing rga5 significantly increases the endogenous GA1 level and plant height of rice, whereas the expression of antisense rga5 decreases the GA1 level and plant height of rice dramatically.

  10. Additive effect of polymorphisms in the β2 -adrenoceptor and NADPH oxidase p22 phox genes contributes to the loss of estimated glomerular filtration rate in Chinese.

    Science.gov (United States)

    Wang, Tao; Zhang, Yan; Ma, JingTao; Feng, Zhen; Niu, Kai; Liu, Bing

    2014-09-01

    Because increased oxidative stress may mediate the detrimental actions of enhanced sympathetic nervous activity on renal function and vice versa, we investigated the effect of the polymorphic Arg16Gly in the β2 -adrenoceptor (ADRB2) gene, Trp64Arg in the β3 -adrenoceptor (ADRB3) gene and C242T in the NADPH oxidase p22phox (CYBA) gene on estimated glomerular filtration rate (eGFR) in a Chinese population. Initially recruited from different outpatient services of HeBei General Hospital in northern China, 668 individuals were finally included in the study, with complete demographic information. Laboratory tests were performed and estimated glomerular filtration rate (eGFR) was derived from the Modification of Diet in Renal Disease (MDRD) equation for the Chinese population. Plasma noradrenaline levels and genotype were determined by HPLC and the TaqMan method, respectively. Only across the Arg16Gly polymorphism did eGFR show significant difference: it was lower in individuals with the Gly16Gly variation, who also had the highest plasma noradrenaline levels. This polymorphism remained a significant determinant of eGFR after multivariate analysis. Of importance, the multifactor dimensionality reduction method further detected a significant synergism between the Arg16Gly and C242T polymorphisms in reducing eGFR. These observations clarify the effects of the studied polymorphisms on eGFR and exemplify gene-gene interactions influencing renal function.

  11. Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Sarah T Miyata

    Full Text Available The Vibrio cholerae type VI secretion system (T6SS assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.

  12. W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila.

    Directory of Open Access Journals (Sweden)

    Wenke Zhou

    Full Text Available We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a "founder" knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W and neomycin resistance (Neo. In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.

  13. Population structure of the Monocelis lineata (Proseriata, Monocelididae species complex assessed by phylogenetic analysis of the mitochondrial Cytochrome c Oxidase subunit I (COI gene

    Directory of Open Access Journals (Sweden)

    Daria Sanna

    2009-01-01

    Full Text Available Monocelis lineata consists of a complex of sibling species, widespread in the Mediterranean and Atlantic Ocean. Previous genetic analysis placed in evidence at least four sibling species. Nevertheless, this research was not conclusive enough to fully resolve the complex or to infer the phylogeny/phylogeography of the group. We designed specific primers aiming at obtaining partial sequences of the mtDNA gene Cytochrome c Oxidase subunit I (COI of M. lineata, and have identified 25 different haplotypes in 32 analyzed individuals. The dendrogram generated by Neighbor-Joining analysis confirmed the differentiation between Atlantic and Mediterranean siblings, as well as the occurrence of at least two Mediterranean sibling species. Thus validated, the method here presented appears as a valuable tool in population genetics and biodiversity surveys on the Monocelis lineata complex.

  14. Daucus carota L.--an old model for cell reprogramming gains new importance through a novel expansion pattern of alternative oxidase (AOX) genes.

    Science.gov (United States)

    Costa, J H; Cardoso, H G; Campos, M D; Zavattieri, A; Frederico, A M; Fernandes de Melo, D; Arnholdt-Schmitt, B

    2009-08-01

    The paper highlights Daucus carota L. as an ideal model to complement plant stress research on Arabidopsis thaliana L. Recently, alternative oxidase (AOX) is discussed as functional marker candidate for cell reprogramming upon stress. Carrot is the most studied species for cell reprogramming and our current research reveals that it is the only one that has expanded both AOX sub-family genes. We point to recently published, but not discussed results on conserved differences in the vicinity of the most active functional site of AOX1 and AOX2, which indicate the importance of studying AOX sequence polymorphism, structure and functionality. Thus, stress-inducible experimental systems of D. carota are especially appropriate to bring research on stress tolerance a significant step forward.

  15. The Characteristics of Cytochrome C Oxidase Gene Subunit I in Wild Silkmoth Cricula trifenestrata Helfer and Its Evaluation for Species Marker

    Directory of Open Access Journals (Sweden)

    Suriana

    2012-08-01

    Full Text Available The study was conducted to assess the characteristics of partial gene of cytochrome C oxidase subunit I (COI of wild silkmoth Cricula trifenestrata, and to detect the diagnostic sites from these gene for evaluation as species marker. A total of fifteen larvae of C. tifenestrata were collected from Bogor, Purwakarta, and Bantul Regencies. Genomic DNA was extracted from silk gland of individual larvae, then amplified by PCR method and sequenced. DNA sequencing was done to characterize their nucleotide and amino acid contents. The results showed that 595 nucleotides at the 5 ‘end of COI gene of C. tifenestrata was conserved at the species level, but varies at the family level. Nucleotide dominated by thymine and adenine bases (± 70%. There were 25 diagnostic sites for C. tifenestrata, and four diagnostic sites for genus level. One hundred eigthty nine (189 amino acids were alignment, and only one percent of the genes was varied among species. The 107th amino acid (valine and 138th (threonine were diagnostics amino acid for C. tifenestrata. Based on nucleotides and amino acids sequences, the phylogeny showed that C. tifenestrata lied on the same nodes with Antheraea, so the Saturniidae family is monophyletic.

  16. Cloning and Expression of an Alternative Oxidase Gene from Lycopersicon esculentum%番茄交替氧化酶基因的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    宋从凤; Broth wayne; 王金生; 胡晋生

    2004-01-01

    A full-length cDNA gene (LeAoxlau) was isolated from a cDNA library made from ripening fruit probing with alternative oxidase (AOX) gene fragments, obtained by degenerate primer PCR. Sequence analysis showed that LeAoxlau was 1 418 bp long and contained a 1 077-bp open reading frame encoding a about 40 kD precursor protein which is processed to a mature protein of 32 kD. Southern blot analysis suggested LeAoxlau is present as a single copy in the genome of tomato. RT-PCR analysis indicated LeAoxlau was expressed in roots, stems, leaves and cotyledons of tomato plants. A recombinant construct containing the open reading frame sequence of the LeAoxlau was transformed into Escherichia coli to express the alternative oxidase precursor protein.%利用简并PCR扩增产物做探针筛选番茄cDNA基因文库获得一个全长交替氧化酶cDNA基因LeAoxlau.经序列分析得出,该基因全长1 418bp,编码区序列长1 077 bp,编码约40 kD的前体蛋白.该蛋白在转运到线粒体时被加工成32kD的成熟蛋白.Southern印迹杂交分析结果显示该基因以单拷贝形式存在于番茄的基因组中RT-PCR显示,该基因在在番茄植株的根、茎、叶和子叶中表达.重组表达实验表明该基因能在大肠杆菌中表达.

  17. Genetic Differences of Mitten Crabs Based on RFLP Analysis on Mitochondrial Cytochrome Oxidase Subunit I (COI) Gene

    Institute of Scientific and Technical Information of China (English)

    HU Pengfei; WANG Qian; DAI Wei; WANG Xiaomei

    2008-01-01

    The genetic differences of 15 mitten crab populations from 6 river systems in mainland China and 1 population from Russia were studied based on RFLP analysis of mitochondrial cytochrome oxidase subunit I (COI).The results showed that Tas I-RFLP pattern could be used as a genetic marker to distinguish Eriocheir hepuensis from Eriocheir sinensis, Eriocheirjaponica and Eriocheir leptognathus;genetic distances among 13 populations ofEriocheir sinensis range from 0 to 0.015, indicating that they were different geographic strains;the subspecies status ofEriocheir sinensis and Eriocheir hepuensis (population from Nanliujiang) were considered owning to their genetic distances of 0.02-0.044,indicating that genetic divergence between them was low; Eriocheir leptognathus (population from Nanpaihe, Tianjin) was the most distant taxon with genetic distances value of 0.147-0.195,which could be defined as genetic distances between species in genus Eriocheir.

  18. Mitochondrial encephalomyopathy with cytochrome c oxidase deficiency caused by a novel mutation in the MTCO1 gene.

    Science.gov (United States)

    Debray, François-Guillaume; Seneca, Sara; Gonce, Michel; Vancampenhaut, Kim; Bianchi, Elettra; Boemer, François; Weekers, Laurent; Smet, Joél; Van Coster, Rudy

    2014-07-01

    Cytochrome c oxidase (COX) deficiency is one of the most common respiratory chain deficiencies. A woman was presented at the age of 18y with acute loss of consciousness, non-convulsive status epilepticus, slow neurological deterioration, transient cortical blindness, exercise intolerance, muscle weakness, hearing loss, cataract and cognitive decline. Muscle biopsy revealed ragged-red fibers, COX negative fibers and a significant decreased activity of complex IV in a homogenate. Using next generation massive parallel sequencing of the mtDNA, a novel heteroplasmic mutation was identified in MTCO1, m.7402delC, causing frameshift and a premature termination codon. Single fiber PCR showed co-segregation of high mutant load in COX negative fibers. Mutation in mitochondrially encoded complex IV subunits should be considered in mitochondrial encephalomyopathies and COX negative fibers after the common mtDNA mutations have been excluded.

  19. Antagonistic control of a dual-input mammalian gene switch by food additives.

    Science.gov (United States)

    Xie, Mingqi; Ye, Haifeng; Hamri, Ghislaine Charpin-El; Fussenegger, Martin

    2014-08-01

    Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.

  20. Gene engineered construction of urate oxidase highly-expressing Lactococcus lactis%高产尿酸氧化酶乳酸工程菌的构建

    Institute of Scientific and Technical Information of China (English)

    张彦新; 曾雪芳; 刘芳; 蒋云生

    2015-01-01

    目的 将尿酸氧化酶基因克隆到乳酸乳球菌(Llactis)NZ9000中,使之能启动nisA放大系统增加尿酸氧化酶活性,构建一株高效分解尿酸的基因工程菌.方法 根据GenBank上已知的产朊假丝酵母菌尿酸氧化酶基因序列(Uricase,E12709)设计引物,PCR扩增尿酸氧化酶基因片段,将其克隆入质粒PNZ8048、PMG36e,构建重组质粒PNZ8048-U、PMG36e-U,重组质粒电转化L.lactis NZ9000构建基因工程菌L.lactis NZ9000-PNZ8048-U、L.lactis NZ9000-PMG36e-U,经十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(SDS-PAGE)鉴定重组菌体裂解液中的尿酸氧化酶,测定尿酸氧化酶的活性和在高尿酸患者血清中降解尿酸的能力.结果 构建的重组质粒PNZ8048-U、PMG36e-U经酶切和测序显示,尿酸氧化酶基因片段长度为0.9 kb,基因片段序列与GenBank上尿酸氧化酶基因序列完全一致.构建重组基因工二程菌L.lactis NZ9000-PNZ8048-U、L.lactis NZ9000-PMG36e-U,均表达相对分子质量约为34 000的重组蛋白,与从尿酸氧化酶基因序列推测的303个氨基酸的理论分子量相符.体外测定菌酶液活性,L.lactis NZ9000-PNZ8048-U组酶活性为(1.92±0.14)u/ml,产朊假丝酵母菌组酶活性为(0.55±0.05)u/ml,L.lactis NZ9000-PMG36e-U组酶活性为(0.29±0.06) u/ml.高尿酸血症患者血清培养结果显示加入生理盐水的对照组尿酸值(620.0±58.7) μmol/L,L.lactis NZ9000-PNZ8048-U组(321.0±46.2) μmol/L,L.lactis NZ9000-PMG36e-U组(568.0±47.3)μmol/L,产朊假丝酵母菌组(406.0±42.4)μmol/L,其他3组与对照组比较,差异均有统计学意义(均P<0.05).结论 成功构建基因工程菌L.lactis NZ9000-PNZ8048-U,重组菌酶活性较基因来源菌产朊假丝酵母菌高,并可高效分解高尿酸患者血清中的尿酸.%Objective To construct a bacterial strain with genectically-engineered high-efficiency in uric acid degradation,through cloning the urate oxidase gene into Lactococcus lactis NZ9000 so that it can

  1. Synthetic liver X receptor agonist T0901317 inhibits semicarbazide-sensitive amine oxidase gene expression and activity in apolipoprotein E knockout mice

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Dai; Xiang Ou; Xinrui Hao; Dongli Cao; Yaling Tang; Yanwei Hu; Xiaoxu Li; Chaoke Tang

    2008-01-01

    Semicarbazide-sensitive amine oxidase(SSAO)catalyzes oxidative deamination of primary aromatic and aliphatic amines.Increased SSAO activity has been found in atherosclerosis and diabetes mellitus.We hypothesize that the anti-atherogenic effect of liver X receptors(LXRs)might be related to the inhibition of SSAD gene expression and its activity.In this study,we investigated the effect of LXR agonist T0901317 on SSAO gene expression and its activity in apolipoprotein E knockout(apoE-/-)mice.Male apoE-/-mice(8 weeks old) were randomly divided into four groups:basal control group;vehicle group;prevention group;and treatment group.SSAO gene expression was analyzed by real-time quantitative polymerase chain reaction and its activity was determined.The activity of superoxide dismutase and content of malondialdehy de in the aorta and liver were also determined.In T0901317-treated mice,SSAO gene expression was significantly decreased in the aorta,liver,small intestine,and brain.SSAO activities in serum and in these tissues were also inhibited.The amount of superoxide dismutase in the aorta and liver of the prevention group and treatment group was significantly higher compared with the vehicle group(P<0.05).Malondialdehyde in the tissues of these two groups was significantly lower compared with the vehicle group(P<0.05).Our results showed that T0901317 inhibits SSAO gene expression and its activity in atherogenic apoE-/-mice.The atheroprotective effect of LXR agonist T0901317 is related to the inhibition of SSAO gene expression and its activity.

  2. Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria.

    Science.gov (United States)

    Yim, W J; Kim, K Y; Lee, Y W; Sundaram, S P; Lee, Y; Sa, T M

    2014-07-15

    Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.

  3. Partial protoporphyrinogen oxidase (PPOX gene deletions, due to different Alu-mediated mechanisms, identified by MLPA analysis in patients with variegate porphyria

    Directory of Open Access Journals (Sweden)

    Barbaro Michela

    2013-01-01

    Full Text Available Abstract Variegate porphyria (VP is an autosomal dominantly inherited hepatic porphyria. The genetic defect in the PPOX gene leads to a partial defect of protoporphyrinogen oxidase, the penultimate enzyme of heme biosynthesis. Affected individuals can develop cutaneous symptoms in sun-exposed areas of the skin and/or neuropsychiatric acute attacks. The identification of the genetic defect in VP families is of crucial importance to detect the carrier status which allows counseling to prevent potentially life threatening neurovisceral attacks, usually triggered by factors such as certain drugs, alcohol or fasting. In a total of 31 Swedish VP families sequence analysis had identified a genetic defect in 26. In the remaining five families an extended genetic investigation was necessary. After the development of a synthetic probe set, MLPA analysis to screen for single exon deletions/duplications was performed. We describe here, for the first time, two partial deletions within the PPOX gene detected by MLPA analysis. One deletion affects exon 5 and 6 (c.339-197_616+320del1099 and has been identified in four families, most probably after a founder effect. The other extends from exon 5 to exon 9 (c.339-350_987+229del2609 and was found in one family. We show that both deletions are mediated by Alu repeats. Our findings emphasize the usefulness of MLPA analysis as a complement to PPOX gene sequencing analysis for comprehensive genetic diagnostics in patients with VP.

  4. Phylogenetic relationships within Taenia taeniaeformis variants and other taeniid cestodes inferred from the nucleotide sequence of the cytochrome c oxidase subunit I gene.

    Science.gov (United States)

    Okamoto, M; Bessho, Y; Kamiya, M; Kurosawa, T; Horii, T

    1995-01-01

    Nucleotide sequence variations in a region of the mitochondrial cytochrome c oxidase subunit I (COI) gene (391 bp) were examined within seven species of the genus Taenia and two species of the genus Echinococcus, including ten isolates of T. taeniaeformis and six isolates of E. multilocularis. More than a 12% rate of nucleotide differences between taeniid species was found, allowing the species to be distinguished. In E. multilocularis, no sequence variation was observed among isolates, regardless of the host (gray red-backed vole, tundra vole, pig, Norway rat) or area (Japan, Alaska) from which each metacestode had been isolated. In contrast, six distinct sequences were detected among the ten T. taeniaeformis isolates examined. The level of nucleotide variation in the COI gene within T. taeniaeformis isolates except for one isolate from the gray red-backed vole (TtACR), which has been proposed as a distinct strain or a different species, was about 0.3%-4.1%, whereas the COI gene sequence for TtACR differed from those of the other isolates, with levels being 9.0%-9.5%. Phylogenetic trees were then inferred from these sequence data using two different algorithms.

  5. Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

    Science.gov (United States)

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

    2013-11-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons.

  6. Effects upon metabolic pathways and energy production by Sb(III) and As(III)/Sb(III)-oxidase gene aioA in Agrobacterium tumefaciens GW4.

    Science.gov (United States)

    Li, Jingxin; Yang, Birong; Shi, Manman; Yuan, Kai; Guo, Wei; Li, Mingshun; Wang, Gejiao

    2017-01-01

    Agrobacterium tumefaciens GW4 is a heterotrophic arsenite [As(III)]/antimonite [Sb(III)]-oxidizing strain. The As(III) oxidase AioAB is responsible for As(III) oxidation in the periplasm and it is also involved in Sb(III) oxidation in Agrobacterium tumefaciens 5A. In addition, Sb(III) oxidase AnoA and cellular H2O2 are also responsible for Sb(III) oxidation in strain GW4. However, the deletion of aioA increased the Sb(III) oxidation efficiency in strain GW4. In the present study, we found that the cell mobility to Sb(III), ATP and NADH contents and heat release were also increased by Sb(III) and more significantly in the aioA mutant. Proteomics and transcriptional analyses showed that proteins/genes involved in Sb(III) oxidation and resistance, stress responses, carbon metabolism, cell mobility, phosphonate and phosphinate metabolism, and amino acid and nucleotide metabolism were induced by Sb(III) and were more significantly induced in the aioA mutant. The results suggested that Sb(III) oxidation may produce energy. In addition, without periplasmic AioAB, more Sb(III) would enter bacterial cells, however, the cytoplasmic AnoA and the oxidative stress response proteins were significantly up-regulated, which may contribute to the increased Sb(III) oxidation efficiency. Moreover, the carbon metabolism was also activated to generate more energy against Sb(III) stress. The generated energy may be used in Sb transportation, DNA repair, amino acid synthesis, and cell mobility, and may be released in the form of heat.

  7. Dual Targeting and Retrograde Translocation: Regulators of Plant Nuclear Gene Expression Can Be Sequestered by Plastids

    Directory of Open Access Journals (Sweden)

    Karin Krupinska

    2012-09-01

    Full Text Available Changes in the developmental or metabolic state of plastids can trigger profound changes in the transcript profiles of nuclear genes. Many nuclear transcription factors were shown to be controlled by signals generated in the organelles. In addition to the many different compounds for which an involvement in retrograde signaling is discussed, accumulating evidence suggests a role for proteins in plastid-to-nucleus communication. These proteins might be sequestered in the plastids before they act as transcriptional regulators in the nucleus. Indeed, several proteins exhibiting a dual localization in the plastids and the nucleus are promising candidates for such a direct signal transduction involving regulatory protein storage in the plastids. Among such proteins, the nuclear transcription factor WHIRLY1 stands out as being the only protein for which an export from plastids and translocation to the nucleus has been experimentally demonstrated. Other proteins, however, strongly support the notion that this pathway might be more common than currently believed.

  8. Cloning and expression of zebrafish genes encoding the heme synthesis enzymes uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO).

    Science.gov (United States)

    Hanaoka, Ryuki; Dawid, Igor B; Kawahara, Atsuo

    2007-02-01

    Heme is synthesized from glycine and succinyl CoA by eight heme synthesis enzymes. Although genetic defects in any of these enzymes are known to cause severe human blood diseases, their developmental expression in mammals is unknown. In this paper, we report two zebrafish heme synthesis enzymes, uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO) that are well conserved in comparison to their human counterparts. Both UROS and PPO formed pairs of bilateral stripes in the lateral plate mesoderm at the 15-somite stage. At 24 h post-fertilization (hpf), UROS and PPO were predominantly expressed in the intermediate cell mass (ICM) that is the major site of primitive hematopoiesis. The expression of UROS and PPO was drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche and vlad tepes/gata 1 are required for the induction and maintenance of UROS and PPO expression in the ICM.

  9. Single-gene dual-color reporter cell line to analyze RNA synthesis in vivo.

    Science.gov (United States)

    Palangat, Murali; Larson, Daniel R

    2016-07-01

    RNA synthesis occurs through the multi-step process of transcription which consists of initiation, elongation, termination, and cleavage of the nascent RNA. In recent years, post-initiation events have attracted considerable attention as regulatory steps in gene expression. In particular, changes in elongation rate have been proposed to alter RNA fate either through changes in RNA secondary structure or recruitment of trans-acting factors, but systematic approaches for perturbing and measuring elongation rate are currently lacking. Here, we describe a system for precisely measuring elongation dynamics for single nascent transcripts at a single gene locus in human cell lines. The system is based on observing the production of fluorescently labeled RNA stem loops which flank a region of interest. The region of interest can be altered using flp recombinases, thus allowing one to study the effects of cis-acting sequences on transcription rate. The dual-color RNAs which are made during this process are exported and translated, thus enabling visualization of each step in gene expression.

  10. Inhibition of development of experimental abdominal aortic aneurysm by c-jun N-terminal protein kinase inhibitor combined with lysyl oxidase gene modified smooth muscle progenitor cells.

    Science.gov (United States)

    Chen, Feng; Zhang, ZhenDong; Zhu, XianHua

    2015-11-05

    Chronic inflammation, imbalance between the extracellular matrix synthesis and degradation, and loss of vascular smooth muscle cells (SMCs) contribute to the development of abdominal aortic aneurysm (AAA). The purpose of this study was to investigate the effect of the therapy with periaortic incubation of c-Jun N-terminal protein kinase inhibitor SP600125 infused from an osmotic pump and subadventitial injection of lysyl oxidase (LOX) gene modified autologous smooth muscle progenitor cells (SPCs) on treatment of AAA in a rabbit model. Obvious dilation of the abdominal aorta in the control group was caused by periaortic incubation of calcium chloride and elastase. But the progression of aortic dilation was significantly decreased after the treatment with SP600125 and LOX gene modified SPCs compared to the treatment with phosphate-buffered saline. This therapy could inhibit matrix metalloproteinases expression, enhance elastin synthesis, improve preservation of elastic laminar integrity, benefit SPCs survival and restore SMCs population. It seemed that this method might provide a novel therapeutic strategy to treat AAA.

  11. Characterization of the polyphenol oxidase gene family reveals a novel microRNA involved in posttranscriptional regulation of PPOs in Salvia miltiorrhiza

    Science.gov (United States)

    Li, Caili; Li, Dongqiao; Li, Jiang; Shao, Fenjuan; Lu, Shanfa

    2017-01-01

    Salvia miltiorrhiza is a well-known material of traditional Chinese medicine. Understanding the regulatory mechanisms of phenolic acid biosynthesis and metabolism are important for S. miltiorrhiza quality improvement. We report here that S. miltiorrhiza contains 19 polyphenol oxidases (PPOs), forming the largest PPO gene family in plant species to our knowledge. Analysis of gene structures and sequence features revealed the conservation and divergence of SmPPOs. SmPPOs were differentially expressed in plant tissues and eight of them were predominantly expressed in phloem and xylem, indicating that some SmPPOs are functionally redundant, whereas the others are associated with different physiological processes. Expression patterns of eighteen SmPPOs were significantly altered under MeJA treatment, and twelve were yeast extract and Ag+-responsive, suggesting the majority of SmPPOs are stress-responsive. Analysis of high-throughput small RNA sequences and degradome data showed that miR1444-mediated regulation of PPOs existing in P. trichocarpa is absent from S. miltiorrhiza. Instead, a subset of SmPPOs was posttranscriptionally regulated by a novel miRNA, termed Smi-miR12112. It indicates the specificity and significance of miRNA-mediated regulation of PPOs. The results shed light on the regulation of SmPPO expression and suggest the complexity of SmPPO-associated phenolic acid biosynthesis and metabolism. PMID:28304398

  12. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    Science.gov (United States)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  13. A mutation in the FAM36A gene, the human ortholog of COX20, impairs cytochrome c oxidase assembly and is associated with ataxia and muscle hypotonia.

    Science.gov (United States)

    Szklarczyk, Radek; Wanschers, Bas F J; Nijtmans, Leo G; Rodenburg, Richard J; Zschocke, Johannes; Dikow, Nicola; van den Brand, Mariël A M; Hendriks-Franssen, Marthe G M; Gilissen, Christian; Veltman, Joris A; Nooteboom, Marco; Koopman, Werner J H; Willems, Peter H G M; Smeitink, Jan A M; Huynen, Martijn A; van den Heuvel, Lambertus P

    2013-02-15

    The mitochondrial respiratory chain complex IV (cytochrome c oxidase) is a multi-subunit enzyme that transfers electrons from cytochrome c to molecular oxygen, yielding water. Its biogenesis requires concerted expression of mitochondria- and nuclear-encoded subunits and assembly factors. In this report, we describe a homozygous missense mutation in FAM36A from a patient who displays ataxia and muscle hypotonia. The FAM36A gene is a remote, putative ortholog of the fungal complex IV assembly factor COX20. Messenger RNA (mRNA) and protein co-expression analyses support the involvement of FAM36A in complex IV function in mammals. The c.154A>C mutation in the FAM36A gene, a mutation that is absent in sequenced exomes, leads to a reduced activity and lower levels of complex IV and its protein subunits. The FAM36A protein is nearly absent in patient's fibroblasts. Cells affected by the mutation accumulate subassemblies of complex IV that contain COX1 but are almost devoid of COX2 protein. We observe co-purification of FAM36A and COX2 proteins, supporting that the FAM36A defect hampers the early step of complex IV assembly at the incorporation of the COX2 subunit. Lentiviral complementation of patient's fibroblasts with wild-type FAM36A increases the complex IV activity as well as the amount of holocomplex IV and of individual subunits. These results establish the function of the human gene FAM36A/COX20 in complex IV assembly and support a causal role of the gene in complex IV deficiency.

  14. Cytochrome oxidase 1 gene sequence analysis in six flatfish species (Teleostei, Pleuronectidae) of Far East Russia with inferences in phylogeny and taxonomy.

    Science.gov (United States)

    Kartavtsev, Yuri Ph; Sharina, Svetlana N; Goto, Tadasuke; Chichvarkhin, Anton Y; Balanov, Andrey A; Vinnikov, Kirill A; Ivankov, Vyacheslav N; Hanzawa, Naoto

    2008-12-01

    Mitochondrial DNA at the cytochrome oxidase 1 (Co-1) gene region was sequenced for six flatfish species (in total, 11 sequences of at least 539 base pairs) from the Far East of Russia and compared with other sequences of Pleuronectiformes, comprising altogether 26 flatfish sequences and two outgroup sequences (Perciformes). An analysis of the protein-coding Co-1 gene revealed a statistically substantiated bias in (T + C):(A + G) content, supporting earlier findings. Average scores of the p-distances for different scales of the evolutionary history at the Co-1 gene revealed a clear pattern of increased nucleotide diversity at four different levels: (1) intraspecies, (2) intragenus, (3) intrafamily, and (4) intra-order. Scores of average p-distances of the four categories of comparison in flatfishes were (1) 0.17 +/- 0.09%, (2) 10.60 +/- 1.57%, (3) 12.40 +/- 0.27%, and (4) 19.93 +/- 0.05%, respectively (mean +/- standard error). These data jointly with current knowledge support the concept that speciation in the order Pleuronectiformes mostly follows a geographic mode through accumulation of numerous small genetic changes over a long period of time. A phylogenetic tree for 26 sequences of flatfishes and two other fishes belonging to ray-finned fishes (Actinopterigii) was developed using the Co-1 gene and four different analytical approaches: neighbour-joining, Bayesian (BA), maximum parsimony (MP), and maximum likelihood. The analysis revealed a monophyletic origin for the representatives of Pleuronectidae, which is the principal flatfish family investigated (73-100% support level in our MP and BA analyses). According to the current and literary data, the monophyletic origin for the six compared flatfish families was well supported. Species identification on a per-individual basis (barcoding tagging) was high.

  15. The effects of child maltreatment on early signs of antisocial behavior: genetic moderation by tryptophan hydroxylase, serotonin transporter, and monoamine oxidase A genes.

    Science.gov (United States)

    Cicchetti, Dante; Rogosch, Fred A; Thibodeau, Eric L

    2012-08-01

    Gene-environment interaction effects in predicting antisocial behavior in late childhood were investigated among maltreated and nonmaltreated low-income children (N = 627, M age = 11.27). Variants in three genes were examined: tryptophan hydroxylase 1 (TPH1), serotonin transporter linked polymorphic region (5-HTTLPR), and monoamine oxidase A (MAOA) upstream variable number tandem repeat. In addition to child maltreatment status, we considered the impact of maltreatment subtypes, developmental timing of maltreatment, and chronicity. Indicators of antisocial behavior were obtained from self-, peer, and adult counselor reports. In a series of analyses of covariance, child maltreatment and its parameters demonstrated strong main effects on early antisocial behavior as assessed by all report forms. Genetic effects operated primarily in the context of gene-environment interactions, moderating the impact of child maltreatment on outcomes. Across the three genes, among nonmaltreated children no differences in antisocial behavior were found based on genetic variation. In contrast, among maltreated children specific polymorphisms of TPH1, 5-HTTLPR, and MAOA were each related to heightened self-report of antisocial behavior; the interaction of 5-HTTLPR and developmental timing of maltreatment also indicated more severe antisocial outcomes for children with early onset and recurrent maltreatment based on genotype. TPH1 and 5-HTTLPR interacted with maltreatment subtype to predict peer reports of antisocial behavior; genetic variation contributed to larger differences in antisocial behavior among abused children. The TPH1 and 5-HTTLPR polymorphisms also moderated the effects of maltreatment subtype on adult reports of antisocial behavior; again, the genetic effects were strongest for children who were abused. In addition, TPH1 moderated the effect of developmental timing of maltreatment and chronicity on adult reports of antisocial behavior. The findings elucidate how genetic

  16. Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance.

    Science.gov (United States)

    Matsunaga, Etsuko; Nanto, Kazuya; Oishi, Masatoshi; Ebinuma, Hiroyasu; Morishita, Yoshihiko; Sakurai, Nozomu; Suzuki, Hideyuki; Shibata, Daisuke; Shimada, Teruhisa

    2012-01-01

    Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.

  17. Mitochondrial DNA diversity in the acanthocephalan Prosthenorchis elegans in Colombia based on cytochrome c oxidase I (COI) gene sequence.

    Science.gov (United States)

    Falla, Ana Carolina; Brieva, Claudia; Bloor, Paul

    2015-12-01

    Prosthenorchis elegans is a member of the Phylum Acanthocephala and is an important parasite affecting New World Primates in the wild in South America and in captivity around the world. It is of significant management concern due to its pathogenicity and mode of transmission through intermediate hosts. Current diagnosis of P. elegans is based on the detection of eggs by coprological examination. However, this technique lacks both specificity and sensitivity, since eggs of most members of the genus are morphologically indistinguishable and shed intermittently, making differential diagnosis difficult, and coprological examinations are often negative in animals severely infected at death. We examined sequence variation in 633 bp of mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) sequence in 37 isolates of P. elegans from New World monkeys (Saguinus leucopus and Cebus albifrons) in Colombia held in rescue centers and from the wild. Intraspecific divergence ranged from 0.0 to 1.6% and was comparable with corresponding values within other species of acanthocephalans. Furthermore, comparisons of patterns of sequence divergence within the Acanthocephala suggest that Prosthenorchis represents a separate genus within the Oligacanthorhynchida. Six distinct haplotypes were identified within P. elegans which grouped into one of two well-supported mtDNA haplogroups. No association between haplogroup/haplotype, holding facility and species was found. This information will help pave the way to the development of molecular-based diagnostic tools for the detection of P. elegans as well as furthering research into the life cycle, intermediate hosts and epidemiological aspects of the species.

  18. Mitochondrial DNA diversity in the acanthocephalan Prosthenorchis elegans in Colombia based on cytochrome c oxidase I (COI gene sequence

    Directory of Open Access Journals (Sweden)

    Ana Carolina Falla

    2015-12-01

    Full Text Available Prosthenorchis elegans is a member of the Phylum Acanthocephala and is an important parasite affecting New World Primates in the wild in South America and in captivity around the world. It is of significant management concern due to its pathogenicity and mode of transmission through intermediate hosts. Current diagnosis of P. elegans is based on the detection of eggs by coprological examination. However, this technique lacks both specificity and sensitivity, since eggs of most members of the genus are morphologically indistinguishable and shed intermittently, making differential diagnosis difficult, and coprological examinations are often negative in animals severely infected at death. We examined sequence variation in 633 bp of mitochondrial DNA (mtDNA cytochrome c oxidase I (COI sequence in 37 isolates of P. elegans from New World monkeys (Saguinus leucopus and Cebus albifrons in Colombia held in rescue centers and from the wild. Intraspecific divergence ranged from 0.0 to 1.6% and was comparable with corresponding values within other species of acanthocephalans. Furthermore, comparisons of patterns of sequence divergence within the Acanthocephala suggest that Prosthenorchis represents a separate genus within the Oligacanthorhynchida. Six distinct haplotypes were identified within P. elegans which grouped into one of two well-supported mtDNA haplogroups. No association between haplogroup/haplotype, holding facility and species was found. This information will help pave the way to the development of molecular-based diagnostic tools for the detection of P. elegans as well as furthering research into the life cycle, intermediate hosts and epidemiological aspects of the species.

  19. Inhibition of hepatocelluar carcinoma MAT2A and MAT2beta gene expressions by single and dual small interfering RNA

    Directory of Open Access Journals (Sweden)

    Sun Quan

    2008-11-01

    Full Text Available Abstract RNA interference (RNAi has been successfully applied in suppression of hepatic cancer genes. In hepatocelluar carcinoma cell, one methionine adenosyltransferase (MAT isozyme, MATII was found to have two catalytic subunits which were encoded by MAT2A and MAT2β respectively. During tumorigeness of hepatocelluar carcinoma, expressions of the two genes were discovered to be increased combining with a switch of MAT (form MATI to MATII, To figure out the role played by MATII in hepatic cancer, In this study, for the first time we established a dual small interfering RNA (siRNA expression system, which could simultaneously express two different siRNA molecules specifically targeting two genes. To test the effectiveness of this system, we applied this approach to express simultaneously two different siRNA duplexes that specifically target MAT2A and MAT2β genes of hepatocelluar carcinoma respectively in HepG2 cell. Results indicated that dual siRNA could simultaneously inhibit the expression of MAT2A and MAT2β gene by 89.5% and 97.8% respectively, In addition, dual siRNA molecules were able to significantly suppress growth of hepatocelluar carcinoma cell in vitro as well as induce apoptosis which was involved in arrest cell cycle at the G1/S checkpoint and the expressions of p21, p27 and Bax.

  20. A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit.

    Science.gov (United States)

    Barad, Shiri; Horowitz, Sigal Brown; Moscovitz, Oren; Lichter, Amnon; Sherman, Amir; Prusky, Dov

    2012-06-01

    Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.

  1. Transcriptional regulation of human dual specificity protein phosphatase 1 (DUSP1 gene by glucocorticoids.

    Directory of Open Access Journals (Sweden)

    Lauren E Shipp

    Full Text Available BACKGROUND: Glucocorticoids are potent anti-inflammatory agents commonly used to treat inflammatory diseases. They convey signals through the intracellular glucocorticoid receptor (GR, which upon binding to ligands, associates with genomic glucocorticoid response elements (GREs to regulate transcription of associated genes. One mechanism by which glucocorticoids inhibit inflammation is through induction of the dual specificity phosphatase-1 (DUSP1, a.k.a. mitogen-activated protein kinase phosphatase-1, MKP-1 gene. METHODOLOGY/PRINCIPAL FINDINGS: We found that glucocorticoids rapidly increased transcription of DUSP1 within 10 minutes in A549 human lung adenocarcinoma cells. Using chromatin immunoprecipitation (ChIP scanning, we located a GR binding region between -1421 and -1118 upstream of the DUSP1 transcription start site. This region is active in a reporter system, and mutagenesis analyses identified a functional GRE located between -1337 and -1323. We found that glucocorticoids increased DNase I hypersensitivity, reduced nucleosome density, and increased histone H3 and H4 acetylation within genomic regions surrounding the GRE. ChIP experiments showed that p300 was recruited to the DUSP1 GRE, and RNA interference experiments demonstrated that reduction of p300 decreased glucocorticoid-stimulated DUSP1 gene expression and histone H3 hyperacetylation. Furthermore, overexpression of p300 potentiated glucocorticoid-stimulated activity of a reporter gene containing the DUSP1 GRE, and this coactivation effect was compromised when the histone acetyltransferase domain was mutated. ChIP-reChIP experiments using GR followed by p300 antibodies showed significant enrichment of the DUSP1 GRE upon glucocorticoid treatment, suggesting that GR and p300 are in the same protein complex recruited to the DUSP1 GRE. CONCLUSIONS/SIGNIFICANCE: Our studies identified a functional GRE for the DUSP1 gene. Moreover, the transcriptional activation of DUSP1 by

  2. D-amino acid oxidase activator gene (DAOA) variation affects cerebrospinal fluid homovanillic acid concentrations in healthy Caucasians

    DEFF Research Database (Denmark)

    Andreou, Dimitrios; Saetre, Peter; Werge, Thomas;

    2012-01-01

    3918342 and rs1421292, were significantly associated with CSF HVA concentrations. Rs3918342 was found to be nominally associated with CSF 5-HIAA concentrations. None of the polymorphisms were significantly associated with MHPG concentrations. Our results indicate that DAOA gene variation affects dopamine...

  3. Association of a Monoamine Oxidase-A Gene Promoter Polymorphism with ADHD and Anxiety in Boys with Autism Spectrum Disorder

    Science.gov (United States)

    Roohi, Jasmin; DeVincent, Carla J.; Hatchwell, Eli; Gadow, Kenneth D.

    2009-01-01

    The aim of the present study was to examine the association between a variable number tandem repeat (VNTR) functional polymorphism in the promoter region of the MAO-A gene and severity of ADHD and anxiety in boys with ASD. Parents and teachers completed a DSM-IV-referenced rating scale for 5- to 14-year-old boys with ASD (n = 43). Planned…

  4. Genetic structure of the snakehead murrel, Channa striata (channidae) based on the cytochrome c oxidase subunit I gene: Influence of historical and geomorphological factors.

