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Sample records for acyl-coa oxidase aco

  1. Short-term exposures of fish to perfluorooctane sulfonate: acute effects on fatty acyl-coa oxidase activity, oxidative stress, and circulating sex steroids.

    PubMed

    Oakes, Ken D; Sibley, Paul K; Martin, Jon W; MacLean, Dan D; Solomon, Keith R; Mabury, Scott A; Van Der Kraak, Glen J

    2005-05-01

    This study investigated the effects of exposure to waterborne perfluorooctane sulfonate (PFOS) on oxidative stress and reproductive endpoints in fish. Exposures utilized species commonly used in toxicological testing, including the fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss), as well as relatively insensitive taxa such as creek chub (Semotilus atromaculatus), spottail shiner (Notropis hudsonius), and white sucker (Catostomus commersoni). In all fish species, short-term (14-28 d) exposure to PFOS produced only modest mortality at concentrations consistent with environmental spill scenarios. However, PFOS consistently increased hepatic fatty acyl-CoA oxidase activity and increased oxidative damage, as quantified using the 2-thiobarbituric acid-reactive substances assay. Plasma testosterone, 11-ketotestosterone, and 17beta-estradiol titers were often elevated with PFOS exposure. Vitellogenin, the egg yolk precursor protein, was occasionally altered in the plasma with PFOS exposure, but responses varied with maturity. Oviposition frequency and egg deposition in fathead minnow were not significantly impaired with PFOS exposure, despite a trend toward progressive impairment with increasing exposure concentrations. Although short-term PFOS exposure produced significant impacts on biochemical and reproductive endpoints in fish at concentrations consistent with environmental spills, the impact of long-term exposure to environmentally relevant concentrations of PFOS is unclear.

  2. 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.

    PubMed

    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.

  3. Pear ACO genes encoding putative 1-aminocyclopropane-1-carboxylate oxidase homologs are functionally expressed during fruit ripening and involved in response to salicylic acid.

    PubMed

    Shi, Hai-Yan; Zhang, Yu-Xing

    2012-10-01

    1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final reaction of the ethylene biosynthetic pathway, converting ACC into ethylene. Past studies have shown a possible link between ACC oxidase and salicylic acid during fruit ripening in pear, but the relationship has received no more than modest study at the gene expression level. In this study, two cDNA clones encoding putative ACC oxidase, PpACO1 and PpACO2, were isolated from a cDNA library constructed by our own laboratory and produced using mRNA from mesocarp of pear (Pyrus pyrifolia Nakai. cv.Whangkeumbae). One cDNA clone, designated PpACO1 (GenBank accession No. JN807390), comprised an open reading frame of 945 bp encoding a protein of 314 amino acids. The other cDNA, designated PpACO2 (GenBank accession No. JN807392), encodes a protein with 322 amino acids that shares high similarity with the known plant ACOs. Using PCR amplification techniques, two genomic clones corresponding to PpACO1 and PpACO2 were isolated and shown to contain independently three introns with typical GT/AG boundaries defining the splice junctions. The PpACO1 gene product shared 99 % identity with an ACC oxidase from pear (Pyrus × bretschneideri Rehd.cv.Yali), and phylogenetic analyses clearly placed the gene product in the ACC oxidase cluster of the pear 2-oxoglutarate-dependent dioxygenase superfamily tree. Quantitative RT-PCR analysis indicated that the two PpACO genes are differentially expressed in pear tissues. PpACO1 and PpACO2 were predominantly expressed in fruit. The transcripts of PpACO1 were accumulated at relatively low levels in early fruit, but strongly high levels in fruit ripening and senescence stages, while the transcripts of PpACO2 were accumulated at higher levels in early fruit and much lower levels with further fruit cell development than the transcripts of PpACO1. In addition, PpACO1 gene was down-regulated in fruit by salicylic acid (SA). Nevertheless, PpACO2 gene was dramatically up-regulated in

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

    PubMed

    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.

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

    PubMed Central

    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. PMID

  6. Cloning and expression of the 1-aminocyclopropane-1-carboxylic oxidase gene from Agrostis stolonifera.

    PubMed

    Xiao, G Z; Li, L J; Teng, K; Chao, Y H; Han, L B

    2016-11-03

    A gene encoding 1-aminocyclopropane-1-carboxylic oxidase (ACO), which catalyzes the terminal step in ethylene biosynthesis, was isolated from Agrostis stolonifera. The AsACO gene is composed of 975 bp, encoding 324 amino acids. Three exons interspersed by two introns form AsACO gDNA. A BLAST search of the nucleotide sequence revealed a high level of similarity (79-91%) between AsACO and ACO genes of other plants. A phylogenetic tree was constructed via BLAST in the NCBI, and revealed the highest homology with wheat TaACO. The calculated molecular mass and predicted isoelectric point of AsACO were 36.25 and 4.89 kDa, respectively. Analysis of subcellular localization revealed that AsACO is located in the nucleus and cytoplasm. The Fe(II)-binding cofactors and cosubstrate were identified, pertaining to the ACO family. The expression patterns of AsACO were determined by quantitative real time PCR. AsACO expression was highest in the stem, and was strongly up-regulated in response to ethephon, methyl jasmonate, salicylic acid, and cold temperature, but down-regulated in response to drought and NaCl treatment. The protein encoded by AsACO exhibited ACC oxidase activity in vitro. Taken together, these findings suggest that AsACO contains domains common to the ACO family, and is induced in response to exogenous hormones. Conversely, some abiotic stress conditions can inhibit AsACO expression.

  7. Characterization and expression profiles of MaACS and MaACO genes from mulberry (Morus alba L.).

    PubMed

    Liu, Chang-ying; Lü, Rui-hua; Li, Jun; Zhao, Ai-chun; Wang, Xi-ling; Diane, Umuhoza; Wang, Xiao-hong; Wang, Chuan-hong; Yu, Ya-sheng; Han, Shu-mei; Lu, Cheng; Yu, Mao-de

    2014-07-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) are encoded by multigene families and are involved in fruit ripening by catalyzing the production of ethylene throughout the development of fruit. However, there are no reports on ACS or ACO genes in mulberry, partly because of the limited molecular research background. In this study, we have obtained five ACS gene sequences and two ACO gene sequences from Morus Genome Database. Sequence alignment and phylogenetic analysis of MaACO1 and MaACO2 showed that their amino acids are conserved compared with ACO proteins from other species. MaACS1 and MaACS2 are type I, MaACS3 and MaACS4 are type II, and MaACS5 is type III, with different C-terminal sequences. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) expression analysis showed that the transcripts of MaACS genes were strongly expressed in fruit, and more weakly in other tissues. The expression of MaACO1 and MaACO2 showed different patterns in various mulberry tissues. MaACS and MaACO genes demonstrated two patterns throughout the development of mulberry fruit, and both of them were strongly up-regulated by abscisic acid (ABA) and ethephon.

  8. ACOs Holding Commercial Contracts Are Larger And More Efficient Than Noncommercial ACOs

    PubMed Central

    Peiris, David; Phipps-Taylor, Madeleine C.; Stachowski, Courtney A.; Kao, Lee-Sien; Shortell, Stephen M.; Lewis, Valerie A.; Rosenthal, Meredith B.; Colla, Carrie H.

    2016-01-01

    Accountable care organizations (ACOs) have diverse contracting arrangements and have displayed wide variations in their performance. Using data from national surveys of 399 ACOs, we examined differences between the 228 commercial ACOs (those with commercial payer contracts) and the 171 noncommercial ACOs (those with only public contracts, such as with Medicare or Medicaid). Commercial ACOs were significantly larger and more integrated with hospitals, and had lower benchmark expenditures and higher quality scores, compared to noncommercial ACOs. Among all of the ACOs, there was low uptake of quality and efficiency activities. However, commercial ACOs reported more use of disease monitoring tools, patient satisfaction data, and quality improvement methods than did noncommercial ACOs. Few ACOs reported having high-level performance monitoring capabilities. About two-thirds of the ACOs had established processes for distributing any savings accrued, and these ACOs allocated approximately the same amount of savings to the ACOs themselves, participating member organizations, and physicians. Our findings demonstrate that ACO delivery systems remain at a nascent stage. Structural differences between commercial and noncommercial ACOs are important factors to consider as public policy efforts continue to evolve. PMID:27702959

  9. Including Language Access into Medicaid ACO Design.

    PubMed

    Gershon, Rachel; Morris, Lisa; Ferguson, Warren

    2016-09-01

    Quality health care relies upon communication in a patient's preferred language. Language access in health care occurs when individuals are: (1) Welcomed by providers regardless of language ability; and (2) Offered quality language services as part of their care. Federal law generally requires access to health care and quality language services for deaf and Limited English Proficient (LEP) patients in health care settings, but these patients still find it hard to access health care and quality language services.Meanwhile, several states are implementing Medicaid Accountable Care Organization (ACO) initiatives to reduce health care costs and improve health care quality. Alternative payment methods used in these initiatives can give Accountable Care Organizations more flexibility to design linguistically accessible care, but they can also put ACOs at increased financial risk for the cost of care. If these new payment methods do not account for differences in patient language needs, ACO initiatives could have the unintended consequence of rewarding ACOs who do not reach out to deaf and LEP communities or offer quality language services.We reviewed public documents related to Medicaid ACO initiatives in six states. Some of these documents address language access. More could be done, however, to pay for language access efforts. This article describes Medicaid ACO initiatives and explores how different payment tools could be leveraged to reward ACOs for increased access to care and quality language services. We find that a combination of payment tools might be helpful to encourage both access and quality.

  10. ACO model should encourage efficient care delivery.

    PubMed

    Toussaint, John; Krueger, David; Shortell, Stephen M; Milstein, Arnold; Cutler, David M

    2015-09-01

    The independent Office of the Actuary for CMS certified that the Pioneer ACO model has met the stringent criteria for expansion to a larger population. Significant savings have accrued and quality targets have been met, so the program as a whole appears to be working. Ironically, 13 of the initial 32 enrollees have left. We attribute this to the design of the ACO models which inadequately support efficient care delivery. Using Bellin-ThedaCare Healthcare Partners as an example, we will focus on correctible flaws in four core elements of the ACO payment model: finance spending and targets, attribution, and quality performance.

  11. 42 CFR 425.316 - Monitoring of ACOs.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... satisfy the eligibility and program requirements under this part, CMS monitors and assesses the... to monitor and assess the performance of ACOs, ACO participants, and ACO providers/suppliers...). (b) Monitoring ACO avoidance of at-risk beneficiaries. (1) CMS may use one or more of the...

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

    PubMed Central

    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

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

    PubMed

    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.

  14. Certain Organizational Characteristics Affect ACO Preventive Care Quality Performance.

    PubMed

    Ticse, Caroline

    2016-06-01

    Key findings. (1) ACOs at provider workforce extremes--few primary care providers or many specialists--performed worse on measures of preventive care quality relative to those with more PCPs and fewer specialists. (2) Upfront investment in ACO formation is associated with higher performance in preventive care quality. (3) ACOs with a higher proportion of minority beneficiaries performed worse on disease prevention measures than did ACOs with a lower proportion of minority beneficiaries. (4) ACOs facing barriers to quality performance may benefit from organizational characteristics such as electronic health record capabilities and hospital inclusion in the ACO.

  15. Cloning, identification and expression analysis of ACC oxidase gene involved in ethylene production pathway.

    PubMed

    Jafari, Zohreh; Haddad, Raheem; Hosseini, Ramin; Garoosi, Ghasemali

    2013-02-01

    1-aminocyclopropane-1-carboxylic acid oxidase (ACO) enzyme is a member of the Fe II-dependent family of oxidases/oxygenases which require Fe(2+) as a cofactor, ascorbate as a cosubstrate and CO(2) as an activator. This enzyme catalyses the terminal step in the plant signaling of ethylene biosynthetic pathway. A 948 bp fragment of the ACO1 gene cDNA sequence was cloned from tomato (Lycopersicon esculentum) fruit tissues by using reverse transcriptase-polymerase chain reaction (RT-PCR) with two PCR primers designed according to the sequence of a tomato cDNA clone (X58273). The BLAST search showed a high level of similarity (77-98 %) between ACO1 and ACO genes of other plants. The calculated molecular mass and predicted isoelectric point of LeACO1 were 35.8 kDa and 5.13, respectively. The three-dimensional structure studies illustrated that the LeACO1 protein folds into a compact jelly-roll motif comprised of 8 α-helices, 12 β-strands and several long loops. The cosubstrate was located in a cofactor-binding pocket referred to as a 2-His-1-carboxylate facial triad. Semi-quantitative RT-PCR analysis of gene expression revealed that the LeACO1 was expressed in fruit tissues at different ripening stages.

  16. High fat fed heart failure animals have enhanced mitochondrial function and acyl-coa dehydrogenase activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously shown that administration of high fat in heart failure (HF) increased mitochondrial respiration and did not alter left ventricular (LV) function. PPARalpha is a nuclear transcription factor that activates expression of genes involved in fatty acid uptake and utilization. We hypoth...

  17. Isolation and characterization of Aconitate hydratase 4 (Aco4) from soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aconitase catalyzes the reversible isomerization of two tricarboxylic acids citrate and isocitrate during the Krebs cycle. Five aconitase genes namely Aco1, Aco2, Aco3, Aco4 and Aco5 have been identified in soybean. Previously, Aco4 was mapped on chromosome 11. The purpose of this investigation was ...

  18. ACC oxidase genes expressed in the wood-forming tissues of loblolly pine (Pinus taeda L.) include a pair of nearly identical paralogs (NIPs).

    PubMed

    Yuan, S; Wang, Y; Dean, J F D

    2010-03-15

    1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final reaction of the ethylene biosynthetic pathway, converting the unusual cyclic amino acid, ACC, into ethylene. Past studies have shown a possible link between ethylene and compression wood formation in conifers, but the relationship has received no more than modest study at the gene expression level. In this study, a cDNA clone encoding a putative ACC oxidase, PtACO1, was isolated from a cDNA library produced using mRNA from lignifying xylem of loblolly pine (Pinus taeda) trunk wood. The cDNA clone comprised an open reading frame of 1461 bp encoding a protein of 333 amino acids. Using PCR amplification techniques, a genomic clone corresponding to PtACO1 was isolated and shown to contain three introns with typical GT/AG boundaries defining the splice junctions. The PtACO1 gene product shared 70% identity with an ACC oxidase from European white birch (Betula pendula), and phylogenetic analyses clearly placed the gene product in the ACC oxidase cluster of the Arabidopsis thaliana 2-oxoglutarate-dependent dioxygenase superfamily tree. The PtACO1 sequence was used to identify additional ACC oxidase clones from loblolly pine root cDNA libraries characterized as part of an expressed sequence tag (EST) discovery project. The PtACO1 sequence was also used to recover additional paralogous sequences from genomic DNA, one of which (PtACO2) turned out to be >98% identical to PtACO1 in the nucleotide coding sequence, leading to its classification as a "nearly identical paralog" (NIP). Quantitative PCR analyses showed that the expression level of PtACO1-like transcripts varied in different tissues, as well as in response to hormonal treatments and bending. Possible roles for PtACO1 in compression wood formation in loblolly pine and the discovery of its NIP are discussed in light of these results.

  19. Silencing of ACO decreases reproduction and energy metabolism in triazophos-treated female brown plant hoppers, Nilaparvata lugens Stål (Hemiptera: Delphacidae).

    PubMed

    Liu, Zong-Yu; Jiang, Yi-Ping; Li, Lei; You, Lin-Lin; Wu, You; Xu, Bin; Ge, Lin-Quan; Wu, Jin-Cai

    2016-03-01

    The brown plant hopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a major pest affecting rice in Asia, and outbreaks of this pest are closely linked to pesticide-induced stimulation of reproduction. Therefore, the BPH is a classic example of a resurgent pest. However, the effects of different genes on the regulation of pesticide-induced reproductive stimulation in the BPH are unclear. In this study, the regulatory effects of acyl-coenzyme A oxidase (ACO) on the reproduction and biochemistry of the BPH were investigated with gene silencing. The number of eggs laid per female by triazophos (TZP)+dsACO BPH females was significantly lower than those of TZP-treated (without ACO silencing) or TZP+GFP females (negative control), with the number of eggs decreasing by 30.8% (from 529.5 to 366.3) and 32.0% (from 540.5 to 366.3), respectively. The preoviposition period, oviposition period, and longevity of the TZP-treated females were also influenced by dsACO treatment. Additionally, the amounts of crude fat, protein, and some fatty acids (oleic acid, palmitic acid, linoleic acid, stearic acid, and myristoleic acid) in TZP+dsACO females were significantly lower than in TZP-treated females. Thus, ACO is one of the key genes regulating the TZP-induced stimulation of reproduction in BPH females.

  20. Asthma-COPD overlap syndrome (ACOS): A diagnostic challenge.

    PubMed

    Tho, Nguyen Van; Park, Hye Yun; Nakano, Yasutaka

    2016-04-01

    Asthma-chronic obstructive pulmonary disease (COPD) overlap syndrome (ACOS) is characterized by persistent airflow limitation with several features usually associated with asthma and several features usually associated with COPD. ACOS may be a special phenotype of a spectrum of chronic obstructive airway diseases, in which asthma and COPD are at the two opposite ends. The prevalence of ACOS varies considerably due to differing criteria being applied for diagnosis. Patients with ACOS utilize a large proportion of medical resources. They are associated with more frequent adverse outcomes than those with asthma or COPD alone. ACOS is currently a diagnostic challenge for physicians because there are no specific biomarkers to differentiate ACOS from asthma or COPD. The approach to diagnosing ACOS depends on the population from which the patient originated. The management of ACOS should be individualized to ensure the most effective treatment with minimal side effects. In this paper, we review the diagnostic criteria of ACOS used in previous studies, propose practical approaches to diagnosing and managing ACOS and raise some research questions related to ACOS.

  1. Isolation of an 1-aminocyclopropane-1-carboxylate oxidase gene from mulberry (Morus alba L.) and analysis of the function of this gene in plant development and stresses response.

    PubMed

    Pan, Gang; Lou, Chengfu

    2008-07-31

    Mulberry (Morus alba) is an important crop tree involved in sericulture and pharmaceuticals. To further understand the development and the environmental adaptability mechanism of mulberry, a cDNA of the gene MaACO1 encoding 1-aminocyclopropane-1-carboxylate oxidase was isolated from mulberry. This was used to investigate stress-responsive expression in mulberry. Developmental expression of ACC oxidase in mulberry leaves and spatial expression in mulberry flowers were also investigated. Damage and low-temperature treatment promoted the expression of MaACO1 in mulberry. In leaves, expression of the MaACO1 gene increased in cotyledons and the lowest leaves with leaf development, but showed reduced levels in emerging leaves. In flowers, the pollinated stigma showed the highest expression level, followed by the unpollinated stigma, ovary, and immature flowers. These results suggest that high MaACO1 expression may be predominantly associated with tissue aging or senescence in mulberry.

  2. Methyl jasmonate-induced defense responses are associated with elevation of 1-aminocyclopropane-1-carboxylate oxidase in Lycopersicon esculentum fruit.

    PubMed

    Yu, Mengmeng; Shen, Lin; Zhang, Aijun; Sheng, Jiping

    2011-10-15

    It has been known that methyl jasmonate (MeJA) interacts with ethylene to elicit resistance. In green mature tomato fruits (Lycopersicon esculentum cv. Lichun), 0.02mM MeJA increased the activity of 1-aminocyclopropane-1-carboxylate oxidase (ACO), and consequently influenced the last step of ethylene biosynthesis. Fruits treated with a combination of 0.02 MeJA and 0.02 α-aminoisobutyric acid (AIB, a competitive inhibitor of ACO) exhibited a lower ethylene production comparing to that by 0.02mM MeJA alone. The increased activities of defense enzymes and subsequent control of disease incidence caused by Botrytis cinerea with 0.2mM MeJA treatment was impaired by AIB as well. A close relationship (P<0.05) was found between the activity alterations of ACO and that of chitinase (CHI) and β-1,3-glucanase (GLU). In addition, this study further detected the changes of gene expressions and enzyme kinetics of ACO to different concentrations of MeJA. LeACO1 was found the principal member from the ACO gene family to respond to MeJA. Accumulation of LeACO1/3/4 transcripts followed the concentration pattern of MeJA treatments, where the largest elevations were reached by 0.2mM. For kinetic analysis, K(m) values of ACO stepped up during the experiment and reached the maximums at 0.2mM MeJA with ascending concentrations of treatments. V(max) exhibited a gradual increase from 3h to 24h, and the largest induction appeared with 1.0mM MeJA. The results suggested that ACO is involved in MeJA-induced resistance in tomato, and the concentration influence of MeJA on ACO was attributable to the variation of gene transcripts and enzymatic properties.

  3. The Accountable Care Organization (ACO) model: building blocks for success.

    PubMed

    Lowell, Kristina Hanson; Bertko, John

    2010-01-01

    The Accountable Care Organization (ACO) model has received significant attention among policymakers and leaders in the healthcare community in the context of the ongoing debate over health reform, not only because of the unsustainable path on which the country now finds itself but also because it directly focuses on what must be a key goal of the healthcare system: higher value. The model offers a promising approach for achieving this goal. This article provides an overview of the ACO model and its role in the current policy context, highlights the key elements that will be common to all ACOs, and provides details of several challenges that may arise throughout the implementation process, including a host of technical, legal, and operational challenges. These challenges range from issues such as the organizational form and management of the ACO to analytic challenges such as the calculation of spending benchmarks and the selection of quality measures.

  4. Characterizing the population of Asteroids in Cometary Orbits (ACOs)

    NASA Astrophysics Data System (ADS)

    Tancredi, Gonzalo; Licandro, Javier; Alí-Lagoa, Victor; Martino, Silvia; Vieira Monteiro, Filipe; Silva, Jose Sergio; Lazzaro, Daniela

    2015-08-01

    The classification criterion between asteroids and comets has evolved in recent decades, but the main phenomenological distinction remains unchanged: comets are active objects as they present gas and dust ejection from the surface at some point of their orbits, while asteroids are inert objects as they do not show any kind of large scale gas and dust ejection.To identify the transitional objects several classification schemes based on the orbital elements have been used. They are usually based on the Tisserand’s parameter (TJ). Tancredi (2014) presents a much more restrictive criterion to identify ACOs that ensured that the objects have a dynamical evolution similar to the population of periodic comets. After applying the criteriaa to the sample of over half a million asteroids already discovered, we obtain 316 ACOs that are further classified in subclasses similar to the cometary classification: 203 objects belong to the Jupiter Family group; 72 objects are classified as Centaurs; and 56 objects have Halley Type Orbits (also known as Damocloids). These are the best-known extinct/dormant comets candidates from a dynamical point of view.We study the physical properties of this sample of ACOs. Two results will be presented:- We look for the ACOs detected by the NASA’s WISE and by fitting a thermal model to their observations, we derive: the effective diameter, beaming parameter and the visible geometric albedo, using the method described in Al-Lagoa et al (2013). We obtain these parameters for 37 of 203 ACOs in JFC orbits and 13 of 56 Damocloids. We also compute the Cumulative Size Distribution (CSDs) of these populations and compare them with the CSDs of JF Comets and Centaurs.- We have been monitoring the observable ACOs since 12/2014 up to 06/2015. Every other month we select all the ACOs with elongations >90deg and estimated magnitudes V<21. We try to observe them with the 1m IMPACTON telescope of the Observatório Astronômico do Sertão de Itaparica (OASI

  5. 1-Aminocyclopropane-1-Carboxylate Oxidase Activity Limits Ethylene Biosynthesis in Rumex palustris during Submergence

    PubMed Central

    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

  6. Multiobjective optimization of evacuation routes in stadium using superposed potential field network based ACO.

    PubMed

    Kou, Jialiang; Xiong, Shengwu; Fang, Zhixiang; Zong, Xinlu; Chen, Zhong

    2013-01-01

    Multiobjective evacuation routes optimization problem is defined to find out optimal evacuation routes for a group of evacuees under multiple evacuation objectives. For improving the evacuation efficiency, we abstracted the evacuation zone as a superposed potential field network (SPFN), and we presented SPFN-based ACO algorithm (SPFN-ACO) to solve this problem based on the proposed model. In Wuhan Sports Center case, we compared SPFN-ACO algorithm with HMERP-ACO algorithm and traditional ACO algorithm under three evacuation objectives, namely, total evacuation time, total evacuation route length, and cumulative congestion degree. The experimental results show that SPFN-ACO algorithm has a better performance while comparing with HMERP-ACO algorithm and traditional ACO algorithm for solving multi-objective evacuation routes optimization problem.

  7. Molecular Evolution and Expression Divergence of the Aconitase (ACO) Gene Family in Land Plants

    PubMed Central

    Wang, Yi-Ming; Yang, Qi; Liu, Yan-Jing; Yang, Hai-Ling

    2016-01-01

    Aconitase (ACO) is a key enzyme that catalyzes the isomerization of citrate to isocitrate in the tricarboxylic acid (TCA) and glyoxylate cycles. The function of ACOs has been well studied in model plants, such as Arabidopsis. In contrast, the evolutionary patterns of the ACO family in land plants are poorly understood. In this study, we systematically examined the molecular evolution and expression divergence of the ACO gene family in 12 land plant species. Thirty-six ACO genes were identified from the 12 land plant species representing the four major land plant lineages: Bryophytes, lycophytes, gymnosperms, and angiosperms. All of these ACOs belong to the cytosolic isoform. Three gene duplication events contributed to the expansion of the ACO family in angiosperms. The ancestor of angiosperms may have contained only one ACO gene. One gene duplication event split angiosperm ACOs into two distinct clades. Two clades showed a divergence in selective pressure and gene expression patterns. The cis-acting elements that function in light responsiveness were most abundant in the promoter region of the ACO genes, indicating that plant ACO genes might participate in light regulatory pathways. Our findings provide comprehensive insights into the ACO gene family in land plants. PMID:28018410

  8. Cloning and expression of 1-aminocyclopropane-1-carboxylate oxidase cDNA induced by thidiazuron during somatic embryogenesis of alfalfa (Medicago sativa).

    PubMed

    Feng, Bi-Hong; Wu, Bei; Zhang, Chun-Rong; Huang, Xia; Chen, Yun-Feng; Huang, Xue-Lin

    2012-01-15

    Embryogenic callus (EC) induced from petioles of alfalfa (Medicago sativa L. cv. Jinnan) on B5h medium turned green, compact and non-embryogenic when the kinetin (KN) in the medium was replaced partially or completely by thidiazuron (TDZ). The application of CoCl₂, which is an inhibitor of 1-aminocyclopropane-1-carboxylate oxidase (ACO), counteracted the effect of TDZ. Ethylene has been shown to be involved in the modulation of TDZ-induced morphogenesis responses. However, very little is known about the genes involved in ethylene formation during somatic embryogenesis (SE). To investigate whether ethylene mediated by ACO is involved in the effect of TDZ on inhibition of embryogenic competence of the alfalfa callus. In this study we cloned full-length ACO cDNA from the alfalfa callus, named MsACO, and observed changes in this gene expression during callus formation and induction of SE under treatment with TDZ or TDZ plus CoCl₂. RNA blot analysis showed that during the EC subcultural period, the expression level of MsACO in EC was significantly increased on the 2nd day, rose to the highest level on the 8th day and remained at this high level until the 21st day. However, the ACO expression in the TDZ (0.93 μM)-treated callus was higher than in the EC especially on the 8th day. Moreover the ACO expression level increased with increasing TDZ concentration during the subcultural/maintenance period of the callus. It is worth noting that comparing the treatment with TDZ alone, the treatment with 0.93 μM TDZ plus 50 μM CoCl₂ reduced both of the ACO gene expressions and ACO activity in the treated callus. These results indicate that the effect of TDZ could be counteracted by CoCl₂ either on the ACO gene expression level or ACO activity. Thus, a TDZ inhibitory effect on embryogenic competence of alfalfa callus could be mediated by ACO gene expression.

  9. Mechanistic determinates of the acute coagulopathy of trauma (ACoT) in patients requiring emergency surgery

    PubMed Central

    Sixta, Sherry L; Hatch, Quinton M; Matijevic, Nena; Wade, Charles E; Holcomb, John B; Cotton, Bryan A

    2012-01-01

    Introduction: The development of acute coagulopathy of trauma (ACoT) is associated with a significant increase in mortality. However, the contributory mechanisms behind ACoT have yet to be clearly defined. The purpose of this study was to evaluate the influence of multiple variables, including base deficit and injury severity, on development of ACoT within a subset of critically ill trauma patients. Methods: A retrospective review of all trauma laparotomies between 01/2004-12/2009 was performed. ACoT (+) was defined as an arrival INR ≥1.5, ACoT (-) defined as INR<1.5. Univariate and multivariate analyses were performed. Results: Of 1218 patients, 337 (27%) were ACoT (+) and 881 (73%) were ACoT (-) upon presentation. Groups were similar in demographics, ED fluid administration, GCS scores, and admission temperatures. Admission base deficit (8.5 vs. 4, p<0.001) and ISS (median 25 vs. 16, p<0.001) were higher in the ACoT (+) group, as were intra-operative RBC (median 4 vs. 0 U) and plasma (3 vs. 0 U) transfusions; both p<0.001. Multiple-linear regression revealed INR values were independently associated with arrival base deficit and pre-hospital fluid volumes (both p<0.001). On logistic regression, the development of ACoT (+) was associated with base deficit (OR 0.92, p=0.013) as well as ISS (OR 1.05, p<0.001). However, blunt mechanism alone was not an independent predictor of ACoT. Conclusion: The current study revealed that ACoT is independently associated with both shock (base deficit) and tissue injury. Additionally, tissue injury is a significant contributor to the development of early ACoT regardless of blunt or penetrating mechanism. PMID:23272297

  10. First national survey of ACOs finds that physicians are playing strong leadership and ownership roles.

    PubMed

    Colla, Carrie H; Lewis, Valerie A; Shortell, Stephen M; Fisher, Elliott S

    2014-06-01

    The extent to which physicians lead, own, and govern accountable care organizations (ACOs) is unknown. However, physicians' involvement in ACOs will influence how clinicians and patients perceive the ACO model, how effective these organizations are at improving quality and costs, and how future ACOs will be organized. From October 2012 to May 2013 we fielded the National Survey of Accountable Care Organizations, the first such survey of public and private ACOs. We found that 51 percent of ACOs were physician-led, with another 33 percent jointly led by physicians and hospitals. In 78 percent of ACOs, physicians constituted a majority of the governing board, and physicians owned 40 percent of ACOs. The broad reach of physician leadership has important implications for the future evolution of ACOs. It seems likely that the challenge of fundamentally changing care delivery as the country moves away from fee-for-service payment will not be accomplished without strong, effective leadership from physicians.

  11. 42 CFR 425.306 - Participation agreement and exclusivity of ACO participant TINs.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... participant TINs. 425.306 Section 425.306 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT... participant TINs. (a) For purposes of the Shared Savings Program, each ACO participant TIN is required to commit to a participation agreement with CMS. (b) Each ACO participant TIN upon which...

  12. 42 CFR 425.306 - Participation agreement and exclusivity of ACO participant TINs.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... participant TINs. 425.306 Section 425.306 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT... participant TINs. (a) For purposes of the Shared Savings Program, each ACO participant TIN is required to commit to a participation agreement with CMS. (b) Each ACO participant TIN upon which...

  13. 42 CFR 425.306 - Participation agreement and exclusivity of ACO participant TINs.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... participant TINs. 425.306 Section 425.306 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT... participant TINs. (a) For purposes of the Shared Savings Program, each ACO participant TIN is required to commit to a participation agreement with CMS. (b) Each ACO participant TIN upon which...

  14. Report on the 10th International Conference of the Asian Clinical Oncology Society (ACOS 2012).

    PubMed

    Kim, Yeul Hong; Yang, Han-Kwang; Kim, Tae Won; Lee, Jung Shin; Seong, Jinsil; Lee, Woo Yong; Ahn, Yong Chan; Lim, Ho Yeong; Won, Jong-Ho; Park, Kyong Hwa; Cho, Kyung Sam

    2013-04-01

    The 10th International Conference of the Asian Clinical Oncology Society (ACOS 2012) in conjunction with the 38th Annual Meeting of the Korean Cancer Association, was held on June 13 to 15 (3 days) 2012 at COEX Convention and Exhibition Center in Seoul, Korea. ACOS has a 20-year history starting from the first conference in Osaka, Japan, which was chaired by Prof. Tetsuo Taguchi and the ACOS conferences have since been conducted in Asian countries every 2 years. Under the theme of "Work Together to Make a Difference for Cancer Therapy in Asia", the 10th ACOS was prepared to discuss various subjects through a high-quality academic program, exhibition, and social events. The ACOS 2012 Committee was composed of the ACOS Organizing Committee, Honorary Advisors, Local Advisors, and ACOS 2012 Organizing Committee. The comprehensive academic program had a total of 92 sessions (3 Plenary Lectures, 1 Award Lectures, 1 Memorial Lectures, 9 Special Lectures, 15 Symposia, 1 Debate & Summary Sessions, 1 Case Conferences, 19 Educational Lectures, 1 Research & Development Session, 18 Satellite Symposia, 9 Meet the Professors, 14 Oral Presentations) and a total 292 presentations were delivered throughout the entire program. Amongst Free Papers, 462 research papers (110 oral presentations and 352 poster presentations) were selected to be presented. This conference was the largest of all ACOS conferences in its scale with around 1,500 participants from 30 countries. Furthermore, despite strict new financial policies and requirements governing fundraising alongside global economic stagnation, a total of 14 companies participated as sponsors and an additional 35 companies purchased 76 exhibition booths. Lastly, the conference social events provided attendees with a variety of opportunities to experience and enjoy Korea's rich culture and traditions during the Opening Ceremony, Welcome Reception, Invitee Dinner, Banquet, and Closing Ceremony. Overall, ACOS 2012 reinforced and promoted

  15. Succinate oxidase in Neurospora.

    PubMed

    West, D J; Woodward, D O

    1973-02-01

    Two kinetically distinct states of succinate oxidase have been detected in the mitochondria of Neruospora crassa. One state has a K(m) for succinate of 4.1 x 10(-3)m, and the other has a K(m) for succinate of 3.5 x 10(-4)m. The high K(m) state was found in freshly extracted mitochondria from either 20- or 72-hr mycelium. However, the succinate oxidase activity in mitochondria from 20-hr mycelium rapidly deteriorated in vitro, leaving a stable residual activity with the lower K(m) for succinate. Adenosine triphosphate (ATP) plus Mg(2+) stabilized the high K(m) state in these preparations. The high K(m) state of succinate oxidase was further characterized by a two- to threefold increase in activity over the pH range 6.6 to 8.0 and by classical competitive inhibition by fumarate and malonate. By contrast, the low K(m) state of succinate oxidase showed a relatively flat response to pH over the range 6.6 to 8.0 and a nonclassical pattern of inhibition by fumarate and malonate, as shown by nonlinear plots of reciprocal velocity versus reciprocal substrate concentration in the presence of inhibitor or reciprocal velocity versus inhibitor concentration at fixed substrate concentrations. The relationship of mycelial age to the in vitro stability of succinate oxidase is considered with reference to probable changes in the relative pool sizes of extra- and intramitochondrial ATP in response to changes in the rate of glycolysis.

  16. 1-Aminocyclopropane-1-Carboxylate Oxidase Induction in Tomato Flower Pedicel Phloem and Abscission Related Processes Are Differentially Sensitive to Ethylene

    PubMed Central

    Chersicola, Marko; Kladnik, Aleš; Tušek Žnidarič, Magda; Mrak, Tanja; Gruden, Kristina; Dermastia, Marina

    2017-01-01

    Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0–12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on

  17. Hospitals Participating In ACOs Tend To Be Large And Urban, Allowing Access To Capital And Data.

    PubMed

    Colla, Carrie H; Lewis, Valerie A; Tierney, Emily; Muhlestein, David B

    2016-03-01

    Relationships between physicians and hospitals have changed considerably over the past decade, as hospitals and physician groups have integrated and new public and private payment policies have created financial interdependence. The extent to which accountable care organizations (ACOs) involve hospitals in their operations may prove to be vitally important, because managing hospital care is a key part of improving health care quality and lowering cost growth. Using primary data on ACO composition and capabilities paired with hospital characteristics, we found that 20 percent of US hospitals were part of an ACO in 2014. Hospitals that were in urban areas, were nonprofit, or had a smaller share of Medicare patients were more likely to participate in ACOs, compared to hospitals that were in more rural areas, were for-profit or government owned, or had a larger share of Medicare patients, respectively. Qualitative data identified the following advantages of including a hospital in an ACO: the availability of start-up capital, advanced data sharing, and engagement of providers across the care continuum. Although the 63 percent of ACOs that included hospitals offered more comprehensive services compared to ACOs without hospitals, we found no differences between the two groups in their ability to manage hospital-related aspects of patient care.

  18. Layered ACO-OFDM for intensity-modulated direct-detection optical wireless transmission.

    PubMed

    Wang, Qi; Qian, Chen; Guo, Xuhan; Wang, Zhaocheng; Cunningham, David G; White, Ian H

    2015-05-04

    Layered asymmetrically clipped optical orthogonal frequency division multiplexing (ACO-OFDM) with high spectral efficiency is proposed in this paper for optical wireless transmission employing intensity modulation with direct detection. In contrast to the conventional ACO-OFDM, which only utilizes odd subcarriers for modulation, leading to an obvious spectral efficiency loss, in layered ACO-OFDM, the subcarriers are divided into different layers and modulated by different kinds of ACO-OFDM, which are combined for simultaneous transmission. In this way, more subcarriers are used for data transmission and the spectral efficiency is improved. An iterative receiver is also proposed for layered ACO-OFDM, where the negative clipping distortion of each layer is subtracted once it is detected so that the signals from different layers can be recovered. Theoretical analysis shows that the proposed scheme can improve the spectral efficiency by up to 2 times compared with conventional ACO-OFDM approaches with the same modulation order. Meanwhile, simulation results confirm a considerable signal-to-noise ratio gain over ACO-OFDM at the same spectral efficiency.

  19. PAPR reduction scheme for ACO-OFDM based visible light communication systems

    NASA Astrophysics Data System (ADS)

    zhang, Tian; Ghassemlooy, Zabih; Ma, Chunyang; Guo, Shuxu

    2017-01-01

    In this paper, we proposed a novel peak to average power ratio (PAPR) reduction scheme for the asymmetrically clipped optical orthogonal frequency division multiplexing (ACO-OFDM) visible light communications system. We implement the Toeplitz matrix based Gaussian blur method to reduce the high PAPR of ACO-OFDM at the transmitter and use the orthogonal matching pursuit algorithm to recover the original ACO-OFDM frame at the receiver. Simulation results show that for the 256-subcarrier ACO-OFDM system a 6 dB improvement in PAPR is achieved compared with the original ACO-OFDM in term of the complementary cumulative distribution function, while maintaining a competitive bit-error rate performance compared with the ideal ACO-OFDM lower bound. We also demonstrated the optimal parameter C of 2 for the recovery algorithm based on the tradeoff between the data rate and recovery accuracy. The recovery results show that using the proposed scheme the ACO-OFDM can faithfully be reconstructed judging by the very low value for the reconstruct error of 0.06.

  20. Optimizing the Replication of Multi-Quality Web Applications Using ACO and WoLF

    DTIC Science & Technology

    2006-09-14

    similar approach was used to merge WoLF with ACS- TSP (an ACO algorithm) to solve a Traveling Salesman Problem in [26]. The variables used for policy update...OPTIMIZING THE REPLICATION OF MULTI-QUALITY WEB APPLICATIONS USING ACO AND WOLF THESIS Judson C Dressler, Second Lieutenant, USAF AFIT/GCS/ENG/06-05...Air Force, Department of Defense, or the U.S. Government. AFIT/GCS/ENG/06-05 OPTIMIZING THE REPLICATION OF MULTI-QUALITY WEB APPLICATIONS USING ACO AND

  1. ACOs in real life: a reflection on the Medicare Shared Savings Program.

    PubMed

    Behm, Craig R

    2015-01-01

    The Medicare Shared Savings Program introduced Accountable Care Organizations (ACOs) as one potential method for meeting the often-cited triple aim of better individual care, improved population health, and lower cost. Built on concepts originating from HMOs and then Medicare Advantage plans, ACOs provide incentives based on total cost of care rather than any individual provider's cost. Early quality and cost results are mixed, and, more importantly, so is physician response. The ACO program still has potential to be a bright spot for the future of healthcare, but until there is widespread physician engagement, achieving the triple aim is likely to remain elusive.

  2. Presenting Provenance Based on User Roles - Experiences from the ACOS System

    NASA Astrophysics Data System (ADS)

    West, P.; Michaelis, J.; Fox, P. A.; Zednik, S.; McGuinness, D. L.

    2010-12-01

    One goal of provenance is to provide users an understanding of the steps a system took to generate data products. Here, the level of detail captured by provenance becomes an important consideration. As detail is added, more questions can be hypothetically addressed. However, presenting significant provenance detail may also overwhelm end users, for one of two reasons: (i) the detail presented is irrelevant to the objectives, or (ii) the detail requires background knowledge a user lacks. Both of these challenges are present for data generated by the Mauna Loa Solar Observatory’s (MLSO) Advanced Coronal Observing System (ACOS). In ACOS, photometer-based readings are taken of solar activity and subsequently processed into data products consumable by end users. To fully understand these sequences of steps, background knowledge corresponding to various areas (e.g., astronomy, digital imaging, and ACOS specific techniques) is required by end users. This makes reviewing provenance difficult for users outside the ACOS development team, where varying degrees of background may be expected (ranging from outside domain experts in Solar Physics to citizen scientists). Likewise, even when steps taken by ACOS are understandable, they may provide undesired detail to an end user if presented. The work with ACOS involved the development of a Semantic Web based framework to selectively present provenance detail for data products in ACOS. Here, provenance is captured according to two sets of ontologies, the Proof Markup Language, which is an ontology based domain-independent provenance model, and a step ontology, designed to capture hierarchies of provenance steps. Used in combination, these ontology sets enable the creation of multiple levels of provenance, ranging from coarse to fine grained detail. In this setting, users may choose to expand/collapse provenance steps to view desired details. However, the specific provenance details a user initially sees is defined through

  3. CACONET: Ant Colony Optimization (ACO) Based Clustering Algorithm for VANET

    PubMed Central

    Bajwa, Khalid Bashir; Khan, Salabat; Chaudary, Nadeem Majeed; Akram, Adeel

    2016-01-01

    A vehicular ad hoc network (VANET) is a wirelessly connected network of vehicular nodes. A number of techniques, such as message ferrying, data aggregation, and vehicular node clustering aim to improve communication efficiency in VANETs. Cluster heads (CHs), selected in the process of clustering, manage inter-cluster and intra-cluster communication. The lifetime of clusters and number of CHs determines the efficiency of network. In this paper a Clustering algorithm based on Ant Colony Optimization (ACO) for VANETs (CACONET) is proposed. CACONET forms optimized clusters for robust communication. CACONET is compared empirically with state-of-the-art baseline techniques like Multi-Objective Particle Swarm Optimization (MOPSO) and Comprehensive Learning Particle Swarm Optimization (CLPSO). Experiments varying the grid size of the network, the transmission range of nodes, and number of nodes in the network were performed to evaluate the comparative effectiveness of these algorithms. For optimized clustering, the parameters considered are the transmission range, direction and speed of the nodes. The results indicate that CACONET significantly outperforms MOPSO and CLPSO. PMID:27149517

  4. CACONET: Ant Colony Optimization (ACO) Based Clustering Algorithm for VANET.

    PubMed

    Aadil, Farhan; Bajwa, Khalid Bashir; Khan, Salabat; Chaudary, Nadeem Majeed; Akram, Adeel

    2016-01-01

    A vehicular ad hoc network (VANET) is a wirelessly connected network of vehicular nodes. A number of techniques, such as message ferrying, data aggregation, and vehicular node clustering aim to improve communication efficiency in VANETs. Cluster heads (CHs), selected in the process of clustering, manage inter-cluster and intra-cluster communication. The lifetime of clusters and number of CHs determines the efficiency of network. In this paper a Clustering algorithm based on Ant Colony Optimization (ACO) for VANETs (CACONET) is proposed. CACONET forms optimized clusters for robust communication. CACONET is compared empirically with state-of-the-art baseline techniques like Multi-Objective Particle Swarm Optimization (MOPSO) and Comprehensive Learning Particle Swarm Optimization (CLPSO). Experiments varying the grid size of the network, the transmission range of nodes, and number of nodes in the network were performed to evaluate the comparative effectiveness of these algorithms. For optimized clustering, the parameters considered are the transmission range, direction and speed of the nodes. The results indicate that CACONET significantly outperforms MOPSO and CLPSO.