    Science.gov (United States)

    Jamsari, Amirul Firdaus Jamaluddin; Jamaluddin, Jamsari Amirul Firdaus; Pau, Tan Min; Siti-Azizah, Mohd Nor

    2011-01-01

    Nucleotide sequences of a partial cytochrome c oxidase subunit I gene were used to assess the manner in which historical processes and geomorphological effects may have influenced genetic structuring and phylogeographic patterns in Channa striata. Assaying was based on individuals from twelve populations in four river systems, which were separated into two regions, the eastern and western, of the biodiversely rich state of Perak in central Peninsular Malaysia. In 238 specimens, a total of 368-bp sequences with ten polymorphic sites and eleven unique haplotypes were detected. Data on all the twelve populations revealed incomplete divergence due to past historical coalescence and the short period of separation. Nevertheless, SAMOVA and F(ST) revealed geographical structuring existed to a certain extent in both regions. For the eastern region, the data also showed that the upstream populations were genetically significantly different compared to the mid- and downstream ones. It is inferred that physical barriers and historical processes played a dominant role in structuring the genetic dispersal of the species. A further inference is that the Grik, Tanjung Rambutan and Sungkai are potential candidates for conservation and aquaculture programmes since they contained most of the total diversity in this area.

  5. Genetic structure of the snakehead murrel, Channa striata (channidae based on the cytochrome c oxidase subunit I gene: influence of historical and geomorphological factors

    Directory of Open Access Journals (Sweden)

    Jamsari Amirul Firdaus Jamaluddin

    2011-01-01

    Full Text Available Nucleotide sequences of a partial cytochrome c oxidase subunit I gene were used to assess the manner in which historical processes and geomorphological effects may have influenced genetic structuring and phylogeographic patterns in Channa striata. Assaying was based on individuals from twelve populations in four river systems, which were separated into two regions, the eastern and western, of the biodiversely rich state of Perak in central Peninsular Malaysia. In 238 specimens, a total of 368-bp sequences with ten polymorphic sites and eleven unique haplotypes were detected. Data on all the twelve populations revealed incomplete divergence due to past historical coalescence and the short period of separation. Nevertheless, SAMOVA and F ST revealed geographical structuring existed to a certain extent in both regions. For the eastern region, the data also showed that the upstream populations were genetically significantly different compared to the mid- and downstream ones. It is inferred that physical barriers and historical processes played a dominant role in structuring the genetic dispersal of the species. A further inference is that the Grik, Tanjung Rambutan and Sungkai are potential candidates for conservation and aquaculture programmes since they contained most of the total diversity in this area.

  6. A role for NADPH oxidase in antigen presentation

    Directory of Open Access Journals (Sweden)

    Gail J Gardiner

    2013-09-01

    Full Text Available The nicotinamide adenine dinucleotide phosphate (NADPH oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.-. This radical is an important precursor of hydrogen peroxide (H2O2 and other reactive oxygen species (ROS needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD, a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC crosspresentation by major histocompatibility complex class I molecules (MHC-I through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules (MHC-II.

  7. A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome.

    Science.gov (United States)

    Tiranti, V; Corona, P; Greco, M; Taanman, J W; Carrara, F; Lamantea, E; Nijtmans, L; Uziel, G; Zeviani, M

    2000-11-01

    We report on a novel frameshift mutation in the mtDNA gene encoding cytochrome c oxidase (COX) subunit III. The proband is an 11-year-old girl with a negative family history and an apparently healthy younger brother. Since 4 years of age, she has developed a progressive spastic paraparesis associated with ophthalmoparesis and moderate mental retardation. The presence of severe lactic acidosis and Leigh-like lesions of putamina prompted us to perform muscle and skin biopsies. In both, a profound, isolated defect of COX was found by histochemical and biochemical assays. Sequence analysis of muscle mtDNA resulted in the identification of a virtually homoplasmic frameshift mutation in the COIII gene, due to the insertion of an extra C at nucleotide position 9537 of mtDNA. Although the 9537C(ins) does not impair transcription of COIII, no full-length COX III protein was detected in mtDNA translation assays in vivo. Western blot analysis of two-dimensional blue-native electrophoresis showed a reduction of specific crossreacting material and the accumulation of early-assembly intermediates of COX, whereas the fully assembled complex was absent. One of these intermediates had an electrophoretic mobility different from those seen in controls, suggesting the presence of a qualitative abnormality of COX assembly. Immunostaining with specific antibodies failed to detect the presence of several smaller subunits in the complex lacking COX III, in spite of the demonstration that these subunits were present in the crude mitochondrial fraction of patient's cultured fibroblasts. Taken together, the data indicate a role for COX III in the incorporation and maintenance of smaller COX subunits within the complex.

  8. [Genes of insecticidal crystal proteins with dual specificity in Bacillus thuringiensis strains, isolated in the Crimea territory].

    Science.gov (United States)

    Rymar, S Iu; Isakova, I A; Kuznietsova, L M; Kordium, V A

    2006-01-01

    The insecticidal crystal proteins of 15 B. thuringiensis strains, isolated in the Crimea territory that are toxical for some Lepidoptera and Colorado potato beetle larvae were identified by PAGE electrophoresis. Ten strains produced the crystal proteins with high molecular weight (> 120 kD). PCR with use of broad specificity primers and DNA of these B. thuringiensis strains as template demonstrated the specific PCR products (1000 bp). Amplified DNA fragments were cloned and sequenced. The nucleotide sequence analysis revealed that the genomes of ten strains of B. thuringiensis carried Cry1B genes, which are responsible for production of the insecticidal crystal proteins with dual specificity. The influence of the solubilization conditions on the structure and toxicity of Cry1B protein for Colorado potato beetle larvae was shown. The dual toxicity of studied B. thuringiensis strains is explained by the Cry1B genes presence in their genomes. These strains may be used to develop the broad specificity bioinsecticides.

  9. The Diamine Oxidase Gene Is Associated with Hypersensitivity Response to Non-Steroidal Anti-Inflammatory Drugs

    Science.gov (United States)

    Agúndez, José A. G.; Ayuso, Pedro; Cornejo-García, José A.; Blanca, Miguel; Torres, María J.; Doña, Inmaculada; Salas, María; Blanca-López, Natalia; Canto, Gabriela; Rondon, Carmen; Campo, Paloma; Laguna, José J.; Fernández, Javier; Martínez, Carmen; García-Martín, Elena

    2012-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are the drugs most frequently involved in hypersensitivity drug reactions. Histamine is released in the allergic response to NSAIDs and is responsible for some of the clinical symptoms. The aim of this study is to analyze clinical association of functional polymorphisms in the genes coding for enzymes involved in histamine homeostasis with hypersensitivity response to NSAIDs. We studied a cohort of 442 unrelated Caucasian patients with hypersensitivity to NSAIDs. Patients who experienced three or more episodes with two or more different NSAIDs were included. If this requirement was not met diagnosis was established by challenge. A total of 414 healthy unrelated controls ethnically matched with patients and from the same geographic area were recruited. Analyses of the SNPs rs17740607, rs2073440, rs1801105, rs2052129, rs10156191, rs1049742 and rs1049793 in the HDC, HNMT and DAO genes were carried out by means of TaqMan assays. The detrimental DAO 16 Met allele (rs10156191), which causes decreased metabolic capacity, is overrepresented among patients with crossed-hypersensitivity to NSAIDs with an OR  = 1.7 (95% CI  = 1.3–2.1; Pc  = 0.0003) with a gene-dose effect (P = 0.0001). The association was replicated in two populations from different geographic areas (Pc  = 0.008 and Pc  = 0.004, respectively). Conclusions and implications The DAO polymorphism rs10156191 which causes impaired metabolism of circulating histamine is associated with the clinical response in crossed-hypersensitivity to NSAIDs and could be used as a biomarker of response. PMID:23152756

  10. Evidence that the methylation state of the monoamine oxidase A (MAOA) gene predicts brain activity of MAO A enzyme in healthy men

    National Research Council Canada - National Science Library

    Shumay, Elena; Logan, Jean; Volkow, Nora D; Fowler, Joanna S

    2012-01-01

    ...). PET brain imaging of monoamine oxidase A (MAO A)-an enzyme metabolizing neurotransmitters-revealed that MAO A levels vary widely between healthy men and this variability was not explained by the common MAOA genotype (VNTR genotype...

  11. Construction and expression of retroviruses encoding dual drug resistance genes in human umbilical cord blood CD34+ cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A series of retroviral vectors encoding human mdr1 gene alone as well as in combination with either human mgmt gene or human mutant Ser31-dhfr gene are engineered. The resultant retroviruses are used to transduce human umbilical cord blood CD34+ cells. It has been shown that expression of dual drug resistance genes in transduced cells confers a broad range of resistance to both kinds of corresponding drugs. These data suggest a rationale for the use of such double chemoresistance gene constructs in an in vivo model in which transduced hematopoietic cells will acquire multiple protection against the cytotoxic side effects of combination chemotherapy and may have future application in chemoprotection of normal tissues, thus killing tumor cells more effectively.

  12. Sex-specific associations of variants in regulatory regions of NADPH oxidase-2 (CYBB) and glutathione peroxidase 4 (GPX4) genes with kidney disease in type 1 diabetes.

    Science.gov (United States)

    Monteiro, M B; Patente, T A; Mohammedi, K; Queiroz, M S; Azevedo, M J; Canani, L H; Parisi, M C; Marre, M; Velho, G; Corrêa-Giannella, M L

    2013-10-01

    Oxidative stress is involved in the pathophysiology of diabetic nephropathy. The superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase 2 (NOX2, encoded by the CYBB gene) and the antioxidant enzyme glutathione peroxidase 4 (GPX4) play opposing roles in the balance of cellular redox status. In the present study, we investigated associations of single nucleotide polymorphisms (SNPs) in the regulatory regions of CYBB and GPX4 with kidney disease in patients with type 1 diabetes. Two functional SNPs, rs6610650 (CYBB promoter region, chromosome X) and rs713041 (GPX4 3'untranslated region, chromosome 19), were genotyped in 451 patients with type 1 diabetes from a Brazilian cohort (diabetic nephropathy: 44.6%) and in 945 French/Belgian patients with type 1 diabetes from Genesis and GENEDIAB cohorts (diabetic nephropathy: 62.3%). The minor A-allele of CYBB rs6610650 was associated with lower estimated glomerular filtration rate (eGFR) in Brazilian women, and with the prevalence of established/advanced nephropathy in French/Belgian women (odds ratio 1.75, 95% CI 1.11-2.78, p = 0.016). The minor T-allele of GPX4 rs713041 was inversely associated with the prevalence of established/advanced nephropathy in Brazilian men (odds ratio 0.30, 95% CI 0.13-0.68, p = 0.004), and associated with higher eGFR in French/Belgian men. In conclusion, these heterogeneous results suggest that neither CYBB nor GPX4 are major genetic determinants of diabetic nephropathy, but nevertheless, they could modulate in a gender-specific manner the risk for renal disease in patients with type 1 diabetes.

  13. Phylogenetic position of Linguatula arctica and Linguatula serrata (Pentastomida) as inferred from the nuclear 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit I gene.

    Science.gov (United States)

    Gjerde, Bjørn

    2013-10-01

    Genomic DNA was isolated from a Linguatula serrata female expelled from a dog imported to Norway from Romania and from four Linguatula arctica females collected from semi-domesticated reindeer from northern Norway and subjected to PCR amplification of the complete nuclear 18S rRNA gene and a 1,045-bp portion of the mitochondrial cytochrome c oxidase subunit I gene (cox1). The two species differed at two of 1,830 nucleotide positions (99.9% identity) of the complete 18S rRNA gene sequences and at 102 of 1,045 nucleotide positions (90.2% identity) of the partial cox1 sequences. The four isolates of L. arctica showed no genetic variation in either gene. The new cox1 primers may facilitate the diagnosis of various developmental stages of L. arctica and L. serrata in their hosts. In separate phylogenetic analyses using the maximum likelihood method on sequence data from either gene, L. arctica and L. serrata clustered with members of the order Cephalobaenida rather than with members of the order Porocephalida, in which the genus Linguatula is currently placed based on morphological characters. The phylogenetic relationship of L. arctica, L. serrata and other pentastomids to other metazoan groups could not be clearly resolved, but the pentastomids did not seem to have a sister relationship to crustaceans of the subclass Branchiura as found in other studies. A more extensive taxon sampling, including molecular characterisation of more pentastomid taxa across different genera, seems to be necessary in order to estimate the true relationship of the Pentastomida to other metazoan groups.

  14. An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design.

    Science.gov (United States)

    Zhao, Yongping; Zhang, Congsheng; Liu, Wenwen; Gao, Wei; Liu, Changlin; Song, Gaoyuan; Li, Wen-Xue; Mao, Long; Chen, Beijiu; Xu, Yunbi; Li, Xinhai; Xie, Chuanxiao

    2016-04-01

    Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement.

  15. STEADY-STATE TRANSCRIPT LEVELS OF CYTOCHROME-C-OXIDASE GENES DURING HUMAN MYOGENESIS INDICATE SUBUNIT SWITCHING OF SUBUNIT VIA AND COEXPRESSION OF SUBUNIT VIIA ISOFORMS

    NARCIS (Netherlands)

    TAANMAN, JW; HERZBERG, NH; DEVRIES, H; BOLHUIS, PA; VANDENBOGERT, C

    1992-01-01

    Steady-state levels of the mitochondrial rRNAs, of mRNAs for mitochondrially and nuclear-encoded subunits of cytochrome c oxidase and for the beta-subunit of ATP synthase were assessed by Northern blot hybridizations during the in vitro differentiation of human myoblasts. Transcript levels of the so

  16. Subcellular localization of vanillyl-alcohol oxidase in Penicillium simplicissimum

    NARCIS (Netherlands)

    Fraaije, MW; Sjollema, KA; Veenhuis, M; van Berkel, WJH; Berkel, Willem J.H. van

    1998-01-01

    Growth of Penicillium simplicissimum on anisyl alcohol, veratryl alcohol or 3-(methoxymethyl)phenol, is associated with the synthesis of relatively large amounts of the hydrogen peroxide producing flavoprotein vanillyl-alcohol oxidase (VAO), Immunocytochemistry revealed that the enzyme has a dual lo

  17. Dual DNA methylation patterns in the CNS reveal developmentally poised chromatin and monoallelic expression of critical genes.

    Science.gov (United States)

    Wang, Jinhui; Valo, Zuzana; Bowers, Chauncey W; Smith, David D; Liu, Zheng; Singer-Sam, Judith

    2010-11-04

    As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay). We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F(1) hybrid clonal neural stem cell (NSC) lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1-2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment.

  18. Dual DNA methylation patterns in the CNS reveal developmentally poised chromatin and monoallelic expression of critical genes.

    Directory of Open Access Journals (Sweden)

    Jinhui Wang

    Full Text Available As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay. We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F(1 hybrid clonal neural stem cell (NSC lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1-2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment.

  19. Mitochondrial cytochrome c oxidase deficiency.

    Science.gov (United States)

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-03-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance of studying different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy.

  20. Characterization of polyphenol oxidase from plants

    Institute of Scientific and Technical Information of China (English)

    LEI Dongfeng; FENG Yi; JIANG Dazong

    2004-01-01

    Polyphenol oxidase (PPO) which can mediate browning reaction is a bifunctional copper-containing enzyme encoded by plant nucleolus gene. It usually leads to excessive browning reaction which reduces the coercial profits of fruits and vegetables. In this paper, PPO genes and enzymes in plants are characterized systematically, and the latest progress is reviewed. Some clonings of PPOs genes are reported; the specific temporal and spatial expression pattern of PPOs genes is described; the model of the structure of the precursor form of catechol oxidase is introduced; the possible functions of PPOs in defending against pathogen, wounding, surrounding stress and other inducing factors are demonstrated; the induction and activation of latent PPOs in some plants is elucidated; the scheme of browning inhibition by L-cysteine is clarified; the mechanism of suicide inhibition of latent PPO and kinetic synergism are established. Furthermore, the area for future study is also discussed.

  1. CHD5 is required for neurogenesis and has a dual role in facilitating gene expression and polycomb gene repression

    DEFF Research Database (Denmark)

    Egan, Chris M; Nyman, Ulrika; Skotte, Julie

    2013-01-01

    differentiation and leads to an accumulation of undifferentiated progenitors. CHD5 binds a large cohort of genes and is required for facilitating the activation of neuronal genes. It also binds a cohort of Polycomb targets and is required for the maintenance of H3K27me3 on these genes. Interestingly...

  2. multicopper oxidases important for human iron metabolism

    Directory of Open Access Journals (Sweden)

    Diana Wierzbicka

    2014-01-01

    Full Text Available Multi-copper oxidases are a group of proteins which demonstrate enzymatic activity and are capable of oxidizing their substrates with the concomitant reduction of dioxygen to two water molecules. For some multi-copper oxidases there has been demonstrated ferroxidase activity which is related to their specific structure characterized by the presence of copper centres and iron-binding sites. Three multi-copper oxidases have been included in this group: ceruloplasmin, hephaestin and zyklopen. Multi copper oxidases which are expressed in different tissues are capable of oxidizing a wide spectrum of substrates. Multi-copper oxidases are capable of oxidizing a wide spectrum of substrates. Ceruloplasmin exhibits antioxidant activity as well as being involved in many other biological processes. The observations of phenotypic effects of absence or low expression of multi-copper ferroxidase-coding genes suggest that the main role of these proteins is taking part in iron metabolism. The main role of ceruloplasmin in iron turnover is oxidizing Fe2+ into Fe3+, a process which is essential for iron binding to transferrin (the main iron-transporting protein, as well as to ferritin (the main iron-storage protein. The function of hephaestin as ferroxidase is essential for iron binding to apotransferrin in the lamina propria of the intestinal mucosa, a process that is important for further transport of iron to the liver by the portal vein. Available data indicate that zyklopen is responsible for the placental iron transport. The presence of three multi-copper oxidases with ferroxidase activity emphasizes the significance of oxidation for iron metabolism. The distribution of multi-copper ferroxidases in many tissues ensures the proper iron turnover in the body as well as preventing toxic effects related to the presence of Fe2+ ions. These ions contribute to generation of free radicals, including the highly reactive hydroxyl radical, through the Fenton and Haber

  3. Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran

    Science.gov (United States)

    Reaghi, Saber; Haghighi, Ali; Harandi, Majid Fasihi; Spotin, Adel; Arzamani, Kourosh; Rouhani, Soheila

    2016-01-01

    Aim: Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I [ND1] and cytochrome oxidase subunit I) gene marker. Materials and Methods: In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed. Results: Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference. Conclusion: F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world. PMID:27733809

  4. Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.

    Science.gov (United States)

    Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R

    2011-01-01

    The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  5. RET and GDNF gene scanning in Hirschprung patients using two dual denaturing gel systems

    NARCIS (Netherlands)

    Hofstra, RMW; Wu, Y; Stulp, RP; Elfferich, P; Osinga, J; Maas, SM; Siderius, L; Brooks, AS; Von der Ende, JJ; Heydendael, VMR; Severijnen, RSVM; Bax, KMA; Meijers, C; Buys, CHCM

    2000-01-01

    Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal obstruction due to an absence of intramural ganglia along variable lengths of the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF gene, and encoding one of the RET ligands, either alone or in co

  6. Identification of Bovine, Pig and Duck Meat Species in Mixtures and in Meat Products on the Basis of the mtDNA Cytochrome Oxidase Subunit I (COI Gene Sequence

    Directory of Open Access Journals (Sweden)

    Spychaj Anita

    2016-03-01

    Full Text Available The aim of this study was to develop a method using PCR and self-designed primers on the basis of the mtDNA cytochrome oxidase subunit I (COI gene sequence to enable direct identification of the meat of three species of animals, i.e. bovines, pigs and ducks, in the single type sample, in meat mixtures and meat products. The mixtures comprised up to six meat species including apart from beef, pork and duck also chicken, turkey and goose meat. The obtained results indicate the possibility of qualitative identification of the aforementioned meat species in all types of investigated food products. The maximum length of PCR products did not exceed 300 bp, which was supposed to favour the amplification of DNA from meat products which are usually thermally processed and/or exposed to high pressure. PCR primers hybridised selectively with bovine, pig and duck DNA, showing total species specificity.

  7. Monoamine Oxidase A (MAOA) and Catechol-O-Methyltransferase (COMT) Gene Polymorphisms Interact with Maternal Parenting in Association with Adolescent Reactive Aggression but not Proactive Aggression: Evidence of Differential Susceptibility.

    Science.gov (United States)

    Zhang, Wenxin; Cao, Cong; Wang, Meiping; Ji, Linqin; Cao, Yanmiao

    2016-04-01

    To date, whether and how gene-environment (G × E) interactions operate differently across distinct subtypes of aggression remains untested. More recently, in contrast with the diathesis-stress hypothesis, an alternative hypothesis of differential susceptibility proposes that individuals could be differentially susceptible to environments depending on their genotypes in a "for better and for worse" manner. The current study examined interactions between monoamine oxidase A (MAOA) T941G and catechol-O-methyltransferase (COMT) Val158Met polymorphisms with maternal parenting on two types of aggression: reactive and proactive. Moreover, whether these potential G × E interactions would be consistent with the diathesis-stress versus the differential susceptibility hypothesis was tested. Within the sample of 1399 Chinese Han adolescents (47.2 % girls, M age = 12.32 years, SD = 0.50), MAOA and COMT genes both interacted with positive parenting in their associations with reactive but not proactive aggression. Adolescents with T alleles/TT homozygotes of MAOA gene or Met alleles of COMT gene exhibited more reactive aggression when exposed to low positive parenting, but less reactive aggression when exposed to high positive parenting. These findings provide the first evidence for distinct G × E interaction effects on reactive versus proactive aggression and lend further support for the differential susceptibility hypothesis.

  8. Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-12-10

    D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.

  9. Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.

    Science.gov (United States)

    Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

    2014-09-01

    Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2.

  10. Dual RNA-seq of parasite and host reveals gene expression dynamics during filarial worm-mosquito interactions.

    Directory of Open Access Journals (Sweden)

    Young-Jun Choi

    2014-05-01

    Full Text Available BACKGROUND: Parasite biology, by its very nature, cannot be understood without integrating it with that of the host, nor can the host response be adequately explained without considering the activity of the parasite. However, due to experimental limitations, molecular studies of parasite-host systems have been predominantly one-sided investigations focusing on either of the partners involved. Here, we conducted a dual RNA-seq time course analysis of filarial worm parasite and host mosquito to better understand the parasite processes underlying development in and interaction with the host tissue, from the establishment of infection to the development of infective-stage larva. METHODOLOGY/PRINCIPAL FINDINGS: Using the Brugia malayi-Aedes aegypti system, we report parasite gene transcription dynamics, which exhibited a highly ordered developmental program consisting of a series of cyclical and state-transitioning temporal patterns. In addition, we contextualized these parasite data in relation to the concurrent dynamics of the host transcriptome. Comparative analyses using uninfected tissues and different host strains revealed the influence of parasite development on host gene transcription as well as the influence of the host environment on parasite gene transcription. We also critically evaluated the life-cycle transcriptome of B. malayi by comparing developmental stages in the mosquito relative to those in the mammalian host, providing insight into gene expression changes underpinning the mosquito-borne parasitic lifestyle of this heteroxenous parasite. CONCLUSIONS/SIGNIFICANCE: The data presented herein provide the research community with information to design wet lab experiments and select candidates for future study to more fully dissect the whole set of molecular interactions of both organisms in this mosquito-filarial worm symbiotic relationship. Furthermore, characterization of the transcriptional program over the complete life cycle of

  11. Primary mechanism of the role of dual oxidase-1 causing airway allergic diseases in human bronchial epithelium%双重氧化酶1引起变应性疾病发生机制的初步研究

    Institute of Scientific and Technical Information of China (English)

    王丽芬; 黄志纯; 吴修法; 王海飞

    2013-01-01

    能依赖于脂筏来源的神经酰胺.%Objective To investigate the role of dual oxidase-1 (DUOX-1) inducing airway hyperresponsiveness in human bronchial epithelium.Methods The human bronchial epithelial cells were divided into several groups:control group,tumor necrosis factor-α (TNF-α) group,methyl-β-cyclodextrin (M-β-CD) + TNF-α group,desipramine (DES) + TNF-α group,diphenylene iodonium (DPI) + TNF-α group and apocynin (APO) + TNF-α group.Fractionation was performed by sucrose gradient centrifugation and the protein DUOX-1 was measured by western blotting.The lipid raft clusters and its colocalization with DUOX-1 were confocal analysed.The intracellular reactive oxygen species (ROS) accumulation was measured by fluorescence of reactive oxygen probe of intracellular measurement.Sigmastat 3.02 software was used to analyze the data.Results (1) Detection of ROS,control group:1.00± 0.00 ; TNF-α group:1.95± 0.16 ; M-β-CD + TNF-α group:0.91 ± 0.16 ; DES + TNF-α group:1.49± 0.20 ; DPI + TNF-α group:1.03 ± 0.16 ; APO + TNF-α group:1.47 ± 0.26.The difference was statistically significant (F =3.83,P <0.05).(2) Extracts in rafts to lipid rafts region represents the ratio of total protein,protein content DUOX-1each group,control group:0.21 ± 0.02; TNF-α group:0.49 ± 0.04; M-β-CD + TNF-α group:0.08 ±0.02 ; DES + TNF-α group:0.09 ± 0.03 ; the difference was statistically significant (F =3.96,P < 0.05).(3) DUOX-1 protein fluorescence values,control group:1.72 ± 0.21; TNF-α group:8.11 ± 1.23; M-β-CD + TNF-α group:1.51 ± 0.32; DES + TNF-α group:1.43 ± 0.11 ; the difference was statistically significant (F =4.87,P < 0.05).(4) DUOX-1 gene detection,control group:1.00 ± 0.00 ScrRNA +TNF-α group:1.75 ± 0.04; DUOX-1siRNA + TNF-αgroup:1.15 ± 0.02; the difference was statistically significant (F =4.19,P < 0.05).Conclusion TNF-α can induce DUOX-1 expression increasing in lipid raft,then the DUOX-1 can be activated to increase reactive

  12. [Molecular phylogeny and the time of divergence of minges (Chironomidae, Nematocera, Diptera) inferred from a partial nucleotide sequence of the cytochrome oxidase I gene (COI)].

    Science.gov (United States)

    Demin, A G; Polukonova, N V; Miuge, N S

    2011-10-01

    This is the first study to infer the phylogenetic structure of minges of the subfamily Chironominae from the amino acid sequence of cytochrome oxidase I (COI). The subdivision of Chironominae into two tribes, Chironomini and Tanytarsini, has been confirmed. The segregation of the genera Pseudochironomus and Riethia into a separate tribe has not been confirmed. Stenochironomus gibbus forms a branch considerably deviating from the subfamily Chironominae. The genus Micropsectra is formed by a large polyphyletic cluster that also includes the genera Virgotanytharsus, Reotanytharsus, Kenopsectra, and Parapsectra. Tanytarsus is the basal genus of the tribe Tanytarsini. The times of divergence of the main taxa of Chironominae have been estimated. The calculated time of divergence of the genus Chironomus disproves the assumption that it is phylogenetically old.

  13. Isolated sulfite oxidase deficiency.

    Science.gov (United States)

    Relinque, B; Bardallo, L; Granero, M; Jiménez, P J; Luna, S

    2015-03-10

    Sulfite oxidase deficiency is an uncommon metabolic disease. Only few cases of its isolated form have been reported in the literature. We report a case of severe neonatal onset. A newborn baby of 41 weeks gestational age, weighted at birth of 3240 grams and had an Apgar score of 6-10-10. Fifty-three hours after being born, the baby started with seizures that were refractory to antiepileptic treatment. Brain function was monitored using a-EEG. Laboratory and imaging tests were performed. All of them were consistent with sulfite oxidase deficiency. The diagnosis was confirmed by genetic testing. We highlight the importance of this disease as part of the differential diagnosis of seizures during the neonatal period, as well as the importance of the therapeutic support based on dietary restrictions. It's also remarkable the possibility of prenatal diagnosis by quantifying enzyme activity and it's also possible carrying out DNA mutational analysis.

  14. Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation.

    Science.gov (United States)

    Mick, David U; Fox, Thomas D; Rehling, Peter

    2011-01-01

    Mitochondria maintain genome and translation machinery to synthesize a small subset of subunits of the oxidative phosphorylation system. To build up functional enzymes, these organellar gene products must assemble with imported subunits that are encoded in the nucleus. New findings on the early steps of cytochrome c oxidase assembly reveal how the mitochondrial translation of its core component, cytochrome c oxidase subunit 1 (Cox1), is directly coupled to the assembly of this respiratory complex.

  15. Dual control by a single gene of secondary sexual characters and mating preferences in medaka

    Directory of Open Access Journals (Sweden)

    Oda Shoji

    2009-09-01

    Full Text Available Abstract Background Animals utilize a wide variety of tactics to attract reproductive partners. Behavioral experiments often indicate an important role for visual cues in fish, but their molecular basis remains almost entirely unknown. Studies on model species (such as zebrafish and medaka allow investigations into this fundamental question in behavioral and evolutionary biology. Results Through mate-choice experiences using several laboratory strains of various body colors, we successfully identified one medaka mutant (color interfere; ci that is distinctly unattractive to reproductive partners. This unattractiveness seems to be due to reduced orange pigment cells (xanthophores in the skin. The ci strain carries a mutation on the somatolactin alpha (SLa gene, therefore we expected over-expression of SLa to make medaka hyper-attractive. Indeed, extremely strong mating preferences were detected in a choice between the ci and SLa-transgenic (Actb-SLa:GFP medaka. Intriguingly, however, the strains showed opposite biases; that is, the mutant and transgenic medaka liked to mate with partners from their own strain, similar to becoming sexually isolated. Conclusion This study spotlighted SLa as a novel mate-choice gene in fish. In addition, these results are the first demonstration of a single gene that can pleiotropically and harmoniously change both secondary sexual characters and mating preferences. Although theoretical models have long suggested joint evolution of linked genes on a chromosome, a mutation on a gene-regulatory region (that is, switching on/off of a single gene might be sufficient to trigger two 'runaway' processes in different directions to promote (sympatric speciation.

  16. Versatile dual reporter gene systems for investigating stop codon readthrough in plants.

    Directory of Open Access Journals (Sweden)

    Nga T Lao

    Full Text Available BACKGROUND: Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA in an mRNA. However, many viruses (and some cellular mRNAs contain "stop" codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a "readthrough", or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context. METHODOLOGY: We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP. CONCLUSIONS: We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a 'leaky' UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV readthrough context.

  17. Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes.

    Science.gov (United States)

    Bour, Sandy; Daviaud, Danièle; Gres, Sandra; Lefort, Corinne; Prévot, Danielle; Zorzano, Antonio; Wabitsch, Martin; Saulnier-Blache, Jean-Sébastien; Valet, Philippe; Carpéné, Christian

    2007-08-01

    A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.

  18. Differential gene expression along the animal-vegetal axis in the ascidian embryo is maintained by a dual functional protein Foxd.

    Science.gov (United States)

    Tokuhiro, Shin-Ichi; Tokuoka, Miki; Kobayashi, Kenji; Kubo, Atsushi; Oda-Ishii, Izumi; Satou, Yutaka

    2017-05-01

    In many animal embryos, a specific gene expression pattern is established along the animal-vegetal axis soon after zygotic transcription begins. In the embryo of the ascidian Ciona intestinalis, soon after the division that separates animal and vegetal hemispheres into distinct blastomeres, maternal Gata.a and β-catenin activate specific genes in the animal and vegetal blastomeres, respectively. On the basis of these initial distinct gene expression patterns, gene regulatory networks promote animal cells to become ectodermal tissues and vegetal cells to become endomesodermal tissues and a part of the nerve cord. In the vegetal hemisphere, β-catenin directly activates Foxd, an essential transcription factor gene for specifying endomesodermal fates. In the present study, we found that Foxd also represses the expression of genes that are activated specifically in the animal hemisphere, including Dmrt1, Prdm1-r.a (Bz1), Prdm1-r.b (Bz2), and Otx. A reporter assay showed that Dmrt1 expression was directly repressed by Foxd, and a chromatin immunoprecipitation assay showed that Foxd was bound to the upstream regions of Dmrt1, Prdm1-r.a, Prdm1-r.b, and Otx. Thus, Foxd has a dual function of activating specific gene expression in the vegetal hemisphere and of repressing the expression of genes that are normally expressed in the animal hemisphere. This dual function stabilizes the initial patterning along the animal-vegetal axis by β-catenin and Gata.a.

  19. Dual histone reader ZMYND8 inhibits cancer cell invasion by positively regulating epithelial genes.

    Science.gov (United States)

    Basu, Moitri; Sengupta, Isha; Khan, Md Wasim; Srivastava, Dushyant Kumar; Chakrabarti, Partha; Roy, Siddhartha; Das, Chandrima

    2017-05-19

    Enhanced migratory potential and invasiveness of cancer cells contribute crucially to cancer progression. These phenotypes are achieved by precise alteration of invasion-associated genes through local epigenetic modifications which are recognized by a class of proteins termed a chromatin reader. ZMYND8 [zinc finger MYND (myeloid, Nervy and DEAF-1)-type containing 8], a key component of the transcription regulatory network, has recently been shown to be a novel reader of H3.1K36Me2/H4K16Ac marks. Through differential gene expression analysis upon silencing this chromatin reader, we identified a subset of genes involved in cell proliferation and invasion/migration regulated by ZMYND8. Detailed analysis uncovered its antiproliferative activity through BrdU incorporation, alteration in the expression of proliferation markers, and cell cycle regulating genes and cell viability assays. In addition, performing wound healing and invasion/migration assays, its anti-invasive nature is evident. Interestingly, epithelial-mesenchymal transition (EMT), a key mechanism of cellular invasion, is regulated by ZMYND8 where we identified its selective enrichment on promoters of CLDN1/CDH1 genes, rich in H3K36Me2/H4K16Ac marks, leading to their up-regulation. Thus, the presence of ZMYND8 could be implicated in maintaining the epithelial phenotype of cells. Furthermore, syngeneic mice, injected with ZMYND8-overexpressed invasive breast cancer cells, showed reduction in tumor volume and weight. In concert with this, we observed a significant down-regulation of ZMYND8 in invasive ductal and lobular breast cancer tissues compared with normal tissue. Taken together, our study elucidates a novel function of ZMYND8 in regulating EMT and invasion of cancer cells, possibly through its chromatin reader function. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  20. 家蚕磷酸吡哆醇氧化酶基因的表达谱分析%Expression profiling of pyridoxine 5’-phosphate oxidase gene in Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    葛俊楠; 张剑韵; 黄龙全

    2011-01-01

    [Aim] The expression profile of gene encoding pyridoxine phosphate oxidase (PNPO) , which is a key enzyme related to VB6 metabolism, was analyzed in different developmental stages and tissues of the 5th instar larvae of Bombyx mori. [ Methods ] The recombinant plasmid pET-22b ( + ) -PNPO was transformed to Escherichia coli Rosetta for induction, expression and purification of PNPO, and then the polyclonal antibody was prepared. The expression profile of PNPO gene was analyzed by Real-time PCR and Western blot, respectively. [ Results ] The highest translation level of PNPO gene was found in the 5th instar larvae. The transcription level in tissues of 5th instar larvae was in sequence of testis > head > tnidgut > Malpighian tubules > ovary > cuticle > fat body > silk glands. However, the highest translation level was found in testis, and then was in head, midgut, and malpighian tubules, respectively. [ Conclusion ] The expression profile PNPO gene in B. Mori has been defined by this study.%目的 了解家蚕Bombyx mori维生素B6关键代谢酶磷酸吡哆醇氧化酶(pyridoxine-5'-phosphate oxidase,PNPO)基因在家蚕不同发育阶段及5龄幼虫不同组织中的表达差异.方法 将家蚕PNPO基因的重组表达质粒pET-22b(+)-PNPO转化入大肠杆菌Escherichia coli Rosetta中诱导表达,纯化蛋白制备多克隆抗体.分别采用荧光定量PCR和Western blot方法对家蚕PNPO基因进行了转录水平和翻译水平的表达分析.结果 在家蚕发育水平上,5龄幼虫的PNPO翻译量为最高.PNPO基因在5龄幼虫各组织中的转录水平由高到低依次为精巢、头、中肠、马氏管、卵巢、表皮、脂肪体、丝腺;翻译量也以精巢为最高,其次是头、中肠和马氏管.结论 明确了PNPO在家蚕各发育阶段及5龄幼虫各组织中的表达情况.

  1. MegaTevs: single-chain dual nucleases for efficient gene disruption.

    Science.gov (United States)

    Wolfs, Jason M; DaSilva, Matthew; Meister, Sarah E; Wang, Xu; Schild-Poulter, Caroline; Edgell, David R

    2014-07-01

    Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications.

  2. Dual AAV/IL-10 Plus STAT3 Anti-Inflammatory Gene Delivery Lowers Atherosclerosis in LDLR KO Mice, but without Increased Benefit

    Directory of Open Access Journals (Sweden)

    Maohua Cao

    2012-01-01

    Full Text Available Both IL-10 and STAT3 are in the same signal transduction pathway, with IL-10-bound IL10 receptor (R acting through STAT3 for anti-inflammatory effect. To investigate possible therapeutic synergism, we delivered both full-length wild-type human (h STAT3 and hIL-10 genes by separate adenoassociated virus type 8 (AAV8 tail vein injection into LDLR KO on HCD. Compared to control Neo gene-treated animals, individual hSTAT3 and hIL-10 delivery resulted in significant reduction in atherogenesis, as determined by larger aortic lumen size, thinner aortic wall thickness, and lower blood velocity (all statistically significant. However, dual hSTAT3/hIL-10 delivery offered no improvement in therapeutic effect. Plasma cholesterol levels in dual hSTAT3/hIL-10-treated animals were statistically higher compared to hIL-10 alone. While no advantage was seen in this case, we consider that the dual gene approach has intrinsic merit, but properly chosen partnered genes must be used.

  3. Improved detection of malaria cases in island settings of Vanuatu and Kenya by PCR that targets the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene.

    Science.gov (United States)

    Isozumi, Rie; Fukui, Mayumi; Kaneko, Akira; Chan, Chim W; Kawamoto, Fumihiko; Kimura, Masatsugu

    2015-06-01

    Detection of sub-microscopic parasitemia is crucial for all malaria elimination programs. PCR-based methods have proven to be sensitive, but two rounds of amplification (nested PCR) are often needed to detect the presence of Plasmodium DNA. To simplify the detection process, we designed a nested PCR method whereby only the primary PCR is required for the detection of the four major human Plasmodium species. Primers designed for the detection of the fifth species, Plasmodium knowlesi, were not included in this study due to the absence of appropriate field samples. Compared to the standard 18S rDNA PCR method, our cytochrome c oxidase III (cox3) method detected 10-50% more cases while maintaining high sensitivities (1.00) for all four Plasmodium species in our samples from Vanuatu (n=77) and Kenya (n=76). Improvement in detection efficiency was more substantial for samples with sub-microscopic parasitemia (54%) than those with observable parasitemia (10-16%). Our method will contribute to improved malaria surveillance in low endemicity settings. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Mutations in mitochondrial-encoded cytochrome c oxidase subunits I, II, and III genes detected in Alzheimer's disease using single-strand conformation polymorphism.

    Science.gov (United States)

    Hamblet, Natasha S; Ragland, Brian; Ali, Mervat; Conyers, Barbara; Castora, Frank J

    2006-02-01

    A "mitochondrial hypothesis" of late onset Alzheimer's disease (AD) has been proposed. Biochemical studies indicate that there is a significant decrease in cytochrome oxidase (CO) activity as well as perturbed CO I and CO III mRNA levels in platelets and brain tissue from Alzheimer's patients. Using the electrophoretic mutation detection technique SSCP and DNA sequencing, we have identified 20 point mutations in the mitochondrial-encoded CO subunits (CO I, II, and III) in AD and age-matched control brain samples. Eight of the mutations are new variants of the mitochondrial genome. The efficiency of SSCP in detecting mutations in the CO subunits was estimated to be 80% when compared to dideoxy sequencing. One of the mutations (at position 9,861) results in a phenylalanine-->leucine substitution at a highly conserved residue in CO III. CO activity was reduced by an average of 35% in all AD brains compared to age-matched control samples, which agrees with previous reports. CO activity in one of the AD brain samples carrying the 9,861 mutation decreased by 80% relative to control brain samples, suggesting that the phenotypic expression of this mutation may result in reduced CO activity and compromised mitochondrial function.

  5. Lysyl Oxidase Gene G473A Polymorphism and Cigarette Smoking in Association with a High Risk of Lung and Colorectal Cancers in a North Chinese Population

    Directory of Open Access Journals (Sweden)

    Guoli Wang

    2016-06-01

    Full Text Available The relationship among the lysyl oxidase (LOX G473A single nucleotide polymorphism (SNP, cigarette smoking and lung, colorectal, colon and rectum cancer susceptibility was studied in 200 cases of lung cancer, 335 cases of colorectal cancer including 130 cases of colon cancer and 205 cases of rectum cancer, and 335 healthy people in Tangshan, China. Peripheral blood DNA samples were collected, DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP performed, followed by multivariate logistic regression analysis. In comparison to LOX473GG genotype carriers, individuals with LOX473AA exhibited a higher susceptibility to lung, colon-rectum, colon, and rectum cancers with OR values amounting to 3.84-, 2.74-, 2.75-, and 2.74-fold of the control, respectively. In the LOX 473AA-positive population, females were more susceptible than males to carcinogenesis with OR values (female vs. male: 5.25 vs. 3.23, 2.29 vs. 1.51, 2.27 vs. 1.45, and 2.25 vs. 1.53, respectively, for lung, colon-rectum combined, colon, and rectum cancers. LOX G473A polymorphism apparently elevated human sensitivity to cigarette smoking carcinogens for eliciting cancers in the lung and colon only. Thus, LOX G473A polymorphism positively correlates with carcinogenesis and it may be used as an ideal intrinsic biomarker for prediction or diagnosis of carcinogenesis in humans.

  6. Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus

    OpenAIRE

    Trask, Shane D.; Taraporewala, Zenobia F.; Boehme, Karl W.; Dermody, Terence S.; Patton, John T.

    2010-01-01

    Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to is...

  7. The correlation between dual-color chromogenic in situ hybridization and fluorescence in situ hybridization in assessing HER2 gene amplification in breast cancer

    DEFF Research Database (Denmark)

    Pedersen, Marianne; Rasmussen, Birgitte Bruun

    2009-01-01

    obtained by the 2 methods showed excellent correlation (correlation coefficient 0.95). In conclusion, it is possible by dual-color CISH method to demonstrate HER2 genes and chromosome 17 genes, in the same tissue section and reliably assess HER2 status. The CISH method is a very promising alternative...... invasive breast carcinomas, both FISH and CISH detected HER2 amplification in 24 cases and nonamplification was detected in 47 cases. One case showed a discrepancy between FISH and CISH. The concordance between CISH and FISH was found to be almost perfect (98.6%). The correlation between the HER2 ratios...

  8. 西瓜交替氧化酶AOX2基因的克隆与分析%Cloning and analysis of the alternative oxidase-2(AOX2) gene in watermelon

    Institute of Scientific and Technical Information of China (English)

    李严曼; 朱磊; 杨景华; 张明方

    2011-01-01

    In order to study the possible functions of the alternative oxidase gene family in watermelon plants,high-quality RNA was extracted from cold-tolerant watermelon(Citrullus lanatus) germplasm IVSM No.9 and then cDNA was obtained through reverse transcription kit.A cDNA fragment of alternative oxidase was amplified using degenerate oligonucleotide primers designed on the basis of the conserved domain of alternative oxidase genes in other plants.According to the cDNA fragment sequence,the 5' and 3' end fragment sequences were obtained using RACE technology respectively.The full length cDNA of ClAOX2 was obtained by DNAMAN5.22 from the above three sequences.The cDNA fragment exhibited high homology to the alternative oxidase-2(AOX2) of other species and could be referred to as ClAOX2.Then the full lengths of cDNA and DNA were obtained by PCR according to the known cDNA sequence.The full-length cDNA of ClAOX2 was 1 263 bp in size with a 1050 bp open reading frame(ORF).The full-length DNA was 1 983 bp which has consisted of four exons interrupted by three introns.The accession number of ClAOX2 in GenBank database is ADD84880.Real-time PCR showed that the ClAOX2 gene expressed in root,stem,leaf,flower,and fruit of watermelon seedling and significantly higher in the fruit.These results demonstrated that ClAOX2 may play a role during fruit development.%为了研究西瓜交替氧化酶基因家族在西瓜植株中可能发挥的功能,以西瓜(Citrullus lanatus)耐冷种质IVSM9为材料,根据植物不同物种交替氧化酶基因核苷酸保守区序列设计兼并引物,得到西瓜交替氧化酶(alterna-tive oxidase)AOX基因的中间片段。在已知序列的基础上,分别设计5’和3’末端扩增的特异引物,采用cDNA末端快速扩增(RACE)的方法得到西瓜交替氧化酶基因的5’端和3’端序列,用DNAMAN5.22软件对3个序列进行拼接和分析,获得全长序列。得到的基因片段同其

  9. 巴西橡胶树乙醇酸氧化酶HbGOX1基因的鉴定与表达分析%Identification and Expression of Glycolate Oxidase HbGOX1 Gene from Hevea brasiliensis

    Institute of Scientific and Technical Information of China (English)

    程汉; 陈相黄华孙

    2016-01-01

    Photorespiratory pathway is an important metabolic pathway in plant,which consumes the products of photo-synthesis and reduces crop final yield. Glycolate oxidase is the key regulatory enzyme of this pathway, while the study in rubber tree is absence. In this study, the cDNA sequence of HbGOX1 gene was identified and characterized, and the peptide sequence was analyzed bioinformatically. The expression pattern of HbGOX1 gene was further explored under cold stress. The results showed that this gene was negatively regulated by cold stress. This study provided some fundenmental information for the roles of photorespiration in rubber biosynthesis in Hevea brasiliensis.%光呼吸途径是植物中重要的反应途径,它通过消耗光合作用产物影响作物的产量。乙醇酸氧化酶是该途径的关键调控酶,在橡胶树中尚未见研究报道。本研究分离和鉴定橡胶树HbGOX1基因的全长cDNA序列,对其编码的蛋白质进行生物信息学分析,并通过qPCR技术进一步研究HbGOX1基因在低温胁迫下的表达情况。结果发现,该基因受低温胁迫负调控。此结果为进一步揭示橡胶树光呼吸途径在橡胶合成中的作用奠定了基础。

  10. Analytical specificity and sensitivity of the novel dual-target GeneProof Neisseria gonorrhoeae PCR kit for detection of N. gonorrhoeae.

    Science.gov (United States)

    Golparian, Daniel; Hellmark, Bengt; Unemo, Magnus

    2015-11-01

    Detection of Neisseria gonorrhoeae relies increasingly on nucleic acid amplification tests (NAATs). The specificity of many gonococcal NAATs has been suboptimal and supplementary testing remains recommended in Europe and several additional countries. The novel dual-target GeneProof Neisseria gonorrhoeae PCR kit, targeting porA pseudogene and 16S rRNA gene, showed a high specificity and sensitivity when isolates of non-gonococcal Neisseria and related species (n = 144), and gonococci (n = 104) were tested. However, rare gonococcal porA mutants were only detected in the 16S rRNA gene target and two non-gonococcal isolates showed a low-level cross-reactivity in the 16S rRNA gene target. The detection limit for both targets was 1.5 copies per reaction.

  11. Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus.

    Science.gov (United States)

    Foster, Alexander; Barnes, Nicole; Speight, Robert; Morris, Peter C; Keane, Mark A

    2013-04-10

    While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH4Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations>1mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis-Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (KM)=190μM and maximum rate (kcat)=21.8s(-1) for the oxidative deamination of putrescine with a lower KM (=60μM) and comparable kcat (=18.2s(-1)) for the copper oxidase. MALDI-TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.

  12. 以线粒体COⅠ基因探讨萧氏松茎象的分类地位%Phylogenetic Relationship of Hylobitelus xiaoi inferred from Cytochrome C Oxidasegene

    Institute of Scientific and Technical Information of China (English)

    栾丰刚; 丁俊杰; 何龙喜; 王建国

    2013-01-01

    用蛋白酶K消化法提取萧氏松茎象总DNA,并对细胞色素C氧化酶亚基Ⅰ(cytochrome c oxidasegene,COⅠ)序列分析发现,CO Ⅰ序列4种核苷酸量平均为A=30.2%,C=17.3%,G=15.4%,T=37.0%.将所获得的5个不同地区萧氏松茎象个体CO Ⅰ序列排序后,比较形成了一个含1 209个位点的矩阵,其中1 174个不变位点,35个变异位点,所占比例为2.89%,序列的A+T含量较高.遗传距离、核酸和氨基酸变异及系统发育树分析表明:萧氏松茎象与树皮象属亲缘关系最近,且分子水平上萧氏松茎象与树皮象属之间较之树皮象属内种间无明显差异,对目前的分类地位提出质疑.

  13. Transcriptional coupling of synaptic transmission and energy metabolism: role of nuclear respiratory factor 1 in co-regulating neuronal nitric oxide synthase and cytochrome c oxidase genes in neurons.

    Science.gov (United States)

    Dhar, Shilpa S; Liang, Huan Ling; Wong-Riley, Margaret T T

    2009-10-01

    Neuronal activity is highly dependent on energy metabolism; yet, the two processes have traditionally been regarded as independently regulated at the transcriptional level. Recently, we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1) co-regulates an important energy-generating enzyme, cytochrome c oxidase, as well as critical subunits of glutamatergic receptors. The present study tests our hypothesis that the co-regulation extends to the next level of glutamatergic synapses, namely, neuronal nitric oxide synthase, which generates nitric oxide as a downstream signaling molecule. Using in silico analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, promoter mutations, and NRF-1 silencing, we documented that NRF-1 functionally bound to Nos1, but not Nos2 (inducible) and Nos3 (endothelial) gene promoters. Both COX and Nos1 transcripts were up-regulated by depolarizing KCl treatment and down-regulated by TTX-mediated impulse blockade in neurons. However, NRF-1 silencing blocked the up-regulation of both Nos1 and COX induced by KCl depolarization, and over-expression of NRF-1 rescued both Nos1 and COX transcripts down-regulated by TTX. These findings are consistent with our hypothesis that synaptic neuronal transmission and energy metabolism are tightly coupled at the molecular level.

  14. Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

    Directory of Open Access Journals (Sweden)

    Zheng Zhou

    Full Text Available Molybdenum cofactor (Moco is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1 is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L. Cnx1 gene (GmCnx1 was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR and aldehydeoxidase (AO of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 μg g(-1 h(-1 and 30 pmol L(-1, respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants.In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV. DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

  15. 四川宜宾产桃花水母的线粒体CO I序列片段分析%Sequence analyses of mitochondrial cytochrome c oxidase subunitⅠgene fragment of freshwater jellyfish from Yibin, Sichuan

    Institute of Scientific and Technical Information of China (English)

    蔡延森; 刘芹; 王译伟; 马明义; 税青林

    2014-01-01

    目的:针对桃花水母形态分类中存在的一些混乱和矛盾,引入分子生物学方法阐明其分类难题。方法:采用PCR和DNA测序技术,对四川宜宾产桃花水母标本的线粒体细胞色素C氧化酶亚基Ⅰ基因(mitochondrial cytochrome c oxidase subunitⅠ,COⅠ)片段进行了扩增与测序,并与GenBank中已有的淡水水母目COⅠ基因序列进行比对分析,利用MEGA 5计算它们的遗传距离,构建NJ树。结果:该桃花水母标本与索氏桃花水母Craspedacusta sowerbii的COI基因相似度极高,同源性在99%以上,遗传距离为0.003,在进化树中与索氏桃花水母聚为同一支。结论:该标本属于索氏桃花水母。%objective:Molecular biological method was introduced to clarify the difficult classification problem of freshwater jellyfish (Craspedacusta sowerbii). Methods:The mitochondrial cytochrome c oxidase subunit I (COI) gene of freshwater jellyfish (from Yibin, Sichuan) was amplified by polymerase chain reaction (PCR) and then sequenced. Aligning with other Limnomedusae species retrieved from GenBank, genetic distance among species was calculated by using the Kimura-2-parameter (K2P) method in MEGA 5. Neighbor-Joining (NJ) tree based on K2P distances was created to provide a graphic representation of the divergence patterns for among-and within-species. Results:The result showed that COI sequence of freshwater jellyfish was highly similar to those of C. sowerbii (similarity 99%). The genetic distance between the sample and C. sowerbii was 0.003. The NJ tree showed 100%degree of confidence cluster of our sample and C. sowerbii from Germany. Conclusion:Our sample is C. sowerbii.

  16. 苹果内根-贝壳杉烯氧化酶基因的克隆及序列分析%Cloning and sequence analysis of ent-Kaurene Oxidase Gene in apple( Malus×domestica )

    Institute of Scientific and Technical Information of China (English)

    田伟; 田义轲; 王彩虹; 宋伟; 李节法; 殷豪

    2011-01-01

    内根-贝壳杉烯氧化酶(KO)是赤霉素合成代谢的关键酶.以苹果(Malus×domestica Borkh.)品种富士(Fuji)为供试材料,应用同源克隆和RACE方法从其茎尖中克隆到KO的cDNA全长序列,命名为MdKO,GenBank登录号:AY563549,其长度为1 859 bp.MdKO的开放性阅读框(ORF)编码514个氨基酸残基,推断其相对分子量为58.9 ku,等电点为7.63.氨基酸同源性分析表明,MdKO与已报道的其他植物的KO氨基酸序列具有较高的相似性;氨基酸聚类分析表明,苹果和梨首先聚类,其次是葡萄;序列结构分析表明MdKO属于细胞色素超家族P450系,具有细胞色素P450的血红素结构域FXXGXRXCXG和跨膜结构域;亚细胞定位分析发现MdKO可能位于内质网膜上.%Ent-Kaurene oxidase(KO)is a critical enzyme in the pathway of gibberellins biosynthesis. In this research, ent-kaurene oxidase gene in apple, designated as MdKO(GenBank accession number:AY563549), was isolated from the apical tissue of Fuji apple stem. The full cDNA was 1 859 bp in length with an open reading frame(ORF)of 1 545 bp encoding a protein of 514 amino acids. The protein molecular weight and isoelectric point were predicted to be 58.9 ku and 7.63, respectively. Amino acids homology analysis indicated a high similarity with its counterparts in other species. Amino acids cluster analysis showed that KO from pear was clustered together with MdKO firstly, and followed by that from grape. Sequence analysis exhibited that MdKO belonged to cytochrome P450 superfamily and contained cysteine heme-iron ligand signature (FXXGXRXCXG)and transmembrane region. Moreover, MdKO was located in endoplasmic reticulum membrane.

  17. Identification of Single Nucleotide Polymorphism (SNP in Mono Amine Oxidase A (MAO-A Gene as a genetic marker for aggressiveness in sheep

    Directory of Open Access Journals (Sweden)

    Eko Handiwirawan

    2012-12-01

    Full Text Available In the population, there are aggressive sheep in a small number which requires special management those specific animal house and routine management. The purpose of this study was to identify the variation of DNA marker SNP (single nucleotide polymorphism as a genetic marker for the aggressive trait in several of sheep breed. The identification of point mutations in exon 8 of MAO-A gene associated with aggressive behavior in sheep may be further useful to become of DNA markers for the aggressive trait in sheep. Five of sheep breed were used, i.e.: Barbados Black belly Cross sheep (BC, Composite Garut (KG, Local Garut (LG, Composite Sumatra (KS and St. Cross Croix (SC. Duration of ten behavior traits, blood serotonin concentrations and DNA sequence of exon 8 of MAO-A gene from the sheep aggressive and nonaggressive were observed. PROC GLM of SAS Ver. 9.0 program was used to analyze variable behavior and blood serotonin concentrations. DNA polymorphism in exon 8 of MAO-A gene was analyzed using the MEGA software Ver. 4.0. The results show that the percentage of the aggressive rams of each breed was less than 10 percent; except for the KS sheep is higher (23%. Based on the duration of behavior, aggressive sheep group was not significantly different with non aggressive sheep group, except duration of care giving and drinking behavior. It is known that concentration of blood serotonin in aggressive and non aggressive rams was not significantly different. The aggressive trait in sheep has a mechanism or a different cause like that occurs in mice and humans. In this study, aggressive behavior in sheep was not associated with a mutation in exon 8 of MAO-A gene.

  18. Association of sequence variants in CKM (creatine kinase, muscle) and COX4I2 (cytochrome c oxidase, subunit 4, isoform 2) genes with racing performance in Thoroughbred horses.

    Science.gov (United States)

    Gu, J; MacHugh, D E; McGivney, B A; Park, S D E; Katz, L M; Hill, E W

    2010-11-01

    The wild progenitors of the domestic horse were subject to natural selection for speed and stamina for millennia. Uniquely, this process has been augmented in Thoroughbreds, which have undergone at least 3 centuries of intense artificial selection for athletic phenotypes. While the phenotypic adaptations to exercise are well described, only a small number of the underlying genetic variants contributing to these phenotypes have been reported. A panel of candidate performance-related genes was examined for DNA sequence variation in Thoroughbreds and the association with racecourse performance investigated. Eighteen candidate genes were chosen for their putative roles in exercise. Re-sequencing in Thoroughbred samples was successful for primer sets in 13 of these genes. SNPs identified in this study and from the EquCab2.0 SNP database were genotyped in 2 sets of Thoroughbred samples (n = 150 and 148) and a series of population-based case-control investigations were performed by separating the samples into discrete cohorts on the basis of retrospective racecourse performance. Twenty novel SNPs were detected in 3 genes: ACTN3, CKM and COX4I2. Genotype frequency distributions for 3 SNPs in CKM and COX4I2 were significantly (P race. These associations were not validated when an additional (n = 130) independent set of samples was genotyped, but when analyses included all samples (n = 278) the significance of association at COX4I2 g.22684390C > T was confirmed (P horse industries, it is vital that rigour is applied to studies generating these data and that adequate and appropriate sample sets, particularly for independent replication, are used. © 2010 EVJ Ltd.

  19. Lysyl oxidase in colorectal cancer.

    Science.gov (United States)

    Cox, Thomas R; Erler, Janine T

    2013-11-15

    Colorectal cancer is the third most prevalent form of cancer worldwide and fourth-leading cause of cancer-related mortality, leading to ~600,000 deaths annually, predominantly affecting the developed world. Lysyl oxidase is a secreted, extracellular matrix-modifying enzyme previously suggested to act as a tumor suppressor in colorectal cancer. However, emerging evidence has rapidly implicated lysyl oxidase in promoting metastasis of solid tumors and in particular colorectal cancer at multiple stages, affecting tumor cell proliferation, invasion, and angiogenesis. This emerging research has stimulated significant interest in lysyl oxidase as a strong candidate for developing and deploying inhibitors as functional efficacious cancer therapeutics. In this review, we discuss the rapidly expanding body of knowledge concerning lysyl oxidase in solid tumor progression, highlighting recent advancements in the field of colorectal cancer.

  20. [A case of lambda-expressing pulmonary MALT lymphoma with dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene].

    Science.gov (United States)

    Oh, Hye Ryong; Lee, Mi Ja; Park, Geon; Moon, Dae Soo; Park, Young Jin; Jang, Sook Jin

    2009-06-01

    A 70-yr-old woman was hospitalized with a history of dry cough. Bronchial endoscopy and transbronchial lung biopsy were performed. However, the findings of histopathology and immunohistochemistry were not sufficient to decide whether the lesion was benign or malignant, because of the presence of crush artifacts in the biopsy specimens. We performed B-cell clonality studies using BIOMED-2 multiplex PCR (InVivoScribe Technologies, USA) to detect clonal rearrangements in the immunoglobulin gene. The results of multiplex PCR showed clonal rearrangements of both kappa and lambda immunoglobulin light chain genes. The findings of immunochemistry revealed that the lesion expressed lambda light chain, but not kappa light chain. Based on the clinical, pathologic, and molecular findings, this case was diagnosed as pulmonary MALT lymphoma. We report the first case in Korea of lambda-expressing MALT lymphoma that is shown to have dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene by multiplex PCR.

  1. [Phylogenetic analysis of Altai osmans of the genus Oreoleuciscus (Pisces, Cyprinidae, Leuciscinae), based on the analysis of the cytochrome oxidase 1 gene (Co-1) sequence].

    Science.gov (United States)

    Batishcheva, N M; Kartavtsev, Iu F; Bogutskaia, N G

    2011-10-01

    Molecular genetic analysis of Altai osmans of the genus Oreoleuciscus from two different parts of the range was carried out. In this study, based on the mitochondrial Co-1 gene sequence, a total of 25 fish specimens belonging to four genera were examined: (1) O. humilis, 2 specimens; O. potanini, 13 specimens; (2) Pseudaspius leptocephalus, 1 specimen; (3) Tribolodon brandtii, T. hakonensis, and T. sachalinensis from the GenBank database, 8 speciens; and (4) Leuciscus waleckii, 1 specimen (used as an outgroup). The p-distances were very low both within and between the species: (1) 0.20 +/- 0.03%; (2) 0.40 +/- 0.12%; and (1-2) 0.80 +/- 0.04%. To visualize the relationships among all of the species examined, the neighbor joining (NJ), maximum parsimony (MP), Bayesian (BA), and maximum likelihood (ML) trees were constructed. The results obtained using these methods were very similar. It was demonstrated that species assignment of the individuals (barcoding) with the help Co-1 gene was effective, despite of very low divergence of the two osman taxa, which was comparable with typical intraspecific values in other animal groups. Taxonomic status of O. potanini and O. humilis requires further investigation with paying attention to low genetic distances between these species along with the lack of material from sympatric parts of the ranges.

  2. [Phylogenetic and taxonomic analysis of flatfish species (Teleostei, Pleuronectiformes) inferred from the primary nucleotide sequence of cytochrome oxidase 1 gene (Co-1)].

    Science.gov (United States)

    Sharina, S N; Kartavtsev, Iu F

    2010-03-01

    Seventeen nucleotide sequences of Co-1 gene from 13 Pleuronectiformes species and 2 Perciformes species served as the outgroup were examined. For divergence comparison, the initial stage involved calculation of pairwise p-distances for all investigated sequences. This allowed to evaluate the nucleotide diversity on four phylogenetically different levels: (1) intraspecific, (2) intrageneric, (3) intrafamilial, and (4) intraordinal. The values of p-distances for the Co-1 gene for the four mentioned categories were (1) 0.93 +/- 0.73%, (2) 11.72 +/- 1.86%, (3) 12.10 +/- 1.10%, and (4) 20.20 +/- 0.22%, respectively. An increase in the level of genetic divergence along with an enhancement in taxon rank was previously reported for different species, which might be explained by prevalence of geographic speciation model in nature. Phylogenetic trees were constructed using four approaches: maximum parsimony, Bayesian, maximum likelihood, and neighbor-joining. These trees demonstrated similar results confirming the monophyletic origin of the families studied. The examined representatives of the flatfish species and genera were shown to be sufficiently divergent genetically.

  3. Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Ran Peng; Yuanfen Zhai; Hua Ding; Tianyuan Di; Ting Zhang; Bing Li; Weide Shen; Zhengguo Wei

    2012-01-01

    In general,for real-time quantitative polymerase chain reaction (qPCR),normalization strategies use a reference gene as a control and to avoid the introduction of experi-mental errors expression of this gene should not vary in response to changing conditions.However,the expression of many reference genes has been reported to vary consid-erably and,without appropriate normalization,the expression profile of a target gene can be misinterpreted.In this study,the expression levels of seven commonly used reference genes (ACT,GAPDH,28srRNA,RPL3,α-tubulin,UBC,and TBP) were detected at different development time points and in response to treatment with 20-hydroxyecdysone (20E) and with rntin.The expression stability was analyzed using geNorm and NormFinder software.Significant variations were found among normal tissues and between experimentally treated tissues.The dual spike-in strategy also revealed significant variations of the expression levels of the reference genes among normal tissues and between experimentally treated tissues.Glutathione-S-transferase sigma 1 (GSTs1),which has a high expression level in fat body and is related to the mechanism of resistance,was used as a target gene to validate the feasibility and difference of these two approaches.

  4. Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals

    DEFF Research Database (Denmark)

    Galuszka, P.; Frebort, I.; Sebela, M.

    2001-01-01

    wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N-6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested...

  5. A fragmented adeno-associated viral dual vector strategy for treatment of diseases caused by mutations in large genes leads to expression of hybrid transcripts

    Science.gov (United States)

    McClements, Michelle E.; Charbel Issa, Peter; Blouin, Véronique; MacLaren, Robert E.

    2017-01-01

    Objective Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems. Methods Oversized (>8kb) transgene constructs containing ABCA4 coding sequence were packaged as truncated fragments <5kb in size into various AAV serotypes. In vitro transductions with these fAAV vector preparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and western blot techniques. Results Transductions with fAAV vector preparations yielded ABCA4 mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full length ABCA4 CDS with additional hybrid ABCA4 variants, indicating truncated transgenes without regions of overlap were joining and forming stable hybrid transgenes. In contrast, an ABCA4 overlapping dual vector system (OV) with a defined complementary region generated only full length mRNA transcripts plus detectable ABCA4 protein. Conclusion Despite previous success shown with the fAAV approach, the lack of repeatability and identification of stable hybrid transcripts capable of protein production suggests there is more refinement required before considering this approach in a clinical setting. PMID:28239514

  6. Molecular cloning and expression analysis of cytochrome c oxidase subunit II from Sitophilus zeamais.

    Science.gov (United States)

    Hou, Chang-Liang; Wang, Jing-Bo; Wu, Hua; Liu, Jia-Yu; Ma, Zhi-Qing; Feng, Jun-Tao; Zhang, Xing

    2016-09-30

    Cytochrome c oxidase subunit II (COX II) containing a dual core CuA active site is one of the core subunits of mitochondrial Cytochrome c oxidase (Cco), which plays a significant role in the physiological process. In this report, the full-length cDNA of COXII gene was cloned from Sitophilus zeamais, which had an open reading frame (ORF) of 684 bp encoding 227 amino acids residues. The predicted COXII protein had a molecular mass of 26.2 kDa with pI value of 6.37. multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXII had high sequence identity with the COXII of other insect species. The gene was subcloned into the expression vector pET-32a, and induced by isopropyl β-d-thiogalactopyranoside (IPTG) in E. coli Transetta (DE3) expression system. Finally the recombinant COXII with 6-His tag was purified using affinity chromatography with Ni(2+)-NTA agarose. Western Blotting (WB) showed the recombinant protein was about 44 kD, and the concentration of fusion protein was 50 μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXII could catalyze the oxidation of substrate Cytochrome C (Cyt c), and influenced by allyl isothiocyanate (AITC). By using molecular docking method, It was found that a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results suggested that tag-free COXII was functional and one of the action sites of AITC, which will be helpful to carry out a point mutation in binding sites for the future research.

  7. Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design

    OpenAIRE

    de Gier, Jan-Willem L.; Schepper, Mike; Reijnders, Willem N.M.; Dyck, Stef J. van; Slotboom, Dirk Jan; Warne, Antony; Saraste, Matti; Krab, Klaas; Finel, Moshe; Stouthamer, Adriaan H.; Van Spanning, Rob J. M.; van der Oost, John

    1996-01-01

    In Paracoccus denitrificans the aa3-type cytochrome c oxidase and the bb3-type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3-type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen-sen...

  8. Multiple origins of the phenol reaction negative phenotype in foxtail millet, Setaria italica (L.) P. Beauv., were caused by independent loss-of-function mutations of the polyphenol oxidase (Si7PPO) gene during domestication.

    Science.gov (United States)

    Inoue, Takahiko; Yuo, Takahisa; Ohta, Takeshi; Hitomi, Eriko; Ichitani, Katsuyuki; Kawase, Makoto; Taketa, Shin; Fukunaga, Kenji

    2015-08-01

    Foxtail millet shows variation in positive phenol color reaction (Phr) and negative Phr in grains, but predominant accessions of this crop are negative reaction type, and the molecular genetic basis of the Phr reaction remains unresolved. In this article, we isolated polyphenol oxidase (PPO) gene responsible for Phr using genome sequence information and investigated molecular genetic basis of negative Phr and crop evolution of foxtail millet. First of all, we searched for PPO gene homologs in a foxtail millet genome database using a rice PPO gene as a query and successfully found three copies of the PPO gene. One of the PPO gene homologs on chromosome 7 showed the highest similarity with PPO genes expressed in hulls (grains) of other cereal species including rice, wheat, and barley and was designated as Si7PPO. Phr phenotypes and Si7PPO genotypes completely co-segregated in a segregating population. We also analyzed the genetic variation conferring negative Phr reaction. Of 480 accessions of the landraces investigated, 87 (18.1 %) showed positive Phr and 393 (81.9 %) showed negative Phr. In the 393 Phr negative accessions, three types of loss-of-function Si7PPO gene were predominant and independently found in various locations. One of them has an SNP in exon 1 resulting in a premature stop codon and was designated as stop codon type, another has an insertion of a transposon (Si7PPO-TE1) in intron 2 and was designated as TE1-insertion type, and the other has a 6-bp duplication in exon 3 resulting in the duplication of 2 amino acids and was designated as 6-bp duplication type. As a rare variant of the stop codon type, one accession additionally has an insertion of a transposon, Si7PPO-TE2, in intron 2 and was designated as "stop codon +TE2 insertion type". The geographical distribution of accessions with positive Phr and those with three major types of negative Phr was also investigated. Accessions with positive Phr were found in subtropical and tropical regions at

  9. Alanyl-tRNA synthetase genes of Vanderwaltozyma polyspora arose from duplication of a dual-functional predecessor of mitochondrial origin.

    Science.gov (United States)

    Chang, Chia-Pei; Tseng, Yi-Kuan; Ko, Chou-Yuan; Wang, Chien-Chia

    2012-01-01

    In eukaryotes, the cytoplasmic and mitochondrial forms of a given aminoacyl-tRNA synthetase (aaRS) are typically encoded by two orthologous nuclear genes, one of eukaryotic origin and the other of mitochondrial origin. We herein report a novel scenario of aaRS evolution in yeast. While all other yeast species studied possess a single nuclear gene encoding both forms of alanyl-tRNA synthetase (AlaRS), Vanderwaltozyma polyspora, a yeast species descended from the same whole-genome duplication event as Saccharomyces cerevisiae, contains two distinct nuclear AlaRS genes, one specifying the cytoplasmic form and the other its mitochondrial counterpart. The protein sequences of these two isoforms are very similar to each other. The isoforms are actively expressed in vivo and are exclusively localized in their respective cellular compartments. Despite the presence of a promising AUG initiator candidate, the gene encoding the mitochondrial form is actually initiated from upstream non-AUG codons. A phylogenetic analysis further revealed that all yeast AlaRS genes, including those in V. polyspora, are of mitochondrial origin. These findings underscore the possibility that contemporary AlaRS genes in V. polyspora arose relatively recently from duplication of a dual-functional predecessor of mitochondrial origin.

  10. Phenylboronic acid-sugar grafted polymer architecture as a dual stimuli-responsive gene carrier for targeted anti-angiogenic tumor therapy.

    Science.gov (United States)

    Kim, Jinhwan; Lee, Yeong Mi; Kim, Hyunwoo; Park, Dongsik; Kim, Jihoon; Kim, Won Jong

    2016-01-01

    We present a cationic polymer architecture composed of phenylboronic acid (PBA), sugar-installed polyethylenimine (PEI), and polyethylene glycol (PEG). The chemical bonding of PBA with the diol in the sugar enabled the crosslinking of low-molecular-weight (MW) PEI to form high-MW PEI, resulting in strong interaction with anionic DNA for gene delivery. Inside the cell, the binding of PBA and sugar was disrupted by either acidic endosomal pH or intracellular ATP, so gene payloads were released effectively. This dual stimuli-responsive gene release drove the polymer to deliver DNA for high transfection efficiency with low cytotoxicity. In addition, PBA moiety with PEGylation facilitated the binding of polymer/DNA polyplexes to sialylated glycoprotein which is overexpressed on the tumor cell membrane, and thus provided high tumor targeting ability. Therapeutic application of our polymer was demonstrated as an anti-angiogenic gene delivery agent for tumor growth inhibition. Our judicious designed polymer structure based on PBA provides enormous potential as a gene delivery agent for effective gene therapy by stimuli-responsiveness and tumor targeting.

  11. Molecular identification of sibling species of Sclerodermus (Hymenoptera: Bethylidae that parasitize buprestid and cerambycid beetles by using partial sequences of mitochondrial DNA cytochrome oxidase subunit 1 and 28S ribosomal RNA gene.

    Directory of Open Access Journals (Sweden)

    Yuan Jiang

    Full Text Available The species belonging to Sclerodermus (Hymenoptera: Bethylidae are currently the most important insect natural enemies of wood borer pests, mainly buprestid and cerambycid beetles, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. To address this issue, we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I (COI and 28S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: S. guani Xiao et Wu, S. sichuanensis Xiao, S. pupariae Yang et Yao, and Sclerodermus spp. (Nos. 1-5. A 594-bp fragment of COI and 750-bp fragment of 28S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences (Kimura-2-parameter distances between congeneric species averaged to 4.5%, and intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp. (No. 5 averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining (NJ tree. The NJ tree inferred from the 28S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae, and Sclerodermus spp. (Nos. 1-4 clustered together and only Sclerodermus sp. (No. 5 clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp. (No. 5 might be a new species of Sclerodermus.

  12. Expression of the Aspergillus niger Glucose Oxidase Gene in Pichia pastoris SMD1168%黑曲霉葡萄糖氧化酶在毕赤酵母SMD1168中的表达

    Institute of Scientific and Technical Information of China (English)

    郭元芳; 孙高英; 郝建荣; 彭炳银; 毕文祥; 鲍晓明

    2013-01-01

    目的:在毕赤酵母SMD1168中用3-磷酸甘油醛脱氢酶基因启动子(glyceraldehyde-3-phosphate dehygrogenase gene promoter,pGAP)表达黑曲霉葡萄糖氧化酶(glucose oxidase,GOD).方法:将黑曲霉accc30161的GOD基因插入具有pGAP的pGAPZαA质粒中,构建黑曲霉GOD毕赤酵母表达载体,并用菌落PCR、重组质粒琼脂糖凝胶电泳、限制性酶切分析及测序等方法对其进行验证.然后用重组质粒电转化毕赤酵母SMD1168,并用PCR扩增分析观察转化效果,用SDS-PAGE及酶活检测观察重组酵母黑曲霉GOD的表达和活性.结果:用黑曲霉accc30161的GOD基因成功构建了GOD表达载体pGAPZαA-GOD.转化后,pGAPZαA-GOD相关DNA片段已整合进重组毕赤酵母SMD1168-GOD基因组中.SMD1168-GOD可高表达具有活性的GOD,在30℃、pH 6的条件下,其培养液上清GOD酶活可达107.18 u/mL.结论:重组蛋白缺陷性毕赤酵母SMD1168-GOD可以利用pGAP高效表达黑曲霉GOD.

  13. Lysyl oxidase in colorectal cancer

    DEFF Research Database (Denmark)

    Cox, Thomas R; Erler, Janine T

    2013-01-01

    Colorectal cancer is the third most prevalent form of cancer worldwide and fourth-leading cause of cancer-related mortality, leading to ~600,000 deaths annually, predominantly affecting the developed world. Lysyl oxidase is a secreted, extracellular matrix-modifying enzyme previously suggested...... to act as a tumor suppressor in colorectal cancer. However, emerging evidence has rapidly implicated lysyl oxidase in promoting metastasis of solid tumors and in particular colorectal cancer at multiple stages, affecting tumor cell proliferation, invasion, and angiogenesis. This emerging research has...... advancements in the field of colorectal cancer....

  14. Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis.

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

    Full Text Available Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.

  15. Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria.

    Science.gov (United States)

    Matsutani, Minenosuke; Fukushima, Kota; Kayama, Chiho; Arimitsu, Misato; Hirakawa, Hideki; Toyama, Hirohide; Adachi, Osao; Yakushi, Toshiharu; Matsushita, Kazunobu

    2014-10-01

    The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Flavoprotein oxidases : classification and applications

    NARCIS (Netherlands)

    Dijkman, Willem P.; de Gonzalo, Gonzalo; Mattevi, Andrea; Fraaije, Marco W.

    2013-01-01

    This review provides an overview of oxidases that utilise a flavin cofactor for catalysis. This class of oxidative flavoenzymes has shown to harbour a large number of biotechnologically interesting enzymes. Applications range from their use as biocatalysts for the synthesis of pharmaceutical compoun

  17. DYRK1A (Dual-Specificity Tyrosine-Phosphorylated and -Regulated Kinase 1A: A Gene with Dosage Effect During Development and Neurogenesis

    Directory of Open Access Journals (Sweden)

    M. Dierssen

    2006-01-01

    Full Text Available DYRKs (dual-specificity tyrosine-regulated kinases are an emerging family of evolutionarily conserved dual-specificity kinases that play key roles in cell proliferation, survival, and development. The research in the last years suggests a relevant conserved function during neuronal development, related to proliferation and/or differentiation for DYRK1A. It is expressed in neural progenitor cells and has been proposed to participate in the signaling mechanisms that regulate dendrite differentiation. In Drosophila, disruption of the homolog minibrain gene results in flies with reduced neuroblast proliferation, decreased numbers of central brain neurons, and learning/memory deficits. Knockout DYRK1A mice are embryonic lethal, and heterozygotes show decreased viability and region-specific reductions in brain size. In humans, DYRK1A has been proposed to be involved in the neurodevelopmental alterations associated with Down syndrome. The large number of protein interaction and putative substrates described for DYRK1A suggest multiple pathways and functions to be involved in its developmental function. This review focuses on the functional role that DYRK1A plays in brain development.

  18. Chromate reduction by rabbit liver aldehyde oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Banks, R.B.; Cooke, R.T. Jr.