  5. Contributions of Relational Coordination to Care Management in ACOs: Views of Managerial and Clinical Leaders

    PubMed Central

    Rundall, Thomas G.; Wu, Frances M.; Lewis, Valerie A.; Schoenherr, Karen E.; Shortell, Stephen M.

    2017-01-01

    Background The accountable care organization (ACO) is a new type of health care organization incentivized to improve quality of care, improve population health, and reduce the total cost of care. An ACO’s success in meeting these objectives will depend greatly upon its ability to improve patient care management. Numerous studies have found relational coordination to be positively associated with key measures of organizational performance in health care organizations, including quality and efficiency. Purpose The purpose of this paper is twofold: (1) identify the extent to which ACO leaders are aware of the dimensions of relational coordination, and (2) identify the ways these leaders believe the dimensions influenced care management practices in their organization. Methodology/Approach We performed content analysis of interviews with managerial and clinical leaders from a diverse group of 11 ACOs to assess awareness of relational coordination and identify the ways that dimensions of relational coordination were perceived to influence development of care management practices. Findings ACO leaders mentioned four relational coordination dimensions: shared goals, frequency of communication, timeliness of communication, and problem solving communication. Three dimensions – shared knowledge of team members’ tasks, mutual respect, and accuracy of communication – were not mentioned. Our analysis identified numerous ways leaders believed the four mentioned dimensions contributed to the development of care management, including contributions to standardization of care, patient engagement, coordination of care, and care planning. Discussion We propose two hypotheses for future research on relational coordination and care management. Practice Implications If relational coordination is to have a beneficial influence on ACO performance, organizational leaders must become more aware of relational coordination and its various dimensions and become cognizant of relational

  6. MGA trajectory planning with an ACO-inspired algorithm

    NASA Astrophysics Data System (ADS)

    Ceriotti, Matteo; Vasile, Massimiliano

    2010-11-01

    Given a set of celestial bodies, the problem of finding an optimal sequence of swing-bys, deep space manoeuvres (DSM) and transfer arcs connecting the elements of the set is combinatorial in nature. The number of possible paths grows exponentially with the number of celestial bodies. Therefore, the design of an optimal multiple gravity assist (MGA) trajectory is a NP-hard mixed combinatorial-continuous problem. Its automated solution would greatly improve the design of future space missions, allowing the assessment of a large number of alternative mission options in a short time. This work proposes to formulate the complete automated design of a multiple gravity assist trajectory as an autonomous planning and scheduling problem. The resulting scheduled plan will provide the optimal planetary sequence and a good estimation of the set of associated optimal trajectories. The trajectory model consists of a sequence of celestial bodies connected by two-dimensional transfer arcs containing one DSM. For each transfer arc, the position of the planet and the spacecraft, at the time of arrival, are matched by varying the pericentre of the preceding swing-by, or the magnitude of the launch excess velocity, for the first arc. For each departure date, this model generates a full tree of possible transfers from the departure to the destination planet. Each leaf of the tree represents a planetary encounter and a possible way to reach that planet. An algorithm inspired by ant colony optimization (ACO) is devised to explore the space of possible plans. The ants explore the tree from departure to destination adding one node at the time: every time an ant is at a node, a probability function is used to select a feasible direction. This approach to automatic trajectory planning is applied to the design of optimal transfers to Saturn and among the Galilean moons of Jupiter. Solutions are compared to those found through more traditional genetic-algorithm techniques.

  7. A Rare Case of Short-Chain Acyl-COA Dehydrogenase Deficiency: The Apparent Rarity of the Disorder Results in Under Diagnosis.

    PubMed

    Reddy, G Shilpa; Sujatha, M

    2011-07-01

    Short-chain acyl-CoA dehydrogenase (ACAD) deficiency is an extremely rare inherited mitochondrial disorder of fat metabolism. This belongs to a group of diseases known as fatty acid oxidation disorders. Screening programmes have provided evidence that all the fatty acid oxidation disorders combined are among the most common inborn errors of metabolism. Mitochondrial beta oxidation of fatty acids is an essential energy producing pathway. It is a particularly important pathway during prolonged periods of starvation and during periods of reduced caloric intake due to gastrointestinal illness or increased energy expenditure during febrile illness. The most common presentation is an acute episode of life threatening coma and hypoglycemia induced by a period of fasting due to defective hepatic ketogenesis. Here, the case of a 4 month old female patient who had seizures since the third day of her birth and persistent hypoglycemia is described. She was born to parents of second degree consanguinity after 10 years of infertility treatment. There was history of delayed cry after birth. Metabolic screening for TSH, galactosemia, 17-OHP, G6PD, cystic fibrosis, biotinidase were normal. Tandem mass spectrometric (TMS) screening for blood amino acids, organic acids, fatty acids showed elevated butyryl carnitine (C4) as 3.40 μmol/L (normal <2.00 μmol/L), hexanoyl carnitine (C6) as 0.92 μmol/L (normal <0.72 μmol/L), C4/C3 as 2.93 μmol/L (normal <1.18 μmol/L). The child was started immediately on carnitor syrup (carnitine) 1/2 ml twice daily. Limitation of fasting stress and dietary fat was advised. Baby responded well by gaining weight and seizures were controlled. Until now, less than 25 patients have been reported worldwide. The limited number of patients diagnosed until now is due to the rarity of the disorder resulting in under diagnosis.

  8. Increased long chain acyl-Coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles.

    PubMed

    Nchoutmboube, Jules A; Viktorova, Ekaterina G; Scott, Alison J; Ford, Lauren A; Pei, Zhengtong; Watkins, Paul A; Ernst, Robert K; Belov, George A

    2013-01-01

    All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be relevant for other (+)RNA viruses as well.

  9. Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis.

    PubMed

    Lü, Shiyou; Song, Tao; Kosma, Dylan K; Parsons, Eugene P; Rowland, Owen; Jenks, Matthew A

    2009-08-01

    Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C(24)) were elevated more than 155%. The C(16) cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C(18) monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C(20)-C(30), with highest activity for C(30) acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C(16) but not C(18) cutin monomers are reduced in lacs1, and C(16) acids are the next most preferred acid (behind C(30)) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C(16) monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C(16)) fatty acids for cutin synthesis.

  10. A Few Thoughts about ACO Antitrust Issues from a Local Enforcement Perspective.

    PubMed

    Foote, Kathleen E; Varanini, Emilio E

    2015-08-01

    Accountable care organizations (ACOs), joint ventures of commercial insurers and various groups of medical providers such as physicians, specialists, and hospitals whose development in California has been quickened by the Affordable Care Act, carry with them both promise and pitfalls. On the positive side of the ledger, ACOs may improve the quality of medical care even as they lower the costs of that care. On the negative side of the ledger, ACOs may lead to a gain in market power for their participations, allowing those participants to increase the prices they charge to commercial insurers. It is thus a key question for antitrust enforcers to figure out how to separate the sheep from the goats. This article, representing our personal views as state antitrust enforcers in the California attorney general's office, offers our reflection on a number of ACO articles and studies in this special issue through the prism of this key question and sets out a number of additional issues that we believe warrant study in conjunction with ACOs.

  11. Lysyl oxidase in colorectal cancer.

    PubMed

    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.

  12. Respiratory burst oxidase of fertilization.

    PubMed Central

    Heinecke, J W; Shapiro, B M

    1989-01-01

    Partially reduced oxygen species are toxic, yet sea urchin eggs synthesize H2O2 in a "respiratory burst" at fertilization, as an extracellular oxidant to crosslink their protective surface envelopes. To study the biochemical mechanism for H2O2 production, we have isolated an NADPH-specific oxidase fraction from homogenates of unfertilized Strongylocentrotus purpuratus eggs that produces H2O2 when stimulated with Ca2+ and MgATP2-. Concentrations of free Ca2+ previously implicated in regulation of egg activation modulate the activity of the oxidase. Inhibitors were used to test the relevance of this oxidase to the respiratory burst of fertilization. Procaine, two phenothiazines, and N-ethylmaleimide (but not iodoacetamide) inhibited H2O2 production by the oxidase fraction and oxygen consumption by activated eggs. The ATP requirement suggested that protein kinase activity might regulate the respiratory burst of fertilization; consonant with this hypothesis, H-7 and staurosporine were inhibitory. The respiratory burst oxidase of fertilization is an NADPH:O2 oxidoreductase that appears to be regulated by a protein kinase; although it bears a remarkable resemblance to the neutrophil oxidase, unlike the latter it does not form O2- as its initial product. PMID:2537493

  13. NADPH Oxidases in Vascular Pathology

    PubMed Central

    Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

  14. Glutathione Regulates 1-Aminocyclopropane-1-Carboxylate Synthase Transcription via WRKY33 and 1-Aminocyclopropane-1-Carboxylate Oxidase by Modulating Messenger RNA Stability to Induce Ethylene Synthesis during Stress.

    PubMed

    Datta, Riddhi; Kumar, Deepak; Sultana, Asma; Hazra, Saptarshi; Bhattacharyya, Dipto; Chattopadhyay, Sharmila

    2015-12-01

    Glutathione (GSH) plays a fundamental role in plant defense-signaling network. Recently, we have established the involvement of GSH with ethylene (ET) to combat environmental stress. However, the mechanism of GSH-ET interplay still remains unexplored. Here, we demonstrate that GSH induces ET biosynthesis by modulating the transcriptional and posttranscriptional regulations of its key enzymes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Transgenic Arabidopsis (Arabidopsis thaliana) plants with enhanced GSH content (AtECS) exhibited remarkable up-regulation of ACS2, ACS6, and ACO1 at transcript as well as protein levels, while they were down-regulated in the GSH-depleted phytoalexin deficient2-1 (pad2-1) mutant. We further observed that GSH induced ACS2 and ACS6 transcription in a WRKY33-dependent manner, while ACO1 transcription remained unaffected. On the other hand, the messenger RNA stability for ACO1 was found to be increased by GSH, which explains our above observations. In addition, we also identified the ACO1 protein to be a subject for S-glutathionylation, which is consistent with our in silico data. However, S-glutathionylation of ACS2 and ACS6 proteins was not detected. Further, the AtECS plants exhibited resistance to necrotrophic infection and salt stress, while the pad2-1 mutant was sensitive. Exogenously applied GSH could improve stress tolerance in wild-type plants but not in the ET-signaling mutant ethylene insensitive2-1, indicating that GSH-mediated resistance to these stresses occurs via an ET-mediated pathway. Together, our investigation reveals a dual-level regulation of ET biosynthesis by GSH during stress.

  15. Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase.

    PubMed

    McDonald, Allison E; Amirsadeghi, Sasan; Vanlerberghe, Greg C

    2003-12-01

    The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.

  16. [Alternative oxidase in industrial fungi].

    PubMed

    Gu, Shuai; Liu, Qiang; He, Hao; Li, Shuang

    2015-01-01

    Filamentous fungi have been used in industrial fermentation extensively. Based on non-phosphorylating electron transport process, alternative respiration pathway (ARP) acts as an energy overflow, which can balance carbon metabolism and electron transport, allow the continuance of tricarboxylic acid cycle without the formation of ATP, and permit the turnover of carbon skeletons. Alternative respiration pathway also plays an important role in the stress response of fungi and the physiological function of conditioned pathogen. Alternative oxidase (AOX) is the terminal oxidase responsible for the activity of alternative respiration pathway, which exists widely in higher plants, parts of fungi and algae. Owing to the property that alternative oxidase (AOX) is sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of cytochrome respiration, alternative respiration pathway by AOX is also named as cyanide-resistant respiration (CRR). In recent years, the study of the alternative respiration pathway and alternative oxidase has been a hot topic in the area involving cellular respiration metabolism. In this review we summarized the latest research advances about the functions of alternative respiration pathway and alternative oxidase in industrial fungi.

  17. Post-Acute Care and ACOs — Who Will Be Accountable?

    PubMed Central

    McWilliams, J Michael; Chernew, Michael E; Zaslavsky, Alan M; Landon, Bruce E

    2013-01-01

    Objective To determine how the inclusion of post-acute evaluation and management (E&M) services as primary care affects assignment of Medicare beneficiaries to accountable care organizations (ACOs). Data Sources Medicare claims for a random 5 percent sample of 2009 Medicare beneficiaries linked to American Medical Association Group Practice data identifying provider groups sufficiently large to be eligible for ACO program participation. Study Design We calculated the fraction of community-dwelling beneficiaries whose assignment shifted, as a consequence of including post-acute E&M services, from the group providing their outpatient primary care to a different group providing their inpatient post-acute care. Principal Findings Assignment shifts occurred for 27.6 percent of 25,992 community-dwelling beneficiaries with at least one post-acute skilled nursing facility stay, and they were more common for those incurring higher Medicare spending. Those whose assignment shifted constituted only 1.3 percent of all community-dwelling beneficiaries cared for by large ACO-eligible organizations (n = 535,138), but they accounted for 8.4 percent of total Medicare spending for this population. Conclusions Under current Medicare assignment rules, ACOs may not be accountable for an influential group of post-acute patients, suggesting missed opportunities to improve care coordination and reduce inappropriate readmissions. PMID:23350910

  18. 42 CFR 425.404 - Special assignment conditions for ACOs including FQHCs and RHCs.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Special assignment conditions for ACOs including FQHCs and RHCs. 425.404 Section 425.404 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) MEDICARE SHARED...

  19. Expression of alternative oxidase in tomato

    SciTech Connect

    Kakefuda, M.; McIntosh, L. )

    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.

  20. Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism.

    PubMed Central

    Priefert, H; Hein, S; Krüger, N; Zeh, K; Schmidt, B; Steinbüchel, A

    1991-01-01

    Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude extracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of alpha and beta subunits (Mrs, 35,500 and 36,000, respectively), and a tetrameric alpha 2 beta 2 structure was most likely for the native protein. The molecular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were determined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the alpha and beta subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, acoB, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, and the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together with the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading frame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial sigma 54-dependent promoter consensus sequence. The amino acid sequences deduced from acoA and acoB for the alpha subunit (Mr, 35,243) and the beta subunit (Mr, 35,788) exhibited significant homologies to the primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whereas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltransferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed. Images

  1. Down-regulation of acyl-CoA oxidase gene expression and increased NF-kappaB activity in etomoxir-induced cardiac hypertrophy.

    PubMed

    Cabrero, Agatha; Merlos, Manuel; Laguna, Juan C; Carrera, Manuel Vázquez

    2003-02-01

    Activation of nuclear factor-kappaB (NF-kappaB) is required for hypertrophic growth of cardiomyocytes. Etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) that activates peroxisome proliferator-activated receptor alpha (PPARalpha) and induces cardiac hypertrophy through an unknown mechanism. We studied the mRNA expression of genes involved in fatty acid oxidation in the heart of mice treated for 1 or 10 days with etomoxir (100 mg/kg/day). Etomoxir administration for 1 day significantly increased (4.4-fold induction) the mRNA expression of acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in peroxisomal beta-oxidation. In contrast, etomoxir treatment for 10 days dramatically decreased ACO mRNA levels by 96%. The reduction in ACO expression in the hearts of 10-day etomoxir-treated mice was accompanied by an increase in the mRNA expression of the antioxidant enzyme glutathione peroxidase and the cardiac marker of oxidative stress bax. Moreover, the activity of the redox-regulated transcription factor NF-kappaB was increased in heart after 10 days of etomoxir treatment. Overall, the findings here presented show that etomoxir treatment may induce cardiac hypertrophy via increased cellular oxidative stress and NF-kappaB activation.

  2. Tailoring Systems Engineering Processes in a Conceptual Design Environment: A Case Study at NASA Marshall Spaceflight Center's ACO

    NASA Technical Reports Server (NTRS)

    Mulqueen, John; Maples, C. Dauphne; Fabisinski, Leo, III

    2012-01-01

    This paper provides an overview of Systems Engineering as it is applied in a conceptual design space systems department at the National Aeronautics and Space Administration (NASA) Marshall Spaceflight Center (MSFC) Advanced Concepts Office (ACO). Engineering work performed in the NASA MFSC's ACO is targeted toward the Exploratory Research and Concepts Development life cycle stages, as defined in the International Council on Systems Engineering (INCOSE) System Engineering Handbook. This paper addresses three ACO Systems Engineering tools that correspond to three INCOSE Technical Processes: Stakeholder Requirements Definition, Requirements Analysis, and Integration, as well as one Project Process Risk Management. These processes are used to facilitate, streamline, and manage systems engineering processes tailored for the earliest two life cycle stages, which is the environment in which ACO engineers work. The role of systems engineers and systems engineering as performed in ACO is explored in this paper. The need for tailoring Systems Engineering processes, tools, and products in the ever-changing engineering services ACO provides to its customers is addressed.

  3. Solving optimum operation of single pump unit problem with ant colony optimization (ACO) algorithm

    NASA Astrophysics Data System (ADS)

    Yuan, Y.; Liu, C.

    2012-11-01

    For pumping stations, the effective scheduling of daily pump operations from solutions to the optimum design operation problem is one of the greatest potential areas for energy cost-savings, there are some difficulties in solving this problem with traditional optimization methods due to the multimodality of the solution region. In this case, an ACO model for optimum operation of pumping unit is proposed and the solution method by ants searching is presented by rationally setting the object function and constrained conditions. A weighted directed graph was constructed and feasible solutions may be found by iteratively searching of artificial ants, and then the optimal solution can be obtained by applying the rule of state transition and the pheromone updating. An example calculation was conducted and the minimum cost was found as 4.9979. The result of ant colony algorithm was compared with the result from dynamic programming or evolutionary solving method in commercial software under the same discrete condition. The result of ACO is better and the computing time is shorter which indicates that ACO algorithm can provide a high application value to the field of optimal operation of pumping stations and related fields.

  4. [Alternative oxidase - never ending story].

    PubMed

    Szal, Bożena; Rychter, Anna M

    2016-01-01

    Investigations of plant cyanide resistant respiration lead to the discovery in mitochondrial respiratory chain of the second terminal oxidase, alternative oxidase (AOX). AOX transfers electrons from reduced ubiquinone to oxygen omitting two coupling places thus lowering energetic efficiency of respiration. The presence of AOX was shown in all plants and also in some fungi, mollusca and protista. In termogenic plants the activity of AOX is connected with heat production. In other organisms AOX activity is important for maintaining metabolic homeostasis (carbon metabolism, cell redox state and energy demand) and ROS homeostasis. In this article structure of plant AOX protein and the regulation on molecular levels was described. Possible role of AOX as stress marker was pointed and the possibility of using AOX in human gene therapy was discussed.

  5. Lysyl oxidase in cancer research.

    PubMed

    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.

  6. The terminal oxidases of Paracoccus denitrificans.

    PubMed

    de Gier, J W; Lübben, M; Reijnders, W N; Tipker, C A; Slotboom, D J; van Spanning, R J; Stouthamer, A H; van der Oost, J

    1994-07-01

    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 (delta ctaDI, delta ctaDII), 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-containing oxidase, called cytochrome bb3, is the only quinol oxidase expressed under the conditions used. In a triple oxidase mutant (delta ctaDI, delta ctaDII, cyoB::KmR) an alternative cytochrome c oxidase has been characterized; this cbb3-type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.

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

    PubMed

    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.

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

    PubMed

    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.

  9. Glutathione Regulates 1-Aminocyclopropane-1-Carboxylate Synthase Transcription via WRKY33 and 1-Aminocyclopropane-1-Carboxylate Oxidase by Modulating Messenger RNA Stability to Induce Ethylene Synthesis during Stress1[OPEN

    PubMed Central

    Kumar, Deepak; Hazra, Saptarshi; Chattopadhyay, Sharmila

    2015-01-01

    Glutathione (GSH) plays a fundamental role in plant defense-signaling network. Recently, we have established the involvement of GSH with ethylene (ET) to combat environmental stress. However, the mechanism of GSH-ET interplay still remains unexplored. Here, we demonstrate that GSH induces ET biosynthesis by modulating the transcriptional and posttranscriptional regulations of its key enzymes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Transgenic Arabidopsis (Arabidopsis thaliana) plants with enhanced GSH content (AtECS) exhibited remarkable up-regulation of ACS2, ACS6, and ACO1 at transcript as well as protein levels, while they were down-regulated in the GSH-depleted phytoalexin deficient2-1 (pad2-1) mutant. We further observed that GSH induced ACS2 and ACS6 transcription in a WRKY33-dependent manner, while ACO1 transcription remained unaffected. On the other hand, the messenger RNA stability for ACO1 was found to be increased by GSH, which explains our above observations. In addition, we also identified the ACO1 protein to be a subject for S-glutathionylation, which is consistent with our in silico data. However, S-glutathionylation of ACS2 and ACS6 proteins was not detected. Further, the AtECS plants exhibited resistance to necrotrophic infection and salt stress, while the pad2-1 mutant was sensitive. Exogenously applied GSH could improve stress tolerance in wild-type plants but not in the ET-signaling mutant ethylene insensitive2-1, indicating that GSH-mediated resistance to these stresses occurs via an ET-mediated pathway. Together, our investigation reveals a dual-level regulation of ET biosynthesis by GSH during stress. PMID:26463088

  10. Monoamine Oxidase Inhibitors: Clinical Review

    PubMed Central

    Remick, Ronald A.; Froese, Colleen

    1990-01-01

    Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984

  11. Mitochondrial cytochrome c oxidase deficiency.

    PubMed

    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.

  12. Immunological comparison of sulfite oxidase

    SciTech Connect

    Pollock, V.; Barber, M.J. )

    1991-03-11

    Polyclonal antibodies (rabbit), elicited against FPLC-purified chicken and rat liver sulfite oxidase (SO), have been examined for inhibition and binding to purified chicken (C), rat (R), bovine (B), alligator (A) and shark (S) liver enzymes. Anti-CSO IgG cross-reacted with all five enzymes, with varying affinities, in the order CSO=ASO{gt}RSO{gt}BSO{gt}SSO. Anti-ROS IgG also cross-reacted with all five enzymes in the order RSO{gt}CSO=ASO{gt}BSO{gt}SSO. Anti-CSO IgG inhibited sulfite:cyt. c reductase (S:CR), sulfite:ferricyanide reductase (S:FR) and sulfite:dichlorophenolindophenol reductase (S:DR) activities of CSO to different extents (S:CR{gt}S:FR=S:DR). Similar differential inhibition was found for anti-ROS IgG and RSO S:CR, S:FR and S:DR activities. Anti-CSO IgG inhibited S:CR activities in the order CSO=ASO{much gt}SSO{gt}BSO. RSO was uninhibited. For anti-RSO IgG the inhibition order was RSO{gt}SSO{gt}BSO{gt}ASO. CSO was uninhibited. Anti-CSO and RSO IgGs partially inhibited Chlorella nitrate reductase (NR). Minor cross-reactivity was found for xanthine oxidase. Common antigenic determinants for all five SO's and NR are indicated.