    1986-05-29

    Chromate was reduced during the oxidation of 1-methylnicotinamide chlorine by partially purified rabbit liver aldehyde oxidase. In addition to l-methylnicotinamide, several other electron donor substrates for aldehyde oxidase were able to support the enzymatic chromate reduction. The reduction required the presence of both enzyme and the electron donor substrate. The rate of the chromate reduction was retarded by inhibitors or aldehyde oxidase but was not affected by substrates or inhibitors of xanthine oxidase. These results are consistent with the involvement of aldehyde oxidase in the reduction of chromate by rabbit liver cytosolic enzyme preparations.

  19. CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

    Directory of Open Access Journals (Sweden)

    Jianmei Su

    Full Text Available Multicopper oxidases (MCOs are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II oxidation, the cotA gene from a highly active Mn(II-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0 supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II-oxidizing activity. The specific activity of purified CotA towards Mn(II was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II were 14.85±1.17 mM, 3.01×10(-6±0.21 M·min(-1 and 0.32±0.02 s(-1, respectively. Moreover, the Mn(II-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II oxidation mechanisms, but also offers

  20. Peroxisomal Polyamine Oxidase and NADPH-Oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Efthimios A. Andronis

    2014-04-01

    Full Text Available Homeostasis of reactive oxygen species (ROS in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd and spermine (Spm to putrescine (Put and Spd, respectively is catalyzed by two peroxisomal PA oxidases (AtPAO. However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI. Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions (O2.-, but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX. On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and O2.-. These results suggest that the ratio of O2.-/H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of O2.- by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed.

  1. Multiple amine oxidases in cucumber seedlings.

    Science.gov (United States)

    Percival, F W; Purves, W K

    1974-10-01

    Cell-free extracts of cucumber (Cucumis sativus L. cv. National Pickling) seedlings were found to have amine oxidase activity when assayed with tryptamine as a substrate. Studies of the effect of lowered pH on the extract indicated that this activity was heterogeneous, and three amine oxidases could be separated by ion exchange chromatography. The partially purified enzymes were tested for their activities with several substrates and for their sensitivities to various amine oxidase inhibitors. One of the enzymes may be a monoamine oxidase, although it is inhibited by some diamine oxidase inhibitors. The other two enzymes have properties more characteristic of the diamine oxidases. The possible relationship of the amine oxidases to indoleacetic acid biosynthesis in cucumber seedlings is discussed.

  2. A stringent dual control system overseeing transcription and activity of the Cre recombinase for the liver-specific conditional gene knock-out mouse model

    Institute of Scientific and Technical Information of China (English)

    Yu Wu; Yinghua He; Hongyu Zhang; Xinlan Dai; Xiaoyu Zhou; Jun Gu; Guan Wang; Jingde Zhu

    2008-01-01

    Liver cancer is one of the most threatening diseases in Chinese population. Just like in other tissues, tumor initiation and development in liver involve multiple steps of genetic and epigenetic alterations with several unknown details. However, unlike in other tissues, a tis- sue specific inducible Cre recombinase system that allows temporal and spatial deletion of a target DNA fragment is still not available for in vivo functional gene annotation in hepatocytes. In our pursuit to establish such a mouse model, we designed a dual inducible Cre transgene system and tested it in cultured cells. By combining a CCAAT/enhancer binding protein β (C/EBP β) promoter derived Tet-off expression system and the estrogen receptor (ER) mediated functional control, we show a desirable profile of both hepatocyte-specificity and regulability of the Cre expression in a series of critical assessments in the cell culture system, which provides confidence in continua- tion of our ongoing pursuit in mouse.

  3. Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L.

    Science.gov (United States)

    Pimenta Lange, Maria João; Liebrandt, Anja; Arnold, Linda; Chmielewska, Sara-Miriam; Felsberger, André; Freier, Eduard; Heuer, Monika; Zur, Doreen; Lange, Theo

    2013-06-01

    Cucurbits have been used widely to elucidate gibberellin (GA) biosynthesis. With the recent availability of the genome sequence for the economically important cucurbit Cucumis sativus, sequence data became available for all genes potentially involved in GA biosynthesis for this species. Sixteen cDNAs were cloned from root and shoot of 3-d to 7-d old seedlings and from mature seeds of C. sativus. Two cDNAs code for GA 7-oxidases (CsGA7ox1, and -2), five for GA 20-oxidases (CsGA20ox1, -2, -3, -4, and -5), four for GA 3-oxidases (CsGA3ox1, -2, -3, and -4), and another five for GA 2-oxidases (CsGA2ox1, -2, -3, -4, and -5). Their enzymatic activities were investigated by heterologous expression of the cDNAs in Escherichia coli and incubation of the cell lysates with (14)C-labelled, D2-labelled, or unlabelled GA-substrates. The two GA 7-oxidases converted GA12-aldehyde to GA12 efficiently. CsGA7ox1 converted GA12 to GA14, to 15α-hydroxyGA12, and further to 15α-hydroxyGA14. CsGA7ox2 converted GA12 to its 12α-hydroxylated analogue GA111. All five GA 20-oxidases converted GA12 to GA9 as a major product, and to GA25 as a minor product. The four GA 3-oxidases oxidized the C19-GA GA9 to GA4 as the only product. In addition, three of them (CsGA3ox2, -3, and -4) converted the C20-GA GA12 to GA14. The GA 2-oxidases CsGA2ox1, -2, -3, and -4 oxidized the C19-GAs GA9 and GA4 to GA34 and GA51, respectively. CsGA2ox2, -3, and -4 converted GA51 and GA34 further to respective GA-catabolites. In addition to C19-GAs, CsGA2ox4 also converted the C20-GA GA12 to GA110. In contrast, CsGA2ox5 oxidized only the C20 GA12 to GA110 as the sole product. As shown for CsGA20ox1 and CsGA3ox1, similar reactions were catalysed with 13-hydroxlyated GAs as substrates. It is likely that these enzymes are also responsible for the biosynthesis of 13-hydroxylated GAs in vivo that occur at low levels in cucumber.

  4. Regulation of cytochrome c- and quinol oxidases, and piezotolerance of their activities in the deep-sea piezophile Shewanella violacea DSS12 in response to growth conditions.

    Science.gov (United States)

    Ohke, Yoshie; Sakoda, Ayaka; Kato, Chiaki; Sambongi, Yoshihiro; Kawamoto, Jun; Kurihara, Tatsuo; Tamegai, Hideyuki

    2013-01-01

    The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O2 concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O2 concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O2 concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.

  5. Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

    Science.gov (United States)

    Gong, Haixia; Liu, Menglin; Klomp, Jeff; Merrill, Bradley J.; Rehman, Jalees; Malik, Asrar B.

    2017-01-01

    Human endothelial cells (ECs) are widely used to study mechanisms of angiogenesis, inflammation, and endothelial permeability. Targeted gene disruption induced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) nuclease gene editing is potentially an important tool for definitively establishing the functional roles of individual genes in ECs. We showed that co-delivery of adenovirus encoding EGFP-tagged Cas9 and lentivirus encoding a single guide RNA (sgRNA) in primary human lung microvascular ECs (HLMVECs) disrupted the expression of the Tie2 gene and protein. Tie2 disruption increased basal endothelial permeability and prevented permeability recovery following injury induced by the inflammatory stimulus thrombin. Thus, gene deletion via viral co-delivery of CRISPR-Cas9 in primary human ECs provides a novel platform to investigate signaling mechanisms of normal and perturbed EC function without the need for clonal expansion. PMID:28198371

  6. Inventory control: cytochrome oxidase assembly regulates mitochondrial translation

    Science.gov (United States)

    Mick, David U.; Fox, Thomas D.; Rehling, Peter

    2012-01-01

    Mitochondria maintain a genome and translation-machinery to synthesize a small subset of subunits of the oxidative phosphorylation system. These organellar gene products must assemble with imported subunits that are encoded in the nucleus to build up functional enzymes. New findings on the early steps in cytochrome oxidase assembly reveal how the mitochondrial translation of its core component Cox1 is directly coupled to the assembly of this respiratory complex. PMID:21179059

  7. Lysyl oxidase in cancer research.

    Science.gov (United States)

    Perryman, Lara; Erler, Janine T

    2014-01-01

    Metastasis is the main reason for cancer-associated deaths and therapies are desperately needed to target the progression of cancer. Lysyl oxidase (LOX) plays a pivotal role in cancer progression, including metastasis, and is therefore is an attractive therapeutic target. In this review we will breakdown the process of cancer progression and the various roles that LOX plays has in the advancement of cancer. We will highlight why LOX is an exciting therapeutic target for the future.

  8. Differences in Gene Regulation by Dual Ligands of Nuclear Receptors Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) in HepG2 Cells Stably Expressing CAR/PXR.

    Science.gov (United States)

    Kanno, Yuichiro; Tanuma, Nobuaki; Yazawa, Saki; Zhao, Shuai; Inaba, Miki; Nakamura, Satoshi; Nemoto, Kiyomitsu; Inouye, Yoshio

    2016-08-01

    The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate various genes involved in xenobiotics and drug metabolism. In many cases, CAR/PXR share ligands termed dual ligands of CAR/PXR. It is difficult to investigate the effect of CAR/PXR dual ligands in cell lines because CAR and PXR expression is scarcely detected in cultured cell lines. Here, we established a tetracycline-inducible human CAR and stably human PXR-overexpressing HepG2 cell line (HepTR/hCAR/hPXR) to examine CAR/PXR dual ligands. In the present study, we investigated the regulation of CYP2B6, CYP2C9, CYP3A4, and UDP-glucuronosyl transferase, which are target genes of CAR/PXR, by dual ligands of CAR/PXR in two transfectants. Activation of CAR and PXR in cells treated with a high dose of CITCO [6-(4-chlorophenyl)-imidazo(2,1-b)thiazole-5-carbaldehyde] or cotreated with rifampicin and tetracycline resulted in synergistic enhancement of CYP3A4, but not CYP2B6, CYP2C9, or UGT1A1, mRNA expression in HepTR/hCAR/hPXR cells. In contrast, this synergistic effect was not observed in HepTR/hCAR cells. These observations were also demonstrated in human primary hepatocytes. Taken together, our results suggest that dual ligands of CAR/PXR show distinct gene regulation patterns by cross-talk between CAR and PXR. Furthermore, the two newly established cell lines are useful tools to investigate dual ligands of CAR/PXR.

  9. The snf1 gene of Ustilago maydis acts as a dual regulator of cell wall degrading enzymes.

    Science.gov (United States)

    Nadal, Marina; Garcia-Pedrajas, Maria D; Gold, Scott E

    2010-12-01

    Many fungal plant pathogens are known to produce extracellular enzymes that degrade cell wall elements required for host penetration and infection. Due to gene redundancy, single gene deletions generally do not address the importance of these enzymes in pathogenicity. Cell wall degrading enzymes (CWDEs) in fungi are often subject to carbon catabolite repression at the transcriptional level such that, when glucose is available, CWDE-encoding genes, along with many other genes, are repressed. In Saccharomyces cerevisiae, one of the main players controlling this process is SNF1, which encodes a protein kinase. In this yeast, Snf1p is required to release glucose repression when this sugar is depleted from the growth medium. We have employed a reverse genetic approach to explore the role of the SNF1 ortholog as a potential regulator of CWDE gene expression in Ustilago maydis. We identified U. maydis snf1 and deleted it from the fungal genome. Consistent with our hypothesis, the relative expression of an endoglucanase and a pectinase was higher in the wild type than in the Δsnf1 mutant strain when glucose was depleted from the growth medium. However, when cells were grown in derepressive conditions, the relative expression of two xylanase genes was unexpectedly higher in the Δsnf1 strain than in the wild type, indicating that, in this case, snf1 negatively regulated the expression of these genes. Additionally, we found that, contrary to several other fungal species, U. maydis Snf1 was not required for utilization of alternative carbon sources. Also, unlike in ascomycete plant pathogens, deletion of snf1 did not profoundly affect virulence in U. maydis.

  10. Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals

    Directory of Open Access Journals (Sweden)

    Wentao Wu

    2017-10-01

    Full Text Available The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three (Physcomitrella patens to 63 (Glycine max. The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.

  11. Portrait of ependymoma recurrence in children: biomarkers of tumor progression identified by dual-color microarray-based gene expression analysis.

    Directory of Open Access Journals (Sweden)

    Matthieu Peyre

    Full Text Available BACKGROUND: Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11 or in neural development (CD133, Wnt and Notch pathways. Metallothionein (MT genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002. Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03. Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression. CONCLUSIONS/SIGNIFICANCE: The most frequent molecular events associated with ependymoma recurrence were over-expression of kinetochore proteins and down-regulation of

  12. Effects of iodonium-class flavin dehydrogenase inhibitors on growth, reactive oxygen production, cell cycle progression, NADPH oxidase 1 levels, and gene expression in human colon cancer cells and xenografts.

    Science.gov (United States)

    Doroshow, James H; Gaur, Shikha; Markel, Susan; Lu, Jiamo; van Balgooy, Josephus; Synold, Timothy W; Xi, Bixin; Wu, Xiwei; Juhasz, Agnes

    2013-04-01

    Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10-250nM for DPI, 0.5-2.5μM for DTI, and 155nM to 10μM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced

  13. Dual and Opposing Roles of MicroRNA-124 in Epilepsy Are Mediated through Inflammatory and NRSF-Dependent Gene Networks

    Directory of Open Access Journals (Sweden)

    Gary P. Brennan

    2016-03-01

    Full Text Available Insult-provoked transformation of neuronal networks into epileptic ones involves multiple mechanisms. Intervention studies have identified both dysregulated inflammatory pathways and NRSF-mediated repression of crucial neuronal genes as contributors to epileptogenesis. However, it remains unclear how epilepsy-provoking insults (e.g., prolonged seizures induce both inflammation and NRSF and whether common mechanisms exist. We examined miR-124 as a candidate dual regulator of NRSF and inflammatory pathways. Status epilepticus (SE led to reduced miR-124 expression via SIRT1—and, in turn, miR-124 repression—via C/EBPα upregulated NRSF. We tested whether augmenting miR-124 after SE would abort epileptogenesis by preventing inflammation and NRSF upregulation. SE-sustaining animals developed epilepsy, but supplementing miR-124 did not modify epileptogenesis. Examining this result further, we found that synthetic miR-124 not only effectively blocked NRSF upregulation and rescued NRSF target genes, but also augmented microglia activation and inflammatory cytokines. Thus, miR-124 attenuates epileptogenesis via NRSF while promoting epilepsy via inflammation.

  14. pH and reduction dual-responsive dipeptide cationic lipids with α-tocopherol hydrophobic tail for efficient gene delivery.

    Science.gov (United States)

    Liu, Qiang; Su, Rong-Chuan; Yi, Wen-Jing; Zheng, Li-Ting; Lu, Shan-Shan; Zhao, Zhi-Gang

    2017-03-31

    A series of tocopherol-based cationic lipid 3a-3f bearing a pH-sensitive imidazole moiety in the dipeptide headgroup and a reduction-responsive disulfide linkage were designed and synthesized. Acid-base titration of these lipids showed good buffering capacities. The liposomes formed from 3 and co-lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) could efficiently bind and condense DNA into nanoparticles. Gel binding and HPLC assays confirmed the encapsulated DNA could release from lipoplexes 3 upon addition of 10 mM glutathione (GSH). MTT assays in HEK 293 cells demonstrated that lipoplexes 3 had low cytotoxicity. The in vitro gene transfection studies showed cationic dipeptide headgroups clearly affected the transfection efficiency (TE), and arginine-histidine based dipeptide lipid 3f give the best TE, which was 30.4 times higher than Lipofectamine 3000 in the presence of 10% serum. Cell-uptake assays indicated that basic amino acid containing dipeptide cationic lipids exhibited more efficient cell uptake than serine and aromatic amino acids based dipeptide lipids. Confocal laser scanning microscopy (CLSM) studies corroborated that 3 could efficiently deliver and release DNA into the nuclei of HeLa cells. These results suggest that tocopherol-based dipeptide cationic lipids with pH and reduction dual-sensitive characteristics might be promising non-viral gene delivery vectors.

  15. Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B.

    Science.gov (United States)

    Hagenow, S; Stasiak, A; Ramsay, R R; Stark, H

    2017-01-13

    Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer's disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands.

  16. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium Botulinum Strain PS-5.

    Science.gov (United States)

    Singh, Ajay K; Sachdeva, Amita; Degrasse, Jeffrey A; Croley, Timothy R; Stanker, Larry H; Hodge, David; Sharma, Shashi K

    2013-04-01

    Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography-Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.

  17. 共培养下后交叉韧带成纤维细胞中赖氨酰氧化酶的基因表达**☆%Lysyl oxidase gene expressions in the posterior cruciate ligament fibroblasts co-cultured with synovial cells

    Institute of Scientific and Technical Information of China (English)

    张艳君; 梅虎; 蒋稼欢; 谢静; 尹琳; 陈荣富; 许春明; 王春莉; 宋国立

    2013-01-01

    to improve the healing ability of injured posterior cruciate ligament, we need to find new ways for regeneration and repair of injured posterior cruciate ligament. Previous studies have demonstrated that lysyl oxidases play an important role in the tissue repair mechanism, but the effect of lysyl oxidases from injured posterior cruciate ligament on the process of wound repair remains unclear. OBJECTIVE: To investigate the expressions of lysyl oxidases in the posterior cruciate ligament fibroblasts co-cultured with synovial cel s. METHODS: The fourth passage of posterior cruciate ligament fibroblasts and synovial cel s were placed in 6-wel plates and Transwel , respectively. Two groups were designed as fol ows, posterior cruciate ligament fibroblasts group as control and co-cultured group termed as test group. At 6 hours after co-culture, total RNA was isolated and the expressions of lysyl oxidases in the posterior cruciate ligament fibroblasts were analyzed by semi-quantitative reverse-transcription PCR and quantitative real-time PCR. RESULTS AND CONCLUSION: The results revealed that co-culture contributed to up-regulations of lysyl oxidases compared with the control group, and gene levels were up to 1.1 folds in lysyl oxidase, 1.4 folds in lysyl oxidase-like 1 protein, 1.1 folds in lysyl oxidase-like 2 protein, 1.3 folds in lysyl oxidase-like 3 protein, 1.1 folds in lysyl oxidase-like 4 protein in co-cultured posterior cruciate ligament cel s (P < 0.05). The differential expression of lysyl oxidases in co-cultured posterior cruciate ligament cel s implies that cel-cel interaction and crosstalk are related with posterior cruciate ligament wound healing and have significant potential value and clinical usage for cure of injured posterior cruciate ligament.

  18. Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.

    OpenAIRE

    Reynafarje, B; Alexandre, A.; Davies, P.; Lehninger, A L

    1982-01-01

    The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 i...

  19. Dual monitoring using {sup 124}I-FIAU and bioluminescence for HSV1-tk suicide gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, T. S.; Kim, J. H.; Kwon, H. C. [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)] (and others)

    2007-07-01

    Herpes simplex virus type I thymidine kinase (HSV-tk) is the most common reporter gene and is used in cancer gene therapy with a prodrug nucleoside analog, ganciclovir (GCV). The aim of this study is to evaluate therapeutic efficacy of suicide gene therapy with 2'-fluoro-2'-deoxy-1-D-arabinofuranosyl-5-[{sup 124}I] iodouracil ({sup 124}I - FIAU) and bioluminescence in retrovirally HSV -tk and firefly luciferase transduced hepatoma model. The HSV -tk and firefly luciferase (Luc) was retrovirally transduced and expressed in MCA rat Morris hepatoma cells. Nude mice with subcutaneous tumors, MCA and MCA-TK-Luc, were subjected to GCV treatment (50mg/Kg/d intraperitoneally) for 5 day. PET imaging and biodistribution with ({sup 124}I-FIAU) were performed at before and after initiation of therapy with GCV. Bioluminescent signal was also measured during GCV treatment. Before GCV treatment, no significant difference in tumor volume was found in tumors between MCA and MCA-TK-Luc. After GCV treatment, tumor volume of MCA-TK-Luc markedly reduced compared to that of MCA. In biodistribution study, {sup 124}I-FIAU uptake after GCV therapy significantly decreased compared with pretreatment levels (34.8 13.67 %ID/g vs 7.6 2.59 %ID/g) and bioluminescent signal was also significantly decreased compared with pretreatment levels. In small animal PET imaging, {sup 124}I-FIAU selectively localized in HSV -tk expressing tumor and the therapeutic efficacy of GCV treatment was evaluated by {sup 124}I-FIAU PET imaging. {sup 124}I-FIAU PET and bioluminescence imaging in HSV-tk suicide gene therapy were effective to evaluate the therapeutic response. {sup 124}I-FIAU may serve as an efficient and selective agent for monitoring of transduced HSV1-tk gene expression in vivo in clinical trials.

  20. Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

    NARCIS (Netherlands)

    van Hellemond, Erik W.; van Dijk, Marianne; Heuts, Dominic P. H. M.; Janssen, Dick B.; Fraaije, Marco W.

    2008-01-01

    A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting

  1. Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

    NARCIS (Netherlands)

    van Hellemond, Erik W.; van Dijk, Marianne; Heuts, Dominic P. H. M.; Janssen, Dick B.; Fraaije, Marco W.

    A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting

  2. Dual-site phosphorylation of the control of virulence regulator impacts group a streptococcal global gene expression and pathogenesis.

    Directory of Open Access Journals (Sweden)

    Nicola Horstmann

    2014-05-01

    Full Text Available Phosphorylation relays are a major mechanism by which bacteria alter transcription in response to environmental signals, but understanding of the functional consequences of bacterial response regulator phosphorylation is limited. We sought to characterize how phosphorylation of the control of virulence regulator (CovR protein from the major human pathogen group A Streptococcus (GAS influences GAS global gene expression and pathogenesis. CovR mainly serves to repress GAS virulence factor-encoding genes and has been shown to homodimerize following phosphorylation on aspartate-53 (D53 in vitro. We discovered that CovR is phosphorylated in vivo and that such phosphorylation is partially heat-stable, suggesting additional phosphorylation at non-aspartate residues. Using mass spectroscopy along with targeted mutagenesis, we identified threonine-65 (T65 as an additional CovR phosphorylation site under control of the serine/threonine kinase (Stk. Phosphorylation on T65, as mimicked by the recombinant CovR T65E variant, abolished in vitro CovR D53 phosphorylation. Similarly, isoallelic GAS strains that were either unable to be phosphorylated at D53 (CovR-D53A or had functional constitutive phosphorylation at T65 (CovR-T65E had essentially an identical gene repression profile to each other and to a CovR-inactivated strain. However, the CovR-D53A and CovR-T65E isoallelic strains retained the ability to positively influence gene expression that was abolished in the CovR-inactivated strain. Consistent with these observations, the CovR-D53A and CovR-T65E strains were hypervirulent compared to the CovR-inactivated strain in a mouse model of invasive GAS disease. Surprisingly, an isoalleic strain unable to be phosphorylated at CovR T65 (CovR-T65A was hypervirulent compared to the wild-type strain, as auto-regulation of covR gene expression resulted in lower covR gene transcript and CovR protein levels in the CovR-T65A strain. Taken together, these data

  3. Gold Nanocluster Decorated Polypeptide/DNA Complexes for NIR Light and Redox Dual-Responsive Gene Transfection.

    Science.gov (United States)

    Lei, Qi; Hu, Jing-Jing; Rong, Lei; Cheng, Han; Sun, Yun-Xia; Zhang, Xian-Zheng

    2016-08-20

    Endo/lysosomal escape and subsequent nuclear translocation are recognized as the two major challenges for efficient gene transfection. Herein, nuclear localization signal (NLS) peptide sequences and oligomeric lysine sequences were crosslinked via disulfide bonds to obtain glutathione (GSH) reducible polypeptide (pNLS). The pNLS could condense DNA into compact positive-charged complexes with redox sensitivity, and then gold nanoclusters (AuNC) were further decorated to the surface via electrostatic interactions obtaining versatile pNLS/DNA/AuNC complexes. The AuNC could generate reactive oxygen species (ROS) under NIR-irradiation and accelerate the endo/lysosomal escape of the complexes, and then the pNLS sequence degraded by GSH in cytoplasm would release the DNA and facilitate the subsequent nuclear translocation for enhanced gene transfection.

  4. Origin and evolution of lysyl oxidases.

    Science.gov (United States)

    Grau-Bové, Xavier; Ruiz-Trillo, Iñaki; Rodriguez-Pascual, Fernando

    2015-05-29

    Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize primary amine substrates to reactive aldehydes. The best-studied role of LOX enzymes is the remodeling of the extracellular matrix (ECM) in animals by cross-linking collagens and elastin, although intracellular functions have been reported as well. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, showing a highly conserved catalytic carboxy terminal domain and more divergence in the rest of the sequence. Here we have surveyed a wide selection of genomes in order to infer the evolutionary history of LOX. We identified LOX proteins not only in animals, but also in many other eukaryotes, as well as in bacteria and archaea - which reveals a pre-metazoan origin for this gene family. LOX genes expanded during metazoan evolution resulting in two superfamilies, LOXL2/L3/L4 and LOX/L1/L5. Considering the current knowledge on the function of mammalian LOX isoforms in ECM remodeling, we propose that LOXL2/L3/L4 members might have preferentially been involved in making cross-linked collagen IV-based basement membrane, whereas the diversification of LOX/L1/L5 forms contributed to chordate/vertebrate-specific ECM innovations, such as elastin and fibronectin. Our work provides a novel view on the evolution of this family of enzymes.

  5. Genome-Wide Association Studies Candidate Gene to Dual Modifier of Nonalcoholic Steatohepatitis and Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Clint L. Miller, PhD

    2016-12-01

    Full Text Available Nonalcoholic steatohepatitis is a common disease involving chronic accumulation of fat and inflammation in the liver, often leading to advanced fibrosis, cirrhosis, and cancer. It is known that nonalcoholic steatohepatitis shares many features with atherosclerosis; however, there are still no effective therapeutics. In a recent study published in Nature, investigators demonstrated that mice lacking a high-density lipoprotein–associated gene were surprisingly protected from both steatohepatitis and atherosclerosis through the stabilization of the liver X receptor. This work reveals a timely candidate target for 2 highly prevalent cardiovascular diseases.

  6. Crystallization of recombinant 1-amino cyclo propane-1-carboxylate (Acc) oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, L.; Arni, R.K. [UNESP, Sao Jose do Rio Preto, SP (Brazil). Dept. de Fisica; Dilley, D. [Michigan State Univ., East Lansing, MI (United States). Dept. of Biophysics

    1996-12-31

    Full text. Ethylene is an important harmone in plant biology because it activates gene expression with consequences at all phases of plant growth and development spanning seed germination to fruit ripening and senesense of plant organs. In climacteric fruits, the sharp increase in ethylene production at the onset of ripening is throught to trigger the changes in colour, aroma, texture and flavour. The final step in ethylene biosynthesis is catalyzed by ACC oxidase. Biothechnological methods have been used to inhibit ethylene biosynthesis and ripening in tomato by down-regulating ACC synthase and ACC oxidase gene expression using the antisense RNA strategy. A similar goal has been achieved by overexpressing a bacterial ACC deaminase or a viral-S-adenosylmethionine hydrolase gene, which reduces the availability of the ethylene precursors., ACC and S-adenosylmethionine, respectively. C0{sub 2} at concentrations commonly found in the intracellular space of plant tissues is required to active ACC oxidase to produce ethylene and can elevate enzyme activity 20-fold in a concentration dependent manner. Consequently, the intracellular ethylene level is modulated from low inactive levels when C0{sub 2} is not limiting and this may alter gene expression. ACC oxidase undergoes catalytic inactivation as the reaction to make ethylene procedes and this too may involve CO{sub 2}. It has been suggested that CO{sub 2}acts as a modulator of ACC oxidase activity and therby helps regulate ethylene levels in the cell and thus may explain many ethylene related phenomena in plant biology. CO{sub 2} is know to affect O{sub 2} binding in hemoglobin and ribulose bisphosphate carboxylase-oxygenase (Rubisco). Catalytic inactivation is a common phenomena in enzyme turnover, ACC oxidase is a Fe{sup +2}/ascorbate requiring enzyme and this makes it a prime candidate for metal ion oxidation-based inactivation. Charentais melon with an antisense ACC oxidase cDNA. A trangenic line exhibits reduction

  7. Direct and accurate measurement of CAG repeat configuration in the ataxin-1 (ATXN-1) gene by "dual-fluorescence labeled PCR-restriction fragment length analysis".

    Science.gov (United States)

    Lin, Jiang X; Ishikawa, Kinya; Sakamoto, Masaki; Tsunemi, Taiji; Ishiguro, Taro; Amino, Takeshi; Toru, Shuta; Kondo, Ikuko; Mizusawa, Hidehiro

    2008-01-01

    Spinocerebellar ataxia type 1 (SCA1; OMIM: #164400) is an autosomal dominant cerebellar ataxia caused by an expansion of CAG repeat, which encodes polyglutamine, in the ataxin-1 (ATXN1) gene. Length of polyglutamine in the ATXN1 protein is the critical determinant of pathogenesis of this disease. Molecular diagnosis of SCA1 is usually undertaken by assessing the length of CAG repeat configuration using primers spanning this configuration. However, this conventional method may potentially lead to misdiagnosis in assessing polyglutamine-encoding CAG repeat length, since CAT interruptions may be present within the CAG repeat configuration, not only in normal controls but also in neurologically symptomatic subjects. We developed a new method for assessing actual CAG repeat numbers not interrupted by CAT sequences. Polymerase chain reaction using a primer pair labeled with two different fluorescences followed by restriction enzyme digestion with SfaNI which recognizes the sequence "GCATC(N)(5)", lengths of actual CAG repeats that encode polyglutamine were directly detected. We named this method "dual fluorescence labeled PCR-restriction fragment length analysis". We found that numbers of actual CAG repeat encoding polyglutamine do not overlap between our cohorts of normal chromosomes (n=385) and SCA1 chromosomes (n=5). We conclude that the present method is a useful way for molecular diagnosis of SCA1.

  8. Dual targeting of ANGPT1 and TGFBR2 genes by miR-204 controls angiogenesis in breast cancer

    Science.gov (United States)

    Flores-Pérez, Ali; Marchat, Laurence A.; Rodríguez-Cuevas, Sergio; Bautista-Piña, Verónica; Hidalgo-Miranda, Alfredo; Ocampo, Elena Aréchaga; Martínez, Mónica Sierra; Palma-Flores, Carlos; Fonseca-Sánchez, Miguel A.; Astudillo-de la Vega, Horacio; Ruíz-García, Erika; González-Barrios, Juan Antonio; Pérez-Plasencia, Carlos; Streber, María L.; López-Camarillo, César

    2016-01-01

    Deregulated expression of microRNAs has been associated with angiogenesis. Studying the miRNome of locally advanced breast tumors we unsuspectedly found a dramatically repression of miR-204, a small non-coding RNA with no previous involvement in tumor angiogenesis. Downregulation of miR-204 was confirmed in an independent cohort of patients and breast cancer cell lines. Gain-of-function analysis indicates that ectopic expression of miR-204 impairs cell proliferation, anchorage-independent growth, migration, invasion, and the formation of 3D capillary networks in vitro. Likewise, in vivo vascularization and angiogenesis were suppressed by miR-204 in a nu/nu mice model. Genome-wide profiling of MDA-MB-231 cells expressing miR-204 revealed changes in the expression of hundred cancer-related genes. Of these, we focused on the study of pro-angiogenic ANGPT1 and TGFβR2. Functional analysis using luciferase reporter and rescue assays confirmed that ANGPT1 and TGFβR2 are novel effectors downstream of miR-204. Accordingly, an inverse correlation between miR-204 and ANGPT1/TGFβR2 expression was found in breast tumors. Knockdown of TGFβR2, but not ANGPT1, impairs cell proliferation and migration whereas inhibition of both genes inhibits angiogenesis. Taken altogether, our findings reveal a novel role for miR-204/ANGPT1/TGFβR2 axis in tumor angiogenesis. We propose that therapeutic manipulation of miR-204 levels may represent a promising approach in breast cancer. PMID:27703260

  9. Dual diagnosis

    OpenAIRE

    2013-01-01

    Dual diagnosis denotes intertwining of intellectual disabilities with mental disorders. With the help of systematic examination of literature, intellectual disabilities are determined (they are characterized by subaverage intellectual activity and difficulties in adaptive skills), along side mental disorders. Their influence is seen in changes of thinking, perception, emotionality, behaviour and cognition. Mental disorders often occur with people with intellectual disabilities (data differs f...

  10. 基于线粒体CO Ⅰ基因的闪蛱蝶亚科系统发育关系研究%Phylogeny of Apaturinae Butterflies (Lepidoptera: Nymphalidae) Based on Mitochondrial Cytochrome OxidaseGene

    Institute of Scientific and Technical Information of China (English)

    张敏; 曹天文; 张睿; 郭亚平; 段毅豪; 马恩波

    2007-01-01

    The phylogenetic relationships of genera in the subfamily Apaturinae were examined using mtDNA sequence data from 1,471 bp of cytochrome oxidase subunit Ⅰ (COI). The mitochondrial COI gene from a total of 16 species in 11 genera were sequenced to obtain mtDNA data, along with those of 4 species obtained from GenBank, to construct the MP and the NJ trees using Athyma jina, Penthema adelma, Polyura nepenthes, and Charaxes bernardus as outgroups. The transitions at the third codon positions of the COI data set were found saturated, but they were retained for analysis, because they contain the majority of the phylogenetic information. The impacts of equal weight assumptions for all characters in the parsimonious analysis were assessed by potential alternations in clades in response to different transition/transversion weighting schemes. The results indicated four distinct major groups in Apaturinae. Moreover, several well supported and stable clades were found in the Apaturinae. The study also identified undetermined taxon groups whose positions were weakly supported and were subject to changes under different weighting schemes. Within the Apaturinae, the clustering results are approximately identical to the classical morphological classification. The mtDNA data suggest the genus Mimathyma as a monophyletic group. Lelecella limenitoides and Dilipa fenestra have close relationship with very strong support in all phylogenetic trees. It also supports the taxonomic revision of removing several species from Apatura to other genera, namely Mimathyma schrenckii, M. chevana, M. nycteis, Chitoria subcaerulea, C. fasciola, C. pallas, and Helcyra subalba.%测定了重要林业害虫闪蛱蝶亚科Apaturinae 11属17种蝶类的线粒体细胞色素氧化酶Ⅰ的部分序列,并结合由GenBank下载的该亚科4种蝶类的相应序列进行分析,探讨了闪蛱蝶亚科各属间的系统发育关系.以玉杵带蛱蝶Athyma jina,白斑眼蝶Penthema adelma

  11. Residual NADPH Oxidase Activity and Isolated Lung Involvement in X-Linked Chronic Granulomatous Disease

    Directory of Open Access Journals (Sweden)

    Maria J. Gutierrez

    2012-01-01

    Full Text Available Chronic granulomatous disease (CGD is characterized by inherited immune defects resulting from mutations in the NADPH oxidase complex genes. The X-linked type of CGD is caused by defects in the CYBB gene that encodes gp91-phox, a fundamental component of the NADPH oxidase complex. This mutation originates the most common and severe form of CGD, which typically has absence of NADPH oxidase function and aggressive multisystemic infections. We present the case of a 9-year-old child with a rare CYBB mutation that preserves some NADPH oxidase activity, resulting in an atypical mild form of X-linked CGD with isolated lung involvement. Although the clinical picture and partially preserved oxidase function suggested an autosomal recessive form of CGD, genetic testing demonstrated a mutation in the exon 3 of CYBB gene (c.252 G>A, p.Ala84Ala, an uncommon X-linked CGD variant that affects splicing. Atypical presentation and diagnostic difficulties are discussed. This case highlights that the diagnosis of mild forms of X-linked CGD caused by rare CYBB mutations and partially preserved NADPH function should be considered early in the evaluation of atypical and recurrent lung infections.

  12. A desregulação dos genes relógio modifica o estado redox das células β pancreáticas e modula a secreção de insulina estimulada pela glicose via NADPH oxidase.

    OpenAIRE

    2015-01-01

    Os genes relógio são responsáveis pelo ritmo circadiano e homeostase de diversos sistemas biológicos, incluindo o pâncreas endócrino. Nas células β são de grande importância para a regulação do metabolismo e da secreção de insulina (SI), e sua ausência pode levar ao desenvolvimento do diabetes. A NADPH oxidase (NOX) é um complexo enzimático responsável pela produção do ânion superóxido através da redução do oxigênio molecular. Em ilhotas pancreáticas, a NOX participa da regulação do meta...

  13. Oxidase-based biocatalytic processes

    DEFF Research Database (Denmark)

    Ramesh, Hemalata; Woodley, John; Krühne, Ulrich

    the reaction species (substrate and product volatility for example) and the process (such as oxygen supply, ability to control pH) and are classified as reaction-related and process-related constraintsrespectively. Although the development of biocatalyst and process engineering tools offers a number...... ofsolutions to overcome the limitations, it is often complicated to identify the key limitation of the system that prevents economic scale-up. Hence, development of a systematic method for identifying the limitations during early-stage development of a biocatalytic process and potentially the order in which...... theyneed to be tackled would offer a valuable tool for process development.Biocatalytic oxidationsare potentially of great value because of theselective chemistry that they offer,resulting in higher yieldscompared to thoseachievable through chemical catalysis. Oxidases areparticularly...

  14. Cucumber Seedling Indoleacetaldehyde Oxidase 1

    Science.gov (United States)

    Bower, Peter J.; Brown, Hugh M.; Purves, William K.

    1978-01-01

    Extracts of light-grown Cucumis sativus L. seedlings catalyzed the oxidation of indole-3-acetaldehyde to indole-3-acetic acid. No added cofactors were required. Inhibitor studies indicated that the enzyme is a metalloflavoprotein. While indole-3-aldehyde, benzaldehyde, and phenylacetaldehyde partially inhibited the oxidation of indole-3-acetaldehyde, suggesting that they may serve as alternative substrates, it is proposed that indoleacetaldehyde is the major substrate in vivo. 2,4-Dichlorophenoxyacetic acid strongly inhibited the indoleacetaldehyde oxidase activity, and it is proposed that this enzyme may be subject in vivo to feedback inhibition by indole-3-acetic acid. The enzyme was activated by brief heating or by treatment with mercaptoethanol. PMID:16660220

  15. A broad distribution of the alternative oxidase in microsporidian parasites.

    Directory of Open Access Journals (Sweden)

    Bryony A P Williams

    2010-02-01

    Full Text Available Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX, a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1 as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.

  16. Modular assembly of yeast cytochrome oxidase.

    Science.gov (United States)

    McStay, Gavin P; Su, Chen Hsien; Tzagoloff, Alexander

    2013-02-01

    Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high-molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.

  17. Dual Pressure from Antiretroviral Therapy and Cell-Mediated Immune Response on the Human Immunodeficiency Virus Type 1 Protease Gene

    Science.gov (United States)

    Karlsson, Annika C.; Deeks, Steven G.; Barbour, Jason D.; Heiken, Brandon D.; Younger, Sophie R.; Hoh, Rebecca; Lane, Meghan; Sällberg, Matti; Ortiz, Gabriel M.; Demarest, James F.; Liegler, Teri; Grant, Robert M.; Martin, Jeffrey N.; Nixon, Douglas F.

    2003-01-01

    Human immunodeficiency virus (HIV)-specific CD8+ T-lymphocyte pressure can lead to the development of viral escape mutants, with consequent loss of immune control. Antiretroviral drugs also exert selection pressures on HIV, leading to the emergence of drug resistance mutations and increased levels of viral replication. We have determined a minimal epitope of HIV protease, amino acids 76 to 84, towards which a CD8+ T-lymphocyte response is directed. This epitope, which is HLA-A2 restricted, includes two amino acids that commonly mutate (V82A and I84V) in the face of protease inhibitor therapy. Among 29 HIV-infected patients who were treated with protease inhibitors and who had developed resistance to these drugs, we show that the wild-type PR82V76-84 epitope is commonly recognized by cytotoxic T lymphocytes (CTL) in HLA-A2-positive patients and that the CTL directed to this epitope are of high avidity. In contrast, the mutant PR82A76-84 epitope is generally not recognized by wild-type-specific CTL, or when recognized it is of low to moderate avidity, suggesting that the protease inhibitor-selected V82A mutation acts both as a CTL and protease inhibitor escape mutant. Paradoxically, the absence of a mutation at position 82 was associated with the presence of a high-avidity CD8+ T-cell response to the wild-type virus sequence. Our results indicate that both HIV type 1-specific CD8+ T cells and antiretroviral drugs provide complex pressures on the same amino acid sequence of the HIV protease gene and, thus, can influence viral sequence evolution. PMID:12767994

  18. REGULATION OF THE EXPRESSION OF MITOCHONDRIAL PROTEINS - RELATIONSHIP BETWEEN MTDNA COPY NUMBER AND CYTOCHROME-C-OXIDASE ACTIVITY IN HUMAN-CELLS AND TISSUES

    NARCIS (Netherlands)

    VANDENBOGERT, C; DEVRIES, H; HOLTROP, M; MUUS, P; DEKKER, HL; VANGALEN, MJM; BOLHUIS, PA; TAANMAN, JW

    1993-01-01

    The relationship between the relative amounts of nuclear and mitochondrial genes for cytochrome-c oxidase subunits and their transcripts and cytochrome-c oxidase activity was investigated in several human tissues and cell lines to get more insight into the regulation of the expression of this mitoch

  19. QCD Dual

    DEFF Research Database (Denmark)

    Sannino, Francesco

    2009-01-01

    We uncover a novel solution of the 't Hooft anomaly matching conditions for QCD. Interestingly in the perturbative regime the new gauge theory, if interpreted as a possible QCD dual, predicts the critical number of flavors above which QCD in the nonperturbative regime, develops an infrared stable...... fixed point. Remarkably this value is identical to the maximum bound predicted in the nonpertubative regime via the all-orders conjectured beta function for nonsupersymmetric gauge theories.......We uncover a novel solution of the 't Hooft anomaly matching conditions for QCD. Interestingly in the perturbative regime the new gauge theory, if interpreted as a possible QCD dual, predicts the critical number of flavors above which QCD in the nonperturbative regime, develops an infrared stable...

  20. Growth of transplastomic cells expressing D-amino acid oxidase in chloroplasts is tolerant to D-alanine and inhibited by D-valine.

    Science.gov (United States)

    Gisby, Martin F; Mudd, Elisabeth A; Day, Anil

    2012-12-01

    Dual-conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. Plastid genomes are relatively small and conserved DNA molecules that can be manipulated precisely by homologous recombination. High-yield expression of recombinant products and maternal inheritance of plastid-encoded traits make plastids attractive sites for modification. Here, we describe the cloning and expression of a dao gene encoding D-amino acid oxidase from Schizosaccharomyces pombe in tobacco (Nicotiana tabacum) plastids. The results provide genetic evidence for the uptake of D-amino acids into plastids, which contain a target that is inhibited by D-alanine. Importantly, this nonantibiotic-based selection system allows the use of cheap and widely available D-amino acids, which are relatively nontoxic to animals and microbes, to either select against (D-valine) or for (D-alanine) cells containing transgenic plastids. Positive/negative selection with d-amino acids was effective in vitro and against transplastomic seedlings grown in soil. The dual functionality of dao is highly suited to the polyploid plastid compartment, where it can be used to provide tolerance against potential D-alanine-based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop D-valine-based methods to manage the spread of transgenic plastids tagged with dao.

  1. AvrXa3:A novel member of avrBs3 gene family from Xanthomonas oryzae pv.oryzae has a dual function

    Institute of Scientific and Technical Information of China (English)

    LI Ping; LONG Juying; HUANG Yingchun; ZHANG Yan; WANG Jinsheng

    2004-01-01

    Two positive clones pUAV45 and pUAV47 were identified from the cDNA library of JxoⅢ, a race 3 strain of Xanthomonas oryzae pv. Oryzae Dye (Xoo) in Japanese system, using Tn5 based technology. pUAV45 clone contained a 25.4 kb Xoo genomic DNA insert. Southern blot analysis with avrXa10 as the probe showed that DNA insert in pUAV45 shares homology with avrX10. Furthermore within the Xoo insert, a smaller 5.7 kb KpnI fragment (pUAVSk) was identified through hybridization with avrXa10. The transformation of pUAV45 and pUAV5k into the strain Pxo99 (race 6 in Philippine system) led to the decrease of Pxo99pathogenicity on rice cultivar Wase Aikoku 3 (Xa3) and the increase of the pathogen pathogenicity on Cas209 (Xa10). The result of sequence analysis showed that there is a 2598 bp open reading frame (ORF) within the 5.7 kb Kpn 1 fragment (pUAVSk). The ORF shared high identity (97 % ) with avrXa10. The deduced sequence of the ORF contained 8.5 tandem repeat units of 34-amino-acids, one leucine zipper (LZ), three nuclear localization signal (NLS) motifs, and an acidic activation transcriptional domain (AAD) at C-terminus.We named this ORF avrXa3 and it is classified as a new member of avrBs3 (avr/pth) family with the dual-function determined by alternations of avirulence and aggressiveness on rice cultivars carrying different ‘ R' genes.

  2. 1-Aminocyclopropane-1-Carboxylate Oxidase Activity Limits Ethylene Biosynthesis in Rumex palustris during Submergence

    Science.gov (United States)

    Vriezen, Wim H.; Hulzink, Raymond; Mariani, Celestina; Voesenek, Laurentius A.C.J.

    1999-01-01

    Submergence strongly stimulates petiole elongation in Rumex palustris, and ethylene accumulation initiates and maintains this response in submerged tissues. cDNAs from R. palustris corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (RP-ACO1) were isolated from elongating petioles and used to study the expression of the corresponding gene. An increase in RP-ACO1 messenger was observed in the petioles and lamina of elongating leaves 2 h after the start of submergence. ACC oxidase enzyme activity was measured in homogenates of R. palustris shoots, and a relevant increase was observed within 12 h under water with a maximum after 24 h. We have shown previously that the ethylene production rate of submerged shoots does not increase significantly during the first 24 h of submergence (L.A.C.J. Voesenek, M. Banga, R.H. Thier, C.M. Mudde, F.M. Harren, G.W.M. Barendse, C.W.P.M. Blom [1993] Plant Physiol 103: 783–791), suggesting that under these conditions ACC oxidase activity is inhibited in vivo. We found evidence that this inhibition is caused by a reduction of oxygen levels. We hypothesize that an increased ACC oxidase enzyme concentration counterbalances the reduced enzyme activity caused by low oxygen concentration during submergence, thus sustaining ethylene production under these conditions. Therefore, ethylene biosynthesis seems to be limited at the level of ACC oxidase activity rather than by ACC synthase in R. palustris during submergence. PMID:10482674

  3. The C-terminal region controls correct folding of genus Trametes pyranose 2-oxidases.

    Science.gov (United States)

    Maresová, Helena; Palyzová, Andrea; Kyslík, Pavel

    2007-06-30

    The pyranose 2-oxidases from Trametes ochracea and Trametes pubescens share markedly similar amino acid sequences with identity of 93.4%. When expressed from the recombinant plasmids based on the same vector in the Escherichia coli host strain BL21(DE3) at higher growth temperatures, they differ strikingly in the formation of the inclusion bodies. Upon overexpression in the cultures performed at 28 degrees C, the specific activity of pyranose 2-oxidase from T. pubescens was eight times higher than that from T. ochracea: 93% of pyranose 2-oxidase from T. ochracea and only 15% of that from T. pubescens was present in the form of inclusion bodies. To ascertain the cause of this difference, both cloned genes were shuffled. Site-directed recombination of p2o cDNAs revealed that DNA constructs ending with 3' end of p2o cDNA from T. pubescens code for proteins that are folded into an active form to the greater extent, regardless of the gene expression level. "In silicio" analysis of physico-chemical properties of the protein sequences of pyranose 2-oxidases revealed that the sequence of amino acid residues 368-430, constituting the small, head domain of pyranose 2-oxidase from T. pubescens, affects positively the enzyme folding at higher cultivation temperatures. The domain differs in six amino acid residues from that of T. ochracea.

  4. Vanillyl-alcohol oxidase, a tasteful biocatalyst

    NARCIS (Netherlands)

    Heuvel, Robert H.H. van den; Fraaije, Marco W.; Mattevi, Andrea; Laane, Colja; Berkel, Willem J.H. van

    2001-01-01

    The covalent flavoenzyme vanillyl-alcohol oxidase (VAO) is a versatile biocatalyst. It converts a wide range of phenolic compounds by catalysing oxidation, deamination, demethylation, dehydrogenation and hydroxylation reactions. The production of natural vanillin, 4-hydroxybenzaldehyde, coniferyl al

  5. Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

    Science.gov (United States)

    Choi, Eun-hye; Song, Min-Suk; Park, Su-Jin; Pascua, Philippe Noriel Q; Baek, Yun Hee; Kwon, Hyeok-il; Kim, Eun-Ha; Kim, Semi; Jang, Hyung-Kwan; Poo, Haryoung; Kim, Chul-Joong; Choi, Young Ki

    2015-07-01

    An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-β activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.

  6. Galactose oxidase nanoaggregates: Preparation and characterization

    OpenAIRE

    Mamta Sharma; Minakshi Sharma

    2016-01-01

    Galctose oxidase nanoaggregates have been prepared by chemical desolvation method involving the crosslinkng agent glutaraldehyde. These enzyme nanoagregates have been characterized by transmission electron microscopy(TEM), UV visible spectroscopy, Fourier transform infrared spectroscopy (FTIR). TEM reveals the globular spherical nanostructured form upto the range of 20nm. UV visible spectroscopy of galactose oxidase nanoaggregates shows maximum absorption peak at 237nm. FTIR spectra obtaine...

  7. Pyranose 2-oxidase from Phanerochaete chrysosporium : expression in E. coli and biochemical characterization

    Science.gov (United States)

    Ines Pisanelli; Magdalena Kujawa; Oliver Spadiut; Roman Kittl; Petr Halada; Jindrich Volc; Michael D. Mozuch; Philip Kersten; Dietmar Haltrich; Clemens Peterbauer

    2009-01-01

    The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in...

  8. Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein

    NARCIS (Netherlands)

    Bengtsson, J; Tjalsma, H; Rivolta, C; Hederstedt, L

    1999-01-01

    The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochrome caa(3), which is a cytochrome c oxidase. Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a

  9. 乳糖诱导重组大肠杆菌表达胆固醇氧化酶的研究%Expression Cholesterol Oxidase Gene in Recombinant Escherichia coli Using Lactose as Inducer

    Institute of Scientific and Technical Information of China (English)

    李闯; 孙艳; 张玲; 杨海麟; 王武

    2011-01-01

    Lactose was used to induce cholesterol oxidase (COD) expression in Escherichia coli BL21 (DE3) in this manuscript. Effect of different nutrient and environmental conditions,including media ingredients, lactose concentration, lactose addition time, and the culture temperature on COD expression were carefully investigated. Under the optimum induced conditions, the maximal activity of COD reached at 15.2 U/mL. The results confirmed that lactose could be used as an inducer in the fermentation process.%采用乳糖诱导胆固醇氧化酶(COD)基因在大肠杆菌BL21(DE3)中表达,研究了培养基成分、乳糖浓度、诱导时间和诱导温度对胆固醇氧化酶表达的影响.结果显示,在对诱导条件进行优化控制的前提下,胆固醇氧化酶酶活达到15.2 U/mL.研究结果为乳糖作为诱导剂最终应用于重组基因工程药物的工业化生产提供了有益的参考和借鉴.

  10. H2O2-Forming NADH Oxidase with Diaphorase (Cytochrome) Activity from Archaeoglobus fulgidus

    OpenAIRE

    Reed, David W.; Millstein, Jack; Hartzell, Patricia L.

    2001-01-01

    An enzyme exhibiting NADH oxidase (diaphorase) activity was isolated from the hyperthermophilic sulfate-reducing anaerobe Archaeoglobus fulgidus. N-terminal sequence of the protein indicates that it is coded for by open reading frame AF0395 in the A. fulgidus genome. The gene AF0395 was cloned and its product was purified from Escherichia coli. Like the native NADH oxidase (NoxA2), the recombinant NoxA2 (rNoxA2) has an apparent molecular mass of 47 kDa, requires flavin adenine dinucleotide fo...

  11. Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

    Directory of Open Access Journals (Sweden)

    Sluse F.E.

    1998-01-01

    Full Text Available Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression or short-term (post-translational modification, allosteric activation regulated. Electron distribution (partitioning between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach. Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon and with harmful reactive oxygen species formation.

  12. Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302.

    Science.gov (United States)

    Frébort, I; Matsushita, K; Toyama, H; Lemr, K; Yamada, M; Adachi, O

    1999-01-01

    Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.

  13. Differential Expression of the Three Multicopper Oxidases from Myxococcus xanthus▿

    Science.gov (United States)

    Sánchez-Sutil, María Celestina; Gómez-Santos, Nuria; Moraleda-Muñoz, Aurelio; Martins, Lígia O.; Pérez, Juana; Muñoz-Dorado, José

    2007-01-01

    Myxococcus xanthus is a soil bacterium that undergoes a unique life cycle among the prokaryotes upon starvation, which includes the formation of macroscopic structures, the fruiting bodies, and the differentiation of vegetative rods into coccoid myxospores. This peculiarity offers the opportunity to study the copper response in this bacterium in two different stages. In fact, M. xanthus vegetative rods exhibit 15-fold-greater resistance against copper than developing cells. However, cells preadapted to this metal reach the same levels of resistance during both stages. Analysis of the M. xanthus genome reveals that many of the genes involved in copper resistance are redundant, three of which encode proteins of the multicopper oxidase family (MCO). Each MCO gene exhibits a different expression profile in response to external copper addition. Promoters of cuoA and cuoB respond to Cu(II) ions during growth and development; however, they show a 10-fold-increased copper sensitivity during development. The promoter of cuoC shows copper-independent induction upon starvation, but it is copper up-regulated during growth. Phenotypic analyses of deletion mutants reveal that CuoB is involved in the primary copper-adaptive response; CuoA and CuoC are necessary for the maintenance of copper tolerance; and CuoC is required for normal development. These roles seem to be carried out through cuprous oxidase activity. PMID:17483223

  14. DNA-based diagnosis of isolated sulfite oxidase deficiency by denaturing high-performance liquid chromatography.

    Science.gov (United States)

    Lam, Ching-Wan; Li, Chi-Keung; Lai, Chi-Kong; Tong, Sui-Fan; Chan, Kwok-Yin; Ng, Grace Sui-Fun; Yuen, Yuet-Ping; Cheng, Anna Wai-Fun; Chan, Yan-Wo

    2002-01-01

    Isolated sulfite oxidase deficiency is a rare autosomal recessive disease, characterized by severe neurological abnormalities, seizures, mental retardation, and dislocation of the ocular lenses, that often leads to death in infancy. There is a special demand for prenatal diagnosis, since no effective treatment is available for isolated sulfite oxidase deficiency. Until now, the cDNA sequence of the sulfite oxidase (SUOX) gene has been available, but the genomic sequence of the SUOX gene has not been published. In this study, we have performed a DNA-based diagnosis of isolated sulfite oxidase deficiency in a Chinese patient. To do so, we designed oligonucleotide primers for amplification of the predicted exons and intron-exon boundaries of the SUOX gene obtained from the completed draft version of the human genome. Using overlapping PCR products, we confirmed the flanking intronic sequences of the coding exons and that the entire 466-residue mature peptide is encoded by the last exon of the gene. We then performed mutation detection using denaturing high-performance liquid chromatography (DHPLC). The DHPLC chromatogram of exon 2b showed the presence of heteroduplex peaks only after mixing of the mutant DNA with the wild-type DNA, indicating the presence of a homozygous mutation. Direct DNA sequencing showed a homozygous base substitution at codon 160, changing the codon from CGG to CAG, which changes the amino acid from arginine to glutamine, i.e., R160Q. The DNA-based diagnosis of isolated sulfite oxidase deficiency will enable us to make an accurate determination of carrier status and to perform prenatal diagnosis of this disease. The availability of the genomic sequences of human genes from the completed draft human genome sequence will simplify the development of molecular genetic diagnoses of human diseases from peripheral blood DNA.

  15. Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius.

    Science.gov (United States)

    Yang, Lei; Lübeck, Mette; Lübeck, Peter S

    2014-01-01

    Aspergillus carbonarius has potential as a cell factory for the production of different organic acids. At pH 5.5, A.carbonarius accumulates high amounts of gluconic acid when it grows on glucose based medium whereas at low pH, it produces citric acid. The conversion of glucose to gluconic acid is carried out by secretion of the enzyme, glucose oxidase. In this work, the gene encoding glucose oxidase was identified and deleted from A. carbonarius with the aim of changing the carbon flux towards other organic acids. The effect of genetic engineering was examined by testing glucose oxidase deficient (Δgox) mutants for the production of different organic acids in a defined production medium. The results obtained showed that the gluconic acid accumulation was completely inhibited and increased amounts of citric acid, oxalic acid and malic acid were observed in the Δgox mutants.

  16. Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

    Science.gov (United States)

    Vanhooren, J C; Fransen, M; de Béthune, B; Baumgart, E; Baes, M; Torrekens, S; Van Leuven, F; Mannaerts, G P; Van Veldhoven, P P

    1996-07-15

    The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

  17. NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys

    Directory of Open Access Journals (Sweden)

    Sunil Joshi

    2013-01-01

    Full Text Available A major role of the nicotinamide adenine dinucleotide phosphate (NADPH oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS. These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS. Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease.

  18. Comparative characterization of three D-aspartate oxidases and one D-amino acid oxidase from Caenorhabditis elegans.

    Science.gov (United States)

    Katane, Masumi; Saitoh, Yasuaki; Seida, Yousuke; Sekine, Masae; Furuchi, Takemitsu; Homma, Hiroshi

    2010-06-01

    Previously, we cloned cDNAs for four Caenorhabditis elegans genes (F20 Hp, C47Ap, F18Ep, and Y69Ap genes) that were annotated in the database as encoding D-amino acid oxidase (DAO) or D-aspartate oxidase (DDO) proteins. These genes were expressed in Escherichia coli, and the recombinant C47Ap and F18Ep were shown to have functional DDO activities, while Y69Ap had functional DAO activity. In this study, we improved the E. coli culture conditions for the production of recombinant F20 Hp and, following purification of the protein, revealed that it has functional DDO activity. The kinetic properties of recombinant C47Ap (DDO-1), F18Ep (DDO-2), F20 Hp (DDO-3), and Y69Ap (DAO) were also determined and compared with recombinant human DDO and DAO. In contrast to the low catalytic efficiency of human DDO for D-Glu, all three C. elegans DDOs showed higher catalytic efficiencies for D-Glu than D-Asp or N-methyl-D-Asp. The catalytic efficiency of C. elegans DAO for D-Ser was substantially lower than that of human DAO, while the C. elegans DAO was more efficient at deamination of basic D-amino acids (D-Arg and D-His) than human DAO. Collectively, our results indicate that C. elegans contains at least three genes that encode functional DDOs, and one gene encoding a functional DAO, and that these enzymes have different and distinctive properties.

  19. FASTKD2 Nonsense Mutation in an Infantile Mitochondrial Encephalomyopathy Associated with Cytochrome C Oxidase Deficiency

    OpenAIRE

    2008-01-01

    In two siblings we found a mitochondrial encephalomyopathy, characterized by developmental delay, hemiplegia, convulsions, asymmetrical brain atrophy, and low cytochrome c oxidase (COX) activity in skeletal muscle. The disease locus was identified on chromosome 2 by homozygosity mapping; candidate genes were prioritized for their known or predicted mitochondrial localization and then sequenced in probands and controls. A homozygous nonsense mutation in the KIAA0971 gene segregated with the di...

  20. Gibberellin metabolism in Vitis vinifera L. during bloom and fruit-set: functional characterization and evolution of grapevine gibberellin oxidases.

    Science.gov (United States)

    Giacomelli, Lisa; Rota-Stabelli, Omar; Masuero, Domenico; Acheampong, Atiako Kwame; Moretto, Marco; Caputi, Lorenzo; Vrhovsek, Urska; Moser, Claudio

    2013-11-01

    Gibberellins (GAs) are involved in the regulation of flowering and fruit-set in grapes (Vitis vinifera L.), but the molecular mechanisms behind this process are mostly unknown. In this work, the family of grapevine GA oxidases involved in the biosynthesis and deactivation of GAs was characterized. Six putative GA 20-oxidase (GA20ox), three GA 3-oxidase (GA3ox), and eight GA 2-oxidase (GA2ox) proteins, the latter further divided into five C19-GA 2ox and three C20-GA2ox proteins, were identified. Phylogenetic analyses suggest a common origin of the GA3ox and C19-GA2ox groups and challenge previous evolutionary models. In vitro analysis revealed that all GA3ox and GA20ox enzymes prefer substrates of the non-13-hydroxylation pathway. In addition, ectopic expression of GA2ox genes in Arabidopsis thaliana confirmed the activity of their encoded proteins in vivo. The results show that bioactive GA1 accumulates in opening grapevine flowers, whereas at later developmental stages only GA4 is detected in the setting fruit. By studying the expression pattern of the grapevine GA oxidase genes in different organs, and at different stages of flowering and fruit-set, it is proposed that the pool of bioactive GAs is controlled by a fine regulation of the abundance and localization of GA oxidase transcripts.

  1. Pyruvate oxidase is a determinant of Avery's rough morphology.

    Science.gov (United States)

    Belanger, Aimee E; Clague, Melissa J; Glass, John I; Leblanc, Donald J

    2004-12-01

    In pioneering studies, Avery et al. identified DNA as the hereditary material (A. T. Avery, C. M. MacLeod, and M. McCarty, J. Exp. Med. 79:137-158, 1944). They demonstrated, by means of variation in colony morphology, that this substance could transform their rough type 2 Streptococcus pneumoniae strain R36A into a smooth type 3 strain. It has become accepted as fact, from modern textbook accounts of these experiments, that smooth pneumococci make capsule, while rough strains do not. We found that rough-to-smooth morphology conversion did not occur in rough strains R36A and R6 when the ability to synthesize native type 2 capsule was restored. The continued rough morphology of these encapsulated strains was attributed to a second, since-forgotten, morphology-affecting mutation that was sustained by R36A during strain development. We used a new genome-PCR-based approach to identify spxB, the gene encoding pyruvate oxidase, as the mutated locus in R36A and R6 that, with unencapsulation, gives rise to rough colony morphology, as we know it. The variant spxB allele of R36A and R6 is associated with increased cellular pyruvate oxidase activity relative to the ancestral strain D39. Increased pyruvate oxidase activity alters colony shape by mediating cell death. R36A requires a wild-type spxB allele for the expression of smooth type 2 morphology but not for the expression of smooth type 3 morphology, the phenotype monitored by Avery et al. Thus, the mutated spxB allele did not impact their use of smooth morphology to identify the transforming principle.

  2. Alternative oxidase: distribution, induction, properties, structure, regulation, and functions.

    Science.gov (United States)

    Rogov, A G; Sukhanova, E I; Uralskaya, L A; Aliverdieva, D A; Zvyagilskaya, R A

    2014-12-01

    The respiratory chain in the majority of organisms with aerobic type metabolism features the concomitant existence of the phosphorylating cytochrome pathway and the cyanide- and antimycin A-insensitive oxidative route comprising a so-called alternative oxidase (AOX) as a terminal oxidase. In this review, the history of AOX discovery is described. Considerable evidence is presented that AOX occurs widely in organisms at various levels of organization and is not confined to the plant kingdom. This enzyme has not been found only in Archaea, mammals, some yeasts and protists. Bioinformatics research revealed the sequences characteristic of AOX in representatives of various taxonomic groups. Based on multiple alignments of these sequences, a phylogenetic tree was constructed to infer their possible evolution. The ways of AOX activation, as well as regulatory interactions between AOX and the main respiratory chain are described. Data are summarized concerning the properties of AOX and the AOX-encoding genes whose expression is either constitutive or induced by various factors. Information is presented on the structure of AOX, its active center, and the ubiquinone-binding site. The principal functions of AOX are analyzed, including the cases of cell survival, optimization of respiratory metabolism, protection against excess of reactive oxygen species, and adaptation to variable nutrition sources and to biotic and abiotic stress factors. It is emphasized that different AOX functions complement each other in many instances and are not mutually exclusive. Examples are given to demonstrate that AOX is an important tool to overcome the adverse aftereffects of restricted activity of the main respiratory chain in cells and whole animals. This is the first comprehensive review on alternative oxidases of various organisms ranging from yeasts and protists to vascular plants.

  3. 黄河裸裂尻鱼细胞色素C氧化酶Ⅰ、Ⅱ和Ⅲ亚基基因的克隆及序列特征分析%Cloning and Sequence Analysis of Genes Encoding mtDNA Cytochrome C Oxidase Subunits I, I and Ⅲ in Schizopygopsis pylzovi

    Institute of Scientific and Technical Information of China (English)

    晁燕; 祁得林; 申志新; 王国杰; 杨成

    2011-01-01

    The complete sequences of genes encoding cytochrome C oxidase subunits I, li and ill were cloned in Schizopygopsis pylzovi by RT-PCR method. The sequence analysis showed that the complete sequence of CO I was 1 551 bp, which consisted of the open reading frame (ORF) encoding 516 amino acid residues; the CO Ⅱ gene was 691 bp and contained a 690-nucleotide ORF encoding 230 amino acid residues; and the CO Ⅲ gene was 786 bp, which was composed of the ORF encoding 261 amino acid residues. The homologous analysis of sequences showed high similarity both in DNA and corresponding amino acid sequences of CO I, CO Ⅱ and CO Ⅲ between S. pylzovi and other 9 species from family Cyprinidae. Although there were sequence differences in subunit genes among different species, the corresponding amino acids tended to be consistent, suggesting functional stability of cytochrome C oxidase in electron transfer of mitochondrial respiration chain. S. pylzovi and Sinocyclocheilus grahami clustered together in the phylogenetic trees based on gene and amino acid sequences of CO I , and gene sequences of CO Ⅱ and CO Ⅲ, coupled with the same types, positions and numbers of functional sites in CO I and CO Ⅱ , suggesting that there is a relatively close relationship between them, which is consistent with the conclusion that the subfamily Schizothoracinae is originated from one of the primitive Barbinae fishes widely distributed in warm drainages in Qinghai-Tibetan Plateau during the Tertiary Period.%采用RT-PCR技术克隆获得了黄河裸裂尻鱼(Schizopygopsis pylzovi)CO Ⅰ、Ⅱ、Ⅲ基因的编码序列,并对此进行了初步分析.结果表明,黄河裸裂尻鱼CO I基因全长为1 551 bp,开放阅读框(ORF)由基因全长组成,编码516个氨基酸;COⅡ基因全长为691 bp,开放阅读框为690 bp,编码230个氨基酸;COⅢ基因全长为786 bp,其开放阅读框也是由基因全长组成,编码261个氨基酸.序列同源性分析显示

  4. Cloning and Expression Analysis of ent-kaurene Oxidase Gene CKO in Cucumber%黄瓜贝壳杉烯氧化酶基因CKO的克隆及其表达分析

    Institute of Scientific and Technical Information of China (English)

    胡宏敏; 蒋芳玲; 曹雪; 吴震; 王广龙

    2012-01-01

    With homologous cloning and RACE technology, ent-Kaurene oxidase (KO), a key enzyme in the pathway of gibberellins biosynthesis, was cloned from the shoot tip of' Deltastal' ( Cucumis sativus L. ) and named CKO (GenBank accession number: JN792591 ) . The full eDNA was 1 895 bp in length with an open reading frame (ORF) encoding a protein of 519 amino acids. The protein molecular weight and isoelectric point were predicted to be 59.211 kD and 8.45 respectively. Amino acids homology analysis indicated that the sequence had 84% similarity with that of Momordica charantia. Sequence analysis showed that CKO belonged to cytochrome P450 superfamily and contained cysteine hemeiron ligand signature (FXXGXRXCXG) and transmembrane region. The fluorescent quantitative RT-PCR result showed that the expression level of CKO was the highest in leggy seedling under shading treatment, being 5.18 times of the normal one. While the expression of CKO significantly reduced after spraying paclobutrazol at shading.%以‘戴多星’黄瓜为试材,应用同源克隆和RACE技术从茎尖中得到赤霉素合成的关键酶——贝壳杉烯氧化酶(KO)基因cDNA全长序列,命名为CKO,GenBank登录号为JN792591,其长度为l895bp,开放阅读框(ORF)编码519个氨基酸,相对分子量为59.211kD,等电点(PI)8.45。氨基酸同源性分析发现,CKO与苦瓜的KO同源性最高,达84%:序列结构分析表明,CKO属于细胞色素超家族P450系,具有细胞色素P450的血红素结构域FXXGXRXCXG和跨膜结构域。实时荧光定量分析表明,CKO在遮荫的徒长苗中表达量最高,是正常苗的5.18倍,在遮阴条件下喷施多效唑可降低期表达。

  5. Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm

    NARCIS (Netherlands)

    Luppens, S.B.I.; Kara, D.; Bandounas, L.; Jonker, M.J.; Wittink, F.R.A.; Bruning, O.; Breit, T.M.; ten Cate, J.M.; Crielaard, W.

    2008-01-01

    Introduction: Our previous studies showed that Streptococcus mutans and Veillonella parvula dual-species biofilms have a different acid production profile and a higher resistance to chlorhexidine than their single-species counterparts. The aim of the current study was to test whether the susceptibil

  6. 肿瘤双自杀基因治疗系统pTRKH2-PsT/CD,pTRKH2-PsT/UPRT在厌氧婴儿双歧杆菌中的构建%Construction of dual suicide gene therapy system pTRKH2/CD and pTRKH2/UPRT in Bifidobacterium infantis and its characterization

    Institute of Scientific and Technical Information of China (English)

    Zhuhua Li; Peng Ye; Yanbiao Yang; Guangyu Ran; Shuren Wang

    2009-01-01

    Objective: We recombine the suicide gene CD, UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function. Methods: CD gene, UPRT gene and lactic acid bacteria expression plasmid pTRKH2 were digested by restriction endonuclease BamH I and Sal I, and constructed recombinant plasmids pTRKH2/CD and pTRKH2/UPRT in E. coll. The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation. Identification of pTRKH2/CD and pTRKH2/UPRT was processed by dual restriction endonuclease digesting and sequencing. RT-PCR and SDS-PAGE were used to examine the expression of CD and UPRT genes at RNA and protein levels. The killing effects on Melanoma B16-F10 cells by pTRKH2/CD and pTRKH2/UPRT suicide gene therapy system with 5-FC were examined by MTT assay. Results: The CD gene and UPRT gene was successfully recombined into lactic acid bacteria expression plasmid pTRKH2. After dual endonuclease digestion of plasmid purified from the positively transfected E. co/i, two fragments of 6.9 Kb and 1.3 Kb were found for CD gene and two fragments of 6.9 Kb and 620 bp were found for UPRT gene. The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data. A fragment of 1.3 Kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobac- terium by RT-PCR. A 52 KDa protein for CD gene was identified in whole-cell protein of recombinant Bifidobacterium and a 26 KDa protein for UPRT gene was identified in supernatant fluid of recombinant Bifidobacterium. The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pTRKH2/CD and pTRKH2/UPRT dual suicide gene therapy system on Melanoma B16-F10 cells. Conclusion: CD gene and UPRT gene are suc- cessfully inserted into pTRKH2 and transfected into tumor

  7. 三价砷氧化细菌Acidovorax sp.GW2中As(Ⅲ)氧化酶基因和调控序列的克隆鉴定%Isolation and identification of arsenite oxidase gene and regulatory sequences in an arsenite-oxidizing bacterium Acidovorax sp. GW2

    Institute of Scientific and Technical Information of China (English)

    赵凯; 黄银燕; 王倩; 王革娇

    2011-01-01

    Using reverse transcriptase PCR method and a bacterial fosmid library screening, an arsenite oxidase gene cluster were isolated from an arsenite-oxidizing bacterium Acidovorax sp. GW2. There are seven genes including aoxRSXABCD putatively encoding the transcriptional regulator AoxR of a two-component signal transduction system (68% identity), a periplasmic sensor histidine kinase AoxS (55 % identity), a periplasmic binding protein AoxX (55 % identity), arsenite oxidase AoxAB(74 % and 71% identity, respectively), nitroreductase AoxC (46 % identity) and cytochrome c AoxD (63 % identity) respectively. According to the reverse transcriptase PCR experiments,aoxR and aoxS encoding for a two-component system proteins are co-transcribed and located in opposite to structural genes aoxABCD.aoxX and aoxRS are not in the same operon. Functional analyses through gene knock-out of aoxS, aoxX and aoxD showed that aoxS and aoxX are the essential genes in arsenite oxidation of GW2, and the loss of aoxD did not show significant effects on arsenite oxidation.%通过反向PCR和细菌Fosmid文库筛选,克隆得到1株二三价砷[As(Ⅲ)]氧化细菌Acidovorax sp.GW2的As(Ⅲ)氧化酶Aox基因簇,包括aoxRSXABCD 7个基因,分别预测编码双组分信号传导系统转录调控子AoxR(同源性68%),周质感应组氨酸激酶AoxS(同源性55%),周质结合蛋白AoxX(同源性55%),砷氧化酶AoxAB(同源性分别为74%和71%),硝基还原酶AoxC(同源性46%),细胞色素C AoxD(同源性63%).反转录PCR结果显示,编码双组分系统的aoxRS基因共转录,而与之转录方向相反的结构基因aoxABCD处于同一操纵子中,aoxX基因和aoxRS基因不在同一操纵子中.通过对aoxS、aoxX、aoxD的基因敲除功能研究发现aoxS和aoxX基因为GW2三价砷氧化的必需基因,aoxD的功能丧失减慢了三价砷的氧化速率,但非关键基因.

  8. The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yun-Ling; Li, Li; Wu, Keqiang [Michigan State Univ., East Lansing, MI (United States)] [and others

    1995-07-03

    The biosynthesis of gibberellins (GAs) after GA{sub 12}-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA{sub 53} to GA{sub 44} and GA{sub 19} to GA{sub 20}. The Arabidopsis GA 20-oxidase shares 55% identity and >80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA locus of Arabidopsis. The ga5 semidwarf mutant contains a G {yields} A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA{sub 4} treatment, suggesting end-product repression in the GA biosynthetic pathway. 28 refs., 6 figs.

  9. Evidence for Interplay between Genes and Parenting on Infant Temperament in the First Year of Life: Monoamine Oxidase a Polymorphism Moderates Effects of Maternal Sensitivity on Infant Anger Proneness

    Science.gov (United States)

    Pickles, Andrew; Hill, Jonathan; Breen, Gerome; Quinn, John; Abbott, Kate; Jones, Helen; Sharp, Helen

    2013-01-01

    Background: The low expression polymorphism of the MAOA gene in interaction with adverse environments (G × E) is associated with antisocial behaviour disorders. These have their origins in early life, but it is not known whether MAOA G × E occurs in infants. We therefore examined whether MAOA G × E predicts infant anger proneness, a temperamental…

  10. Polymorphisms in the genes of citohrom oxidase P450 2D6 (CYP2D6, paraoxonase 1 (PON1 and apolipoproteine E (APOE as risk factors for Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Đurić Gordana

    2007-01-01

    Full Text Available Background/Aim. The presence of Parkinson's disease (PD among the members of a family is a clear indication of the significance of genetics in its development. In spite of that, the majority of patients with PD shows a sporadic form of the disease induced as a result of interaction of both environmental and genetic factors. The aim of this study was to examine the effects of polymorphisms in the genes of cytohrome P450 2D6(CYP2D6, paraoxonase 1 (PON 1 and apolipoprotein E (APOE, as risk factors for PD. Methods. We examined 106 patients with PD (65 men and 41 women and 75 ethnically matched control subjects. The mean age at onset of PD in the patients was 46.9±9.4 years (ranging from 30 to 70 years. Genotyping was performed using standard PCR amplification and restriction endonuclease digestion protocols described for known polymorphism in the candidate genes under study. Results. The genotype A/A polymorphisms 2D6* gene of CYP2D6 and genotype M/M polymorphisms L54M gene of PON1 were significantly more frequent in the patients with PD than in the control group. The patients with genotypes A/A and M/M had 3.4 and 3.2 higher risk of PD, respectively than the control group (p = 0.01. The relation between genotypes A/A gene of CYP2D6 and M/M gene of PON1 was modified by the age at onset. The genotypes were associated with early onset of PD (p = 0.001, p = 0.004. The carriers of the A and M alleles in homozygote had 2.4 and 4.2 years respectively earlier onset of PD than carriers of other genotypes with these polymorphisms. The frequency allele ε4 gene of APOE was higher in the PD patients with early onset (20% than in PD with later onset (7.4%, while the genotype ε3/ε3 was associated with PD late onset (p = 0.024. Combined genotype I (carriers of the two risk allels in homozygote and one alleles risk in heterozygote and combined genotype II (carriers of the three alleles risk in homozygote caused early PD. Combined genotype II was detected in 12

  11. Development of a dual-internal-reference technique to improve accuracy when determining bacterial 16S rRNA:16S rRNA gene ratio with application to Escherichia coli liquid and aerosol samples.