  13. Mitochondrial Cytochrome c Oxidase Deficiency

    PubMed Central

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

    2016-01-01

    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 to study 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. PMID:26846578

  14. Dietary inhibitors of monoamine oxidase A.

    PubMed

    Dixon Clarke, Sarah E; Ramsay, Rona R

    2011-07-01

    Inhibition of monoamine oxidase is one way to treat depression and anxiety. The information now available on the pharmacokinetics of flavonoids and of the components of tobacco prompted an exploration of whether a healthy diet (with or without smoking) provides active compounds in amounts sufficient to partially inhibit monoamine oxidase. A literature search was used to identify dietary monoamine oxidase inhibitors, the levels of these compounds in foods, the pharmacokinetics of the absorption and distribution, and tissue levels observed. An estimated daily intake and the expected tissue concentrations were compared with the measured efficacies of the compounds as inhibitors of monoamine oxidases. Norharman, harman and quercetin dietary presence, pharmacokinetics, and tissue levels were consistent with significant levels reaching neuronal monoamine oxidase from the diet or smoking; 1,2,3,4-tetrahydroisoquinoline, eugenol, 1-piperoylpiperidine, and coumarin were not. Quercetin was equipotent with norharman as a monoamine oxidase A inhibitor and its metabolite, isorhamnetin, also inhibits. Total quercetin was the highest of the compounds in the sample diet. Although bioavailability was variable depending on the source, a healthy diet contains amounts of quercetin that might give sufficient amounts in brain to induce, by monoamine oxidase A inhibition, a small decrease in neurotransmitter breakdown.

  15. Structural insights into sulfite oxidase deficiency.

    PubMed

    Karakas, Erkan; Wilson, Heather L; Graf, Tyler N; Xiang, Song; Jaramillo-Busquets, Sandra; Rajagopalan, K V; Kisker, Caroline

    2005-09-30

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  16. Genistein effect on xanthine oxidase activity.

    PubMed

    Sumbayev, V V

    2001-01-01

    Genistein was defined to be an allosteric xanthine oxidase inhibitor in the concentrations 0.1-4.0 microM and xanthine oxidase activator with superoxide scavenging activity in the concentrations 5.0 microM and higher. But the most effective allosteric binding with the highest affinity was observed in the genistein concentrations 0.1-1.0 microM. Intraperitoneum injections of genistein (500 micrograms/kg) during three days with the interval 24 hours decrease xanthine oxidase activity in the liver, lung and brain of the Vistar rats.

  17. Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

    PubMed Central

    Soh, Ju-Ryoun; Shin, Dong-Hwa; Kwon, Dae Young

    2007-01-01

    This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice. PMID:18850232

  18. 42 CFR 425.608 - Determining first year performance for ACOs beginning April 1 or July 1, 2012.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Determining first year performance for ACOs... windows. (i) The first window will be the first 12 months used for interim payment calculation. (ii) The second window will be CY2013. (4) Expenditures for the first performance year are the sum of...

  19. Quantifying the effects of different post-processing quality filters on regionally and temporally averaged ACOS XCO2

    NASA Astrophysics Data System (ADS)

    Fisher, B.; O'Dell, C.; Mandrake, L.

    2013-12-01

    The Atmospheric CO2 Observations from Space (ACOS) group has been producing and distributing total column CO2 (XCO2) products using JAXA/NIES/MOE Greenhouse Gases Observing SATellite (GOSAT) spectra and has accumulated almost 4 years of data with version 3.3. While the ACOS team strives to only process soundings that the retrieval algorithm can handle well, we are conservative in what we reject from processing. Consequently, some soundings get processed which do not yield reliable results. We have developed post-processing filters based on comparisons to a few truth proxies (model means, TCCON, and the southern hemisphere approximation) to flag the less reliable soundings. Here we compare regionally (using TRANSCOM spatial bins) and monthly averaged XCO2 that have been filtered by our normal method (described in the ACOS Level 2 Data User's Guide) and a newer method, which we have named warn levels. Mean XCO2 differences are quantified spatially and temporally to inform possible biases in carbon cycle studies that could potentially be introduced by the application of differing post-processing screening methodologies to the ACOS products.

  20. Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes.

    PubMed

    Finnegan, Patrick M; Umbach, Ann L; Wilce, Jackie A

    2003-12-18

    The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.

  1. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30

  2. Azide inhibition of urate oxidase.

    PubMed

    Gabison, Laure; Colloc'h, Nathalie; Prangé, Thierry

    2014-07-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX-UA or UOX-8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site.

  3. Azide inhibition of urate oxidase

    PubMed Central

    Gabison, Laure; Colloc’h, Nathalie; Prangé, Thierry

    2014-01-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX–UA or UOX–8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site. PMID:25005084

  4. Effect of naphthalene on cytochrome oxidase activity

    SciTech Connect

    Harmon, H.J.

    1988-01-01

    Previous reports have demonstrated that naphthalene inhibits oxygen consumption in Daphnia magna tissue culture cells, and intact mitochondria and submitochondrial particles. These studies were extended to algal mitochondrial respiration as well as photosynthetic activity. The authors were able to demonstrate the specific site of apparent respiratory inhibition to be coenzyme Q (ubiquinone, UQ) and later to demonstrate the molecular basis of this inhibition at ubiquinone. The authors previously could not demonstrate an effect of naphthalene on cytochrome oxidase activity. However, the observation that naphthalene can stimulate respiration in algae prompted the reinvestigation of the effect of naphthalene on the kinetics of cytochrome oxidase. Cytochrome oxidase is a multi-subunit membranous protein responsible for the oxidation of cytochrome c and the reduction of molecular oxygen to water. Because of the complicated nature and mechanism of this enzyme, the potential exists for multiple and possibly opposite effects of naphthalene on its function.

  5. Eight reasons payer interoperability and data sharing are essential in ACOs. Interoperability standards could be a prerequisite to measuring care.

    PubMed

    Mookencherry, Shefali

    2012-01-01

    It makes strategic and business sense for payers and providers to collaborate on how to take substantial cost out of the healthcare delivery system. Acting independently, neither medical groups, hospitals nor health plans have the optimal mix of resources and incentives to significantly reduce costs. Payers have core assets such as marketing, claims data, claims processing, reimbursement systems and capital. It would be cost prohibitive for all but the largest providers to develop these capabilities in order to compete directly with insurers. Likewise, medical groups and hospitals are positioned to foster financial interdependence among providers and coordinate the continuum of patient illnesses and care settings. Payers and providers should commit to reasonable clinical and cost goals, and share resources to minimize expenses and financial risks. It is in the interest of payers to work closely with providers on risk-management strategies because insurers need synergy with ACOs to remain cost competitive. It is in the interest of ACOs to work collaboratively with payers early on to develop reasonable and effective performance benchmarks. Hence, it is essential to have payer interoperability and data sharing integrated in an ACO model.

  6. Purification of the Alpha Glycerophosphate Oxidase From Trypanosomes.

    DTIC Science & Technology

    1992-08-01

    is the purifica- tion of the glycerphosphate oxidase from the terminal oxidase in bloodstream trypanosomes. African trypanosomiasis remains one of the...oxidase from the terminal oxidase in bloodstream trypanosomes. African trypanosomiasis remains one of the major diseases in the world today, affecting...interest as a possible target for drug chemotherapy . At present only suramin and organic arsenicals remain as the mainstay of chemotherapy , despite their

  7. NASA GES DISC support of CO2 Data from OCO-2, ACOS, and AIRS

    NASA Technical Reports Server (NTRS)

    Wei, Jennifer C; Vollmer, Bruce E.; Savtchenko, Andrey K.; Hearty, Thomas J; Albayrak, Rustem Arif; Deshong, Barbara E.

    2013-01-01

    NASA Goddard Earth Sciences Data and Information Services Centers (GES DISC) is the data center assigned to archive and distribute current AIRS, ACOS data and data from the upcoming OCO-2 mission. The GES DISC archives and supports data containing information on CO2 as well as other atmospheric composition, atmospheric dynamics, modeling and precipitation. Along with the data stewardship, an important mission of GES DISC is to facilitate access to and enhance the usability of data as well as to broaden the user base. GES DISC strives to promote the awareness of science content and novelty of the data by working with Science Team members and releasing news articles as appropriate. Analysis of events that are of interest to the general public, and that help in understanding the goals of NASA Earth Observing missions, have been among most popular practices.Users have unrestricted access to a user-friendly search interface, Mirador, that allows temporal, spatial, keyword and event searches, as well as an ontology-driven drill down. Variable subsetting, format conversion, quality screening, and quick browse, are among the services available in Mirador. The majority of the GES DISC data are also accessible through OPeNDAP (Open-source Project for a Network Data Access Protocol) and WMS (Web Map Service). These services add more options for specialized subsetting, format conversion, image viewing and contributing to data interoperability.

  8. Redshift observations of Abell/ACO galaxy clusters in two candidate superclusters

    NASA Astrophysics Data System (ADS)

    Batuski, D. J.; Maurogordato, S.; Balkowski, C.; Olowin, R. P.

    1995-02-01

    The results of spectroscopic observations of five R greater than or equal to 1 clusters of galaxies from the Abell (1958) and Abell et al. (1989) (hereafter ACO) catalogs are presented. The observations were conducted at the ESO 3.6m telescope with the EFOSC spectrograph and PUMA hole-punch aperture plates. Two of the clusters (A2576 and A2628) had been identified as members of one candidate supercluster that appears particularly dense in projection, and the other three clusters (A3802, A3817, and A3834) were in another candidate supercluster, based on redshifts estimated from the magnitudes of tenth brightest galaxies. Our observations confirm very similar redshifts for A2576 and A2628 ( z = 0.1875 and z = 0.1858, respectively) and for A3802 and A3834 (z = 0.1579 and z = 0.1518). From the agreement in redshifts and the proximity on the sky, it is suggestive that both pairs are indeed components of superclusters, although redshifts of other nearby candidates must be measured to determine the significance of the structure present. The fifth cluster, A3817, has a mean redshift of z = 0.2115, and so appears to be background to any possible A3802/A3834 supercluster. We note that the complex of clusters around A2576 and A2628 is a region of high spatial density, even ignoring the many clusters with unmeasured redshift. The region is an exceptional opportunity for large-scale structure study.

  9. The First Mammalian Aldehyde Oxidase Crystal Structure

    PubMed Central

    Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T. P.; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

    2012-01-01

    Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity. PMID:23019336

  10. An oxidase road to platelet adhesion.

    PubMed

    Krause, Diane S

    2016-03-17

    Platelet adhesion to collagen via collagen receptors is an important part of thrombosis. In this issue of Blood, Matsuura et al identify collagen receptors as previously unrecognized targets of the extracellular enzyme lysyl oxidase (LOX), the level of which is increased in myeloproliferative neoplasms (MPNs) and other conditions associated with pathological thromboses.

  11. Polyphenol oxidase activity in annual forage clovers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO)-mediated phenol reactions in red clover (Trifolium pratense L.) bind forage protein and reduce proteolysis, producing beneficial effects on forage protein degradability, silage fermentation, and soil-N cycling. We evaluated PPO activity in seven previously untested annual c...

  12. A colorimetric assay for cytokinin oxidase.

    PubMed

    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.

  13. Exploiting algal NADPH oxidase for biophotovoltaic energy.

    PubMed

    Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K; Bombelli, Paolo; Howe, Christopher J; Merchant, Sabeeha S; Davies, Julia M; Smith, Alison G

    2016-01-01

    Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

  14. Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

    PubMed Central

    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

    2015-01-01

    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. PMID:26680532

  15. Inhibition of rat fat cell lipolysis by monoamine oxidase and semicarbazide-sensitive amine oxidase substrates.

    PubMed

    Visentin, Virgile; Prévot, Danielle; Marti, Luc; Carpéné, Christian

    2003-04-18

    It has been demonstrated that amine oxidase substrates stimulate glucose transport in cardiomyocytes and adipocytes, promote adipogenesis in pre-adipose cell lines and lower blood glucose in diabetic rats. These insulin-like effects are dependent on amine oxidation by semicarbazide-sensitive amine oxidase or by monoamine oxidase. The present study aimed to investigate whether amine oxidase substrates also exhibit another insulin-like property, the inhibition of lipolysis. We therefore tested the influence of tyramine and benzylamine on lipolytic activity in rat adipocytes. These amines did not modify basal lipolysis but dose-dependently counteracted the stimulation induced by lipolytic agents. The response to 10 nM isoprenaline was totally inhibited by tyramine 1 mM. The blockade produced by inhibition of amine oxidase activity or by 1 mM glutathione suggested that the generation of oxidative species, which occurs during amine oxidation, was involved in tyramine antilipolytic effect. Among the products resulting from amine oxidation, only hydrogen peroxide was antilipolytic in a manner that was potentiated by vanadate, as for tyramine or benzylamine. Antilipolytic responses to tyramine and to insulin were sensitive to wortmannin. These data suggest that inhibition of lipolysis is a novel insulin-like effect of amine oxidase substrates which is mediated by hydrogen peroxide generated during amine oxidation.

  16. Changes in oxidative stress in transgenic RNAi ACO1 tomato fruit during ripening

    NASA Astrophysics Data System (ADS)

    Eglous, Najat Mohamed; Ali, Zainon Mohd; Hassan, Maizom; Zainal, Zamri

    2013-11-01

    Tomato (Solanum Lycopersicum L.) is the second most cultivated vegetable in the world and widely used as a system for studying the role of ethylene during fruit ripening. Our objective was to study the oxidative stress and antioxidative metabolism during ripening of non transgenic tomato and transgenic line-21 tomato which reduced ethylene. The line-21 of transgenic tomato plants (RNAi ACO1) had lower ethylene production and longer shelf-life more than 32 days as compared to the wild-type fruits which have very short shelf-life. In this study, tomato fruit were divided into five different stages (MG: mature green 5%, B: breaker 25%, T: turning 50%, O: orange75%, RR: red ripe100%). The activity of lipoxygenase (LOX) and lipid peroxidation (MDA) were measured to assess changes in oxidative stress. The LOX activity and MDA content decreased significantly obtaining 2.6-fold and 1.2-fold, respectively, as compared to the wild type fruit. However, superoxide dismutase (SOD) and catalase (CAT) activities were increased to 1.9 and 1.2 folds from the mature green to the fully ripe stage in transgenic tomatoes. Furthermore, the wild type tomato increases 1.3 in SOD and 1.6 in CAT activities. The overall results indicate that the wild type tomato fruit showed a faster rate of ripening, parallel to decline in the rate of enzymatic antioxidative systems as compared to the transgenic line-21 tomato fruit. In addition, the results show that the antioxidant capacity is improved during the ripening process and is accompanied by an increase in the oxidative stress.

  17. Natural Compounds as Modulators of NADPH Oxidases

    PubMed Central

    2013-01-01

    Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols. PMID:24381714

  18. Lysyl Oxidase and the Tumor Microenvironment

    PubMed Central

    Wang, Tong-Hong; Hsia, Shih-Min; Shieh, Tzong-Ming

    2016-01-01

    The lysyl oxidase (LOX) family of oxidases contains a group of extracellular copper-dependent enzymes that catalyze the cross-linking of collagen and elastin by oxidation, thus maintaining the rigidity and structural stability of the extracellular matrix (ECM). Aberrant expression or activation of LOX alters the cellular microenvironment, leading to many diseases, including atherosclerosis, tissue fibrosis, and cancer. Recently, a number of studies have shown that LOX is overexpressed in most cancers and that it is involved in the regulation of tumor progression and metastasis. In contrast, a few reports have also indicated the tumor-suppressing role of LOX. In this short review, we discuss recent research on the correlations between LOX and cancer. Further, the role of LOX in tumor microenvironment remodeling, tumorigenesis, and metastasis and the underlying mechanisms have also been elucidated. PMID:28036074

  19. Lysyl oxidase mediates hypoxic control of metastasis.

    PubMed

    Erler, Janine T; Giaccia, Amato J

    2006-11-01

    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-induced metastasis. In an orthotopic rodent model of breast cancer, a small-molecule or antibody inhibitor of LOX abolished metastasis, offering preclinical validation of this enzyme as a therapeutic target.

  20. Ligand interactions with galactose oxidase: mechanistic insights.

    PubMed Central

    Whittaker, M M; Whittaker, J W

    1993-01-01

    Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme. PMID:8386015

  1. Imaging Monoamine Oxidase in the Human Brain

    SciTech Connect

    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.

  2. Tetrazolium Oxidase Polymorphism in Rainbow Trout

    PubMed Central

    Cederbaum, Stephen D.; Yoshida, Akira

    1972-01-01

    Tetrazolium oxidase from the blood and liver of rainbow trout was found to be genetically polymorphic. The inheritance pattern of the liver enzyme was compatible only with a one locus-two allele hypothesis. The enzymes in the blood while having an electrophoretically identical polymorphism could differ genotypically from that of the liver in a given fish. The significance of these findings to the understanding of the evolution of the salmonid genome is discussed. PMID:4675090

  3. Arabidopsis alternative oxidase sustains Escherichia coli respiration.

    PubMed Central

    Kumar, A M; Söll, D

    1992-01-01

    Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor. Images PMID:1438286

  4. Xanthine dehydrogenase to xanthine oxidase conversion in ischemic rat intestine

    SciTech Connect

    McKelvey, T.G.; Engerson, T.D.; Elmore, C.R.; Jones, H.P. )

    1990-02-26

    The ischemic conversion of the NADH-producing xanthine dehydrogenase (XDH) to an oxidase form, that produces both superoxide radical and hydrogen peroxide, has been proposed as an important step in initiating oxygen radical-mediated ischemia-reperfusion injury. It has also been reported that two forms of converted oxidase are produced in ischemic rat liver; a reversible xanthine oxidase produced through sulfhydryl oxidation, that can be reconverted to XDH by incubation with 10mM dithiothreitol (Dtt) at 37{degrees}C, and a Dtt-irreversible oxidase produced via proteolysis. The authors report that increased oxidase in the ischemic rat intestine results from significant increases in both the Dtt-reversible and Dtt-irreversible forms of xanthine oxidase. Total oxidase activity (Irreversible + Dtt-reversible) was 19% of the total enzyme activity (XDH + XO) in control ileum and distal jejunum, increased to 26% after 1 hour of ischemia at 37{degrees}C, and significantly to 36% after 1.5 hours. After 3 hours 73% of the activity was in the oxidase form. Irreversible oxidase comprised 15% of the total activity in control intestine, significantly increased to 25% after 2 hours, and further to 42% after 3 hours. Dtt-reversible oxidase was 3% of the total activity in controls, increased to 13% after 1.5 hours, and significantly to 29% after 2 hours.

  5. Substrate specificity of guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase for methyl- and nitrobenzaldehydes.

    PubMed

    Veskoukis, Aristidis S; Kouretas, Demetrios; Panoutsopoulos, Georgios I

    2006-01-01

    Both aldehyde oxidase and xanthine oxidase catalyze the oxidation of a wide range of N-heterocycles and aldehydes. These enzymes are important in the oxidation of N-heterocyclic xenobiotics, whereas their role in the oxidation of xenobiotic aldehydes is usually ignored. The present investigation describes the interaction of methyl- and nitrosubstituted benzaldehydes, in the ortho-, meta- and parapositions, with guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase. The kinetic constants showed that most substituted benzaldehydes are excellent substrates of aldehyde oxidase with lower affinities for xanthine oxidase. Low Km values for aldehyde oxidase were observed with most benzaldehydes tested, with 3-nitrobenzaldehyde having the lowest Km value and 3-methylbenzaldehyde being the best substrate in terms of substrate efficiency (Ks). Additionally, low Km values for xanthine oxidase were found with most benzaldehydes tested. However, all benzaldehydes also had low Vmax values, which made them poor substrates of xanthine oxidase. It is therefore possible that aldehyde oxidase may be critical in the oxidation of xenobiotic and endobiotic derived aldehydes and its role in such reactions should not be ignored.

  6. NAD(P)H oxidase and renal epithelial ion transport

    PubMed Central

    Schreck, Carlos

    2011-01-01

    A fundamental requirement for cellular vitality is the maintenance of plasma ion concentration within strict ranges. It is the function of the kidney to match urinary excretion of ions with daily ion intake and nonrenal losses to maintain a stable ionic milieu. NADPH oxidase is a source of reactive oxygen species (ROS) within many cell types, including the transporting renal epithelia. The focus of this review is to describe the role of NADPH oxidase-derived ROS toward local renal tubular ion transport in each nephron segment and to discuss how NADPH oxidase-derived ROS signaling within the nephron may mediate ion homeostasis. In each case, we will attempt to identify the various subunits of NADPH oxidase and reactive oxygen species involved and the ion transporters, which these affect. We will first review the role of NADPH oxidase on renal Na+ and K+ transport. Finally, we will review the relationship between tubular H+ efflux and NADPH oxidase activity. PMID:21270341

  7. Individual variation in hepatic aldehyde oxidase activity.

    PubMed

    Al-Salmy, H S

    2001-04-01

    Aldehyde oxidase (AO) is a molybdo-flavo enzyme expressed predominantly in the liver, lung, and kidney. AO plays a major role in oxidation of aldehydes, as well as oxidation of various N-heterocyclic compounds of pharmacological and toxicological importance including antiviral (famciclovir), antimalarial (quinine), antitumour (methotrexate), and nicotine. The aim of this study was to investigate cytosolic aldehyde oxidase activity in human liver. Cytosolic AO was characterised using both the metabolism of N-[(2-dimethylamino)ethyl] acridine-4-carboxamide (DACA) and benzaldehyde to form DACA-9(10H)-acridone (quantified by HPLC with fluorescence detection) and benzoic acid (quantified spectrophotometrically). Thirteen livers (10 female, 3 male) were examined. The intrinsic clearance (Vmax/Km) of DACA varied 18-fold (0.03-0.50 m/min/mg). Vmax ranged from 0.20-3.10 nmol/ min/mg, and Km ranged from 3.5-14.2 microM. In the same specimens, the intrinsic clearance for benzaldehyde varied 5-fold (0.40-1.8 ml/min/mg). Vmax ranged from 3.60-12.6 nmol/min/mg and Km ranged from 3.6-14.6 microM. Furthermore, there were no differences in AO activity between male and female human livers, nor was there any relationship to age of donor (range 29-73 years), smoking status, or disease status. In conclusion, our results showed that there are variations in AO activity in human liver. These variations in aldehyde oxidase activity might reflect individual variations or they might be due to AO stability during processing and storage.