    Science.gov (United States)

    Zhen, Huajun; Krumins, Valdis; Fennell, Donna E; Mainelis, Gediminas

    2015-10-01

    Accurate enumeration of rRNA content in microbial cells, e.g. by using the 16S rRNA:16S rRNA gene ratio, is critical to properly understand its relationship to microbial activities. However, few studies have considered possible methodological artifacts that may contribute to the variability of rRNA analysis results. In this study, a technique utilizing genomic DNA and 16S rRNA from an exogenous species (Pseudomonas fluorescens) as dual internal references was developed to improve accuracy when determining the 16S rRNA:16S rRNA gene ratio of a target organism, Escherichia coli. This technique was able to adequately control the variability in sample processing and analysis procedures due to nucleic acid (DNA and RNA) losses, inefficient reverse transcription of RNA, and inefficient PCR amplification. The measured 16S rRNA:16S rRNA gene ratio of E. coli increased by 2-3 fold when E. coli 16S rRNA gene and 16S rRNA quantities were normalized to the sample-specific fractional recoveries of reference (P. fluorescens) 16S rRNA gene and 16S rRNA, respectively. In addition, the intra-sample variation of this ratio, represented by coefficients of variation from replicate samples, decreased significantly after normalization. This technique was applied to investigate the temporal variation of 16S rRNA:16S rRNA gene ratio of E. coli during its non-steady-state growth in a complex liquid medium, and to E. coli aerosols when exposed to particle-free air after their collection on a filter. The 16S rRNA:16S rRNA gene ratio of E. coli increased significantly during its early exponential phase of growth; when E. coli aerosols were exposed to extended filtration stress after sample collection, the ratio also increased. In contrast, no significant temporal trend in E. coli 16S rRNA:16S rRNA gene ratio was observed when the determined ratios were not normalized based on the recoveries of dual references. The developed technique could be widely applied in studies of relationship between

  12. FILOGENIA DE ESPECIES DEL SUBGENERO Parides (LEPIDOPTERA: PAPILIONIDAE BASADA EN SECUENCIAS DEL GEN CITOCROMO OXIDASA I Species phylogeny of the Subgenus Parides (Lepidoptera: Papilionidae Based in Sequences of Citochrome Oxidase I Gene

    Directory of Open Access Journals (Sweden)

    INGRID MARCELA GUTIÉRREZ R

    2012-12-01

    Colombia where the biota of North and South America converge. This work presents a phylogenetic hypothesis of these butterflies and proposes a more robust definition of some taxa. For this, 15 taxa of the subgenus Parides were analyzed as ingroup; species of other two genera of Troidini, closer to Parides , were used as out-group. DNA was extracted using the Pascual et al. (1997 protocol and Quiagen DNAeasy kit. A terminal fragment of Cytochrome Oxidase I gen (476 bp were amplified. We obtained a phylogenetic approximation using maximum parsimony and evaluated the branch support with Jackknife and absolute Bremer support. We also conducted a bayesian analysis. The resulting phylogenetic hypothesis suggested that Parides is a paraphyletic group; the molecular evidence support one species and five subspecies. The analyzed taxa were divided in three principal groups coincident with the Lysander (group 1 and Aeneas (groups 1 and 2 groups proposed by Rothschild and Jordan (1906.

  13. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Science.gov (United States)

    2010-04-01

    ... Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase... ingredient that will react with cytochrome oxidase. When cytochrome oxidase is present, the swab turns a dark...

  14. Bilirubin Oxidase Activity of Bacillus subtilis CotA

    OpenAIRE

    Sakasegawa, S; Ishikawa, H.; Imamura, S.; Sakuraba, H.; Goda, S.; Ohshima, T.

    2006-01-01

    The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.

  15. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis

    NARCIS (Netherlands)

    Kopacz, Malgorzata; Heuts, Dominic P. H. M.; Fraaije, Marco W.

    2014-01-01

    Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single-and double-mixing st

  16. THERMOSTABILITY OF RESPIRATORY TERMINAL OXIDASES IN THE LIPID ENVIRONMENT

    NARCIS (Netherlands)

    Elferink, Marieke G.L.; Bosmal, Tjibbe; Lolkema, Juke S.; Gleiszner, Michael; Driessen, Arnold J.M.; Konings, Wil N.

    1995-01-01

    The effect of the lipid environment on the thermostability of three respiratory terminal oxidases was determined. Cytochrome-e oxidase from beef heart and Bacillus stearothermophilus were used as representative proteins from mesophilic and thermophilic origin, respectively. Quinol oxidase from the a

  17. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis

    NARCIS (Netherlands)

    Kopacz, Malgorzata; Heuts, Dominic P. H. M.; Fraaije, Marco W.

    2014-01-01

    Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single-and double-mixing

  18. Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells

    Science.gov (United States)

    Hakami, Nora Y.; Ranjan, Amaresh K.; Hardikar, Anandwardhan A.; Dusting, Greg J.; Peshavariya, Hitesh M.

    2017-01-01

    Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization.

  19. Occurrence and Biocatalytic Potential of Carbohydrate Oxidases.

    NARCIS (Netherlands)

    Hellemond, van E.W.; Leferink, N.G.H.; Heuts, D.P.H.M.; Fraaije, M.W.; Berkel, van W.J.H.

    2006-01-01

    Carbohydrate oxidases are found in all kingdoms of life but are mostly found in fungi. Their natural role is not always clear. Usage of molecular oxygen as electron acceptor is not a logical choice when the enzyme is part of a catabolic pathway. This chapter provides an overview of the occurrence

  20. A colorimetric assay for cytokinin oxidase.

    Science.gov (United States)

    Libreros-Minotta, C A; Tipton, P A

    1995-11-01

    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  1. Spectrophotometric Assay of Immobilized Glucose Oxidase

    Directory of Open Access Journals (Sweden)

    Nojan Noorbehesht

    2016-06-01

    Full Text Available Enzyme results in change the substrate of product. Each enzyme may act on specific substrates, resulting in product or different products. The enzyme glucose oxidase (GOX is a bio catalyst. It accelerates the process of transforming glucose into hydrogen peroxide (H2O2 . These enzymes are used in the chemical industry, food industry, cosmetics and kits for diagnosis of glucose. There are many researches about immobilizations of Glucose Oxide to increase specifications such as repeated use, recovery, stability, shelf life and other features In this work, glucose oxidase enzyme using covalent bonding is placed on the carrier of carbon nanotubes. In this study, multi-walled carbon nanotubes have been used as adsorbents. Also, carbon nanotubes have been functionalized by sulfuric acid and nitric acid with a high concentration. Glucose oxidase is a biological biocatalyst enzyme. It accelerates changing glucose to H2O2. This enzyme is used in the chemical industry, food industry, cosmetics and glucose diagnostic kits. For example, as a result of ongoing research working focuses on the development of glucose biosensors, GOX in practice as standard enzyme has been revealed for immobilization of oxidative enzyme.GOX correct fixation on the MWNTs carrier is a way to reuse enzyme and miniature of biosensor devices and structures. In this study, a spectrophotometer was used to determine the absorbance of the enzyme glucose oxidase (GOX to review its activities after stabilizing the carbon nanotubes.

  2. Lysyl oxidase mediates hypoxic control of metastasis

    DEFF Research Database (Denmark)

    Erler, Janine Terra; Giaccia, Amato J

    2006-01-01

    Hypoxic cancer cells pose a great challenge to the oncologist because they are especially aggressive, metastatic, and resistant to therapy. Recently, we showed that elevation of the extracellular matrix protein lysyl oxidase (LOX) correlates with metastatic disease and is essential for hypoxia...

  3. 应用双重原位杂交检测胃癌HER2基因扩增%Amplification of HER2 gene in gastric carcinoma detected by dual in-situ hybridization

    Institute of Scientific and Technical Information of China (English)

    冼丽芳; 黄香婷; 文剑明; 叶玉清

    2012-01-01

    Objective To explore the status of HER2 gene amplification and its product HER2 protein expression in gastric carcinoma, so as to aid in patient selection for anti-HER2 targeted chemotherapy.Methods Eighty-five cases of gastric carcinoma biopsy tissues were collected.The status of HER2 gene amplification was detected by dual in situ hybridization (dual-ISH).And HER2 protein was detected by immunohistochemistry.Results HER2 gene amplification was detected in 10/85 ( 11.8% )cases of gastric carcinoma,and no amplification was detected in 75/85 (88.2% ) cases.In the 10 cases with HER2 amplification,HER2 immunoreaction scorings of 3 +,2 + and 0/1 + were present in 7,2 and 1 cases,respectively.In the 75 cases without HER2 amplification,HER2 immunoreaction scorings of 3 +,2 + and 0/1 + were present in 0,18 (24.0% ) and 57 (76.0% ) cases,respectively.Histologically,most gastric carcinoma with amplification of HER2 gene was moderately differentiated tubular adenocarcinoma.Conclusions HER2 gene dual-ISH technique is a reliable and objective method for detecting HER2 gene amplification in gastric carcinoma biopsy. Clinically, only few gastric carcinomas show HER2 gene amplification and are suitable candidates for anti-HER2 targeted chemotherapy.%目的 探讨胃癌HER2基因扩增状态及其基因产物蛋白表达,以协助临床筛选抗HER2靶向治疗患者.方法 收集胃癌活检标本共85例,进行HER2基因扩增和蛋白表达检测.HER2基因扩增状态采用双重原位杂交检测(HER2 dual-ISH detection),而HER2基因蛋白采用免疫组织化学[全程在全自动染色机(Ventana)]进行检测.结果 85例胃癌病例中,有HER2基因扩增10例,扩增率为11.8%,无扩增75例,占88.2%.在有HER2基因扩增的10例病例中,7例HER2蛋白免疫组织化学为3+,2例为2+,1例为-/1+;75例无基因扩增的病例,HER2蛋白免疫组织化学3+为0例、2+为18例(24.0%),-/1+为57例(76.0%).HER2基因扩增与HER2

  4. [Molecular phylogenetic study of several eelpout fishes (Perciformes, Zoarcoidei) from far eastern seas on the basis of the nucleotide sequence of the mitochondrial cytochrome oxidase 1 gene (Co-1)].

    Science.gov (United States)

    Turanov, S V; kartavtseva, Iu F; Zemnukhov, V V

    2012-02-01

    A total of 95 nucleotide sequences of a Co-1 gene fragment of approximately 650 bp were analyzed for fishes of the orders Perciformes and Scorpaeniformes (outgroup). Gene trees based on four algorithms (BA, NJ, MP, and ML) were similar in topology of solved branches. An emphasis was placed on the species and generic levels, but a significant phylogenetic signal was obtained for higher taxonomic ranks as well. For instance, a monophyletic origin was confirmed for the family Zoarcidae and the subfamily Opisthocentrinae (Stichaeidae). The proportion of different nucleotides in the sequences compared (p-distances) significantly increased with increasing taxonomic rank. The p-distances were estimated for four hierarchic levels and were (1) 0.15 0.06% for the within-species hierarchic level, (2) 6.33 0.37% for the within-genus level, (3) 11.83 0.06% for the within-family level, and (4) 15.22 0.05% for the within-order level. The difference in the Co-1 gene fragments between levels (1) and (2) allows almost errorless species identification on the basis of this kind of a molecular bar code.

  5. Spatiotemporal localization of D-amino acid oxidase and D-aspartate oxidases during development in Caenorhabditis elegans.

    Science.gov (United States)

    Saitoh, Yasuaki; Katane, Masumi; Kawata, Tomonori; Maeda, Kazuhiro; Sekine, Masae; Furuchi, Takemitsu; Kobuna, Hiroyuki; Sakamoto, Taro; Inoue, Takao; Arai, Hiroyuki; Nakagawa, Yasuhito; Homma, Hiroshi

    2012-05-01

    Recent investigations have shown that a variety of D-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. D-Amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are degradative enzymes stereospecific for D-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of D-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several D- and L-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and D-amino acids in multicellular organisms.

  6. EFFECTS OF Sulfite oxidase GENE OVEREXPRESSION ON IMPROVEMENT OF OXIDATION OF SULFITE IN TRANSGENIC TOMATO%超表达亚硫酸氧化酶基因对促进番茄果实亚硫酸盐氧化的研究

    Institute of Scientific and Technical Information of China (English)

    吴自明; 韩瑞才; 陈慧珍; 谭雪明; 石庆华; 潘晓华

    2011-01-01

    本研究以亚硫酸氧化酶基因(SO)超表达系(OE)、干扰系(Ri)及野生型(WT)的绿熟番茄果实为材料,经SO2熏蒸后,12℃低温贮藏,研究亚硫酸氧化酶基因的超表达,对番茄贮藏期间相对电导率、亚硫酸盐含量、番茄红素含量及成熟度的变化。结果表明,经SO2熏蒸后,Ri果实较WT果实表现为相对电导率增加,亚硫酸盐残留较多,番茄红素含量增加较慢,红熟加快,接种病菌后的第4天果实组织溃烂,菌丝生长明显,组织病斑较大;相反,OE果实较WT果实表现为相对电导率下降,亚硫酸盐残留较少,番茄红素明显增加,红熟延迟,接种病菌后第4天的果实无明显病斑,软化腐烂较小。亚硫酸氧化酶基因超表达,能够有效减少SO2熏蒸后贮藏番茄中亚硫酸盐的残留。%The sulfite oxidase gene(SO) overexpressed(OE),RNA interferred(Ri) and wild type tomato were used as materials.The mature green unripe tomato fruits were fumigated with SO2,then stored at 12℃.The effects of sulfite oxidase overexpression on the relative electrical conductivity,sulfite content,lyconpene and maturity diffrence in transgenic tomato were studied.The results showed that electrical conductivity and sulfite residual of Ri fruits increased,lycopene content decreased,fruit color change accelerated.Tissue rotting and fungal growth of Ri fruit appeared 4d after B.cinerea inoculation,and surrounding pericarp tissue developed.In contrast,the relative electrical conductivity and sulfite residual of OE fruits decreased,lycopene level remained normal,fruit color change delayed.The growth of fungus in OE fruits was limited to the puncture sites and soft rot symptoms did not develop after B.cinerea inoculation for 4d.The study showed that sulfite oxidase gene overexpression in fruit could decrease sulfite residual,and improve the quality and storage properties of the tomato with SO2 fumigation.

  7. Structure-function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family.

    Science.gov (United States)

    Yin, DeLu Tyler; Urresti, Saioa; Lafond, Mickael; Johnston, Esther M; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H; Davies, Gideon J; Brumer, Harry

    2015-12-18

    Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure-function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications.

  8. Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1.

    Science.gov (United States)

    Bareth, Bettina; Nikolov, Miroslav; Lorenzi, Isotta; Hildenbeutel, Markus; Mick, David U; Helbig, Christin; Urlaub, Henning; Ott, Martin; Rehling, Peter; Dennerlein, Sven

    2016-05-15

    The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.

  9. Virtual and in vitro bioassay screening of phytochemical inhibitors from flavonoids and isoflavones against xanthine oxidase and cyclooxygenase-2 for gout treatment.

    Science.gov (United States)

    Li, Yadi; Frenz, Christopher M; Li, Zhiwen; Chen, Mianhua; Wang, Yurong; Li, Fengjuan; Luo, Cheng; Sun, Jian; bohlin, Lars; Li, Zhenjing; Yang, Hua; Wang, Changlu

    2013-04-01

    Synthetic drugs such as allopurinol and benzbromarone are commonly used to treat the complex pathogenesis of gout, a metabolic disease that results from an inflammation of the joints caused by precipitation of uric acid. We seek to discover novel phytochemicals that could treat gout, by targeting the xanthine oxidase and cyclooxygenase-2 enzymes. In this study, we report the screening of nine compounds of flavonoids from the ZINC and PubChem databases (containing 2092 flavonoids) using the IGEMDOCK software tool against the xanthine oxidase and cyclooxygenase-2 3D protein structures. Each compound was also evaluated by an in vitro bioassay testing the inhibition of xanthine oxidase and cyclooxygenase-2. Myricetin and luteolin were found to be the potential dual inhibitors of xanthine oxidase and cyclooxygenase-2 as demonstrated by IC(50): 62.7 and 3.29 μg/mL (xanthine oxidase)/70.8 and 16.38 μg/mL (cyclooxygenase-2), respectively. In addition, structure-activity relationships and other important factors of the flavonoids binding to the active site of xanthine oxidase and cyclooxygenase-2 were discussed, which is expected for further rational drug design.

  10. A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit.

    Science.gov (United States)

    Garchery, Cécile; Gest, Noé; Do, Phuc T; Alhagdow, Moftah; Baldet, Pierre; Menard, Guillaume; Rothan, Christophe; Massot, Capucine; Gautier, Hélène; Aarrouf, Jawad; Fernie, Alisdair R; Stevens, Rebecca

    2013-01-01

    The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild-type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose:sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species.

  11. Thermostable Xanthine Oxidase Activity from Bacillus pumilus RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization.

    Science.gov (United States)

    Sharma, Nirmal Kant; Thakur, Shikha; Thakur, Neerja; Savitri; Bhalla, Tek Chand

    2016-03-01

    Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg(2+), Ag(+) and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.

  12. The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus.

    Directory of Open Access Journals (Sweden)

    Laurence Prunetti

    Full Text Available The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3-type two-subunit (subunits I and II enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix "subunit IIa", which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA(2 of 59000 Da, subunit II (encoded by coxB(2 of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba(3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.

  13. EFFECT OF CROSSLINKING ON MITOCHONDRIAL CYTOCHROME c OXIDASE

    Energy Technology Data Exchange (ETDEWEB)

    Swanson, Maurice; Packer, Lester

    1979-12-01

    Purified and reconstituted cytochrome {und c} oxidase and mitochondria were crosslinked with biimidates in the presence and absence of cytochrome {und c}. These experiments indicate that oxidase subunit interactions are required for activity and that cytochrome {und c} mobility may be required for electron transport activity. Biimidate treatment of purified and reconstituted oxidase crosslinks all of the oxidase protomers except subunit I when {ge} 20% of the free amines are modified and inhibits steady state oxidase activity. Transient kinetics of ferrocytochrome {und c} oxidation and ferricytochrome {und a} reduction indicates inhibition of electron transfer from heme {und a} to heme {und a}{sub 3}. Crosslinking oxidase molecules to form large aggregates displaying rotational correlation times {ge} 1 ms does not affect oxidase activity. Crosslinking of mitochondria covalently binds the bc{sub 1} and {und aa}{sub 3} complexes to cytochrome {und c}, and inhibits steady-state oxidase activity considerably more than in the case of the purified oxidase. Addition of cytochrome {und c} to the purified oxidase or to {und c}-depleted mitoplasts increases inhibition slightly. Cytochrome {und c} oligomers act as competitive inhibitors of native {und c}, however, crosslinking of cytochrome {und c} to {und c}-depleted mitoplasts or purified oxidase (with dimethyl suberimidate or hetrobifunctional crosslinking reagents) results in a catalytically inactive complex.

  14. Study on the Correlation between Single Nucleotide Polymorphism of Monoamine Oxidase A Gene and Anger Regulation%单胺氧化酶A基因单核苷酸多态性与愤怒调节的关系研究

    Institute of Scientific and Technical Information of China (English)

    闫秀娟; 詹向红; 侯俊林; 刘永; 王淑玲; 李伟; 闫国立; 王友杰; 杨丽萍

    2012-01-01

    目的 研究单胺氧化酶A( monoamine oxidase A,MAOA)基因单核苷酸多态性(single nucleotide polymorphism,SNP)与怒的调节关系.方法 以某学院健康大学生为研究对象,根据状态-特质愤怒表达量表2(State-Trait Anger Expression Inventory-Ⅱ,STAXI-2)特质怒得分筛选受试者,其中高特质怒者225名,低特质怒者221名,经采血后,采用酚-氯仿法提取DNA,聚合酶链反应-连接酶检测反应(PCR-LDR)法对MAOA基因上的4个tag SNP位点(rs5906957、rs2235186、rs1181275和rs5905613)进行基因分型,对高、低特质怒不同性别组MAOA基因4个tag SNP位点不同基因型受试者怒的表达量表得分和怒的控制量表得分进行统计分析.结果 低特质怒女性MAOA基因rs2235186位点不同基因型组控制发怒得分比较,差异有统计学意义(P =0.037),其余3个tag SNP位点的不同基因型受试者在怒的表达及控制得分上比较,差异均无统计学意义(P>0.05).结论 MAOA基因tag SNP rs2235186多态性与我国健康大学生低特质怒女性的控制发怒特质有关.%Objective To study the correlation between single nucleotide polymorphism (SNP) of monoamine oxidase A gene (MAOA) and anger regulation. Methods Enrolled were healthy students from some college , including 225 of the high trait anger and 221 of the low trait anger. Subjects were recruited referring to the State-Trait Anger Expression Inventory 2 (STAXI-2) and their blood sampled. The DNA was extracted using phenol-chloroform method, 4 tag SNPs of MAOA (rs5906957, rs2235186, rs1181275, and rs5905613) were genotyped by PCR-based ligase detection reaction (PCR-LDR). The scores for trait anger expression inventory and the scores for trait anger expression control at the 4 tag SNPs of MAOA in the different sexes groups of the high and the low trait anger were statistical analyzed. Results There was statistical difference in anger control score of locus rs2235186 of MAOA gene group (P=0.037). There was no

  15. Biofabrication Using Pyrrole Electropolymerization for the Immobilization of Glucose Oxidase and Lactate Oxidase on Implanted Microfabricated Biotransducers

    Directory of Open Access Journals (Sweden)

    Christian N. Kotanen

    2014-03-01

    Full Text Available The dual responsive Electrochemical Cell-on-a-Chip Microdisc Electrode Array (ECC MDEA 5037 is a recently developed electrochemical transducer for use in a wireless, implantable biosensor system for the continuous measurement of interstitial glucose and lactate. Fabrication of the biorecognition membrane via pyrrole electropolymerization and both in vitro and in vivo characterization of the resulting biotransducer is described. The influence of EDC-NHS covalent conjugation of glucose oxidase with 4-(3-pyrrolyl butyric acid (monomerization and with 4-sulfobenzoic acid (sulfonization on biosensor performance was examined. As the extent of enzyme conjugation was increased sensitivity decreased for monomerized enzymes but increased for sulfonized enzymes. Implanted biotransducers were examined in a Sprague-Dawley rat hemorrhage model. Resection after 4 h and subsequent in vitro re-characterization showed a decreased sensitivity from 0.68 (±0.40 to 0.22 (±0.17 µA·cm−2·mM−1, an increase in the limit of detection from 0.05 (±0.03 to 0.27 (±0.27 mM and a six-fold increase in the response time from 41 (±18 to 244 (±193 s. This evidence reconfirms the importance of biofouling at the bio-abio interface and the need for mitigation strategies to address the foreign body response.

  16. Expression patterns of Eph genes in the "dual visual development" of the lamprey and their significance in the evolution of vision in vertebrates.

    Science.gov (United States)

    Suzuki, Daichi G; Murakami, Yasunori; Yamazaki, Yuji; Wada, Hiroshi

    2015-01-01

    Image-forming vision is crucial to animals for recognizing objects in their environment. In vertebrates, this type of vision is achieved with paired camera eyes and topographic projection of the optic nerve. Topographic projection is established by an orthogonal gradient of axon guidance molecules, such as Ephs. To explore the evolution of image-forming vision in vertebrates, lampreys, which belong to the basal lineage of vertebrates, are key animals because they show unique "dual visual development." In the embryonic and pre-ammocoete larval stage (the "primary" phase), photoreceptive "ocellus-like" eyes develop, but there is no retinotectal optic nerve projection. In the late ammocoete larval stage (the "secondary" phase), the eyes grow and form into camera eyes, and retinotectal projection is newly formed. After metamorphosis, this retinotectal projection in adult lampreys is topographic, similar to that of gnathostomes. In this study, we explored the involvement of Ephs in lamprey "dual visual development" and establishment of the image-form vision. We found that gnathostome-like orthogonal gradient expression was present in the retina during the "secondary" phase; i.e., EphB showed a gradient of expression along the dorsoventral axis, while EphC was expressed along the anteroposterior axis. However, no orthogonal gradient expression was observed during the "primary" phase. These observations suggest that Ephs are likely recruited de novo for the guidance of topographical "second" optic nerve projection. Transformations during lamprey "dual visual development" may represent "recapitulation" from a protochordate-like ancestor to a gnathostome-like vertebrate ancestor.

  17. Cloning and Expression Analysis of GA20-Oxidase Gene from Sugar Apple (Annona squamosa)%番荔枝GA20氧化酶基因的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    刘锴栋; 袁长春; 黎海利; 刘金祥; 陈燕; 莫亿伟; 李华富

    2015-01-01

    A full-length cDNA sequence of homologousGA20ox gene was cloned by employing homology gene cloning and RACE-PCR from sugar apple (Annona squamosa L.), which was named asAsGA20ox(Gen-Bank accession KR676623). Sequence analysis showed that theAsGA20oxgene has a 1 257 bp open reading frame (ORF) encoding 418 amino acids. Sequence alignment displayed that it was a similarity of 71% and 70% with GA20ox ofElaeis guineensis andPhoenix dactylifera, respectively. The phylogenetic tree showed that sugar appleAsGA20ox andDasypyrum villosum GA20ox are closest in molecular evolution distance, followed byMusa acuminatasubsp. Malaccensis GA20ox. It was speculated that AsGA20ox protein is located in the extracellular matrix and peroxisomal. It do not have a signal peptide. The putative protein molecular weight was 47.2 kDa and its theoretical isoelectric point was 6.21. The real-time PCR results suggested thatAsGA20ox gene showed a high transcription level in lfower buds I, immature seeds, young stems of fruiting branches and pistils. The results laid a solid foundation for further research on AsGA20ox protein structure and the relation-ship between the gene and plant lfower development.%本研究利用同源克隆和RACE-PCR的方法获得番荔枝GA20ox基因的全长cDNA序列,命名为AsGA20ox, GenBank登录号为KR676623。序列分析表明,克隆获得的番荔枝AsGA20ox基因编码区长为1257 bp,编码418个氨基酸。序列比对显示与油棕和海枣等GA20ox的相似度分别为71%和70%。构建类似蛋白系统进化树显示,番荔枝AsGA20ox与簇毛麦、香蕉等分子进化距离较近。预测AsGA20ox蛋白定位在细胞外基质和过氧化物酶体中,不具备信号肽。蛋白质分子量为47.2 kDa,等电点为6.21。实时定量RT-PCR结果表明,在不同的组织、不同的器官中,AsGA20ox的表达量存在差异,其中在花蕾期I、未成熟的种子、结果枝的幼嫩茎段和雌蕊中的表达量相对较高。

  18. NADPH oxidases in lung biology and pathology: host defense enzymes, and more.

    Science.gov (United States)

    van der Vliet, Albert

    2008-03-15

    The deliberate production of reactive oxygen species (ROS) by phagocyte NADPH oxidase is widely appreciated as a critical component of antimicrobial host defense. Recently, additional homologs of NADPH oxidase (NOX) have been discovered throughout the animal and plant kingdoms, which appear to possess diverse functions in addition to host defense, in cell proliferation, differentiation, and in regulation of gene expression. Several of these NOX homologs are also expressed within the respiratory tract, where they participate in innate host defense as well as in epithelial and inflammatory cell signaling and gene expression, and fibroblast and smooth muscle cell proliferation, in response to bacterial or viral infection and environmental stress. Inappropriate expression or activation of NOX/DUOX during various lung pathologies suggests their specific involvement in respiratory disease. This review summarizes the current state of knowledge regarding the general functional properties of mammalian NOX enzymes, and their specific importance in respiratory tract physiology and pathology.

  19. Dynamic 1-Aminocyclopropane-1-Carboxylate-Synthase and -Oxidase Transcript Accumulation Patterns during Pollen Tube Growth in Tobacco Styles1

    Science.gov (United States)

    Weterings, Koen; Pezzotti, Mario; Cornelissen, Marc; Mariani, Celestina

    2002-01-01

    In flowering plants, pollination of the stigma sets off a cascade of responses in the distal flower organs. Ethylene and its biosynthetic precursor 1-aminocyclopropane-1-carboxylate (ACC) play an important role in regulating these responses. Because exogenous application of ethylene or ACC does not invoke the full postpollination syndrome, the pollination signal probably consists of a more complex set of stimuli. We set out to study how and when the pollination signal moves through the style of tobacco (Nicotiana tabacum) by analyzing the expression patterns of pistil-expressed ACC-synthase and -oxidase genes. Results from this analysis showed that pollination induces high ACC-oxidase transcript levels in all cells of the transmitting tissue. ACC-synthase mRNA accumulated only in a subset of transmitting tract cells and to lower levels as compared with ACC-oxidase. More significantly, we found that although ACC-oxidase transcripts accumulate to uniform high levels, the ACC-synthase transcripts accumulate in a wave-like pattern in which the peak coincides with the front of the ingrowing pollen tube tips. This wave of ACC-synthase expression can also be induced by incongruous pollination and (partially) by wounding. This indicates that wounding-like features of pollen tube invasion might be part of the stimuli evoking the postpollination response and that these stimuli are interpreted differently by the regulatory mechanisms of the ACC-synthase and -oxidase genes. PMID:12427986

  20. Xanthine oxidase biosensor for monitoring meat spoilage

    Science.gov (United States)

    Vanegas, D. C.; Gomes, C.; McLamore, E. S.

    2014-05-01

    In this study, we have designed an electrochemical biosensor for real-time detection of specific biomarkers of bacterial metabolism related to meat spoilage (hypoxanthine and xanthine). The selective biosensor was developed by assembling a `sandwich' of nanomaterials and enzymes on a platinum-iridium electrode (1.6 mm tip diameter). The materials deposited on the sensor tip include amorphous platinum nanoclusters (i.e. Pt black), reduced graphene oxide, nanoceria, and xanthine oxidase. Xanthine oxidase was encapsulated in laponite hydrogel and used for the biorecognition of hypoxanthine and xanthine (two molecules involved in the rotting of meat by spoilage microorganisms). The developed biosensor demonstrated good electrochemical performance toward xanthine with sensitivity of 2.14 +/- 1.48 μA/mM, response time of 5.2 +/- 1.5 sec, lower detection limit of 150 +/- 39 nM, and retained at least 88% of its activity after 7 days of continuous use.

  1. Plasma diamine oxidase activity in asthmatic children

    Directory of Open Access Journals (Sweden)

    Kyoichiro Toyoshima

    1996-01-01

    Full Text Available Histamine plays an important role in the development of asthmatic symptoms. Diamine oxidase (DAO histaminase, which inactivates histamine, is located in the intestine and kidney and is released into plasma. Plasma DAO activity in asthmatic children was measured by a recently developed high performance liquid chromatographic method using histamine as the DAO substrate. Diamine oxidase activity was higher in severely asthmatic children than in those with mild asthma. A time course study during the acute exacerbation phase revealed that DAO activity rose during acute asthmatic attacks and then decreased gradually over several days. Although the mechanisms of plasma DAO activity increase during acute asthmatic attacks could not be explained, data showed that plasma DAO activity is an important index of histamine metabolism in asthmatics and may relate to some mechanisms of acute exacerbation of airway inflammation. Consequently, fluctuations in plasma DAO can be used as one of various indices of instability in management of asthma.

  2. Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death.

    Science.gov (United States)

    Chartier, François J-M; Hardy, Émilie J-L; Laprise, Patrick

    2012-09-17

    Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1-3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1-NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1-NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.

  3. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

    Science.gov (United States)

    Folmer, O; Black, M; Hoeh, W; Lutz, R; Vrijenhoek, R

    1994-10-01

    We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

  4. Process characterization of a monoamine oxidase

    DEFF Research Database (Denmark)

    Ramesh, Hemalata; Woodley, John

    2014-01-01

    .e, on biocatalyst development (e.g. improvement of expression levels), process development (e.g. improved oxygen supply, product removal strategies) or biocatalyst stabilization (e.g. through immobilization or directed evolution). This paper presents a systematic method to identify the bottleneck of a potential...... biocatalytic process using a monoamine oxidase to synthesise an intermediate in the manufacture of a drug for treating Hepatitis C (Telaprevir)....

  5. Imaging Monoamine Oxidase in the Human Brain

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-11-10

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets.

  6. The inhibition of monoamine oxidase by esomeprazole

    OpenAIRE

    2013-01-01

    Virtual screening of a library of drugs has suggested that esomeprazole, the S-enantiomer of omeprazole, may possess binding affinities for the active sites of the monoamine oxidase (MAO) A and B enzymes. Based on this finding, the current study examines the MAO inhibitory properties of esomeprazole. Using recombinant human MAO-A and MAO-B, IC50 values for the inhibition of these enzymes by esomeprazole were experimentally determined. To examine the reversibility of MAO inhibition by esomepra...

  7. 利用两种不同引物扩增金龟子COI序列片段的研究与对比%Research and comparison of the scarab mitochondrial cytochrome C oxidase Ⅰ (COI) gene fragment sequence cloned by two different primers

    Institute of Scientific and Technical Information of China (English)

    田雷雷; 席景会; 李克斌; 孙昊雨; 尹姣; 曹雅忠

    2013-01-01

    Universal primer (L1490, H2198) and specific primer (Pat, Jerry) were used for cloning the mitochondrial cytochrome C oxidase I (COI ) gene fragments of four common scarabs. The homologous sequences were compared and partial nucleotide sequences of COI (689 bp, 775 bp) obtained. The genetic distances of these sequences were analysed and the sequences used to produce three kinds of phylogenetic trees. The results show that the stability and differentials of interspecific distance of the sequences amplified with specific primers are clearly higher than those of sequences amplified with a universal primer. The phylogenetic trees constructed from sequences amplified with the specific primer are more consistent with the current morphological taxonomy of these scarab species. We conclude that sequences amplified with specific primer are more accurate for scarab classification.%利用两类不同的引物,即通用引物(L1490,H219S)与特异引物(Pat,Jerry)分别对4种常见金龟子线粒体细胞色素C氧化酶I(COI)基因片段序列进行扩增和测序,获得长度为689 bp与775 bp的序列.对测序结果进行遗传距离分析,并构建了4种金龟子系统进化树.结果表明,特异引物扩增序列的遗传距离在种内稳定性与种间的差异性都明显优于通用引物扩增序列,利用特异引物扩增序列所构建的系统进化树最符合实际情况,因此利用特异引物扩增序列更能够准确的对金龟子进行分类.

  8. Brain Monoamine Oxidase-A Activity Predicts Trait Aggression

    Science.gov (United States)

    Alia-Klein, Nelly; Goldstein, Rita Z.; Kriplani, Aarti; Logan, Jean; Tomasi, Dardo; Williams, Benjamin; Telang, Frank; Shumay, Elena; Biegon, Anat; Craig, Ian W.; Henn, Fritz; Wang, Gene-Jack; Volkow, Nora D.; Fowler, Joanna S.

    2008-01-01

    The genetic deletion of monoamine oxidase A (MAO A, an enzyme which breaks down the monoamine neurotransmitters norepinephrine, serotonin and dopamine) produces aggressive phenotypes across species. Therefore, a common polymorphism in the MAO A gene (MAOA, MIM 309850, referred to as high or low based on transcription in non-neuronal cells) has been investigated in a number of externalizing behavioral and clinical phenotypes. These studies provide evidence linking the low MAOA genotype and violent behavior but only through interaction with severe environmental stressors during childhood. Here, we hypothesized that in healthy adult males the gene product of MAO A in the brain, rather than the gene per se, would be associated with regulating the concentration of brain amines involved in trait aggression. Brain MAO A activity was measured in-vivo in healthy non-smoking men with positron emission tomography using a radioligand specific for MAO A (clorgyline labeled with carbon 11). Trait aggression was measured with the Multidimensional Personality Questionnaire (MPQ). Here we report for the first time that brain MAO A correlates inversely with the MPQ trait measure of aggression (but not with other personality traits) such that the lower the MAO A activity in cortical and subcortical brain regions the higher the self-reported aggression (in both MAOA genotype groups) contributing to more than a third of the variability. Since trait aggression is a measure used to predict antisocial behavior, these results underscore the relevance of MAO A as a neurochemical substrate of aberrant aggression. PMID:18463263

  9. Dual targeting of peroxisomal proteins

    Directory of Open Access Journals (Sweden)

    Julia eAst

    2013-10-01

    Full Text Available Cellular compartmentalization into organelles serves to separate biological processes within the environment of a single cell. While some metabolic reactions are specific to a single organelle, others occur in more than one cellular compartment. Specific targeting of proteins to compartments inside of eukaryotic cells is mediated by defined sequence motifs. To achieve multiple targeting to different compartments cells use a variety of strategies. Here, we focus on mechanisms leading to dual targeting of peroxisomal proteins. In many instances, isoforms of peroxisomal proteins with distinct intracellular localization are encoded by separate genes. But also single genes can give rise to differentially localized proteins. Different isoforms can be generated by use of alternative transcriptional start sites, by differential splicing or ribosomal read-through of stop codons. In all these cases different peptide variants are produced, of which only one carries a peroxisomal targeting signal. Alternatively, peroxisomal proteins contain additional signals that compete for intracellular targeting. Dual localization of proteins residing in both the cytoplasm and in peroxisomes may also result from use of inefficient targeting signals. The recent observation that some bona fide cytoplasmic enzymes were also found in peroxisomes indicates that dual targeting of proteins to both the cytoplasm and the peroxisome might be more widespread. Although current knowledge of proteins exhibiting only partial peroxisomal targeting is far from being complete, we speculate that the metabolic capacity of peroxisomes might be larger than previously assumed.

  10. Purine-induced expression of urate oxidase and enzyme activity in Atlantic salmon (Salmo salar). Cloning of urate oxidase liver cDNA from three teleost species and the African lungfish Protopterus annectens.

    Science.gov (United States)

    Andersen, Øivind; Aas, Turid S; Skugor, Stanko; Takle, Harald; van Nes, Solveig; Grisdale-Helland, Barbara; Helland, Ståle J; Terjesen, Bendik F

    2006-07-01

    The peroxisomal enzyme urate oxidase plays a pivotal role in the degradation of purines in both prokaryotes and eukaryotes. However, knowledge about the purine-induced expression of the encoding gene is lacking in vertebrates. These are the first published sequences of fish urate oxidase, which were predicted from PCR amplified liver cDNAs of Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua), Atlantic halibut (Hippoglossus hippoglossus) and African lungfish (Protopterus annectens). Sequence alignment of different vertebrate urate oxidases revealed amino acid substitutions of putative functional importance in the enzyme of chicken and lungfish. In the adult salmon, expression of urate oxidase mRNA predominated in liver, but was also identified in several nonhepatic organs including brain, but not in skeletal muscle and kidney. Juvenile salmon fed diets containing bacterial protein meal (BPM) rich in nucleic acids showed a significant increase in liver urate oxidase enzyme activity, and urea concentrations in plasma, muscle and liver were elevated. Whereas salmon fed the 18% BPM diet showed a nonsignificant increase in liver mRNA levels of urate oxidase compared with the 0% BPM-fed fish, no further increase in mRNA levels was found in fish receiving 36% BPM. The discrepancy between urate oxidase mRNA and enzyme activity was explained by rapid mRNA degradation or alternatively, post-translational control of the activity. Although variable plasma and liver levels of urate were detected, the substrate increased only slightly in 36% BPM-fed fish, indicating that the uricolytic pathway of Atlantic salmon is intimately regulated to handle high dietary purine levels.

  11. Pathological changes in platelet histamine oxidases in atopic eczema

    Directory of Open Access Journals (Sweden)

    Reinhold Kiehl

    1993-01-01

    Full Text Available Increased plasma histamine levels were associated with significantly lowered diamine and type B monoamine oxidase activities in platelet-rich plasma of atopic eczema (AE patients. The diamine oxidase has almost normal cofactor levels (pyridoxal phosphate and Cu2+ but the cofactor levels for type B monoamine oxidase (flavin adenine dinucleotide and Fe2+ are lowered. The biogenic amines putrescine, cadaverine, spermidine, spermine, tyramine and serotonin in the sera, as well as dopamine and epinephrine in EDTA-plasma were found to be normal. It is unlikely, therefore, that these amines are responsible for the decreased activities of monoamine and diamine oxidase in these patients. The most likely causative factors for the inhibition of the diamine oxidase are nicotine, alcohol, food additives and other environmental chemicals, or perhaps a genetic defect of the diamine oxidase.