  8. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  9. Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase.

    PubMed Central

    Gahl, W A; Vale, A M; Pitot, H C

    1980-01-01

    1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate. PMID:7406861

  10. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  11. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    PubMed

    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.

  12. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity

    PubMed Central

    Peng, Zeyu; Dittmer, Neal T.; Lang, Minglin; Brummett, Lisa M.; Braun, Caroline L.; Davis, Lawrence C.; Kanost, Michael R.; Gorman, Maureen J.

    2015-01-01

    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 surpring 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. PMID:25701385

  13. Dissecting the role of climacteric ethylene in kiwifruit (Actinidia chinensis) ripening using a 1-aminocyclopropane-1-carboxylic acid oxidase knockdown line.

    PubMed

    Atkinson, Ross G; Gunaseelan, Kularajathevan; Wang, Mindy Y; Luo, Luke; Wang, Tianchi; Norling, Cara L; Johnston, Sarah L; Maddumage, Ratnasiri; Schröder, Roswitha; Schaffer, Robert J

    2011-07-01

    During climacteric fruit ripening, autocatalytic (Type II) ethylene production initiates a transcriptional cascade that controls the production of many important fruit quality traits including flavour production and softening. The last step in ethylene biosynthesis is the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by the enzyme ACC oxidase (ACO). Ten independent kiwifruit (Actinidia chinensis) lines were generated targeting suppression of fruit ripening-related ACO genes and the fruit from one of these lines (TK2) did not produce detectable levels of climacteric ethylene. Ripening behaviour in a population of kiwifruit at harvest is asynchronous, so a short burst of exogenous ethylene was used to synchronize ripening in TK2 and control fruit. Following such a treatment, TK2 and control fruit softened to an 'eating-ripe' firmness. Control fruit produced climacteric ethylene and softened beyond eating-ripe by 5 d. In contrast, TK2 fruit maintained an eating-ripe firmness for >25 d and total volatile production was dramatically reduced. Application of continuous exogenous ethylene to the ripening-arrested TK2 fruit re-initiated fruit softening and typical ripe fruit volatiles were detected. A 17 500 gene microarray identified 401 genes that changed after ethylene treatment, including a polygalacturonase and a pectate lyase involved in cell wall breakdown, and a quinone oxidoreductase potentially involved in volatile production. Many of the gene changes were consistent with the softening and flavour changes observed after ethylene treatment. However, a surprisingly large number of genes of unknown function were also observed, which could account for the unique flavour and textural properties of ripe kiwifruit.

  14. Xanthine oxidase inhibitors from Garcinia esculenta twigs.

    PubMed

    Zhu, Lun-Lun; Fu, Wen-Wei; Watanabe, Shimpei; Shao, Yi-Nuo; Tan, Hong-Sheng; Zhang, Hong; Tan, Chang-Heng; Xiu, Yan-Feng; Norimoto, Hisayoshi; Xu, Hong-Xi

    2014-12-01

    The EtOAc-soluble portion of the 80 % (v/v) EtOH extract from the twigs of Garcinia esculenta exhibited strong xanthine oxidase inhibition in vitro. Bioassay-guided purification led to the isolation of 1,3,6,7-tetrahydroxyxanthone (3) and griffipavixanthone (8) as the main xanthine oxidase inhibitors, along with six additional compounds (1, 2, 4-7), including two new compounds (1 and 2). This enzyme inhibition was dose dependent with an IC50 value of approximately 1.2 µM for 3 and 6.3 µM for 8. The inhibitory activity of 3 was stronger than the control allopurinol (IC50 value: 5.3 µM). To our knowledge, compound 8 is the first bixanthone that demonstrated potent XO inhibitory activity in vitro. The structures of the new compounds were established by spectroscopic analysis, and the optical properties and absolute stereochemistry of racemic (±) esculentin A (2) were further determined by the calculation of the DP4 probability and analysis of its MTPA ester derivatives.

  15. Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii.

    PubMed

    Jurtshuk, P; Mueller, T J; Wong, T Y

    1981-09-14

    A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27-70% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a3 oxidase of mammalian mitochondria.

  16. Molecular Evolution of Cytochrome bd Oxidases across Proteobacterial Genomes

    PubMed Central

    Degli Esposti, Mauro; Rosas-Pérez, Tania; Servín-Garcidueñas, Luis Eduardo; Bolaños, Luis Manuel; Rosenblueth, Monica; Martínez-Romero, Esperanza

    2015-01-01

    This work is aimed to resolve the complex molecular evolution of cytochrome bd ubiquinol oxidase, a nearly ubiquitous bacterial enzyme that is involved in redox balance and bioenergetics. Previous studies have created an unclear picture of bd oxidases phylogenesis without considering the existence of diverse types of bd oxidases. Integrated approaches of genomic and protein analysis focused on proteobacteria have generated a molecular classification of diverse types of bd oxidases, which produces a new scenario for interpreting their evolution. A duplication of the original gene cluster of bd oxidase might have occurred in the ancestors of extant α-proteobacteria of the Rhodospirillales order, such as Acidocella, from which the bd-I type of the oxidase might have diffused to other proteobacterial lineages. In contrast, the Cyanide-Insensitive Oxidase type may have differentiated into recognizable subtypes after another gene cluster duplication. These subtypes are widespread in the genomes of α-, β-, and γ-proteobacteria, with occasional instances of lateral gene transfer. In resolving the evolutionary pattern of proteobacterial bd oxidases, this work sheds new light on the basal taxa of α-proteobacteria from which the γ-proteobacterial lineage probably emerged. PMID:25688108

  17. Detection and characterization of a multicopper oxidase from Nitrosomonas europaea.

    PubMed

    Lawton, Thomas J; Rosenzweig, Amy C

    2011-01-01

    Blue copper oxidase (BCO) is a multicopper oxidase (MCO) found in Nitrosomonas europaea as well as in other ammonia-oxidizing organisms. In this chapter, we detail methods used to detect, isolate, and characterize BCO from N. europaea. A method for identifying and classifying MCOs commonly found in nitrifiers based on primary sequence is also described.

  18. Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

    PubMed Central

    Lambowitz, A M; Sabourin, J R; Bertrand, H; Nickels, R; McIntosh, L

    1989-01-01

    Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species. Images PMID:2524649

  19. Lysyl oxidase activity in human normal skins and postburn scars.

    PubMed

    Hayakawa, T; Hino, N; Fuyamada, H; Nagatsu, T; Aoyama, H

    1976-09-06

    Lysyl oxidase activity of human normal skins derived from the frontal thighs of 33 subjects showed large variations and the mean value was 11 455 +/- 7 172 (S.D.) cpm/g of wet weight tissue. The age of lesion affected the lysyl oxidase activity in postburn scars. Granulation tissues showed a fairly low activity; however, the activity increased sharply within 2--3 months, and reached a significantly higher value than that of normal skin. The high level of activity continued for up to 2--3 years, then gradually decreased to normal range after 5 years or so. Lysyl oxidase activity was detected only after 4 M urea treatment of tissues. Benzylamine oxidase activity also showed large variations in both normal skins and postburn scars, with mean values of: 0.128 +/- 0.077 (S.D.) and 0.145 +/- 0.090 (S.D.) mmol/g of wet weight/h, respectively. No correlation was observed between lysyl oxidase and benzylamine oxidase activities. The granulation tissues showed significantly high values of benzylamine oxidase activity in contrast to the low values of lysyl oxidase activity.

  20. Source challenges resulting of the first applications of a UV storage ring FEL on Super-ACO

    SciTech Connect

    Couprie, M.E.; Bakker, R.; Nahon, L. |

    1995-12-31

    Since 1992, significant progresses were achieved on the Super-ACO (S-ACO) storage ring Free Electron Laser (FEL) in the UV. The operation at the nominal energy 800 MeV has several consequences: higher average power in the UV (25 mW at 60 mA and more recently 100 mW at 100 mA available for the users), 10 hours of lasing for the same injection of positrons, providing enough time for performing an user experiment, compatibility with the users of synchrotron radiation (SR) in the temporal structure mode for 120 mA with the possibility of closing the four insertion devices of S-ACO. The main difficulties to extend the FEL optical performances come from the small gain (2%), limiting a rapid extention of the spectral range (either in the laser mode or by coherent harmonic generation from the FEL itself in the undulator) or linewidth narrowing. The installation of a 500 MHz harmonic cavity for bunch length reduction and gain increase is under consideration{hor_ellipsis} The stability of the FEL temporal and spectral was systematically followed versus time, for various scales (from ns to half an hour) with different detectors. The stability of the laser source has been significantly improved with a longitudinal feedback system allowing the jitter of the 25 ps RMS laser micropulse to be reduced from 150-200 ps down to 10-20 ps. the intensity fluctuations to be damped down 1% and the spectral drift to be smaller than the resolution of the scanning Fabry-Perot (0.01{angstrom}) at perfect synchronism. The laser can work during more than 3 consecutive hours without readjustments. In addition, according to the ring current, the positron beam is submitted to coherent modes of synchrotron oscillations. Right now, a Pedersen type longitudinal feedback damps the dipolar modes of such oscillation. The quadrupolar modes in the 120-60 mA range leading to a rather unstable FEL are on the way to be damped with an additional feedback.

  1. Hybridisations Of Simulated Annealing And Modified Simplex Algorithms On A Path Of Steepest Ascent With Multi-Response For Optimal Parameter Settings Of ACO

    NASA Astrophysics Data System (ADS)

    Luangpaiboon, P.

    2009-10-01

    Many entrepreneurs face to extreme conditions for instances; costs, quality, sales and services. Moreover, technology has always been intertwined with our demands. Then almost manufacturers or assembling lines adopt it and come out with more complicated process inevitably. At this stage, products and service improvement need to be shifted from competitors with sustainability. So, a simulated process optimisation is an alternative way for solving huge and complex problems. Metaheuristics are sequential processes that perform exploration and exploitation in the solution space aiming to efficiently find near optimal solutions with natural intelligence as a source of inspiration. One of the most well-known metaheuristics is called Ant Colony Optimisation, ACO. This paper is conducted to give an aid in complicatedness of using ACO in terms of its parameters: number of iterations, ants and moves. Proper levels of these parameters are analysed on eight noisy continuous non-linear continuous response surfaces. Considering the solution space in a specified region, some surfaces contain global optimum and multiple local optimums and some are with a curved ridge. ACO parameters are determined through hybridisations of Modified Simplex and Simulated Annealing methods on the path of Steepest Ascent, SAM. SAM was introduced to recommend preferable levels of ACO parameters via statistically significant regression analysis and Taguchi's signal to noise ratio. Other performance achievements include minimax and mean squared error measures. A series of computational experiments using each algorithm were conducted. Experimental results were analysed in terms of mean, design points and best so far solutions. It was found that results obtained from a hybridisation with stochastic procedures of Simulated Annealing method were better than that using Modified Simplex algorithm. However, the average execution time of experimental runs and number of design points using hybridisations were

  2. The complex roles of NADPH oxidases in fungal infection

    PubMed Central

    Hogan, Deborah; Wheeler, Robert T.

    2014-01-01

    Summary NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signaling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signaling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell. PMID:24905433

  3. The NADH oxidase-Prx system in Amphibacillus xylanus.

    PubMed

    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.

  4. Genotypic Variation in Cytokinin Oxidase from Phaseolus Callus Cultures 1

    PubMed Central

    Kaminek, Miroslav; Armstrong, Donald J.

    1990-01-01

    Genotypic variation in cytokinin oxidase has been detected in enzyme preparations from Phaseolus vulgaris L. cv Great Northern and Phaseolus lunatus L. cv Kingston callus cultures. Although cytokinin oxidase preparations from Great Northern and Kingston callus tissues appear to have very similar substrate specificities, the cytokinin oxidase activities from the two callus tissues were found to differ in a number of other properties. The cytokinin oxidase from P. vulgaris cv Great Northern callus tissue exhibited a pH optimum of 6.5 (bisTris) and had a strong affinity for the lectin concanavalin A. The cytokinin oxidase from P. lunatus cv Kingston callus tissue exhibited a pH optimum of 8.4 (Taps) and did not bind to concanavalin A. The two enzymes also differed in position of elution when chromatographed on DEAE-cellulose. Both cytokinin oxidase activities exhibited enhanced activity and lower pH optima in the presence of copper-imidazole complexes, but the optimum copper-imidazole ratio and the magnitude of enhancement differed for the two activities. In both callus tissues, transient increases in the supply of exogenous cytokinins induced increases in cytokinin oxidase activity. The differences in pH optima and in glycosylation (as evidenced by the observed difference in lectin affinity) of the cytokinin oxidases from Great Northern and Kingston callus tissues suggest that the compartmentation of cytokinin oxidase may differ in the two callus tissues. The possibility that enzyme compartmentation and isozyme variation in cytokinin oxidase may play a role in the regulation of cytokinin degradation in plant tissues is discussed in relation to known differences in the rates of cytokinin degradation in Great Northern and Kingston callus tissues. Images Figure 6 PMID:16667652

  5. Emission “Off-On” effect from europium complexes triggered by AcO anion: Synthesis, characterization and sensing performance

    NASA Astrophysics Data System (ADS)

    Li, Xiaogang; Zhang, Dong; Li, Jing

    2014-06-01

    In this paper, a series of Eu(III) complexes based on four diamine ligands and two diketone ligands were synthesized. Their single crystal structures were studied, where intermolecular π-π stacking was found. The photophysical parameters of these Eu(III) complexes were measured, along with their ligand triplet levels. The energy transfer mechanism between ligand and metal center was discussed in detail. Energy transfer roll-back was found in Eu(III) complexes owing large-conjugated diamine ligands, compromising emissive performance. This energy transfer roll-back, however, could be stopped by the presence of AcO anion, leading to Eu(III) complex emission enhancement. The sensing performance of such Eu(III) complexes was thus investigated in detail. High sensitivity and selectivity were observed.

  6. Emission "Off-On" effect from europium complexes triggered by AcO anion: synthesis, characterization and sensing performance.

    PubMed

    Li, Xiaogang; Zhang, Dong; Li, Jing

    2014-06-05

    In this paper, a series of Eu(III) complexes based on four diamine ligands and two diketone ligands were synthesized. Their single crystal structures were studied, where intermolecular π-π stacking was found. The photophysical parameters of these Eu(III) complexes were measured, along with their ligand triplet levels. The energy transfer mechanism between ligand and metal center was discussed in detail. Energy transfer roll-back was found in Eu(III) complexes owing large-conjugated diamine ligands, compromising emissive performance. This energy transfer roll-back, however, could be stopped by the presence of AcO anion, leading to Eu(III) complex emission enhancement. The sensing performance of such Eu(III) complexes was thus investigated in detail. High sensitivity and selectivity were observed.

  7. Expression of the alternative oxidase complements cytochrome c oxidase deficiency in human cells

    PubMed Central

    Dassa, Emmanuel P; Dufour, Eric; Gonçalves, Sérgio; Paupe, Vincent; Hakkaart, Gertjan A J; Jacobs, Howard T; Rustin, Pierre

    2009-01-01

    Cytochrome c oxidase (COX) deficiency is associated with a wide spectrum of clinical conditions, ranging from early onset devastating encephalomyopathy and cardiomyopathy, to neurological diseases in adulthood and in the elderly. No method of compensating successfully for COX deficiency has been reported so far. In vitro, COX-deficient human cells require additional glucose, pyruvate and uridine for normal growth and are specifically sensitive to oxidative stress. Here, we have tested whether the expression of a mitochondrially targeted, cyanide-resistant, alternative oxidase (AOX) from Ciona intestinalis could alleviate the metabolic abnormalities of COX-deficient human cells either from a patient harbouring a COX15 pathological mutation or rendered deficient by silencing the COX10 gene using shRNA. We demonstrate that the expression of the AOX, well-tolerated by the cells, compensates for both the growth defect and the pronounced oxidant-sensitivity of COX-deficient human cells. PMID:20049701

  8. Alternative oxidase and plastoquinol terminal oxidase in marine prokaryotes of the Sargasso Sea.

    PubMed

    McDonald, Allison E; Vanlerberghe, Greg C

    2005-04-11

    Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world's oceans.

  9. [NADPH oxidases, Nox: new isoenzymes family].

    PubMed

    Chuong Nguyen, Minh Vu; Lardy, Bernard; Paclet, Marie-Hélène; Rousset, Francis; Berthier, Sylvie; Baillet, Athan; Grange, Laurent; Gaudin, Philippe; Morel, Françoise

    2015-01-01

    NADPH oxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acquired from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophysiological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the function of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes.

  10. Visualization of monoamine oxidase in human brain

    SciTech Connect

    Fowler, J.S.; Volkow, N.D.; Wang, G.J.; Pappas, N.; Shea, C.; MacGregor, R.R.; Logan, J.

    1996-12-31

    Monoamine oxidase is a flavin enzyme which exists in two subtypes, MAO A and MAO B. In human brain MAO B predominates and is largely compartmentalized in cell bodies of serotonergic neurons and glia. Regional distribution of MAO B was determined by positron computed tomography with volunteers after the administration of deuterium substituted [11C]L-deprenyl. The basal ganglia and thalamus exhibited the greatest concentrations of MAO B with intermediate levels in the frontal cortex and cingulate gyrus while lowest levels were observed in the parietal and temporal cortices and cerebellum. We observed that brain MAO B increases with are in health normal subjects, however the increases were generally smaller than those revealed with post-mortem studies.

  11. NADPH Oxidases in Lung Health and Disease

    PubMed Central

    Bernard, Karen; Hecker, Louise; Luckhardt, Tracy R.; Cheng, Guangjie

    2014-01-01

    Abstract Significance: The evolution of the lungs and circulatory systems in vertebrates ensured the availability of molecular oxygen (O2; dioxygen) for aerobic cellular metabolism of internal organs in large animals. O2 serves as the physiologic terminal acceptor of mitochondrial electron transfer and of the NADPH oxidase (Nox) family of oxidoreductases to generate primarily water and reactive oxygen species (ROS), respectively. Recent advances: The purposeful generation of ROS by Nox family enzymes suggests important roles in normal physiology and adaptation, most notably in host defense against invading pathogens and in cellular signaling. Critical issues: However, there is emerging evidence that, in the context of chronic stress and/or aging, Nox enzymes contribute to the pathogenesis of a number of lung diseases. Future Directions: Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease. Antioxid. Redox Signal. 20, 2838–2853. PMID:24093231

  12. Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

    PubMed

    Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

    2016-10-01

    Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

  13. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  14. Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase

    SciTech Connect

    Atlow, S.C.; Bonadonna-Aparo, L.; Klibanov, A.M.

    1984-01-01

    A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial wastewater samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.

  15. Characterization of two amine oxidases from Aspergillus carbonarius AIU 205.

    PubMed

    Sugawara, Asami; Matsui, Daisuke; Yamada, Miwa; Asano, Yasuhisa; Isobe, Kimiyasu

    2015-06-01

    We have reported that Aspergillus carbonarius AIU 205, which was isolated by our group, produced three enzymes exhibiting oxidase activity for 4-aminobutanamide (4-ABAD) (J. Biosci. Bioeng., 117, 263-268, 2014). Among three enzymes, characteristics of enzyme I have been revealed, but those of the other two enzymes have not. In this study, we purified enzymes II and III, and compared their characteristics with those of enzyme I. Enzymes II and III also oxidized aliphatic monoamines, aromatic amines, and aliphatic aminoalcohols. In addition, the oxidase activity of both enzymes was strongly inhibited by carbonyl reagents and specific inhibitors for copper-containing amine oxidases. Thus, enzymes II and III were also classified into the copper-containing amine oxidase group (EC 1.4.3.6) along with enzyme I. However, these three enzymes differed from each other in their enzymatic, kinetic, and physicochemical properties. The N-terminal amino acid sequences also differed from each other; that of enzyme I was modified, that of enzyme II was similar to those of peroxisomal copper-containing amine oxidases, and that of enzyme III was similar to those of copper-containing amine oxidases from the genus Aspergillus. Therefore, we concluded that A. carbonarius AIU 205 produced three different types of amine oxidase in the mycelia.

  16. THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

    PubMed Central

    Powers, Wendell H.; Lewis, Stanley; Dawson, Charles R.

    1944-01-01

    1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na2HPO4. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied. PMID:19873382

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

    PubMed

    Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

    2013-01-01

    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 a

  18. Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase.

    PubMed

    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.

  19. [An endogenous inhibitor of monoamine oxidase A (tribulin A) from brain: purification and structure identification].

    PubMed

    Medvedev, A E; Kamyshanskaia, N S; Halket, J; Glover, V; Sandler, A

    1995-05-01

    The endogenous monoamine oxidase inhibitor, tribulin, contains several components which selectively (or nonselectively) inhibit monoamine oxidases A and B. The pig brain tribulin component selectively inhibiting monoamine oxidase A was purified and identified as 4-hydroxyphenylethanol using gas chromatography-mass spectrometry. This compound was also found in the rabbit brain tribulin fraction which selectively inhibits monoamine oxidase A but has no influence on monoamine oxidase B. 4-Hydroxyphenylethanol inhibits monoamine oxidase A in an incompetitive manner with respect to the substrate, serotonin (Ki = 1.4 mM). Possible pathways of 4-hydroxyphenylethanol synthesis and its biological importance as the monoamine oxidase A inhibiting component of tribulin are discussed.