  12. Data on cytochrome c oxidase assembly in mice and human fibroblasts or tissues induced by SURF1 defect.

    Science.gov (United States)

    Kovářová, Nikola; Pecina, Petr; Nůsková, Hana; Vrbacký, Marek; Zeviani, Massimo; Mráček, Tomáš; Viscomi, Carlo; Houštěk, Josef

    2016-06-01

    This paper describes data related to a research article entitled "Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects" [1]. This paper includes data of the quantitative analysis of individual forms of respiratory chain complexes I, III and IV present in SURF1 knockout (SURF1 (-/-) ) and control (SURF1 (+/+) ) mouse fibroblasts and tissues and in fibroblasts of human control and patients with SURF1 gene mutation. Also it includes data demonstrating response of complex IV, cytochrome c oxidase (COX), to reversible inhibition of mitochondrial translation in SURF1 (-/-) mouse and SURF1 patient fibroblast cell lines.

  13. Experimental Evidence for a Revision in the Annotation of Putative Pyridoxamine 5'-Phosphate Oxidases P(N/MP from Fungi.

    Directory of Open Access Journals (Sweden)

    Tatiana Domitrovic

    Full Text Available Pyridoxinamine 5'-phosphate oxidases (P(N/MP oxidases that bind flavin mononucleotide (FMN and oxidize pyridoxine 5'-phosphate or pyridoxamine 5'-phosphate to form pyridoxal 5'-phosphate (PLP are an important class of enzymes that play a central role in cell metabolism. Failure to generate an adequate supply of PLP is very detrimental to most organisms and is often clinically manifested as a neurological disorder in mammals. In this study, we analyzed the function of YLR456W and YPR172W, two homologous genes of unknown function from S. cerevisiae that have been annotated as putative P(N/MP oxidases based on sequence homology. Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN. On the other hand, our analysis confirmed the enzymatic activity of Pdx3, the S. cerevisiae protein previously implicated in PLP biosynthesis by genetic and structural characterization. After a careful sequence analysis comparing the putative and confirmed P(N/MP oxidases, we found that the protein domain (PF01243 that led to the YLR456W and YPR172W annotation is a poor indicator of P(N/MP oxidase activity. We suggest that a combination of two Pfam domains (PF01243 and PF10590 present in Pdx3 and other confirmed P(N/MP oxidases would be a stronger predictor of this molecular function. This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/MP oxidases.

  14. The nature of CuA in cytochrome c oxidase

    OpenAIRE

    Stevens, Tom H.; Martin, Craig T.; Wang, Hsin; Brudvig, Gary W.; Scholes, Charles P.; Chan, Sunney I.

    1982-01-01

    The isolation and purification of yeast cytochrome c oxidase is described. Characterization of the purified protein indicates that it is spectroscopically identical with cytochrome c oxidase isolated from beef heart. Preparations of isotopically substituted yeast cytochrome c oxidase are obtained incorporating [1,3-15N2]histidine or [beta,beta- 2H2]cysteine. Electron paramagnetic resonance and electron nuclear double resonance spectra of the isotopically substituted proteins identify unambigu...

  15. SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes.

    Science.gov (United States)

    Yang, Yin; Wang, Zongdan; Sun, Luan; Shao, Lipei; Yang, Nan; Yu, Dawei; Zhang, Xin; Han, Xiao; Sun, Yujie

    2015-01-01

    Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3'-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.

  16. Identification and characterization of a novel Colletotrichum graminicola raffinose oxidase in the AA5 family.

    Science.gov (United States)

    Andberg, Martina; Mollerup, Filip; Parikka, Kirsti; Koutaniemi, Sanna; Boer, Harry; Juvonen, Minna; Master, Emma; Tenkanen, Maija; Kruus, Kristiina

    2017-08-04

    We describe here the identification and characterization of a copper radical oxidase from Auxiliary Activities Family 5 (AA5_2) that was distinguished by showing preferential activity towards raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase activity from Colletotrichum graminicola, (Cg RaOx; EC 1.1.3.-) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. Cg RaOx was expressed in Pichia pastoris and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on D-galactose at 300 mM substrate concentration. Thus, the substrate preference of Cg RaOx distinguished clearly from the known galactose oxidases. The site of oxidation for raffinose was studied by (1)H NMR and mass spectrometry, and confirmed that the hydroxyl group at the C6 position was oxidized to an aldehyde, and that in addition uronic acid was produced as a side product. A new ESI-MS method for the identification of C-6 oxidized products was developed and the formation mechanism of the uronic acid studied. Cg RaOx presented a novel activity pattern in the AA5 family.Importance Currently there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement ofcomplex cofactors such as FAD or NAD(P). Here we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola As discussed in the paper, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C6-OH carbohydrate oxidase having substrate preference for the

  17. DNA cloning of human liver monoamine oxidase A and B: Molecular basis of differences in enzymatic properties

    Energy Technology Data Exchange (ETDEWEB)

    Back, A.W.J.; Lan, N.C.; Johnson, D.L.; Abell, C.W.; Bembenek, M.E.; Kwan, S.W.; Seeburg, P.H.; Shih, J.C. (Univ. of Heidelberg (West Germany))

    1988-07-01

    The monoamine oxidases play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. Using oligonucleotide probes derived from three sequenced peptide fragments, the authors have isolated cDNA clones that encode the A and B forms of monoamine oxidase and have determined the nucleotide sequences of these cDNAs. Comparison of the deduced amino acid sequences shows that the A and B forms have subunit molecular weights of 59,700 and 58,800, respectively, and have 70% sequence identity. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr, in which the obligatory cofactor FAD is covalently bound to cysteine. Based on differences in primary amino acid sequences and RNA gel blot analysis of mRNAs, the A and B forms of monoamine oxidase appear to be derived from separate genes.

  18. Kinetic Results for Mutations of Conserved Residues H304 and R309 of Human Sulfite Oxidase Point to Mechanistic Complexities

    Science.gov (United States)

    Davis, Amanda C.; Johnson-Winters, Kayunta; Arnold, Anna R.; Tollin, Gordon; Enemark, John H.

    2014-01-01

    Several point mutations in the gene of human sulfite oxidase (hSO) result in isolated sulfite oxidase deficiency, an inherited metabolic disorder. Three conserved residues (H304, R309, K322) are hydrogen bonded to the phosphate group of the molybdenum cofactor, and the R309H and K322R mutations are responsible for isolated sulfite oxidase deficiency. The kinetic effects of the K322R mutation have been previously reported (Rajapakshe et al. 2012, Chem. Biodiversity 9, 1621-1634); here we investigate several mutants of H304 and R309 by steady-state kinetics, laser flash photolysis studies of intramolecular electron transfer (IET), and spectroelectrochemistry. An unexpected result is that all of the mutants show decreased rates of IET but increased steady-state rates of catalysis. However, in all cases the rate of IET is greater than the overall turnover rate, showing that IET is not the rate determining step for any of the mutations. PMID:24968320

  19. A two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a dual cassette reporter system capable of assessing target gene translation and target gene product folding. The present invention further relates to vectors and host cells comprising the dual cassette reporter system. In addition the invention relates to the use...... of the dual cassette reporter system for assessing target gene translation and target gene product folding....

  20. Mimicking a SURF1 allele reveals uncoupling of cytochrome c oxidase assembly from translational regulation in yeast.

    Science.gov (United States)

    Reinhold, Robert; Bareth, Bettina; Balleininger, Martina; Wissel, Mirjam; Rehling, Peter; Mick, David U

    2011-06-15

    Defects in mitochondrial energy metabolism lead to severe human disorders, mainly affecting tissues especially dependent on oxidative phosphorylation, such as muscle and brain. Leigh Syndrome describes a severe encephalomyopathy in infancy, frequently caused by mutations in SURF1. SURF1, termed Shy1 in Saccharomyces cerevisiae, is a conserved assembly factor for the terminal enzyme of the respiratory chain, cytochrome c oxidase. Although the molecular function of SURF1/Shy1 is still enigmatic, loss of function leads to cytochrome c oxidase deficiency and reduced expression of the central subunit Cox1 in yeast. Here, we provide insights into the molecular mechanisms leading to disease through missense mutations in codons of the most conserved amino acids in SURF1. Mutations affecting G(124) do not compromise import of the SURF1 precursor protein but lead to fast turnover of the mature protein within the mitochondria. Interestingly, an Y(274)D exchange neither affects stability nor localization of the protein. Instead, SURF1(Y274D) accumulates in a 200 kDa cytochrome c oxidase assembly intermediate. Using yeast as a model, we demonstrate that the corresponding Shy1(Y344D) is able to overcome the stage where cytochrome c oxidase assembly links to the feedback regulation of mitochondrial Cox1 expression. However, Shy1(Y344D) impairs the assembly at later steps, most apparent at low temperature and exhibits a dominant-negative phenotype upon overexpression. Thus, exchanging the conserved tyrosine (Y(344)) with aspartate in yeast uncouples translational regulation of Cox1 from cytochrome c oxidase assembly and provides evidence for the dual functionality of Shy1.

  1. Dual-vector Delivery of Thr664Cys and Thr1826Cys Mutated BDD-FⅧ Gene%双载体转重链Tyr664Cys和轻链Thr1286Cys突变体BDD-FⅧ基因

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 杨树德; 刘泽隆; 缪静; 屈慧鸽; 迟晓艳

    2012-01-01

    用双载体转运凝血Ⅷ因子基因在甲型血友病基因治疗研究中可克服AV毒载体容量限制,但存在重链分泌低效和链不均衡性问题.为探索重、轻链间二硫键形成对重链分泌的促进作用,该文用双载体转B结构域大部缺失型FⅧ(BDD-FⅧ)的重链和轻链基因,将重链的Tyr664和轻链Thr1826突变为Cys,研究了HEK293细胞共转基因后的基因表达、分泌至培养上清的重链量和凝血生物活性.用Western blot检测细胞裂解液结果显示,非还原条件下有明显的二硫键交联的重、轻链蛋白;链特异性ELISA定量检测细胞分泌的重链为(125±29) ng/mL,明显高于共转野生型重链和轻链基因细胞的(75±23) ng/mL; Coatest法显示细胞分泌的凝血活性为(0.78±0.29) U/mL,也明显高于共转野生型重链和轻链基因细胞(0.34±0.12) U/mL.结果表明,重、轻链间的二硫键形成 可提高双载体转FⅧ基因的功效,为进一步在动物体内转基因提供了实验依据.%Dual-vector co-transfer of coagulation factor VIII(FVIII) has been used as an alternative strategy to overcome packaging limitation of adeno-associated virus (AAV) vectors in hemophilia A gene therapy, but leading to a chain imbalance problem for an inefficient heavy chain secretion. To improve heavy chain secretion, here we aimed to develop a strategy to enhance the interaction of heavy and light chains by introducing a disulfide linking between both chains. A pair of vectors was expressing Tyr664 to Cys mutated heavy chain and Thr1826 to Cys mutated light chain and co-transfected into cultured HEK293 cells to investigate the gene expression, heavy chain and bioactivity secreted in the culture medium. A disufide-crosslinked heavy and light chains dimer was observed from total cellular protein by Western blot under non-reduced condition. An ELISA for the heavy chain demonstrated high levels of heavy chain (125±29) ng/mL in the medium, greater than that secreted by

  2. NADPH oxidase: an enzyme for multicellularity?

    Science.gov (United States)

    Lalucque, Hervé; Silar, Philippe

    2003-01-01

    Multicellularity has evolved several times during the evolution of eukaryotes. One evolutionary pressure that permits multicellularity relates to the division of work, where one group of cells functions as nutrient providers and the other in specialized roles such as defence or reproduction. This requires signalling systems to ensure harmonious development of multicellular structures. Here, we show that NADPH oxidases are specifically present in organisms that differentiate multicellular structures during their life cycle and are absent from unicellular life forms. The biochemical properties of these enzymes make them ideal candidates for a role in intercellular signalling.

  3. NADH/NADPH Oxidase and Vascular Function.

    Science.gov (United States)

    Griendling, K K; Ushio-Fukai, M

    1997-11-01

    The vascular NADH/NADPH oxidase has been shown to be the major source of superoxide in the vessel wall. Recent work has provided insight into its structure and activity in vascular cells. This enzyme is involved in both vascular smooth muscle hypertrophy and in some forms of impaired endothelium-dependent relaxation. Because oxidative stress in general participates in the pathogenesis of hypertension and atherosclerosis, the enzymes that produce reactive oxygen species may be important determinants of the course of vascular disease. (Trends Cardiovasc Med 1997;7:301-307). © 1997, Elsevier Science Inc.

  4. OXIDASE REACTION OF VARIOUS GROUPS OF BACTERIA.

    Science.gov (United States)

    Felton, L D

    1923-08-31

    1. A simple technique is described for studying the oxidase action of bacteria by means of the oxidation of p-aminoleucomalachite green. 2. It is shown that pneumococci under aerobic conditions produced an oxidase when grown on suitable medium. The sera of any of seven different animal species constitute such a medium, the degree of oxidation by the pneumococcus depending upon the animal from which the serum was taken-rat, guinea pig, rabbit, horse, man, cat, and chicken in order of diminishing suitability. 3. Conditions favoring the oxidation of p-aminoleucomalachite green by a single strain of pneumococci are: the presence of a slight amount of hemoglobin, dextrose, H ion concentration on the add side, and heating of fresh serum for 30 minutes at 56 degrees C. Conditions preventing the oxidation are: sterilized meat infusion, 1 per cent peptone, plain broth, a high concentration of hemoglobin, and absence of oxygen. In a quantitative fashion, meat infusion, 1 per cent peptone, and plain broth interfere with the suitability of serum as a substratum of oxidase production by the pneumococcus. 4. Twenty-three microbic species were studied with reference to oxidative power. They were grown upon 10 per cent horse serum, with and without dextrose, upon 10 per cent guinea pig serum, and upon plain broth. Only three of the twenty-three gave evidence of oxidative power as tested by p-aminoleucomalachite green; namely, the pneumococcus, Streptococcus viridans, and Streptococcus haemolyticus. Among the strains, of these three pneumococci gave the most intense reaction, after which Streptococcus viridans and Streptococcus haemolyticus follow in the order named, but with a noticeable variation among the different strains of Streptococcus haemolyticus. 5. Hemolytic streptococci of human and bovine origin were studied. The only variation in the type of reaction was manifested by the streptococci of milk and cheese origin. Strains from these sources showed definitely the least

  5. BiP表达下调改善双载体转断裂FⅧ基因的功效%Down Regulated BiP Expression Improves Efficacy of Split F Ⅷ Gene Delivery by a Dual-Vector

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 刘泽隆; 缪静; 屈慧鸽; 迟晓艳

    2011-01-01

    双载体转凝血Ⅷ因子(FⅧ)基因可作为一种转基因策略克服腺相关病毒(AAV)载体容量限制,但重链分泌的低效性影响转基因功效.为提高重链分泌,本文用RNA干扰技术下调内质网内蛋白伴侣分子免疫球蛋白重链结合蛋白(BiP)的表达,观察对HEK293细胞双载体共转FⅧ基因分泌重链和生物活性的影响.结果显示,RNA干扰可明显下调BiP表达,但不影响细胞生长;ELISA检测BiP下调细胞单独转重链基因时的重链分泌量为98士38 ng/mL,与轻链共转基因时显著升高到157±32 ng/mL,明显高于对照细胞单独转重链基因和共转重链和轻链基因的重链分泌量(分别为29±8 ng/mL和79±19 ng/mL);Cotest法检测显示,BiP下调细胞共转重链和轻链基因细胞分泌的凝血生物活性为0.73士0.23 IU/mL,明显高于对照细胞共转重链和轻链基因(0.39±0.07 IU/mL).结果表明,BiP表达下调通过促进重链分泌,可提高双体共转FⅧ基因的功效,为进一步动物体内双AAV载体转FⅧ基因的甲型血友病基因治疗研究提供了实验依据.%Dual-vector transfer of coagulation factor VI ( F VI) gene has been developped as an alternative strategy to overcome the packaging limitation of adeno-associated virus ( AAV) vectors but its efficacy was adversely affected by an inefficient heavy chain secretion. In this study, we aimed to improve secretion of F VI heavy chain by down-regulating expression of immunoglobulin heavy-chain binding protein (BiP) , an ER chaperon protein with RNAi technology, and observed secreted heavy chain and F VI bioactivity by HEK293 cells after a dual-vector co-tranduction with split FVI gene. The data showed that RNA interference could obviously down-regulate the expression of BiP but no effect was found on cell proliferation. It showed much higher levels of heavy chain secretion from heavy chain gene transfected cell (98 ±38 ng/mL) and heavy plus light chain gene co-transfected cell (157

  6. Enzyme-free and sensitive electrochemical determination of the FLT3 gene based on a dual signal amplified strategy: Controlled nanomaterial multilayers and a target-catalyzed hairpin assembly.

    Science.gov (United States)

    Sun, Yingying; Ren, Qunxiang; Liu, Bo; Qin, Yan; Zhao, Shuang

    2016-04-15

    An isothermal, enzyme-free and sensitive electrochemical DNA sensor was developed for the detection of the FLT3 gene in acute myeloid leukemia (AML). First, aminated multi-walled carbon nanotubes (AMWNTs) and gold nanoparticles (AuNPs) were alternately self-assembled on a gold electrode using a layer-by-layer strategy. Then, the hairpin DNA probe 1 (H1), with a thiol group at the 3' end and a ferrocenyl moiety (Fc) at the 5' end, was immobilized on the AMWNTs/AuNPs multilayer films through Au-S bonding. When the target DNA (TD) appeared, it hybridized with and opened the hairpin structure of H1, and Fc was forced away from the electrode surface, leading to a significant decrease in the current peak of square wave voltammetry. Subsequently, the hairpin DNA probe 2 (H2) bound to H1, freeing the TD to trigger another reaction cycle. The combination of this target-catalyzed hairpin assembly and the LBL assembly of nanomaterials achieved a detection limit of 0.1 pM with a wide linear range of 0.1-1000 pM. The sensor discriminated between mismatched DNA and the target DNA with high selectivity. This dual signal amplification strategy is relatively simple and inexpensive because it does not need any enzymes or sophisticated equipment and successfully assayed the FLT3 gene from real samples.

  7. COPPER AMINE OXIDASE1 (CuA01)of Arabidopsis thaliana Contributes to Abscisic Acid-and Polyamine-Induced Nitric Oxide Biosynthesis and Abscisic Acid Signal Transduction

    Institute of Scientific and Technical Information of China (English)

    Rinukshi Wimalasekera; Corina Villar; Tahmina Begum; Günther F. E. Scherer

    2011-01-01

    Polyamines (PA), polyamine oxidases, copper amine oxidases, and nitric oxide (NO)play important roles in physiology and stress responses in plants. NO biosynthesis as a result of catabolism of PA by polyamine oxidases and copper amine oxidases may explain in part PA-mediated responses. Involvement of a copper amine oxidase gene, COPPER AMINE OXIDASE1 (CuA01), of Arabidopsis was tested for its role in stress responses using the knockouts cuaol.1 and cuaol-2. PA-induced and ABA-induced NO production investigated by fluorometry and fluorescence microscopy showed that the cuaol-1 and cuaol-2 are impaired in NO production, suggesting a function of CuAO1 in PA and ABA-mediated NO production. Furthermore, we found a PA-dependent increase in protein S-nitrosylation. The addition of PA and ABA also resulted in HO increases, cuaol-1 and cuaol-2 showed less sensitivity to exogenous ABA supplementation during ger-mination, seedling establishment, and root growth inhibition as compared to wild-type. In response to ABA treatment,expression levels of the stress-responsive genes RD29A and ADH1 were significantly lower in the knockouts. These obser-vations characterize cuaol-1 and cuaol-2 as ABA-insensitive mutants. Taken together, our findings extend the ABA signal transduction network to include CuAO1 as one potential contributor to enhanced NO production by ABA.

  8. Involvement of NADPH oxidase NtrbohD in the rapid production of H2O2 induced by ABA in cultured tobacco cell line BY-2

    Institute of Scientific and Technical Information of China (English)

    Fushun Hao; Jinguang Zhang; Zhonglian Yu; Jia Chen

    2008-01-01

    The mechanisms for the production of hydrogen peroxide (H2O2) induced by abscisic acid (ABA) were investigated in suspension culture cells of tobacco BY-2 cells. The results showed that the immediate generation of H2O2, which was mainly derived from super-oxide dismutase-catalyzed dismutation of superoxide radical, was significantly induced by ABA. Furthermore, treatment of the cultured tobacco cells with ABA resulted in a time-dependent quick increase in plasma membrane (PM) NADPH oxidase activity, which coincided on time and magnitude with the elevation in ABA-induced accumulation of H2O2. Moreover, these enhanced effects were pronouncedly inhibited by two NADPH oxidase inhibitors, diphenylene iodonium and imidazole, suggesting that PM NADPH oxidase is involved in the rapid accumulation of H2O2 in cultured tobacco cells. In addition, analysis of the expression level of NtrbohD, a PM NADPH oxidase gene in tobacco, by RT-PCR and protein gel blot revealed that the gene at both mRNA and protein levels was upregulated by ABA, indicating that NtrbohD participates in the ABA-stimulated rapid production of H2O2 in tobacco culture cells. Taken together, these findings suggest that ABA induces the rapid accumulation of reactive oxygen species via NADPH oxidase in suspension culture cells of tobacco, and that NADPH oxidase and H2O2 appear to be important components in ABA signal transduction pathway in plants.

  9. Nanoparticle strategies for cancer therapeutics: Nucleic acids, polyamines, bovine serum amine oxidase and iron oxide nanoparticles (Review).

    Science.gov (United States)

    Agostinelli, Enzo; Vianello, Fabio; Magliulo, Giuseppe; Thomas, Thresia; Thomas, T J

    2015-01-01

    Nanotechnology for cancer gene therapy is an emerging field. Nucleic acids, polyamine analogues and cytotoxic products of polyamine oxidation, generated in situ by an enzyme-catalyzed reaction, can be developed for nanotechnology-based cancer therapeutics with reduced systemic toxicity and improved therapeutic efficacy. Nucleic acid-based gene therapy approaches depend on the compaction of DNA/RNA to nanoparticles and polyamine analogues are excellent agents for the condensation of nucleic acids to nanoparticles. Polyamines and amine oxidases are found in higher levels in tumours compared to that of normal tissues. Therefore, the metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, can be targets for antineoplastic therapy since these naturally occurring alkylamines are essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. In particular, polyamine catabolism involves copper-containing amine oxidases. Several studies showed an important role of these enzymes in developmental and disease-related processes in animals through the control of polyamine homeostasis in response to normal cellular signals, drug treatment, and environmental and/or cellular stress. The production of toxic aldehydes and reactive oxygen species (ROS), H2O2 in particular, by these oxidases suggests a mechanism by which amine oxidases can be exploited as antineoplastic drug targets. The combination of bovine serum amine oxidase (BSAO) and polyamines prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. The findings described herein suggest that enzymatically formed cytotoxic agents activate stress signal transduction pathways, leading to apoptotic cell death. Consequently, superparamagnetic nanoparticles or other

  10. Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure

    Directory of Open Access Journals (Sweden)

    Biryukova Irina V

    2008-08-01

    Full Text Available Abstract Background The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on in vitro and in vivo recombination processes have been developed. Results Here, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant E. coli strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-attB sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-attP-site, and second, the λ system is used for excision of inserted vector part, including the plasmid ori-replication and the marker, flanked by λ-attL/R-sites. Conclusion The developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.

  11. Functional characterization of a C-4 sterol methyl oxidase from the endomycorrhizal fungus Glomus intraradices.

    Science.gov (United States)

    Oger, Elodie; Ghignone, Stefano; Campagnac, Estelle; Fontaine, Joël; Grandmougin-Ferjani, Anne; Lanfranco, Luisa

    2009-01-01

    Sterols are crucial components of eukaryotic membranes that control membrane fluidity and permeability. They play an important role in cell signaling, polarity and sorting. Since many steps in the pathway are essential, sterol biosynthesis inhibitors (SBI) are widely used as antifungal agents. This work reports the identification and the characterization of a C-4 sterol methyl oxidase (SMO), the first gene involved in the sterol biosynthetic pathway, so far described from an arbuscular mycorrhizal fungus. The sequence, called GintSMO, shows a primary structure, a hydrophobicity profile and a pattern of histidine-rich motifs which are typical of C-4 methyl sterol oxidases. The complementation assay in a Saccharomyces cerevisiae mutant strain demonstrates that GintSMO encodes a functional SMO. Changes in GintSMO transcript levels and in the amount of the sterol precursor squalene were observed in in vitro grown extraradical structures exposed to the fenpropimorph SBI fungicide.

  12. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    Science.gov (United States)

    Peng, Zeyu; Dittmer, Neal T; Lang, Minglin; Brummett, Lisa M; Braun, Caroline L; Davis, Lawrence C; Kanost, Michael R; Gorman, Maureen J

    2015-04-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.

  13. Dual Income Taxes

    DEFF Research Database (Denmark)

    Sørensen, Peter Birch

    This paper discusses the principles and practices of dual income taxation in the Nordic countries. The first part of the paper explains the rationale and the historical background for the introduction of the dual income tax and describes the current Nordic tax practices. The second part...... of the paper focuses on the problems of taxing income from small businesses and the issue of corporate-personal tax integration under the dual income tax, considering alternative ways of dealing with these challenges. In the third and final part of the paper, I briefly discuss whether introducing a dual income...... tax could be relevant for New Zealand....

  14. Dual Income Taxes

    DEFF Research Database (Denmark)

    Sørensen, Peter Birch

    This paper discusses the principles and practices of dual income taxation in the Nordic countries. The first part of the paper explains the rationale and the historical background for the introduction of the dual income tax and describes the current Nordic tax practices. The second part...... of the paper focuses on the problems of taxing income from small businesses and the issue of corporate-personal tax integration under the dual income tax, considering alternative ways of dealing with these challenges. In the third and final part of the paper, I briefly discuss whether introducing a dual income...... tax could be relevant for New Zealand....

  15. Analyses of numerical aberrations of chromosome 17 and tp53 gene deletion/amplification in human oral squamous cell carcinoma using dual-color fluorescence in situ hybridization

    Directory of Open Access Journals (Sweden)

    Noemi MESZAROS

    2010-05-01

    Full Text Available In Romania, oral and facial cancers represent approximately 5% of all cancers. Deactivation and unregulated expression of oncogenes and tumor suppressor genes may be involved in the pathogenesis of oral squamous cell carcinoma. The genomic change results in numerical and structural chromosomal alterations, particularly in chromosomes 3, 9, 11 and 17. The aim of our study was to identify numerical aberrations of chromosome 17, deletion or amplification of p53 gene and to reveal correlations between abnormalities of chromosome 17and of p53 gene with TNM status and grading in 15 subjects with oral squamous cell carcinoma. 80 % of cases presented chromosome 17 polysomy and only 20% of cases had chromosome 17 monosomy. 46.6 % of samples revealed p53 gene amplification and 33.3 % of them p53 deletion. Polysomy of chromosome 17 was also detected in tumor-adjacent epithelia. The degree of the cytogenetic abnormality did not correlate with the stage of the disease, the histological differentiation of oral squamous cell carcinoma and lymph node metastasis. Molecular cytogenetic techniques, using fluorescence in situ hybridization with chromosome-specific DNA probes, facilitate the confirmation of presumed chromosomal aberrations with high sensitivity and specificity.

  16. Positive selection and multiple losses of the LINE-1-derived L1TD1 gene in mammals suggest a dual role in genome defense and pluripotency.

    Science.gov (United States)

    McLaughlin, Richard N; Young, Janet M; Yang, Lei; Neme, Rafik; Wichman, Holly A; Malik, Harmit S

    2014-09-01

    Mammalian genomes comprise many active and fossilized retroelements. The obligate requirement for retroelement integration affords host genomes an opportunity to 'domesticate' retroelement genes for their own purpose, leading to important innovations in genome defense and placentation. While many such exaptations involve retroviruses, the L1TD1 gene is the only known domesticated gene whose protein-coding sequence is almost entirely derived from a LINE-1 (L1) retroelement. Human L1TD1 has been shown to play an important role in pluripotency maintenance. To investigate how this role was acquired, we traced the origin and evolution of L1TD1. We find that L1TD1 originated in the common ancestor of eutherian mammals, but was lost or pseudogenized multiple times during mammalian evolution. We also find that L1TD1 has evolved under positive selection during primate and mouse evolution, and that one prosimian L1TD1 has 'replenished' itself with a more recent L1 ORF1 from the prosimian genome. These data suggest that L1TD1 has been recurrently selected for functional novelty, perhaps for a role in genome defense. L1TD1 loss is associated with L1 extinction in several megabat lineages, but not in sigmodontine rodents. We hypothesize that L1TD1 could have originally evolved for genome defense against L1 elements. Later, L1TD1 may have become incorporated into pluripotency maintenance in some lineages. Our study highlights the role of retroelement gene domestication in fundamental aspects of mammalian biology, and that such domesticated genes can adopt different functions in different lineages.

  17. The novel and taxonomically restricted Ah24 gene from grain amaranth (Amaranthus hypochondriacus has a dual role in development and defense

    Directory of Open Access Journals (Sweden)

    Julio Armando Massange-Sanchez

    2015-08-01

    Full Text Available Grain amaranths tolerate stress and produce highly nutritious seeds. We have identified several (abiotic stress-responsive genes of unknown function in Amaranthus hypochondriacus, including the so-called Ah24 gene. Ah24 was expressed in young or developing tissues; it was also strongly induced by mechanical damage, insect herbivory and methyl jasmonate and in meristems and newly emerging leaves of severely defoliated plants. Interestingly, an in silico analysis of its 1304 bp promoter region showed a predominance of regulatory boxes involved in development, but not in defense. The Ah24 cDNA encodes a predicted cytosolic protein of 164 amino acids, the localization of which was confirmed by confocal microscopy. Additional in silico analysis identified several other Ah24 homologs, present almost exclusively in plants belonging to the Caryophyllales. The possible function of this gene in planta was examined in transgenic Ah24 overexpressing Arabidopsis thaliana and Nicotiana tabacum plants. Transformed Arabidopsis showed enhanced vegetative growth and increased leaf number with no penalty in one fitness component, such as seed yield, in experimental conditions. Transgenic tobacco plants, which grew and reproduced normally, had increased insect herbivory resistance. Modified vegetative growth in transgenic Arabidopsis coincided with significant changes in the expression of genes controlling phytohormone synthesis or signaling, whereas increased resistance to insect herbivory in transgenic tobacco coincided with higher jasmonic acid and proteinase inhibitor activity levels, plus the accumulation of nicotine and several other putative defense-related metabolites. It is proposed that the primary role of the Ah24 gene in A. hypochondriacus is to contribute to a rapid recovery post-wounding or defoliation, although its participation in defense against insect herbivory is also plausible.

  18. Molecular In Vivo Imaging Using a Noninvasive Cardiac-Specific MLC-2v Promoter Driven Dual-Gene Recombinant Lentivirus Monitoring System.

    Directory of Open Access Journals (Sweden)

    Miao Zhang

    Full Text Available Our study aimed to demonstrate the feasibility of using the sodium/iodide symporter (NIS to monitor vascular endothelial growth factor (VEGF165 expression in vivo.We constructed a recombinant lentivirus plasmid with the MLC-2v promoter driving the sodium/iodide symporter (NIS reporter gene linked to the VEGF165 gene. Expression of NIS and VEGF gene were identified by Western blot. On days 2 and 54, 99mTc-MIBI imaging was used to evaluate changes in myocardial ischemia. Noninvasive 125I micro-SPECT/CT imaging was used to assess the expression of NIS reporter gene dynamically over the next 2 months.Western blot analysis showed that both NIS and VEGF165 were highly expressed in rat cardiomyoblast H9C2 cells transduced with Lenti-MLC-2v-NIS--VEGF165. 125I micro-SPECT/CT reporter imaging showed higher uptake in mouse myocardium transduced with Lenti-MLC-2v-VEGF165-IRES-NIS. NIS expression peaked on day 1 after transduction followed by a progressive decline to negligible levels by day 21. On day 1, mean 125I activity value in group 1 was higher than that in group 2 (P 0.05. In group 1 (test group, 99mTc-MIBI SPECT/CT revealed improvements in perfusion and wall thickening in the apical anterior wall. Mean IOD values of NIS and CD34 were significantly higher in group 1 than group 3 (P<0.05. Our study proved mean I-125 uptake was significantly correlated with mean IOD value of NIS and CD34 (P<0.05.This study demonstrates the feasibility of using the NIS gene to monitor VEGF165 expression in a mouse myocardial ischemia model.

  19. The novel and taxonomically restricted Ah24 gene from grain amaranth (Amaranthus hypochondriacus) has a dual role in development and defense

    Science.gov (United States)

    Massange-Sanchez, Julio A.; Palmeros-Suarez, Paola A.; Martinez-Gallardo, Norma A.; Castrillon-Arbelaez, Paula A.; Avilés-Arnaut, Hamlet; Alatorre-Cobos, Fulgencio; Tiessen, Axel; Délano-Frier, John P.

    2015-01-01

    Grain amaranths tolerate stress and produce highly nutritious seeds. We have identified several (a)biotic stress-responsive genes of unknown function in Amaranthus hypochondriacus, including the so-called Ah24 gene. Ah24 was expressed in young or developing tissues; it was also strongly induced by mechanical damage, insect herbivory and methyl jasmonate and in meristems and newly emerging leaves of severely defoliated plants. Interestingly, an in silico analysis of its 1304 bp promoter region showed a predominance of regulatory boxes involved in development, but not in defense. The Ah24 cDNA encodes a predicted cytosolic protein of 164 amino acids, the localization of which was confirmed by confocal microscopy. Additional in silico analysis identified several other Ah24 homologs, present almost exclusively in plants belonging to the Caryophyllales. The possible function of this gene in planta was examined in transgenic Ah24 overexpressing Arabidopsis thaliana and Nicotiana tabacum plants. Transformed Arabidopsis showed enhanced vegetative growth and increased leaf number with no penalty in one fitness component, such as seed yield, in experimental conditions. Transgenic tobacco plants, which grew and reproduced normally, had increased insect herbivory resistance. Modified vegetative growth in transgenic Arabidopsis coincided with significant changes in the expression of genes controlling phytohormone synthesis or signaling, whereas increased resistance to insect herbivory in transgenic tobacco coincided with higher jasmonic acid and proteinase inhibitor activity levels, plus the accumulation of nicotine and several other putative defense-related metabolites. It is proposed that the primary role of the Ah24 gene in A. hypochondriacus is to contribute to a rapid recovery post-wounding or defoliation, although its participation in defense against insect herbivory is also plausible. PMID:26300899

  20. Positive selection and multiple losses of the LINE-1-derived L1TD1 gene in mammals suggest a dual role in genome defense and pluripotency.

    Directory of Open Access Journals (Sweden)

    Richard N McLaughlin

    2014-09-01

    Full Text Available Mammalian genomes comprise many active and fossilized retroelements. The obligate requirement for retroelement integration affords host genomes an opportunity to 'domesticate' retroelement genes for their own purpose, leading to important innovations in genome defense and placentation. While many such exaptations involve retroviruses, the L1TD1 gene is the only known domesticated gene whose protein-coding sequence is almost entirely derived from a LINE-1 (L1 retroelement. Human L1TD1 has been shown to play an important role in pluripotency maintenance. To investigate how this role was acquired, we traced the origin and evolution of L1TD1. We find that L1TD1 originated in the common ancestor of eutherian mammals, but was lost or pseudogenized multiple times during mammalian evolution. We also find that L1TD1 has evolved under positive selection during primate and mouse evolution, and that one prosimian L1TD1 has 'replenished' itself with a more recent L1 ORF1 from the prosimian genome. These data suggest that L1TD1 has been recurrently selected for functional novelty, perhaps for a role in genome defense. L1TD1 loss is associated with L1 extinction in several megabat lineages, but not in sigmodontine rodents. We hypothesize that L1TD1 could have originally evolved for genome defense against L1 elements. Later, L1TD1 may have become incorporated into pluripotency maintenance in some lineages. Our study highlights the role of retroelement gene domestication in fundamental aspects of mammalian biology, and that such domesticated genes can adopt different functions in different lineages.

  1. Dual-fuel, dual-throat engine preliminary analysis

    Science.gov (United States)

    Obrien, C. J.

    1979-01-01

    A propulsion system analysis of the dual fuel, dual throat engine for launch vehicle applications was conducted. Basic dual throat engine characterization data were obtained to allow vehicle optimization studies to be conducted. A preliminary baseline engine system was defined.

  2. Enzymatic Characterization and In Vivo Function of Five Terminal Oxidases in Pseudomonas aeruginosa

    OpenAIRE

    Arai, Hiroyuki; Kawakami, Takuro; Osamura, Tatsuya; Hirai, Takehiro; Sakai, Yoshiaki; Ishii, Masaharu

    2014-01-01

    The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo3-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa3-type cytochrome c oxidase (aa3), and two cbb3-type cytochrome c oxidases (cbb3-1 and cbb3-2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant str...

  3. Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin

    NARCIS (Netherlands)

    Heuvel, van den R.H.H.; Berg, van den W.A.M.; Rovida, S.; Berkel, van W.J.H.

    2004-01-01

    The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol

  4. Endothelins and NADPH oxidases in the cardiovascular system.

    Science.gov (United States)

    Dammanahalli, Karigowda J; Sun, Zhongjie

    2008-01-01

    1. The endothelin (ET) system and NADPH oxidase play important roles in the regulation of cardiovascular function, as well as in the pathogenesis of hypertension and other cardiovascular diseases. 2. Endothelins activate NADPH oxidases and thereby increase superoxide production, resulting in oxidative stress and cardiovascular dysfunction. Thus, NADPH oxidases may mediate the role of endothelins in some cardiovascular diseases. However, the role of reactive oxygen species (ROS) in mediating ET-induced vasoconstriction and cardiovascular disease remains under debate, as evidenced by conflicting reports from different research teams. Conversely, activation of NADPH oxidase can stimulate ET secretion via ROS generation, which further enhances the cardiovascular effects of NADPH oxidase. However, little is known about how ROS activate the endothelin system. It seems that the relationship between ET-1 and ROS may vary with cardiovascular disorders. 3. Endothelins activate NADPH oxidase via the ET receptor-proline-rich tyrosine kinase-2 (Pyk2)-Rac1 pathway. Rac1 is an important regulator of NADPH oxidase. There is ample evidence supporting direct stimulation by Rac1 of NADPH oxidase activity. In addition, Rac1-induced cardiomyocyte hypertrophy is mediated by the generation of ROS.