  20. Aldehyde-induced xanthine oxidase activity in raw milk.

    PubMed

    Steffensen, Charlotte L; Andersen, Henrik J; Nielsen, Jacob H

    2002-12-04

    In the present study, the aldehyde-induced pro-oxidative activity of xanthine oxidase was followed in an accelerated raw milk system using spin-trap electron spin resonance (ESR) spectroscopy. The aldehydes acetaldehyde, propanal, hexanal, trans-2-hexenal, trans-2-heptenal, trans-2-nonenal, and 3-methyl-2-butenal were all found to initiate radical reactions when added to milk. Formation of superoxide through aldehyde-induced xanthine oxidase activity is suggested as the initial reaction, as all tested aldehydes were shown to trigger superoxide formation in an ultrahigh temperature (UHT) milk model system with added xanthine oxidase. It was found that addition of aldehydes to milk initially increased the ascorbyl radical concentration with a subsequent decay due to ascorbate depletion, which renders the formation of superoxide in milk with added aldehyde. The present study shows for the first time potential acceleration of oxidative events in milk through aldehyde-induced xanthine oxidase activity.

  1. Isolation of oxidase-negative Pseudomonas aeruginosa from sputum culture.

    PubMed

    Hampton, K D; Wasilauskas, B L

    1979-05-01

    Two isolates of Pseudomonas aeruginosa lacking characteristic indophenol oxidase were recovered from a sputum specimen. A discussion of the characteristic biochemical tests and antibiograms along with a possible explanation for this phenomenon is presented.

  2. Polyphenol oxidase produced during encystation of Acanthamoeba castellanii.

    PubMed

    Sykes, D E; Band, R N

    1985-08-01

    Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.

  3. Beyond brown: polyphenol oxidases as enzymes of plant specialized metabolism.

    PubMed

    Sullivan, Michael L

    2014-01-01

    Most cloned and/or characterized plant polyphenol oxidases (PPOs) have catechol oxidase activity (i.e., they oxidize o-diphenols to o-quinones) and are localized or predicted to be localized to plastids. As a class, they have broad substrate specificity and are associated with browning of produce and other plant materials. Because PPOs are often induced by wounding or pathogen attack, they are most generally believed to play important roles in plant defense responses. However, a few well-characterized PPOs appear to have very specific roles in the biosynthesis of specialized metabolites via both tyrosinase (monophenol oxidase) and catechol oxidase activities. Here we detail a few examples of these and explore the possibility that there may be many more "biosynthetic" PPOs.

  4. Stimulation of cellular XTT reduction by cytochrome oxidase inhibitors.

    PubMed

    Kunimoto, S; Nosaka, C; Takeuchi, T

    1999-06-01

    XTT reducing activity by CHO and L1210 cells was found to be stimulated by the presence of cytochrome oxidase inhibitors such as NaN3 or KCN. Among the other respiratory chain inhibitors, antimycin A (a complex III inhibitor) and chlorpromazine inhibited cellular XTT reduction, and rotenone and malonate showed slight inhibition and no effect, respectively. It is suggested that XTT reduction is coupled with the respiratory chain via cytochrome c, which is located between complexes III and IV (cytochrome oxidase).

  5. The Impact of Single Nucleotide Polymorphisms on Human Aldehyde OxidaseS

    PubMed Central

    Hartmann, Tobias; Terao, Mineko; Garattini, Enrico; Teutloff, Christian; Alfaro, Joshua F.; Jones, Jeffrey P.; Leimkühler, Silke

    2012-01-01

    Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 μg/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs. PMID:22279051

  6. Confirmation of a blocked amino terminus of sulfhydryl oxidase

    SciTech Connect

    Janolino, V.G.; Morrison-Rowe, S.J.; Swaisgood, H.E. )

    1990-09-01

    The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with ({sup 14}C)iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 ({plus minus} 5)-kDa band observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked.

  7. Characterization of monomeric substates of ascorbate oxidase.

    PubMed

    Di Venere, Almerinda; Nicolai, Eleonora; Rosato, Nicola; Rossi, Antonello; Finazzi Agrò, Alessandro; Mei, Giampiero

    2011-05-01

    Ascorbate oxidase (AAO) is a large, multidomain, dimeric protein whose folding/unfolding pathway is characterized by a complex, multistep process. Here we used fluorescence correlation spectroscopy to demonstrate the formation of partially folded monomers by pH-induced full dissociation into subunits. Hence, the structural features of monomeric AAO could be studied by fluorescence and CD spectroscopy. We found that the monomers keep their secondary structure, whereas subtle conformational changes in the tertiary structure become apparent. AAO dissociation has also been studied when unfolding the protein by high hydrostatic pressure at different pH values. A strong protein concentration dependence was observed at pH 8, whereas the enzyme was either monomeric or dimeric at pH 10 and 6, respectively. The calculated volume change associated with the unfolding of monomeric AAO, ΔV ∼ -55 mL·mol(-1), is in the range observed for most proteins of the same size. These findings demonstrate that partially folded monomeric species might populate the energy landscape of AAO and that the overall AAO stability is crucially controlled by a few quaternary interactions at the subunits' interface.

  8. The function of ascorbate oxidase in tobacco.

    PubMed

    Pignocchi, Cristina; Fletcher, John M; Wilkinson, Joy E; Barnes, Jeremy D; Foyer, Christine H

    2003-07-01

    The function of the apoplastic enzyme ascorbate oxidase (AO) was investigated in tobacco (Nicotiana tabacum). The abundance of AO mRNA was up-regulated by light. Cytosolic ascorbate peroxidase (APX1) transcripts were also highest in the light. In contrast, L-galactono-gamma-lactone dehydrogenase, stromal APX, and thylakoid APX transcripts remained constant over the day/night cycle. Salicylic acid inhibited growth, increased expression of the pathogenesis-related protein (PR) 1a, and decreased AO transcript abundance. In contrast, the application of auxin enhanced growth and increased AO and PR 1a gene expression. Therefore, AO transcript abundance varied in a manner similar to hormone-mediated changes in plant growth. To study the effects of modified AO expression on growth, transformed tobacco plants expressing AO in the sense and antisense orientations were generated. The resultant large changes in apoplastic AO activity in the transformed tobacco plants had little effect on whole leaf ascorbate (AA) content, but they had dramatic effects on apoplastic AA levels. Enhanced AO activity oxidized the apoplastic AA pool, whereas decreased AO activity increased the amount of AA compared with dehydroascorbate. A relationship was observed between AO activity and plant height and biomass. Native AO transcript levels were no longer subject to light/dark regulation in AO sense and antisense plants. Taken together, these data show that there is an interaction between hormone, redox, and light signals at the level of the apoplast via modulation of ion of AA content.

  9. Monoamine oxidase: Radiotracer chemistry and human studies

    DOE PAGES

    Fowler, Joanna S.; Logan, Jean; Shumay, Elena; ...

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serot onin, norepinephrine and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson’s disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 since the first radiotracers were developed and the first PET images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variablesmore » which have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological and psychiatric disorders; (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers which are currently used and possible new applications.« less

  10. Monoamine oxidase: Radiotracer chemistry and human studies

    SciTech Connect

    Fowler, Joanna S.; Logan, Jean; Shumay, Elena; Alia-Klein, Nelly; Wang, Gene-Jack; Volkow, Nora D.

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serot onin, norepinephrine and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson’s disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 since the first radiotracers were developed and the first PET images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variables which have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological and psychiatric disorders; (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers which are currently used and possible new applications.

  11. Modular assembly of yeast cytochrome oxidase.

    PubMed

    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.

  12. Polyphenol oxidase from yacon roots (Smallanthus sonchifolius).

    PubMed

    Neves, Valdir Augusto; da Silva, Maraiza Aparecida

    2007-03-21

    Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius) was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The enzyme had a molecular weight of 45 490+/-3500 Da and Km values of 0.23, 1.14, 1.34, and 5.0 mM for the substrates caffeic acid, chlorogenic acid, 4-methylcatechol, and catechol, respectively. When assayed with resorcinol, DL-DOPA, pyrogallol, protocatechuic, p-coumaric, ferulic, and cinnamic acids, catechin, and quercetin, the PPO showed no activity. The optimum pH varied from 5.0 to 6.6, depending on substrate. PPO activity was inhibited by various phenolic and nonphenolic compounds. p-Coumaric and cinnamic acids showed competitive inhibition, with Ki values of 0.017 and 0.011 mM, respectively, using chlorogenic acid as substrate. Heat inactivation from 60 to 90 degrees C showed the enzyme to be relatively stable at 60-70 degrees C, with progressive inactivation when incubated at 80 and 90 degrees C. The Ea (apparent activation energy) for inactivation was 93.69 kJ mol-1. Sucrose, maltose, glucose, fructose, and trehalose at high concentrations appeared to protect yacon PPO against thermal inactivation at 75 and 80 degrees C.

  13. Origin and evolution of lysyl oxidases

    PubMed Central

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

    2015-01-01

    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. PMID:26024311

  14. Leflunomide, a Reversible Monoamine Oxidase Inhibitor.

    PubMed

    Petzer, Jacobus P; Petzer, Anél

    2016-01-01

    A screening study aimed at identifying inhibitors of the enzyme, monoamine oxidase (MAO), among clinically used drugs have indicated that the antirheumatic drug, leflunomide, is an inhibitor of both MAO isoforms. Leflunomide inhibits human MAO-A and MAO-B and exhibits IC50 values of 19.1 μM and 13.7 μM, respectively. The corresponding Ki values are 17.7 μM (MAO-A) and 10.1 μM (MAO-B). Dialyses of mixtures of the MAO enzymes and leflunomide show that inhibition of the MAOs by leflunomide is reversible. The principal metabolite of leflunomide, teriflunomide (A77 1726), in contrast is not an MAO inhibitor. This study concludes that, although leflunomide is only moderately potent as an MAO inhibitor, isoxazole derivatives may represent a general class of MAO inhibitors and this heterocycle may find application in MAO inhibitor design. In this respect, MAO inhibitors are used in the clinic for the treatment of depressive illness and Parkinson's disease, and are under investigation as therapy for certain types of cancer, Alzheimer's disease and age-related impairment of cardiac function.

  15. Molecular "wiring" glucose oxidase in supramolecular architecture.

    PubMed

    Deng, Liu; Liu, Ying; Yang, Guocheng; Shang, Li; Wen, Dan; Wang, Fuan; Xu, Zhiai; Dong, Shaojun

    2007-07-01

    Supramolecular organized multilayers were constructed by multiwalled carbon nanotubes modified with ferrocene-derivatized poly(allylamine) redox polymer and glucose oxidase by electrostatic self-assembly. From the analysis of voltammetric signals and fluorescence results, a linear increment of the coverage of enzyme per bilayer was estimated, which demonstrated that the multilayer is constructed in a spatially ordered manner. The cyclic voltammograms obtained from the indium tin oxide (ITO) electrodes coated by the (Fc-PAH@CNT/GOx)n multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers; that is, the sensitivity is tunable by controlling the number of bilayers associated with ITO electrodes. The incorporation of redox-polymer-functionalized carbon nanotubes (CNT) into enzyme films resulted in a 6-10-fold increase in the glucose electrocatalytic current; the bimolecular rate constant of FADH2 oxidation (wiring efficiency) was increased up to 12-fold. Impedance spectroscopy data have yielded the electron diffusion coefficient (De) of this nanostructure to be over 10(-8) cm2 s(-1), which is typically higher than those systems without CNT by at least a factor of 10, indicating that electron transport in the new supramolecular architecture was enhanced by communication of the redox active site of enzyme, redox polymer, and CNT.

  16. MONOAMINE OXIDASE: RADIOTRACER DEVELOPMENT AND HUMAN STUDIES.

    SciTech Connect

    FOWLER,J.S.; LOGAN,J.; VOLKOW,N.D.; WANG,G.J.; MACGREGOR,R.R.; DING,Y.S.

    2000-09-28

    PET is uniquely capable of providing information on biochemical transformations in the living human body. Although most of the studies of monoamine oxidase (MAO) have focused on measurements in the brain, the role of peripheral MAO as a phase 1 enzyme for the metabolism of drugs and xenobiotics is gaining attention (Strolin Benedetti and Tipton, 1998; Castagnoli et al., 1997.). MAO is well suited for this role because its concentration in organs such as kidneys, liver and digestive organs is high sometimes exceeding that in the brain. Knowledge of the distribution of the MAO subtypes within different organs and different cells is important in determining which substrates (and which drugs and xenobiotics) have access to which MAO subtypes. The highly variable subtype distribution with different species makes human studies even more important. In addition, the deleterious side effects of combining MAO inhibitors with other drugs and with foodstuffs makes it important to know the MAO inhibitory potency of different drugs both in the brain and in peripheral organs (Ulus et al., 2000). Clearly PET can play a role in answering these questions, in drug research and development and in discovering some of the factors which contribute to the highly variable MAO levels in different individuals.

  17. The New Jersey Medicaid ACO Demonstration Project: seeking opportunities for better care and lower costs among complex low-income patients.

    PubMed

    Cantor, Joel C; Chakravarty, Sujoy; Tong, Jian; Yedidia, Michael J; Lontok, Oliver; DeLia, Derek

    2014-12-01

    A small but growing number of states are turning to accountable care concepts to improve their Medicaid programs. In 2011 New Jersey enacted the Medicaid Accountable Care Organization (ACO) Demonstration Project to offer local provider coalitions the opportunity to share any savings they generate. Impetus came from initiatives in Camden that aim to reduce costs through improved care coordination among hospital high users and that have received considerable media attention and substantial federal and private grant support. Though broadly similar to Medicare and commercial ACOs, the New Jersey demonstration addresses the unique concerns faced by Medicaid populations. Using hospital all-payer billing data, we estimate savings from care improvement efforts among inpatient and emergency department high users in thirteen communities that are candidates for participation in the New Jersey demonstration. We also examine their characteristics to inform Medicaid accountable care strategies. We find substantial variation in the share of high-user hospital patients across the study communities and high rates of avoidable use and costs among these patients. The potential savings among Medicaid enrollees are considerable, particularly if Medicaid ACOs can develop ways to successfully address the high burden of chronic illness and behavioral health conditions prevalent in the prospective demonstration communities.

  18. Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans.

    PubMed

    Sugio, Tsuyoshi; Hisazumi, Tomohiro; Kanao, Tadayoshi; Kamimura, Kazuo; Takeuchi, Fumiaki; Negishi, Atsunori

    2006-07-01

    It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

  19. Conversion of Escherichia coli pyruvate oxidase to an 'alpha-ketobutyrate oxidase'.

    PubMed Central

    Chang, Y Y; Cronan, J E

    2000-01-01

    Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate ('alpha-ketobutyrate'), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380 had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in K(m) for pyruvate and a 10-fold lower V(max) with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme. PMID:11104678

  20. Temporal and spectral evolution of a storage ring FEL source: Experimental results on Super-ACO and new theoretical approach

    SciTech Connect

    Hara, T.; Couprie, M.E. ||

    1995-12-31

    The Super-ACO FEL source in UV is now used for applications like a time-resolved fluorescence in biology and two colors experiments coupling FEL and Synchrotron Radiation, which are naturally synchronized. The stability of the FEL is then a critical issue for the users. Detailed experimental studies conducted on the temporal characteristics of the laser micropulse showed various phenomena, such as a longitudinal micropulse jitter and a deformation of a longitudinal micropulse distribution. A similar analysis has been performed on the laser spectral evolution with a scanning Fabry-Perot interferometer, showing a spectrum narrowing, and a wavelength drift. A longitudinal feedback system developed after the first user experiment, allowed to reduce significantly the longitudinal jitter, the intensity fluctuation and the spectral drift. Nevertheless, the stability of the FEL is very dependent on any perturbation, and the observed phenomena can not be described by former models like super-mode assuming a stationary regime. A new theoretical model has then been developed, in order to simulate dynamic behaviors. A simple iterative method is employed to obtain the laser spectrum. The access to the temporal distribution requires additional complexity, because the Fourier transformation has to be performed for each pass. The comparison between the experimental data and the simulation results will be given.

  1. Identification of the alternative terminal oxidase of higher plant mitochondria

    PubMed Central

    Elthon, Thomas E.; McIntosh, Lee

    1987-01-01

    In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of Sauromatum guttatum and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in Sauromatum, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed. Images PMID:16593898

  2. Cytokinin Oxidase from Phaseolus vulgaris Callus Tissues 1

    PubMed Central

    Chatfield, J. Mark; Armstrong, Donald J.

    1987-01-01

    The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N6-(Δ2-isopentenyl)-adenine-2,8-3H (i6 Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N6-side chain of i6Ade. PMID:16665511

  3. Pentamines as substrate for human spermine oxidase

    PubMed Central

    Takao, Koichi; Shirahata, Akira; Samejima, Keijiro; Casero, Robert A.; Igarashi, Kazuei; Sugita, Yoshiaki

    2013-01-01

    Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO. PMID:23449327

  4. Monoamine oxidase inhibitory components from Cayratia japonica.

    PubMed

    Han, Xiang Hua; Hong, Seong Su; Hwang, Ji Sang; Lee, Myung Koo; Hwang, Bang Yeon; Ro, Jai Seup

    2007-01-01

    Seven flavonoids were isolated from the whole plants and fruits of Cayratia japonica through the activity-guided isolation of a methanol extract using a monoamine oxidase (MAO) inhibition assay as a monitor. The chemical structures of the isolates were assigned as apigenin-7-O-beta-D-glucuronopyranoside (1), apigenin (2), luteolin (3), luteolin-7-O-beta-D-glucopyranoside (4), (+)-dihydroquercetin (taxifolin) (5), (+)-dihydrokaempferol (aromadendrin) (6) and quercetin (7). Among the isolated compounds, flavones such as apigenin (2) and luteolin (3), as well as the flavonol, quercetin (7) showed potent inhibitory effects against the MAO activity with IC50 values of 6.5, 22.6, and 31.6 microM, respectively. However, the flavone glycosides, apigenin-7-O-beta-D-glucuronopyranoside (1) and luteolin-7-O-beta-D-glucopyranoside (4), showed mild MAO inhibition (IC50 values: 81.7 and 118.6 microM, respectively). The flavanonol derivatives, taxifolin (5) and aromadendrin (6), also showed weak inhibition (IC50 values: 154.7 and 153.1 microM, respectively). Furthermore, quercetin (7) had a more potent inhibitory effect on MAO-A (IC50 value: 2.8 microM) than MAO-B (IC50 value: 90.0 microM). Apigenin (2) and luteolin (3) also preferentially inhibited MAO-A (IC50 values: 1.7 and 4.9 microM, respectively) compared with MAO-B (IC50 values: 12.8 and 59.7 microM, respectively).

  5. Current situation of asthma-COPD overlap syndrome (ACOS) in Chinese patients older than 40 years with airflow limitation: rationale and design for a multicenter, cross-sectional trial (study protocol)

    PubMed Central

    Yao, Wanzhen; Cai, Baiqiang; Chen, Ping; Ling, Xia; Shang, Hongyan

    2016-01-01

    Background Asthma and chronic obstructive pulmonary disease (COPD) are the frequently occurring chronic airway diseases, and the overlapping syndrome observed in the majority of patients has been recently defined as asthma-COPD overlap syndrome (ACOS) by the Global Initiative for Chronic Obstructive Lung (GOLD, 2014) and Global initiative for Asthma (GINA, 2015). The proportion, features, and clinical practice of ACOS still remain elusive in China. We are conducting this multicenter, cross-sectional, observational study (NCT02600221) to investigate the distributions of chronic obstructive diseases in patients >40 years of age with chronic airflow limitation in China along with determination of the main clinical practice and features of these diseases. The study will also explore the factors that may influence the exacerbations and severity of ACOS in Chinese patients (>40 years of age). Methods A total of 2,000 patients (age, ≥40 years; either sex) who are clinically diagnosed as having asthma, COPD/chronic bronchitis/emphysema, or ACOS for at least 12 months with airflow limitation [post-bronchodilator forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC): <0.7] will be enrolled from approximately 20 sites in China between December 2015 and December 2016. The proportion of ACOS among patients older than 40 years based on GINA 2015 and GOLD 2014 definitions is the primary variable. Following were the secondary variables: the proportions of COPD and asthma among the patients, distributions of the severity of airflow limitation, distribution of groups according to GOLD 2011 group definition (A, B, C, D), and the distribution of medication by drug class in patients with ACOS, asthma, and COPD. Acute exacerbation history, hospitalization, and severity of ACOS as evaluated using COPD Assessment Test, Asthma Control Questionnaire-5, and Modified British Medical Research Council in patients with ACOS were also assessed. Implications This will be the

  6. Cation binding site of cytochrome c oxidase: progress report.

    PubMed

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A

    2014-07-01

    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 earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  7. Crystal Structure of Alcohol Oxidase from Pichia pastoris

    PubMed Central

    Valerius, Oliver; Feussner, Ivo; Ficner, Ralf

    2016-01-01

    FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring. PMID:26905908

  8. NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

    PubMed

    Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

    2016-01-01

    NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

  9. Red clover polyphenol oxidase and lipid metabolism.

    PubMed

    Van Ranst, G; Lee, M R F; Fievez, V

    2011-02-01

    Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPO's lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vitro rumen system. It is demonstrated that high (induction of) PPO activity can lead to lower lipolysis in the silage and lower biohydrogenation in the rumen. There are three hypotheses on its working mechanism: (i) protein-bound phenols could directly bind to enzymes (e.g. lipases) as such inhibiting them; (ii) binding of quinones in and between proteins embedded in a lipid membrane (e.g. in the chloroplast) could lead to encapsulation of the lipids; (iii) direct binding of quinones to nucleophilic sites in polar lipids also could lead to protection. There is no exclusive evidence on which mechanism is most important, although there are strong indications that only lipid

  10. Forage polyphenol oxidase and ruminant livestock nutrition

    PubMed Central

    Lee, Michael R. F.