  5. Priming and activation of NADPH oxidases in plants and animals.

    Science.gov (United States)

    Canton, Johnathan; Grinstein, Sergio

    2014-09-01

    In mammals, engagement of Toll-like receptors by microbe-associated molecular patterns enhances the responsiveness of NADPH oxidases. Two recent papers report a similar 'priming' mechanism for the plant oxidase RbohD. Despite lacking structural homology, the functional parallels between plants and animals reveal that a common regulatory logic arose by convergent evolution. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. 利用细胞色素氧化酶亚单位Ⅰ分析青海藏区细粒棘球绦虫系统发育学%Phylogeny of Qinghai Plateau Echinococcus g ranulosus isolates inferred by cytochrome oxidasegene sequences

    Institute of Scientific and Technical Information of China (English)

    曹得萍; 樊海宁; 毋德芳; 赵海龙; 白海燕

    2014-01-01

    目的:囊型包虫病是一种广泛流行且危害严重的人兽共患寄生虫病。本文旨在分析流行于青南地区细粒棘球绦虫系统发育学及基因多态性。方法本文利用线粒体 DNA细胞色素氧化酶亚单位Ⅰ(COX Ⅰ)分子对收集到的流行于青海省的59例包虫病样本进行测序。总计71条序列(其中12条来自GenBank)碱基通过Clustal X软件比对,构建贝叶斯进化树。结果流行于青海省包虫病样本大部分与细粒棘球绦虫G1型聚在一枝,还有三个样本与多房棘球绦虫(AB018440)聚在一枝。虽大部分棘球绦虫基因型属于G1型,但各自的基因型各不相同,具有丰富的多样性。结论流行于青海省细粒棘球绦虫系统发育学比我们想象复杂。%Cystic echinococcosis is one of the most widespread and severe zoonotic helminthic diseases .To understand the phylogeny and genetic polymorphism Echinococcus granulosus (E .granulosus) prevailed in south region of Qinghai Prov-ince ,partial fragment of cytochrome oxidase Ⅰ (COX Ⅰ ) gene sequences were used to infer the phylogenetic relationship of 59 collected samples of E .granulosus in Qinghai Province .Total 72 sequences (13 sequences from GenBank) were aligned using CLUSTAL X ,and then ,Bayesian analyses were performed in Mrbayes-3 .1 .2 .The results revealed that Echinococcus spp .isolates did not form a monophyletic group .The most samples clustered with E . granulosus strain (G1) (AB297617) , but showed high genetic polymorphism .Another three samples clustered with E .multilocularis (AB018440) ,while they showed complex phylogenetic relationships among them ,further indicating that Echinococcus spp .isolates from Qinghai Prov-ince may has a more complex evolutionary history than expected .

  7. D-氨基酸氧化酶激活剂基因多态性与精神分裂症的关联研究%Association of D-amino acid oxidase activator gene polymorphisms with schizophrenia in a Chinese Han sample

    Institute of Scientific and Technical Information of China (English)

    杨丽; 邵静茹; 阮冶; 吴艳瑞; 周利国; 张勇辉; 伍星; 郑冰蓉; 肖春杰

    2011-01-01

    Objective To investigate the association between single nucleotide polymorphisms (SNPs) of D-amino acid oxidase activator (DAOA) gene and schizophrenia in Han population from Yunnan Province.Methods Two hundred and seventy-six schizophrenic patients and 312 healthy controls were recruited.Genotyping was performed using polymerase chain reaction(PCR)-restriction fragment length polymerase(RFLP).Odds ratios(ORs)and 95%confidence intervals(CIs)were calculated to evaluate the relationship between the gene polymorphisms and schizophrenia.Results (1)No significant differences of rs2391191,rs947267 and rs778294 allele frequencies were observed between the patients and controls,and the same results existed in the females.(2)In males,there was no significant difference in the allele frequency of rs2391191(P>0.05)between the two groups.The frequencies of rs947267 C allele and rs778294 A allele(0.406 and 0.184,respectively)were higher in patients compared to that in controls (0.327 and 0.103,respectively)[adjusted OR=1.41,95%CI=1.00-1.99,P=0.049;adjusted OR=1.91,95%CI=1.21-3.20,P=0.006].(3)In males,the frequency of haplotype GAA was significantly higher in controls than that in patients(P=0.008).Conclusions The DAOA gene rs947267 and rs778294 polymorphisms may be associated with schizophrenia in males.Rs947267 C allele and rs778294 A allele are possibly risk factors for schizophrenia in Yunnan Han male population.Haplotype GAA may be associated with schizophrenia as a protective factor in males.%目的 探讨D-氨基酸氧化酶激活剂(D-amino acid oxidase activator,DAOA)基因rs2391191、rs947267、rs778294多态性与云南汉族人群精神分裂症发病的相关性.方法 采用聚合酶链式反应-限制性片段长度多态(PCR-RFLP)方法,对276例精神分裂症患者(患者组)和312名健康对照(对照组)DAOA基因rs2391191、rs947267和rs778294 3个位点的多态进行检测,在混合群体和性别分层群体中进行多态性与精

  8. Modulation of NADPH oxidase activity by known uraemic retention solutes

    DEFF Research Database (Denmark)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera

    2014-01-01

    as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase...... inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. METHODS: Mononuclear leucocytes...... isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium...

  9. The NADH oxidase-Prx system in Amphibacillus xylanus.

    Science.gov (United States)

    Niimura, Youichi

    2007-01-01

    Amphibacillus NADH oxidase belongs to a growing new family of peroxiredoxin-linked oxidoreductases including alkyl hydroperoxide reductase F (AhpF). Like AhpF it displays extremely high hydroperoxide reductase activity in the presence of a Prx, thus making up the NADH oxidase-Prx system. The NADH oxidase primarily catalyzes the reduction of oxygen by NADH to form H2O2, while the Prx immediately reduces H2O2 (or ROOH) to water (or ROH). Consequently, the NADH oxidase-Prx system catalyzes the reduction of both oxygen and hydrogen peroxide to water with NADH as the preferred electron donor. The NADH oxidase-Prx system is widely distributed in aerobically growing bacteria lacking a respiratory chain and catalase, and plays an important role not only in scavenging hydroperoxides but also in regenerating NAD in these bacteria.

  10. Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.

    Science.gov (United States)

    Reynafarje, B; Alexandre, A; Davies, P; Lehninger, A L

    1982-12-01

    The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0.

  11. The Dual Career Family.

    Science.gov (United States)

    Gurtin, Lee

    1980-01-01

    The dual career couple is forced to make a series of choices and compromises that impact the realms of marriage and career. The dilemmas that confront dual career marriages can be overcome only by compromise, accommodation, and mutual understanding on the part of the individuals involved. A revamping of human resources and recruitment programs is…

  12. Dual Credit Report

    Science.gov (United States)

    Light, Noreen

    2016-01-01

    In 2015, legislation to improve access to dual-credit programs and to reduce disparities in access and completion--particularly for low income and underrepresented students--was enacted. The new law focused on expanding access to College in the High School but acknowledged issues in other dual-credit programs and reinforced the notion that cost…

  13. Dual Youla parameterization

    DEFF Research Database (Denmark)

    Niemann, Hans Henrik

    2003-01-01

    A different aspect of using the parameterisation of all systems stabilised by a given controller, i.e. the dual Youla parameterisation, is considered. The relation between system change and the dual Youla parameter is derived in explicit form. A number of standard uncertain model descriptions are...

  14. Self-Dual Gravity

    CERN Document Server

    Krasnov, Kirill

    2016-01-01

    Self-dual gravity is a diffeomorphism invariant theory in four dimensions that describes two propagating polarisations of the graviton and has a negative mass dimension coupling constant. Nevertheless, this theory is not only renormalisable but quantum finite, as we explain. We also collect various facts about self-dual gravity that are scattered across the literature.

  15. Optical dual self functions

    Institute of Scientific and Technical Information of China (English)

    华建文; 刘立人; 王宁

    1997-01-01

    A recipe to construct the exact dual self-Fourier-Fresnel-transform functions is shown, where the Dirac comb function and transformable even periodic function are used. The mathematical proof and examples are given Then this kind of self-transform function is extended to the feasible optical dual self-transform functions.

  16. Dual doubled geometry

    NARCIS (Netherlands)

    Bergshoeff, Eric A.; Riccioni, Fabio; Alvarez-Gaumé, L.

    2011-01-01

    We probe doubled geometry with dual fundamental branes. i.e. solitons. Restricting ourselves first to solitonic branes with more than two transverse directions we find that the doubled geometry requires an effective wrapping rule for the solitonic branes which is dual to the wrapping rule for fundam

  17. Research on Dual Control

    Institute of Scientific and Technical Information of China (English)

    Duan Li; Fucai Qian; Peilin Fu

    2005-01-01

    This paper summarizes recent progress by the authors in developing two solution frameworks for dual control. The first solution framework considers a class of dual control problems where there exists a parameter uncertainty in the observation equation of the LQG problem. An analytical active dual control law is derived by a variance minimization approach. The issue of how to determine an optimal degree of active learning is then addressed, thus achieving an optimality for this class of dual control problems. The second solution framework considers a general class of discrete-time LQG problems with unknown parameters in both state and observation equations. The best possible (partial) closed-loop feedback control law is derived by exploring the future nominal posterior probabilities, thus taking into account the effect of future learning when constructing the optimal nominal dual control.

  18. Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes

    Institute of Scientific and Technical Information of China (English)

    Chunxiao Zhang; Ying Gai; Wenqi Wang; Yanyan Zhu; Xuemei Chen; Xiangning Jiang

    2008-01-01

    The constitutive promoter of cauliflower mosaic virus 35S (CaMV 35S) is a polar unidirectional promoter and is widely used in plantgenetic engineering.In the present study,the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5' end of CaMV 35S promoter in the opposite orientation.To qualitatively and quantitatively esti-mate its bi-directional Wanscriptionai function and activity,two visible reporter genes,gusA β-glucuronidase,GUS) and gfp (green fluo-rescent protein,GFP),were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences,respectively.Stable expression of gusA and gfp genes in transgenic tobacco (Nicotiana tabacum L.) was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants.The expression of two reporter genes showed that the eonsuructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations.The quantitativeestimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excita-tion and emission.The GUS activity varied from 8 to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 μg/mg protein in various lines of transgenic tobacco plants.Higher GUS activity generally coupled with lower GFP content,and vice versa.

  19. Construction of a repeat-free dual color fluorescent in situ hybridization probe for ROS1 gene in non-small cell lung cancer diagnosis

    Institute of Scientific and Technical Information of China (English)

    程弘夏

    2014-01-01

    Objective To establish a repeat-free ROS1 gene fluorescence in situ hybridization(FISH)probe,and to compare its efficacy with those of commercial FISH probes in non-small cell lung cancer.Methods The probe was constructed by combining human Cot-1 DNA genome into double-stranded sequence,and then digested by duples specific nuclease to establish a repeat-free sequene.The final repeat-free ROS1 FISH probe was labeled by red and green fluoresceins.Results Compared

  20. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  1. The moderating effect of monoamine oxidase A gene polymorphism in relationship between childhood abuse of male adolescents and externalizing behaviors%单胺氧化酶A基因多态性对男性青少年儿童期虐待与外化性行为关系的调节效应

    Institute of Scientific and Technical Information of China (English)

    明庆森; 姚树桥; 张芸; 陈海燕

    2012-01-01

    Objective To explore the relationship between male adolescents' childhood abuse and externalizing behaviors,and the moderating effect of monoamine oxidase A (MAOA) gene promoter region variable number tandem repeat (VNTR) polymorphism.Methods Through random cluster sampling,352 Han male middle school students from Changsha were tested by Achenbach child behavior checklist-youth self reports (CBCL-YSR)and childhood trauma questionnaire-28 item short form (CTQ-SF),their MAOA genotypes were also identified.A hierarchical multiple regression analysis was used to test the moderating effect.Results ( 1 ) Compared participates with high activity MAOA gene and that with low activity MAOA gene,there were no significant differences on age ( ( 15.82 ± 1.52) vs ( 15.94 ± 1.62 ) ),externalizing behaviors ( ( 15.13 ± 10.14 ) vs ( 14.33 ± 9.70 ) ),total abuse ( (52.59 ± 10.46) vs (51.39 ± 7.56 ) ),emotional abuse( ( 7.63 ± 3.31 ) vs ( 7.11 ± 2.68 ) ),physical abuse ( ( 6.40 ± 2.82) vs (6.12 ± 2.05 ) ),sexual abuse ( ( 6.42 ± 3.24 ) vs ( 5.94 ± 1.72 ) ),emotional neglect ((13.44±5.12) vs (13.16 ±4.83) ),physical neglect( (10.27 ±2.64) vs (10.44±2.53))(t=-1.789~0.678,P > 0.05 ).(2)Except emotional neglect and physical neglect,emotional abuse,physical abuse and sexual abuse could predict externalizing behaviors( Sβ =0.141 ~0.347,P < 0.01 ).(3) MAOA gene was not directly related to externalizing behaviors( Sβ =- 0.023,P > 0.05 ).There was a significant interaction between MAOA gene and emotional abuse( Sβ =-0.148,P < 0.01 ).The interaction between MAOA gene and physical abuse or sexual abuse showed no statistical significance( Sβ =- 0.067,- 0.005,P > 0.05 ).Conclusion MAOA gene polymorphism can moderate the relationship between male adolescents'childhood emotional abuse and externalizing behaviors.%目的 探讨男性青少年的儿童期虐待与外化性行为的关系以及单胺氧化酶A (MAOA)基因启动子区域可变重复序列(VNTR)多态

  2. NADPH Oxidases in Chronic Liver Diseases

    Directory of Open Access Journals (Sweden)

    Joy X. Jiang

    2014-01-01

    Full Text Available Oxidative stress is a common feature observed in a wide spectrum of chronic liver diseases including viral hepatitis, alcoholic, and nonalcoholic steatohepatitis. The nicotinamide adenine dinucleotide phosphate (NADPH oxidases (NOXs are emerging as major sources of reactive oxygen species (ROS. Several major isoforms are expressed in the liver, including NOX1, NOX2, and NOX4. While the phagocytic NOX2 has been known to play an important role in Kupffer cell and neutrophil phagocytic activity and inflammation, the nonphagocytic NOX homologues are increasingly recognized as key enzymes in oxidative injury and wound healing. In this review, we will summarize the current advances in knowledge on the regulatory pathways of NOX activation, their cellular distribution, and their role in the modulation of redox signaling in liver diseases.

  3. Glucose oxidase immobilization onto carbon nanotube networking

    CERN Document Server

    Karachevtsev, V A; Zarudnev, E S; Karachevtsev, M V; Leontiev, V S; Linnik, A S; Lytvyn, O S; Plokhotnichenko, A M; Stepanian, S G

    2012-01-01

    When elaborating the biosensor based on single-walled carbon nanotubes (SWNTs), it is necessary to solve such an important problem as the immobilization of a target biomolecule on the nanotube surface. In this work, the enzyme (glucose oxidase (GOX)) was immobilized on the surface of a nanotube network, which was created by the deposition of nanotubes from their solution in 1,2-dichlorobenzene by the spray method. 1-Pyrenebutanoic acid succinimide ester (PSE) was used to form the molecular interface, the bifunctional molecule of which provides the covalent binding with the enzyme shell, and its other part (pyrene) is adsorbed onto the nanotube surface. First, the usage of such a molecular interface leaves out the direct adsorption of the enzyme (in this case, its activity decreases) onto the nanotube surface, and, second, it ensures the enzyme localization near the nanotube. The comparison of the resonance Raman (RR) spectrum of pristine nanotubes with their spectrum in the PSE environment evidences the creat...

  4. Characterization of Glucose Oxidase from Penicillium notatum

    Directory of Open Access Journals (Sweden)

    Nabeela Saleem

    2009-01-01

    Full Text Available In the present study glucose oxidase (GOD has been isolated from a culture filtrate of Penicillium notatum. The enzyme was purified by ammonium sulphate precipitation, diethylaminoethyl (DEAE cellulose ion-exchange chromatography and Sephadex gel filtration. This protocol gave 16.47-fold purification and 25 % recovery of the enzyme. The optimum pH and temperature for the activity were 5.4 and 45 °C, respectively. The Km and vmax values for the enzyme were 10.5 mM and 456 U/mg, respectively. A detailed kinetic study of thermal inactivation was carried out. Both enthalpy of activation (ΔH* and entropy of activation (ΔS* decreased at higher temperatures. Moreover, free energy of denaturation (ΔG* increased at higher temperature, making the enzyme thermally stable. A possible explanation for the thermal inactivation of GOD at higher temperatures is also discussed.

  5. Constitutive arsenite oxidase expression detected in arsenic-hypertolerant Pseudomonas xanthomarina S11.

    Science.gov (United States)

    Koechler, Sandrine; Arsène-Ploetze, Florence; Brochier-Armanet, Céline; Goulhen-Chollet, Florence; Heinrich-Salmeron, Audrey; Jost, Bernard; Lièvremont, Didier; Philipps, Muriel; Plewniak, Frédéric; Bertin, Philippe N; Lett, Marie-Claire

    2015-04-01

    Pseudomonas xanthomarina S11 is an arsenite-oxidizing bacterium isolated from an arsenic-contaminated former gold mine in Salsigne, France. This bacterium showed high resistance to arsenite and was able to oxidize arsenite to arsenate at concentrations up to 42.72 mM As[III]. The genome of this strain was sequenced and revealed the presence of three ars clusters. One of them is located on a plasmid and is organized as an "arsenic island" harbouring an aio operon and genes involved in phosphorous metabolism, in addition to the ars genes. Neither the aioXRS genes nor a specific sigma-54-dependent promoter located upstream of aioBA genes, both involved in regulation of arsenite oxidase expression in other arsenite-oxidizing bacteria, could be identified in the genome. This observation is in accordance with the fact that no difference was observed in expression of arsenite oxidase in P. xanthomarina S11, whether or not the strain was grown in the presence of As[III].

  6. Loss of lysyl oxidase-like 3 causes cleft palate and spinal deformity in mice.

    Science.gov (United States)

    Zhang, Jian; Yang, Rui; Liu, Ziyi; Hou, Congzhe; Zong, Wen; Zhang, Aizhen; Sun, Xiaoyang; Gao, Jiangang

    2015-11-01

    In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.

  7. Cell Wall Amine Oxidases: New Players in Root Xylem Differentiation under Stress Conditions.

    Science.gov (United States)

    Ghuge, Sandip A; Tisi, Alessandra; Carucci, Andrea; Rodrigues-Pousada, Renato A; Franchi, Stefano; Tavladoraki, Paraskevi; Angelini, Riccardo; Cona, Alessandra

    2015-07-14

    Polyamines (PAs) are aliphatic polycations present in all living organisms. A growing body of evidence reveals their involvement as regulators in a variety of physiological and pathological events. They are oxidatively deaminated by amine oxidases (AOs), including copper amine oxidases (CuAOs) and flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). The biologically-active hydrogen peroxide (H₂O₂) is a shared compound in all of the AO-catalyzed reactions, and it has been reported to play important roles in PA-mediated developmental and stress-induced processes. In particular, the AO-driven H₂O₂ biosynthesis in the cell wall is well known to be involved in plant wound healing and pathogen attack responses by both triggering peroxidase-mediated wall-stiffening events and signaling modulation of defense gene expression. Extensive investigation by a variety of methodological approaches revealed high levels of expression of cell wall-localized AOs in root xylem tissues and vascular parenchyma of different plant species. Here, the recent progresses in understanding the role of cell wall-localized AOs as mediators of root xylem differentiation during development and/or under stress conditions are reviewed. A number of experimental pieces of evidence supports the involvement of apoplastic H₂O₂ derived from PA oxidation in xylem tissue maturation under stress-simulated conditions.

  8. SK channels mediate NADPH oxidase-independent reactive oxygen species production and apoptosis in granulocytes.

    Science.gov (United States)

    Fay, Alex J; Qian, Xiang; Jan, Yuh Nung; Jan, Lily Yeh

    2006-11-14

    Neutrophils are immune cells that bind to, engulf, and destroy bacterial and fungal pathogens in infected tissue, and their clearance by apoptosis is essential for the resolution of inflammation. Killing involves both oxidative and nonoxidative processes, the oxidative pathway requiring electrogenic production of superoxide by the membrane-bound NADPH oxidase complex. A variety of stimuli, from bacterial chemotactic peptides to complement- or IgG-opsonized microbes, can induce the production of reactive oxygen species (ROS) by neutrophils, presumably by means of NADPH oxidase. We report here that 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of Ca2+-activated potassium channels of small conductance (SK) and intermediate conductance (IK), causes production of superoxide and hydrogen peroxide by neutrophils and granulocyte-differentiated PLB-985 cells. This response can be partially inhibited by the SK blocker apamin, which inhibits a Ca2+-activated K+ current in these cells. Analysis of RNA transcripts indicates that channels encoded by the SK3 gene carry this current. The effects of 1-EBIO and apamin are independent of the NADPH oxidase pathway, as demonstrated by using a PLB-985 cell line lacking the gp91phox subunit. Rather, 1-EBIO and apamin modulate mitochondrial ROS production. Consistent with the enhanced ROS production and K+ efflux mediated by 1-EBIO, we found that this SK opener increased apoptosis of PLB-985 cells. Together, these findings suggest a previously uncharacterized mechanism for the regulation of neutrophil ROS production and programmed cell death.

  9. Cell Wall Amine Oxidases: New Players in Root Xylem Differentiation under Stress Conditions

    Directory of Open Access Journals (Sweden)

    Sandip A. Ghuge

    2015-07-01

    Full Text Available Polyamines (PAs are aliphatic polycations present in all living organisms. A growing body of evidence reveals their involvement as regulators in a variety of physiological and pathological events. They are oxidatively deaminated by amine oxidases (AOs, including copper amine oxidases (CuAOs and flavin adenine dinucleotide (FAD-dependent polyamine oxidases (PAOs. The biologically-active hydrogen peroxide (H2O2 is a shared compound in all of the AO-catalyzed reactions, and it has been reported to play important roles in PA-mediated developmental and stress-induced processes. In particular, the AO-driven H2O2 biosynthesis in the cell wall is well known to be involved in plant wound healing and pathogen attack responses by both triggering peroxidase-mediated wall-stiffening events and signaling modulation of defense gene expression. Extensive investigation by a variety of methodological approaches revealed high levels of expression of cell wall-localized AOs in root xylem tissues and vascular parenchyma of different plant species. Here, the recent progresses in understanding the role of cell wall-localized AOs as mediators of root xylem differentiation during development and/or under stress conditions are reviewed. A number of experimental pieces of evidence supports the involvement of apoplastic H2O2 derived from PA oxidation in xylem tissue maturation under stress-simulated conditions.

  10. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

    Science.gov (United States)

    Gesell, Andreas; Chávez, Maria Luisa Díaz; Kramell, Robert; Piotrowski, Markus; Macheroux, Peter; Kutchan, Toni M

    2011-06-01

    Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

  11. A pH and Redox Dual Responsive 4-Arm Poly(ethylene glycol-block-poly(disulfide histamine Copolymer for Non-Viral Gene Transfection in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Kangkang An

    2014-05-01

    Full Text Available A novel 4-arm poly(ethylene glycol-b-poly(disulfide histamine copolymer was synthesized by Michael addition reaction of poly(ethylene glycol (PEG vinyl sulfone and amine-capped poly(disulfide histamine oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter with almost neutral surface charge (+(5–10 mV. Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment, but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment. The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment. In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0. Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.

  12. Upregulation of Mitochondrial Content in Cytochrome c Oxidase Deficient Fibroblasts.

    Science.gov (United States)

    Kogot-Levin, Aviram; Saada, Ann; Leibowitz, Gil; Soiferman, Devorah; Douiev, Liza; Raz, Itamar; Weksler-Zangen, Sarah

    2016-01-01

    Cytochrome-c-oxidase (COX) deficiency is a frequent cause of mitochondrial disease and is associated with a wide spectrum of clinical phenotypes. We studied mitochondrial function and biogenesis in fibroblasts derived from the Cohen (CDs) rat, an animal model of COX deficiency. COX activity in CDs-fibroblasts was 50% reduced compared to control rat fibroblasts (P<0.01). ROS-production in CDs fibroblasts increased, along with marked mitochondrial fragmentation and decreased mitochondrial membrane-potential, indicating mitochondrial dysfunction. Surprisingly, cellular ATP content, oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were unchanged. To clarify the discrepancy between mitochondrial dysfunction and ATP production, we studied mitochondrial biogenesis and turnover. The content of mitochondria was higher in CDs-fibroblasts. Consistently, AMPK activity and the expression of NRF1-target genes, NRF2 and PGC1-α that mediate mitochondrial biogenesis were increased (P<0.01 vs control fibroblast). In CDs-fibrobalsts, the number of autophagosomes (LC3+ puncta) containing mitochondria in CDs fibroblasts was similar to that in control fibroblasts, suggesting that mitophagy was intact. Altogether, our findings demonstrate that mitochondrial dysfunction and oxidative stress are associated with an increase in mitochondrial biogenesis, resulting in preservation of ATP generation.

  13. Portability of oxidase domains in nonribosomal peptide synthetase modules.

    Science.gov (United States)

    Schneider, Tanya L; Walsh, Christopher T

    2004-12-21

    Oxazole and thiazole rings are present in numerous nonribosomal peptide natural products. Oxidase domains are responsible for catalyzing the oxidation of thiazolines and oxazolines to yield fully aromatic heterocycles. Unlike most domains, the placement of oxidase domains within assembly line modules varies. Noting this tolerance, we investigated the portability of an oxidase domain to a heterologous assembly line. The epimerase domain of PchE, involved in pyochelin biosynthesis, was replaced with the oxidase domain from MtaD, involved in myxothiazol biosynthesis. The chimeric module was expressed in soluble form as a flavin mononucleotide-containing flavoprotein. The functionality of the inserted oxidase domain was assayed within PchE and in transfer of the growing siderophore acyl chain from PchE to the next downstream module. While pyochelin-like product release was not observed downstream, the robust activity of the transplanted oxidase domain and the ability of the chimeric module to produce an advanced intermediate bound to the synthetase underscore the possibility of future engineering within nonribosomal peptide synthetase pathways using oxidase domains.

  14. Structural characterization and regulatory element analysis of the heart isoform of cytochrome c oxidase VIa

    Science.gov (United States)

    Wan, B.; Moreadith, R. W.; Blomqvist, C. G. (Principal Investigator)

    1995-01-01

    In order to investigate the mechanism(s) governing the striated muscle-specific expression of cytochrome c oxidase VIaH we have characterized the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 base pairs (bp), and is comprised of three exons. The 5'-ends of transcripts from the gene are heterogeneous, but the most abundant transcript includes a 5'-untranslated region of 30 nucleotides. When fused to the luciferase reporter gene, the 3.5-kilobase 5'-flanking region of the gene directed the expression of the heterologous protein selectively in differentiated Sol8 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identified a 300-bp fragment sufficient to direct the myotube-specific expression of luciferase in Sol8 cells. The region lacks an apparent TATA element, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mitochondrial proteins are not evident. Mutational analysis, however, identified two cis-elements necessary for the high level expression of the reporter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes suggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several contractile and cytosolic proteins.

  15. Nuclear localization of human spermine oxidase isoforms - possible implications in drug response and disease etiology.

    Science.gov (United States)

    Murray-Stewart, Tracy; Wang, Yanlin; Goodwin, Andrew; Hacker, Amy; Meeker, Alan; Casero, Robert A

    2008-06-01

    The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis.

  16. Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

    Science.gov (United States)

    Zhan, Tao; Zhang, Kai; Chen, Yangyan; Lin, Yongjun; Wu, Gaobing; Zhang, Lili; Yao, Pei; Shao, Zongze; Liu, Ziduo

    2013-01-01

    Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops. PMID:24223901

  17. Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans.

    Science.gov (United States)

    Zhang, Xinxing; Feng, Likui; Chinta, Satya; Singh, Prashant; Wang, Yuting; Nunnery, Joshawna K; Butcher, Rebecca A

    2015-03-31

    Caenorhabditis elegans uses ascaroside pheromones to induce development of the stress-resistant dauer larval stage and to coordinate various behaviors. Peroxisomal β-oxidation cycles are required for the biosynthesis of the fatty acid-derived side chains of the ascarosides. Here we show that three acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, form different protein homo- and heterodimers with distinct substrate preferences. Mutations in the acyl-CoA oxidase genes acox-1, -2, and -3 led to specific defects in ascaroside production. When the acyl-CoA oxidases were expressed alone or in pairs and purified, the resulting acyl-CoA oxidase homo- and heterodimers displayed different side-chain length preferences in an in vitro activity assay. Specifically, an ACOX-1 homodimer controls the production of ascarosides with side chains with nine or fewer carbons, an ACOX-1/ACOX-3 heterodimer controls the production of those with side chains with seven or fewer carbons, and an ACOX-2 homodimer controls the production of those with ω-side chains with less than five carbons. Our results support a biosynthetic model in which β-oxidation enzymes act directly on the CoA-thioesters of ascaroside biosynthetic precursors. Furthermore, we identify environmental conditions, including high temperature and low food availability, that induce the expression of acox-2 and/or acox-3 and lead to corresponding changes in ascaroside production. Thus, our work uncovers an important mechanism by which C. elegans increases the production of the most potent dauer pheromones, those with the shortest side chains, under specific environmental conditions.

  18. Dual Credit/Dual Enrollment and Data Driven Policy Implementation

    Science.gov (United States)

    Lichtenberger, Eric; Witt, M. Allison; Blankenberger, Bob; Franklin, Doug

    2014-01-01

    The use of dual credit has been expanding rapidly. Dual credit is a college course taken by a high school student for which both college and high school credit is given. Previous studies provided limited quantitative evidence that dual credit/dual enrollment is directly connected to positive student outcomes. In this study, predictive statistics…

  19. Direct regulation of cytochrome c oxidase by calcium ions.

    Directory of Open Access Journals (Sweden)

    Tatiana Vygodina

    Full Text Available Cytochrome c oxidase from bovine heart binds Ca(2+ reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+ shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca(2+ on the functional characteristics of cytochrome oxidase was revealed earlier. Here we report that Ca(2+ inhibits cytochrome oxidase activity of isolated bovine heart enzyme by 50-60% with Ki of ∼1 µM, close to Kd of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (∼10 s(-1 or less. No inhibitory effect of Ca(2+ is observed under conventional conditions of cytochrome c oxidase activity assays (turnover number >100 s(-1 at pH 8, which may explain why the effect was not noticed earlier. The inhibition is specific for Ca(2+ and is reversed by EGTA. Na(+ ions that compete with Ca(2+ for binding with the Cation Binding Site, do not affect significantly activity of the enzyme but counteract the inhibitory effect of Ca(2+. The Ca(2+-induced inhibition of cytochrome c oxidase is observed also with the uncoupled mitochondria from several rat tissues. At the same time, calcium ions do not inhibit activity of the homologous bacterial cytochrome oxidases. Possible mechanisms of the inhibition are discussed as well as potential physiological role of Ca(2+ binding with cytochrome oxidase. Ca(2+- binding at the Cation Binding Site is proposed to inhibit proton-transfer through the exit part of the proton conducting pathway H in the mammalian oxidases.

  20. Salmonella induces prominent gene expression in the rat colon

    Directory of Open Access Journals (Sweden)

    Roosing Susanne

    2007-09-01

    Full Text Available Abstract Background Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point. As fructo-oligosaccharides (FOS affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. Results Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase, antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2, inflammation (e.g. calprotectin, oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2 and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9. Furthermore, Salmonella translocation increased serum IFNγ and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap, showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. Conclusion We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in

  1. Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV

    Directory of Open Access Journals (Sweden)

    Dayton Andrew I

    2001-11-01

    Full Text Available Abstract Background Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons. Results We cloned into a Δpre-M/E dengue virus replicon genes for either green fluorescent protein (GFP, HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA. Conclusions Heterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods.

  2. TrmB, a sugar sensing regulator of ABC transporter genes in Pyrococcus furiosus exhibits dual promoter specificity and is controlled by different inducers.

    Science.gov (United States)

    Lee, Sung-Jae; Moulakakis, Christina; Koning, Sonja M; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2005-09-01

    TrmB is the transcriptional repressor for the gene cluster of the trehalose/maltose ABC transporter of the hyperthermophilic archaea Thermococcus litoralis and Pyrococcus furiosus (malE or TM operon), with maltose and trehalose acting as inducers. We found that TrmB (the protein is identical in both organisms) also regulated the transcription of genes encoding a separate maltodextrin ABC transporter in P. furiosus (mdxE or MD operon) with maltotriose, longer maltodextrins and sucrose acting as inducers, but not with maltose or trehalose. In vitro transcription of the malE and the mdxE operons was inhibited by TrmB binding to the different operator sequences. Inhibition of the TM operon was released by maltose and trehalose whereas inhibition of the MD operon was released by maltotriose and larger maltodextrins as well as by sucrose. Scanning mutagenesis of the TM operator revealed the role of the palindromic TACTNNNAGTA sequence for TrmB recognition. TrmB exhibits a broad spectrum of sugar-binding specificity, binding maltose, sucrose, maltotriose and trehalose in decreasing order of affinity, half-maximal binding occurring at 20, 60, 250 and 500 microM substrate concentration respectively. Of all substrates, only maltose shows sigmoidal binding characteristics with a Hill coefficient of 2. As measured by molecular sieve chromatography and cross-linking TrmB behaved as dimer in dilute buffer solution at room temperature. We conclude that TrmB acts as a bifunctional transcriptional regulator acting on two different promoters and being differentially controlled by binding to different sugars. We believe this to represent a novel strategy of prokaryotic transcription regulation.

  3. Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase.

    Science.gov (United States)

    Pastorino, Laura; Dellacasa, Elena; Noor, Mohamed R; Soulimane, Tewfik; Bianchini, Paolo; D'Autilia, Francesca; Antipov, Alexei; Diaspro, Alberto; Tofail, Syed A M; Ruggiero, Carmelina

    2014-01-01

    Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties.

  4. Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase.

    Directory of Open Access Journals (Sweden)

    Laura Pastorino

    Full Text Available Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties.

  5. Dual coil ignition system

    Energy Technology Data Exchange (ETDEWEB)

    Huberts, Garlan J.; Qu, Qiuping; Czekala, Michael Damian

    2017-03-28

    A dual coil ignition system is provided. The dual coil ignition system includes a first inductive ignition coil including a first primary winding and a first secondary winding, and a second inductive ignition coil including a second primary winding and a second secondary winding, the second secondary winding connected in series to the first secondary winding. The dual coil ignition system further includes a diode network including a first diode and a second diode connected between the first secondary winding and the second secondary winding.

  6. Dual energy CT

    DEFF Research Database (Denmark)

    Al-Najami, Issam; Drue, Henrik Christian; Steele, Robert

    2017-01-01

    and inaccurate with existing methods. Dual Energy Computed Tomography (DECT) enables qualitative tissue differentiation by simultaneous scanning with different levels of energy. We aimed to assess the feasibility of DECT in quantifying tumor response to neoadjuvant therapy in loco-advanced rectal cancer. METHODS...... to determine the average quantitative parameters; effective-Z, water- and iodine-concentration, Dual Energy Index (DEI), and Dual Energy Ratio (DER). These parameters were compared to the regression in the resection specimen as measured by the pathologist. RESULTS: Changes in the quantitative parameters...

  7. The Apoplastic Copper AMINE OXIDASE1 Mediates Jasmonic Acid-Induced Protoxylem Differentiation in Arabidopsis Roots.

    Science.gov (United States)

    Ghuge, Sandip A; Carucci, Andrea; Rodrigues-Pousada, Renato A; Tisi, Alessandra; Franchi, Stefano; Tavladoraki, Paraskevi; Angelini, Riccardo; Cona, Alessandra

    2015-06-01

    Polyamines are involved in key developmental processes and stress responses. Copper amine oxidases oxidize the polyamine putrescine (Put), producing an aldehyde, ammonia, and hydrogen peroxide (H2O2). The Arabidopsis (Arabidopsis thaliana) amine oxidase gene At4g14940 (AtAO1) encodes an apoplastic copper amine oxidase expressed at the early stages of vascular tissue differentiation in roots. Here, its role in root development and xylem differentiation was explored by pharmacological and forward/reverse genetic approaches. Analysis of the AtAO1 expression pattern in roots by a promoter::green fluorescent protein-β-glucuronidase fusion revealed strong gene expression in the protoxylem at the transition, elongation, and maturation zones. Methyl jasmonate (MeJA) induced AtAO1 gene expression in vascular tissues, especially at the transition and elongation zones. Early protoxylem differentiation was observed upon MeJA treatment along with Put level decrease and H2O2 accumulation in wild-type roots, whereas Atao1 loss-of-function mutants were unresponsive to the hormone. The H2O2 scavenger N,N(1)-dimethylthiourea reversed the MeJA-induced early protoxylem differentiation in wild-type seedlings. Likewise, Put, which had no effect on Atao1 mutants, induced early protoxylem differentiation in the wild type, this event being counteracted by N,N(1)-dimethylthiourea treatment. Consistently, AtAO1-overexpressing plants showed lower Put levels and early protoxylem differentiation concurrent with H2O2 accumulation in the root zone where the first protoxylem cells with fully developed secondary wall thickenings are found. These results show that the H2O2 produced via AtAO1-driven Put oxidation plays a role in MeJA signaling leading to early protoxylem differentiation in root. © 2015 American Society of Plant Biologists. All Rights Reserved.

  8. Alternative oxidase involvement in Daucus carota somatic embryogenesis.

    Science.gov (United States)

    Frederico, António Miguel; Campos, Maria Doroteia; Cardoso, Hélia Guerra; Imani, Jafargholi; Arnholdt-Schmitt, Birgit

    2009-12-01

    Plant alternative oxidase (AOX) is a mitochondrial inner membrane enzyme involved in alternative respiration. The critical importance of the enzyme during acclimation upon stress of plant cells is not fully understood and is still an issue of intensive research and discussion. Recently, a role of AOX was suggested for the ability of plant cells to change easily its fate upon stress. In order to get new insights about AOX involvement in cell reprogramming, quantitative real-time polymerase chain reaction (PCR) and inhibitor studies were performed during cell redifferentiation and developmental stages of Daucus carota L. somatic embryogenesis. Transcript level analysis shows that D. carota AOX genes (DcAOX1a and DcAOX2a) are differentially expressed during somatic embryogenesis. DcAOX1a shows lower expression levels, being mainly down-regulated, whereas DcAOX2a presented a large up-regulation during initiation of the realization phase of somatic embryogenesis. However, when globular embryos start to develop, both genes are down-regulated, being this state transient for DcAOX2a. In addition, parallel studies were performed using salicylhydroxamic acid (SHAM) in order to inhibit AOX activity during the realization phase of somatic embryogenesis. Embryogenic cells growing in the presence of the inhibitor were unable to develop embryogenic structures and its growth rate was diminished. This effect was reversible and concentration dependent. The results obtained contribute to the hypothesis that AOX activity supports metabolic reorganization as an essential part of cell reprogramming and, thus, enables restructuring and de novo cell differentiation.