    2014-01-01

    Polyphenol oxidase (PPO) is predominately associated with the detrimental effect of browning fruit and vegetables, however, interest within PPO containing forage crops (crops to be fed to animals) has grown since the browning reaction was associated with reduced nitrogen (N) losses in silo and the rumen. The reduction in protein breakdown in silo of red clover (high PPO forage) increased the quality of protein, improving N-use efficiency [feed N into product N (e.g., Milk): NUE] when fed to ruminants. A further benefit of red clover silage feeding is a significant reduction in lipolysis (cleaving of glycerol-based lipid) in silo and an increase in the deposition of beneficial C18 polyunsaturated fatty acid (PUFA) in animal products, which has also been linked to PPO activity. PPOs protection of plant protein and glycerol based-PUFA in silo is related to the deactivation of plant proteases and lipases. This deactivation occurs through PPO catalyzing the conversion of diphenols to quinones which bind with cellular nucleophiles such as protein reforming a protein-bound phenol (PBP). If the protein is an enzyme (e.g., protease or lipase) the complexing denatures the enzyme. However, PPO is inactive in the anaerobic rumen and therefore any subsequent protection of plant protein and glycerol based-PUFA in the rumen must be as a result of events that occurred to the forage pre-ingestion. Reduced activity of plant proteases and lipases would have little effect on NUE and glycerol based-PUFA in the rumen due to the greater concentration of rumen microbial proteases and lipases. The mechanism for PPOs protection of plant protein in the rumen is a consequence of complexing plant protein, rather than protease deactivation per se. These complexed proteins reduce protein digestibility in the rumen and subsequently increase undegraded dietary protein flow to the small intestine. The mechanism for protecting glycerol-based PUFA has yet to be fully elucidated but may be associated

  11. Expression of alternative oxidase in Drosophila ameliorates diverse phenotypes due to cytochrome oxidase deficiency.

    PubMed

    Kemppainen, Kia K; Rinne, Juho; Sriram, Ashwin; Lakanmaa, Matti; Zeb, Akbar; Tuomela, Tea; Popplestone, Anna; Singh, Satpal; Sanz, Alberto; Rustin, Pierre; Jacobs, Howard T

    2014-04-15

    Mitochondrial dysfunction is a significant factor in human disease, ranging from systemic disorders of childhood to cardiomyopathy, ischaemia and neurodegeneration. Cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, is a frequent target. Lower eukaryotes possess alternative respiratory-chain enzymes that provide non-proton-translocating bypasses for respiratory complexes I (single-subunit reduced nicotinamide adenine dinucleotide dehydrogenases, e.g. Ndi1 from yeast) or III + IV [alternative oxidase (AOX)], under conditions of respiratory stress or overload. In previous studies, it was shown that transfer of yeast Ndi1 or Ciona intestinalis AOX to Drosophila was able to overcome the lethality produced by toxins or partial knockdown of complex I or IV. Here, we show that AOX can provide a complete or substantial rescue of a range of phenotypes induced by global or tissue-specific knockdown of different cIV subunits, including integral subunits required for catalysis, as well as peripheral subunits required for multimerization and assembly. AOX was also able to overcome the pupal lethality produced by muscle-specific knockdown of subunit CoVb, although the rescued flies were short lived and had a motility defect. cIV knockdown in neurons was not lethal during development but produced a rapidly progressing locomotor and seizure-sensitivity phenotype, which was substantially alleviated by AOX. Expression of Ndi1 exacerbated the neuronal phenotype produced by cIV knockdown. Ndi1 expressed in place of essential cI subunits produced a distinct residual phenotype of delayed development, bang sensitivity and male sterility. These findings confirm the potential utility of alternative respiratory chain enzymes as tools to combat mitochondrial disease, while indicating important limitations thereof.

  12. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

    SciTech Connect

    Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, Francois; Whittaker, James W. . E-mail: jim@ebs.ogi.edu

    2007-05-18

    High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an {alpha}-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4 x 10{sup 4} U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.

  13. Purification and two-dimensional crystallization of bacterial cytochrome oxidases.

    PubMed

    Warne, A; Wang, D N; Saraste, M

    1995-12-01

    A novel strategy which employes chromatography on an immobilized metal ion has been developed for the purification of bacterial cytochrome c and quinol oxidases. Many bacterial oxidase complexes appear to have a natural affinity to bind to the chelated copper ion. A combination of three different chromatographic principles (anion exchange, metal-affinity and gel filtration) makes an effective tool chest for the preparation of homogeneous and protein-chemically pure bacterial oxidases. These preparations have been used for two-dimensional crystallization. Until now, crystals have been obtained using the Paracococcus denitrificans and Rhodobacter sphaeroides cytochrome aa3 and the Escherichia coli cytochrome bo. The crystals diffract to approximately 2.5 nm in negative stain and have potential for further structural studies.

  14. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

    PubMed Central

    Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, François; Whittaker, James W.

    2007-01-01

    High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an α-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4×104 U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions. PMID:17399681

  15. Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha.

    PubMed

    Veenhuis, M; van Dijken, J P; Harder, W

    1976-12-01

    The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes D-aminoacid oxidase and L-alpha-hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.

  16. Optimization on Paddy Crops in Central Java (with Solver, SVD on Least Square and ACO (Ant Colony Algorithm))

    NASA Astrophysics Data System (ADS)

    Parhusip, H. A.; Trihandaru, S.; Susanto, B.; Prasetyo, S. Y. J.; Agus, Y. H.; Simanjuntak, B. H.

    2017-03-01

    Several algorithms and objective functions on paddy crops have been studied to get optimal paddy crops in Central Java based on the data given from Surakarta and Boyolali. The algorithms are linear solver, least square and Ant Colony Algorithms (ACO) to develop optimization procedures on paddy crops modelled with Modified GSTAR (Generalized Space-Time Autoregressive) and nonlinear models where the nonlinear models are quadratic and power functions. The studied data contain paddy crops from Surakarta and Boyolali determining the best period of planting in the year 1992-2012 for Surakarta where 3 periods for planting are known and the optimal amount of paddy crops in Boyolali in the year 2008-2013. Having these analyses may guide the local agriculture government to give a decision on rice sustainability in its region. The best period for planting in Surakarta is observed, i.e. the best period is in September-December based on the data 1992-2012 by considering the planting area, the cropping area, and the paddy crops are the most important factors to be taken into account. As a result, we can refer the paddy crops in this best period (about 60.4 thousand tons per year) as the optimal results in 1992-2012 where the used objective function is quadratic. According to the research, the optimal paddy crops in Boyolali about 280 thousand tons per year where the studied factors are the amount of rainfalls, the harvested area and the paddy crops in 2008-2013. In this case, linear and power functions are studied to be the objective functions. Compared to all studied algorithms, the linear solver is still recommended to be an optimization tool for a local agriculture government to predict paddy crops in future.

  17. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    SciTech Connect

    Qin, Ling; Mills, Denise A.; Buhrow, Leann; Hiser, Carrie; Ferguson-Miller, Shelagh

    2010-09-02

    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

  18. Purification of xanthine dehydrogenase and sulfite oxidase from chicken liver.

    PubMed

    Ratnam, K; Brody, M S; Hille, R

    1996-05-01

    Xanthine dehydrogenase and sulfite oxidase from chicken liver are oxomolybdenum enzymes which catalyze the oxidation of xanthine to uric acid and sulfite to sulfate, respectively. Independent purification protocols have been previously described for both enzymes. Here we describe a procedure by which xanthine dehydrogenase and sulfite oxidase are purified simultaneously from the same batch of fresh chicken liver. Also, unlike the protocols described earlier, this procedure avoids the use of acetone extraction as well as a heat step, thus minimizing damage to the molybdenum centers of the enzymes.

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

    PubMed

    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.

  20. Expression of terminal oxidases under nutrient-starved conditions in Shewanella oneidensis: detection of the A-type cytochrome c oxidase

    PubMed Central

    Le Laz, Sébastien; kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2016-01-01

    Shewanella species are facultative anaerobic bacteria that colonize redox-stratified habitats where O2 and nutrient concentrations fluctuate. The model species Shewanella oneidensis MR-1 possesses genes coding for three terminal oxidases that can perform O2 respiration: a bd-type quinol oxidase and cytochrome c oxidases of the cbb3-type and the A-type. Whereas the bd- and cbb3-type oxidases are routinely detected, evidence for the expression of the A-type enzyme has so far been lacking. Here, we investigated the effect of nutrient starvation on the expression of these terminal oxidases under different O2 tensions. Our results reveal that the bd-type oxidase plays a significant role under nutrient starvation in aerobic conditions. The expression of the cbb3-type oxidase is also modulated by the nutrient composition of the medium and increases especially under iron-deficiency in exponentially growing cells. Most importantly, under conditions of carbon depletion, high O2 and stationary-growth, we report for the first time the expression of the A-type oxidase in S. oneidensis, indicating that this terminal oxidase is not functionally lost. The physiological role of the A-type oxidase in energy conservation and in the adaptation of S. oneidensis to redox-stratified environments is discussed. PMID:26815910

  1. Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana

    PubMed Central

    Andronis, Efthimios A.; Moschou, Panagiotis N.; Toumi, Imene; Roubelakis-Angelakis, Kalliopi A.

    2014-01-01

    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 to putrescine 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. PMID:24765099

  2. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies.

    PubMed

    Andjelković, Ana; Oliveira, Marcos T; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T

    2015-12-17

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.

  3. Covalent immobilization of ascorbate oxidase onto polycarbonate strip for L-ascorbic acid detection.

    PubMed

    Kannoujia, Dileep Kumar; Kumar, Saroj; Nahar, Pradip

    2012-10-01

    Herein, a simple and rapid method is described for detection of L-ascorbic acid by ascorbate oxidase immobilized onto polycarbonate strip pre-activated by 1-fluoro-2-nitro-4-azidobenzene in photochemical reaction. Covalent attachment of ascorbate oxidase was confirmed by XPS studies. The immobilized-ascorbate oxidase shows higher pH, thermal and storage stability in comparison to free enzyme.

  4. Dopa oxidase activity and ceruloplasmin in the sera of hamsters with melanoma.

    PubMed

    Vachtenheim, J; Pavel, S; Duchon, J

    1981-01-01

    Two simple spectrophotometric assays have been employed for the measurement of dopa oxidase activity and ceruloplasmin polyphenol oxidase activity in the sera from normal hamsters and hamsters bearing melanotic melanoma. Both activities were found to be augmented in tumor animals, the dopa oxidase activity much more prominently. The levels of the enzymes tested increased proportionally to the tumor mass.

  5. Is photography-based taxonomy really inadequate, unnecessary, and potentially harmful for biological sciences? A reply to Ceríaco et al. (2016).

    PubMed

    Thorpe, Stephen E

    2017-01-26

    An opinion piece was recently published in this journal which, remarkably, is less than one page in length, but has 493 authors (signatories) and has gained over 15 thousand reads on ResearchGate in a very short time. Ceríaco et al. (2016) argue for a blanket ban to be imposed on the uncommon practice of basing new species descriptions in zoology on photographic evidence only of the animal. I herein aim to show that their argument is a non sequitur, i.e. their stated reasons do not support their desired conclusion.

  6. Phonon modes of A(Co 1/2Mn 1/2) O 3 ( A=La, Nd, Dy, Ho, Yb)

    NASA Astrophysics Data System (ADS)

    Gao, F.; Lewis, R. A.; Wang, X. L.; Dou, S. X.

    2001-09-01

    Phonon energies in cobaltite/manganites A(Co1/2Mn1/2)O3, where A is a lanthanide, have been determined by far-infrared spectroscopy. The phonon energies systematically shift and split and new modes appear as the mass of the lanthanide is increased through the series A=La, Nd, Dy, Ho, Yb. The behavior of the phonon modes correlates with the magnetic properties of this series of compounds, in particular with the appearance of metamagnetism for the compounds with smaller ions on the A site.

  7. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Oxidase screening test for gonorrhea. 866.2420 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...

  8. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Oxidase screening test for gonorrhea. 866.2420 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...

  9. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Oxidase screening test for gonorrhea. 866.2420 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...

  10. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Oxidase screening test for gonorrhea. 866.2420 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...

  11. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Oxidase screening test for gonorrhea. 866.2420 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...

  12. Amine oxidase activity regulates the development of pulmonary fibrosis.

    PubMed

    Marttila-Ichihara, Fumiko; Elima, Kati; Auvinen, Kaisa; Veres, Tibor Z; Rantakari, Pia; Weston, Christopher; Miyasaka, Masayuki; Adams, David; Jalkanen, Sirpa; Salmi, Marko

    2017-03-01

    In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing-3 (AOC3) is a cell-surface expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene-modified and inhibitor-treated mice in a bleomycin-induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3-deficient mice showed a 30-50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4(+) lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild-type control mice. AOC3 knock-in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild-type mice, a small-molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.-Marttila-Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis.

  13. IRON REGULATES XANTHINE OXIDASE ACTIVITY IN THE LUNG

    EPA Science Inventory

    The iron chelator deferoxamine has been reported to inhibit both xanthine oxidase (XO) and xanthine dehydrogenase activity, but the relationship of this effect to the availability of iron in the cellular and tissue environment remains unexplored. XO and total xanthine oxidoreduct...

  14. Solubilized cytochrome c oxidase from Paracoccus denitrificans is a monomer.

    PubMed

    Ludwig, B; Grabo, M; Gregor, I; Lustig, A; Regenass, M; Rosenbusch, J P

    1982-05-25

    Cytochrome c oxidase purified from the bacterium Paracoccus denitrificans was analyzed by analytical ultracentrifugation. In the detergent octyltetra/pentaoxyethylene (C8E45), the isolated enzyme exhibits a molecular weight of 79,000 to 84,000. The detergent-solubilized enzyme is thus a monomer which contains one copy of each of the two subunits.

  15. The number of nucleotide binding sites in cytochrome C oxidase.

    PubMed

    Rieger, T; Napiwotzki, J; Hüther, F J; Kadenbach, B

    1995-12-05

    The binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), [35S]ATP alpha S and 8-azido-[gamma-32P]ATP to isolated cytochrome c oxidase of bovine heart and liver and to the two-subunit enzyme of Paracoccus dentrificans was studied by measuring the fluorescence change or bound radioactivity, respectively. With TNP-ATP three binding sites were determined at cytochrome c oxidase from bovine heart and liver, both with two dissociation constants Kd of about 0.2 and 0.9 microM. Trypsin treatment of the enzyme from bovine heart, resulted in one binding site with a Kd of 0.3 microM. The two-subunit enzyme of Paracoccus dentrificans had only one binding site with a Kd of 3.6 microM. The binding of [35S]ATP alpha S to cytochrome c oxidase was studied by equilibrium dialysis. With the enzyme of bovine heart seven and the enzyme of liver six high-affinity binding sites with apparent Kd's of 7.5 and 12 microM, respectively, were obtained. The two-subunit enzyme of Paracoccus denitrificans had one binding site with a Kd of 20 microM. The large number of binding sites at cytochrome c oxidase from bovine heart, mainly at nuclear coded subunits, was verified by photoaffinity labelling with 8-azido-[gamma-32P]ATP.

  16. Purification of cytochrome c oxidase by lysine-affinity chromatography.

    PubMed

    Felsch, J; Kotake, S; Copeland, R A

    1992-02-01

    A method for the purification of cytochrome c oxidase that is based on the affinity of this enzyme for polycations such as poly-L-lysine is described. When detergent extracts of bovine cardiac mitochondria were applied to either a poly-L-lysine-agarose or a lysine-Sepharose column at low ionic strength, cytochrome c oxidase was found to adhere tightly, whereas the bulk of the proteins were eluted by washing with the same buffer. The cytochrome c oxidase was eluted by application of a linear potassium chloride gradient to the columns. The resulting enzyme was identical to that obtained by more traditional purification methods in terms of its subunit composition, optical and resonance Raman spectra, and cytochrome c oxidizing activity. When detergent extracts of spheroplasts from Paracoccus denitrificans were applied to these columns, the cytochrome c oxidase from this organism was also found to adhere tightly. Thus this purification method appears applicable to both prokaryotic and eukaryotic forms of the enzyme. The advantages of this new purification method are that it is less labor intensive than the traditional procedure and less expensive than methods based on cytochrome c-affinity chromatography.

  17. HypC, the anthrone oxidase involved in aflatoxin biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Based on gene disruption and enzyme activity, hypC, an open reading frame in the pksA (aflC)/nor-1 (aflD) intergenic region in the aflatoxin biosynthesis cluster, encodes a 17 kDa oxidase that catalyzes the conversion of norsolorinic acid anthrone to norsolorinic acid....

  18. Reducing peanut allergens by high pressure combined with polyphenol oxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) has been shown to reduce major peanut allergens (Ara h 1 and Ara h 2). Because high pressure (HP) can increase enzyme activity, we postulated that further reduction of peanut allergens can be achieved through HP combined with PPO. Peanut extracts were treated with each of th...

  19. The glucose oxidase-peroxidase assay for glucose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

  20. Platinum Nanoparticles: Efficient and Stable Catechol Oxidase Mimetics.

    PubMed

    Liu, Yi; Wu, Haohao; Chong, Yu; Wamer, Wayne G; Xia, Qingsu; Cai, Lining; Nie, Zhihong; Fu, Peter P; Yin, Jun-Jie

    2015-09-09

    Although enzyme-like nanomaterials have been extensively investigated over the past decade, most research has focused on the peroxidase-like, catalase-like, or SOD-like activity of these nanomaterials. Identifying nanomaterials having oxidase-like activities has received less attention. In this study, we demonstrate that platinum nanoparticles (Pt NPs) exhibit catechol oxidase-like activity, oxidizing polyphenols into the corresponding o-quinones. Four unique approaches are employed to demonstrate the catechol oxidase-like activity exerted by Pt NPs. First, UV-vis spectroscopy is used to monitor the oxidation of polyphenols catalyzed by Pt NPs. Second, the oxidized products of polyphenols are identified by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution mass spectrometry (HRMS) identification. Third, electron spin resonance (ESR) oximetry techniques are used to confirm the O2 consumption during the oxidation reaction. Fourth, the intermediate products of semiquinone radicals formed during the oxidation of polyphenols are determined by ESR using spin stabilization. These results indicate Pt NPs possess catechol oxidase-like activity. Because polyphenols and related bioactive substances have been explored as potent antioxidants that could be useful for the prevention of cancer and cardiovascular diseases, and Pt NPs have been widely used in the chemical industry and medical science, it is essential to understand the potential effects of Pt NPs for altering or influencing the antioxidant activity of polyphenols.

  1. Polyphenol oxidase activity in co-ensiled temperate grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) and its o-diphenol substrates have been shown to effectively decrease proteolytic activity during the ensiling of forages such as red clover. Orchardgrass and smooth bromegrass both contain high levels of PPO activity, but lack appropriate levels of o-diphenols to adequately...

  2. The proton pump of heme-copper oxidases.

    PubMed

    Papa, S; Capitanio, N; Glaser, P; Villani, G

    1994-05-01

    Proton pumping heme-copper oxidases represent the terminal, energy-transfer enzymes of respiratory chains in prokaryotes and eukaryotes. The CuB-heme a3 (or heme o) binuclear center, associated with the largest subunit I of cytochrome c and quinol oxidases, is directly involved in the coupling between dioxygen reduction and proton pumping. The role of the other subunits is less clear. The following aspects will be covered in this paper: i) the efficiency of coupling in the mitochondrial aa3 cytochrome c oxidase. In particular, the effect of respiratory rate and protonmotive force on the H+/e- stoichiometry and the role of subunit IV; ii) mutational analysis of the aa3 quinol oxidase of Bacillus subtilis addressed to the role of subunit III, subunit IV and specific residues in subunit I; iii) possible models of the protonmotive catalytic cycle at the binuclear center. The observations available suggest that H+/e- coupling is based on the combination of protonmotive redox catalysis at the binuclear center and co-operative proton transfer in the protein.

  3. Targeting NADPH oxidase and phospholipases A2 in Alzheimer's disease.

    PubMed

    Simonyi, Agnes; He, Yan; Sheng, Wenwen; Sun, Albert Y; Wood, W Gibson; Weisman, Gary A; Sun, Grace Y

    2010-06-01

    Alzheimer's disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Abeta) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Abeta. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Abeta in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A(2) (PLA(2)) and secretory PLA(2). In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Abeta NADPH oxidase and PLA(2) can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.

  4. [L-lysine-alpha-oxidase activity of some Trichoderma species].

    PubMed

    Smirnova, I P; Khaduev, S Kh

    1984-01-01

    Trichoderma cultures were tested for their ability to produce L-lysine-alpha-oxidase. The highest enzyme activity was manifested by T. harzianum (MGU), T. longibrachiatum Rifai VKM F-2025 and T. aureoviride Rifai VKM F-2026. The biosynthesis of the enzyme did not depend on the growth of the cultures and did not vary among the species.

  5. Beyond brown: polyphenol oxidases as enzymes of plant specialized metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most cloned and/or characterized plant polyphenol oxidases (PPOs) have catecholase activity (i.e., they oxidize o-diphenols to o-quinones) and are localized or predicted to be localized to plastids. As a class, they have broad substrate specificity and are associated with browning of produce and oth...

  6. Purification of gibberellin sub 53 -oxidase from spinach

    SciTech Connect

    Wilson, T.M.; Zeevaart, J.A.D. )

    1989-04-01

    Spinach is a long-day rosette plants, in which stem growth is mediated by gibberellins. It has been shown that two enzymatic steps, GA{sub 53}-oxidase and GA{sub 19}-oxidase, are controlled by light. To develop an understanding into this light regulation, purification of GA{sub 53}-oxidase has been undertaken. The original assay relied on the HPLC separation of the product and substrate, but was considered too slow for the development of a purification scheme. A TLC system was developed which in conjunction with improvements to the assay conditions was sensitive and gave rapid results. The partial purification of the GA{sub 53}-oxidase is achieved by a high speed centrifugation, 40-55% ammonium sulfate precipitation, an hydroxyapatite column, Sephadex G-100 column and an anion exchange FPLC column, Mono Q HR10/10, yielding 1000-fold purification and 15% recovery. Monoclonal antibodies to the protein will be raised and used to further characterize the enzyme.

  7. Structure of the trypanosome cyanide-insensitive alternative oxidase

    PubMed Central

    Shiba, Tomoo; Kido, Yasutoshi; Sakamoto, Kimitoshi; Inaoka, Daniel Ken; Tsuge, Chiaki; Tatsumi, Ryoko; Takahashi, Gen; Balogun, Emmanuel Oluwadare; Nara, Takeshi; Aoki, Takashi; Honma, Teruki; Tanaka, Akiko; Inoue, Masayuki; Matsuoka, Shigeru; Saimoto, Hiroyuki; Moore, Anthony L.; Harada, Shigeharu; Kita, Kiyoshi

    2013-01-01

    In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction. PMID:23487766

  8. Expression studies of gibberellin oxidases in developing pumpkin seeds.

    PubMed

    Frisse, Andrea; Pimenta, Maria João; Lange, Theo

    2003-03-01

    Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA(12)-aldehyde, GA(12), GA(15), GA(24), GA(25), and GA(9) to GA(14)-aldehyde, GA(14), GA(37), GA(36), GA(13), and GA(4), respectively. Recombinant 2-ox protein oxidized GA(9), GA(4), and GA(1) to GA(51), GA(34), and GA(8), respectively. Previously cloned GA 7-oxidase revealed additional 3beta-hydroxylation activity of GA(12). Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2beta,3beta-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed.

  9. Oxidative metabolic pathway of lenvatinib mediated by aldehyde oxidase.

    PubMed

    Inoue, Kazuko; Mizuo, Hitoshi; Kawaguchi, Shinki; Fukuda, Katsuyuki; Kusano, Kazutomi; Yoshimura, Tsutomu

    2014-08-01

    Lenvatinib is a multityrosine kinase inhibitor that inhibits vascular endothelial growth factor receptors, and is being developed as an anticancer drug. P450s are involved in one of the elimination pathways of lenvatinib, and mono-oxidized metabolites, such as N-oxide (M3) and desmethylated metabolite (M2), form in rats, dogs, monkeys, and humans. Meanwhile, two other oxidative metabolites are produced only in monkey and human liver S9 fractions, and their structures have been identified using high-resolution mass spectrometry as a quinolinone form of lenvatinib (M3') and a quinolinone form of desmethylated lenvatinib (M2'). The formation of M3' from lenvatinib occurred independently of NADPH and was effectively inhibited by typical inhibitors of aldehyde oxidase, indicating the involvement of aldehyde oxidase, but not P450s, in this pathway. M2' was a dioxidized metabolite arising from a combination of mono-oxidation and desmethylation and could only be produced from M2 in a NADPH-independent manner; M2' could not be generated from M3 or M3'. These results suggested that M2' is formed from lenvatinib by a unique two-step pathway through M2. Although both lenvatinib and M2 were substrates for aldehyde oxidase, an enzyme kinetic study indicated that M2 was a much more favorable substrate than lenvatinib. No inhibitory activities of lenvatinib, M2', or M3' and no significant inhibitory activities of M2 or M3 on aldehyde oxidase were observed, suggesting a low possibility of drug-drug interactions in combination therapy with substrates of aldehyde oxidase.

  10. Inheritance of polyphenol oxidase activity in wheat breeding lines derived from matings of low polyphenol oxidase parents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) in grain plays a major role in time-dependent discoloration of wheat (Triticum aestivum L.) products, especially fresh noodles. Breeding wheat cultivars with low or nil PPO activity can reduce the undesirable product darkening. The low PPO line PI 117635 was crossed to two...

  11. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

  12. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard

    2016-01-01

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

  13. Phenol oxidase activity in secondary transformed peat-moorsh soils

    NASA Astrophysics Data System (ADS)

    Styła, K.; Szajdak, L.

    2009-04-01

    The chemical composition of peat depends on the geobotanical conditions of its formation and on the depth of sampling. The evolution of hydrogenic peat soils is closely related to the genesis of peat and to the changes in water conditions. Due to a number of factors including oscillation of ground water level, different redox potential, changes of aerobic conditions, different plant communities, and root exudes, and products of the degradation of plant remains, peat-moorsh soils may undergo a process of secondary transformation conditions (Sokolowska et al. 2005; Szajdak et al. 2007). Phenol oxidase is one of the few enzymes able to degrade recalcitrant phenolic materials as lignin (Freeman et al. 2004). Phenol oxidase enzymes catalyze polyphenol oxidation in the presence of oxygen (O2) by removing phenolic hydrogen or hydrogenes to from radicals or quinines. These products undergo nucleophilic addition reactions in the presence or absence of free - NH2 group with the eventual production of humic acid-like polymers. The presence of phenol oxidase in soil environments is important in the formation of humic substances a desirable process because the carbon is stored in a stable form (Matocha et al. 2004). The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Poznań, West Polish Lowland). The sites of investigation were located along Wyskoć ditch. The following material was taken from four chosen sites marked as Zbechy, Bridge, Shelterbelt and Hirudo in two layers: cartel (0-50cm) and cattle (50-100cm). The object of this study was to characterize the biochemical properties by the determination of the phenol oxidize activity in two layers of the four different peat-moors soils used as meadow. The phenol oxidase activity was determined spectrophotometrically by measuring quinone formation at λmax=525 nm with catechol as substrate by method of Perucci

  14. Use of a quantitative oxidase test for characterizing oxidative metabolism in bacteria.

    PubMed Central

    Jurtshuk, P; McQuitty, D N

    1976-01-01

    It was possible to quantitate the terminal oxidase(s) reaction using bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(O2) value (microliters of O2 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase. The TMPD oxidase Q(O2) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria exhibited a TMPD oxidase Q(O2) value (after correcting for the endogenous by substraction) of less than or equal 33 and had an uncorrected TMPD/endogenous ratio of less than or equal 5. The TMPD oxidase Q(O2) values were also correlated with the data obtained for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respiratory mechanism had high TMPD oxidase values, whereas fermentative organsims had low TMPD oxidase activity. All exceptions to this are noted. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(O2) values. From the 79 bacterial species (36 genera) examined, it appears that this quantitative oxidase test has taxonomic value that can differentiate the oxidative relationships between bacteria at the subspecies, species, and genera levels. PMID:1275489

  15. Alternative oxidase expression in aged potato tuber slices

    SciTech Connect

    Hiser, C.; Herdies, L.; McIntosh, L. )

    1989-04-01

    Higher plant mitochondria posses a cyanide-resistant, hydroxamate-sensitive alternative pathway of electron transport that does not conserve energy. Aging of potato tuber slices for 24 hours leads to the development of an alternative pathway capacity. We have shown that a monoclonal antibody raised against the alternative pathway terminal oxidase of Sauromatum guttatum crossreacts with a protein of similar size in aged potato slice mitochondria. This protein was partially purified and characterized by two-dimensional gel electrophoresis, and its relative levels parallel the rise in cyanide-resistant respiration. We are using a putative clone of the S. guttatum alternative oxidase gene to isolate the equivalent gene from potato and to examine its expression.

  16. Tyrosinase versus Catechol Oxidase: One Asparagine Makes the Difference.

    PubMed

    Solem, Even; Tuczek, Felix; Decker, Heinz

    2016-02-18

    Tyrosinases mediate the ortho-hydroxylation and two-electron oxidation of monophenols to ortho-quinones. Catechol oxidases only catalyze the oxidation of diphenols. Although it is of significant interest, the origin of the functional discrimination between tyrosinases and catechol oxidases has been unclear. Recently, it has been postulated that a glutamate and an asparagine bind and activate a conserved water molecule towards deprotonation of monophenols. Here we demonstrate for the first time that a polyphenoloxidase, which exhibits only diphenolase activity, can be transformed to a tyrosinase by mutation to introduce an asparagine. The asparagine and a conserved glutamate are necessary to properly orient the conserved water in order to abstract a proton from the monophenol. These results provide direct evidence for the crucial importance of a proton shuttle for tyrosinase activity of type 3 copper proteins, allowing a consistent understanding of their different chemical reactivities.

  17. Properties of purified recombinant human polyamine oxidase, PAOh1/SMO.

    PubMed

    Wang, Yanlin; Murray-Stewart, Tracy; Devereux, Wendy; Hacker, Amy; Frydman, Benjamin; Woster, Patrick M; Casero, Robert A

    2003-05-16

    The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.

  18. Cloning and expression of the potato alternative oxidase gene

    SciTech Connect

    Hiser, C.; McIntosh, L. Michigan State Univ., East Lansing )

    1990-05-01

    Mitochondria from 24-hour-aged potato slices possess an alternative path capacity and a 36kD protein not present in fresh potato mitochondria. This 36kD protein was identified by a monoclonal antibody against the Sauromatum guttatum alternative oxidase. These results suggest de novo synthesis of the 36kD protein during the aging process. To investigate this phenomenon, a clone containing a potato alternative oxidase gene was isolated from a cDNA library using the S. guttatum gene as a probe. This clone shows areas of high homology to the S. guttatum gene. Norther blots of RNA from fresh and 24-hour-aged potato slices are being probed with the potato gene to examine its expression in relation to the appearance of the 36kD protein.

  19. Rapid purification of cytochrome c oxidase from Paracoccus denitrificans.

    PubMed

    Steffens, G C; Pascual, E; Buse, G

    1990-11-23

    Two methods are described for the purification of cytochrome c oxidase from Triton X-100 extracts of the periplasma membrane of Paracoccus denitrificans. The first is a large-scale procedure for the preparation of 100-250 nmol of cytochrome c oxidase (10-20 mg) in 1 week. The second is a rapid procedure for isolating up to 25 nmol in 2-3 days. Owing to the high yields given by fast protein liquid chromatography (FPLC) on Mono Q columns, the overall yield is about 20%, whereas the yield in many other previously published procedures does not exceed 10%. The use of FPLC on Mono Q also offers a considerable saving of time.

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

    PubMed Central

    Ge, Xiuchun; Shi, Xiaoli; Shi, Limei; Liu, Jinlin; Stone, Victoria; Kong, Fanxiang; Kitten, Todd; Xu, Ping

    2016-01-01

    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. PMID:26950587

  1. NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology

    PubMed Central

    Cave, Alison; Grieve, David; Johar, Sofian; Zhang, Min; Shah, Ajay M

    2005-01-01

    Chronic heart failure, secondary to left ventricular hypertrophy or myocardial infarction, is a condition with increasing morbidity and mortality. Although the mechanisms underlying the development and progression of this condition remain a subject of intense interest, there is now growing evidence that redox-sensitive pathways play an important role. This article focuses on the involvement of reactive oxygen species derived from a family of superoxide-generating enzymes, termed NADPH oxidases (NOXs), in the pathophysiology of ventricular hypertrophy, the accompanying interstitial fibrosis and subsequent heart failure. In particular, the apparent ability of the different NADPH oxidase isoforms to define the response of a cell to a range of physiological and pathophysiological stimuli is reviewed. If confirmed, these data would suggest that independently targeting different members of the NOX family may hold the potential for therapeutic intervention in the treatment of cardiac disease. PMID:16321803

  2. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction†

    PubMed Central

    Héroux, Annie; Bozinovski, Dragana M.; Valley, Michael P.; Fitzpatrick, Paul F.; Orville, Allen M.

    2009-01-01

    The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 Å resolution or better are described of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct. The D402N enzyme has no detectable activity with neutral nitroalkanes (Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 23, 8738–8739). The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2’-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062–2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2’-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle. PMID:19265437

  3. Soluble CuA domain of cyanobacterial cytochrome c oxidase.

    PubMed

    Paumann, Martina; Lubura, Borjana; Regelsberger, Günther; Feichtinger, Markus; Köllensberger, Gunda; Jakopitsch, Christa; Furtmüller, Paul G; Peschek, Günter A; Obinger, Christian

    2004-03-12

    The genomes of several cyanobacteria show the existence of gene clusters encoding subunits I, II, and III of aa(3)-type cytochrome c oxidase. The enzyme occurs on both plasma and thylakoid membranes of these oxygenic phototrophic prokaryotes. Here we report the expression and purification of a truncated subunit II copper A (Cu(A)) domain (i.e. the electron entry and donor binding site) of cytochrome c oxidase from the cyanobacterium Synechocystis PCC 6803 in high yield. The water-soluble purple redox-active bimetallic center displays a relatively low standard reduction potential of 216 mV. Its absorption spectrum at pH 7 is similar to that of other soluble fragments from aa(3)-type oxidases, but the insensitivity of both absorbance and circular dichroism spectra to pH suggests that it is less exposed to the aqueous milieu compared with other Cu(A) domains. Oxidation of horse heart cytochrome c by the bimetallic center follows monophasic kinetics. At pH 7 and low ionic strength the bimolecular rate constant is (2.1 +/- 0.3) x 10(4) m-1 s(-1), and the rates decrease upon the increase of ionic strength. Sequence alignment and modeling of cyanobacterial Cu(A) domains show several peculiarities such as: (i) a large insertion located between the second transmembrane region and the putative hydrophobic cytochrome c docking site, (ii) the lack of acidic residues shown to be important in the interaction between cytochrome c and Paracoccus Cu(A) domain, and (iii) an extended C terminus similar to Escherichia coli ubiquinol oxidase.

  4. Steady state equivalence among autocatalytic peroxidase-oxidase reactions.

    PubMed

    Méndez-González, José; Femat, Ricardo

    2016-12-14

    Peroxidase-oxidase is an enzymatic reaction that can exhibit dynamical scenarios such as bistability, sustained oscillations, and Shilnikov chaos. In this work, we apply the chemical reaction network theory approach to find kinetic constants such that the associated mass action kinetics ordinary differential equations induced by three four dimensional structurally different enzymatic reaction systems can support the same steady states for several chemical species despite differences in their chemical nature.

  5. Azachalcones: a new class of potent polyphenol oxidase inhibitors.

    PubMed

    Radhakrishnan, Sini Karanayil; Shimmon, Ronald Gibrial; Conn, Costa; Baker, Anthony T

    2015-04-15

    A library of potent inhibitors of polyphenol oxidase and their structure activity relationships are described. Azachalcone derivatives were synthesized and tested for their tyrosinase inhibitory activity. Their inhibitory activities on mushroom tyrosinase using l-DOPA as a substrate were investigated. Two compounds that are the reduction congeners of the pyridinyl azachalcones strongly inhibited the enzyme activity and were more potent than the positive control kojic acid.

  6. Steady state equivalence among autocatalytic peroxidase-oxidase reactions

    NASA Astrophysics Data System (ADS)

    Méndez-González, José; Femat, Ricardo

    2016-12-01

    Peroxidase-oxidase is an enzymatic reaction that can exhibit dynamical scenarios such as bistability, sustained oscillations, and Shilnikov chaos. In this work, we apply the chemical reaction network theory approach to find kinetic constants such that the associated mass action kinetics ordinary differential equations induced by three four dimensional structurally different enzymatic reaction systems can support the same steady states for several chemical species despite differences in their chemical nature.

  7. Cholesterol oxidase: sources, physical properties and analytical applications.

    PubMed

    MacLachlan, J; Wotherspoon, A T; Ansell, R O; Brooks, C J

    2000-04-01

    Since Flegg (H.M. Flegg, An investigation of the determination of serum cholesterol by an enzymatic method, Ann. Clin. Biochem. 10 (1973) 79-84) and Richmond (W. Richmond, The development of an enzymatic technique for the assay of cholesterol in biological fluids, Scand. J. clin. Lab. Invest. 29 (1972) 25; W. Richmond, Preparation and properties of a bacterial cholesterol oxidase from Nocardia sp. and its application to enzyme assay of total cholesterol in serum, Clinical Chemistry 19 (1973) 1350-1356) first illustrated the suitability of cholesterol oxidase (COD) for the analysis of serum cholesterol, COD has risen to become the most widely used enzyme in clinical laboratories with the exception of glucose oxidase (GOD). The use is widespread because assays incorporating the enzyme are extremely simple, specific, and highly sensitive and thus offer distinct advantages over the Liebermann-Burchard analytical methodologies which employ corrosive reagents and can be prone to unreliable results due to interfering substances such as bilirubin. Individuals can now readily determine their own serum cholesterol levels with a simple disposable test kit. This review discusses COD in some detail and includes the topics: (1) The variety of bacterial sources available; (2) The various extraction/purification protocols utilised in order to obtain protein of sufficient clarification (purity) for use in food/clinical analysis; (3) Significant differences in the properties of the individual enzymes; (4) Substrate specificities of the various enzymes; (5) Examples of biological assays which have employed cholesterol oxidase as an integral part of the analysis, and the various assay protocols; (6) New steroidal products of COD. This review is not a comprehensive description of published work, but is intended to provide an account of recent and current research, and should promote further interest in the application of enzymes to analytical selectivity.

  8. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

    SciTech Connect

    Heroux, A.; Bozinovski, D; Valley, M; Fitzpatrick, P; Orville, A

    2009-01-01

    The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 {angstrom} resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.

  9. Studies of GA sub 53 oxidase from spinach

    SciTech Connect

    Wilson, T.; Zeevaart, J.A.D. )

    1990-05-01

    GA{sub 53} oxidase was purified 1,750-fold with 1% recovery of activity from spinach after exposure to 8 long days. This preparation was injected into balb/c mice and hybridomas from spleen cells were produced. Upon preliminary screening by immunoprecipitation of enzyme activity, three positive cell lines were selected. These are being cloned to select a true monoclonal antibody cell line. This antibody will be used to study the light/dark regulation of this enzyme.

  10. Consistent Evaluation of ACOS-GOSAT, BESD-SCIAMACHY, CarbonTracker, and MACC Through Comparisons to TCCON

    NASA Technical Reports Server (NTRS)

    Kulawik, Susan; Wunch, Debra; O’Dell, Christopher; Frankenberg, Christian; Reuter, Maximilian; Chevallier, Frederic; Oda, Tomohiro; Sherlock, Vanessa; Buchwitz, Michael; Osterman, Greg; Miller, Charles E.; Iraci, Laura T.; Wolf, Joyce

    2016-01-01

    Consistent validation of satellite CO2 estimates is a prerequisite for using multiple satellite CO2 measurements for joint flux inversion, and for establishing an accurate long-term atmospheric CO2 data record. Harmonizing satellite CO2 measurements is particularly important since the differences in instruments, observing geometries, sampling strategies, etc. imbue different measurement characteristics in the various satellite CO2 data products. We focus on validating model and satellite observation attributes that impact flux estimates and CO2 assimilation, including accurate error estimates, correlated and random errors, overall biases, biases by season and latitude, the impact of coincidence criteria, validation of seasonal cycle phase and amplitude, yearly growth, and daily variability. We evaluate dry-air mole fraction (X(sub CO2)) for Greenhouse gases Observing SATellite (GOSAT) (Atmospheric CO2 Observations from Space, ACOS b3.5) and SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY) (Bremen Optimal Estimation DOAS, BESD v2.00.08) as well as the CarbonTracker (CT2013b) simulated CO2 mole fraction fields and the Monitoring Atmospheric Composition and Climate (MACC) CO2 inversion system (v13.1) and compare these to Total Carbon Column Observing Network (TCCON) observations (GGG2012/2014). We find standard deviations of 0.9, 0.9, 1.7, and 2.1 parts per million vs. TCCON for CT2013b, MACC, GOSAT, and SCIAMACHY, respectively, with the single observation errors 1.9 and 0.9 times the predicted errors for GOSAT and SCIAMACHY, respectively. We quantify how satellite error drops with data averaging by interpreting according to (error(sup 2) equals a(sup 2) plus b(sup 2) divided by n (with n being the number of observations averaged, a the systematic (correlated) errors, and b the random (uncorrelated) errors). a and b are estimated by satellites, coincidence criteria, and hemisphere. Biases at individual stations have year

  11. Consistent evaluation of ACOS-GOSAT, BESD-SCIAMACHY, CarbonTracker, and MACC through comparisons to TCCON

    NASA Astrophysics Data System (ADS)

    Kulawik, Susan; Wunch, Debra; O'Dell, Christopher; Frankenberg, Christian; Reuter, Maximilian; Oda, Tomohiro; Chevallier, Frederic; Sherlock, Vanessa; Buchwitz, Michael; Osterman, Greg; Miller, Charles E.; Wennberg, Paul O.; Griffith, David; Morino, Isamu; Dubey, Manvendra K.; Deutscher, Nicholas M.; Notholt, Justus; Hase, Frank; Warneke, Thorsten; Sussmann, Ralf; Robinson, John; Strong, Kimberly; Schneider, Matthias; De Mazière, Martine; Shiomi, Kei; Feist, Dietrich G.; Iraci, Laura T.; Wolf, Joyce

    2016-02-01

    Consistent validation of satellite CO2 estimates is a prerequisite for using multiple satellite CO2 measurements for joint flux inversion, and for establishing an accurate long-term atmospheric CO2 data record. Harmonizing satellite CO2 measurements is particularly important since the differences in instruments, observing geometries, sampling strategies, etc. imbue different measurement characteristics in the various satellite CO2 data products. We focus on validating model and satellite observation attributes that impact flux estimates and CO2 assimilation, including accurate error estimates, correlated and random errors, overall biases, biases by season and latitude, the impact of coincidence criteria, validation of seasonal cycle phase and amplitude, yearly growth, and daily variability. We evaluate dry-air mole fraction (XCO2) for Greenhouse gases Observing SATellite (GOSAT) (Atmospheric CO2 Observations from Space, ACOS b3.5) and SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY) (Bremen Optimal Estimation DOAS, BESD v2.00.08) as well as the CarbonTracker (CT2013b) simulated CO2 mole fraction fields and the Monitoring Atmospheric Composition and Climate (MACC) CO2 inversion system (v13.1) and compare these to Total Carbon Column Observing Network (TCCON) observations (GGG2012/2014). We find standard deviations of 0.9, 0.9, 1.7, and 2.1 ppm vs. TCCON for CT2013b, MACC, GOSAT, and SCIAMACHY, respectively, with the single observation errors 1.9 and 0.9 times the predicted errors for GOSAT and SCIAMACHY, respectively. We quantify how satellite error drops with data averaging by interpreting according to error2 = a2 + b2/n (with n being the number of observations averaged, a the systematic (correlated) errors, and b the random (uncorrelated) errors). a and b are estimated by satellites, coincidence criteria, and hemisphere. Biases at individual stations have year-to-year variability of ˜ 0.3 ppm, with biases larger than the TCCON

  12. Safety evaluation of glucose oxidase from Penicillium chrysogenum.

    PubMed

    Konishi, Tetsuya; Aoshima, Takuya; Mizuhashi, Fukutaro; Choi, Sharon S H; Roberts, Ashley

    2013-06-01

    Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) is used in the food and beverage industry as a preservative and stabilizer and is commonly derived from the fungus Aspergillus niger. Although the safety of glucose oxidase preparations from A. niger is well-established, the use of preparations derived from other fungal species is of interest; however, an assessment of their safety is warranted. Here, we report on the safety of a glucose oxidase preparation derived from the fungus Penicillium chrysogenum (designated as PGO) for commercial use in food processing, as well as an ingredient in food. In a repeated dose 90-day oral toxicity study conducted in rats, PGO was without compound-related adverse effects at doses of up to 15,600U/kg body weight/day, equivalent to 193mg total organic solids/kg body weight/day. In addition, PGO was non-genotoxic in a series of genotoxicity tests, including a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and a combined in vivo mammalian erythrocyte micronucleus test and comet assay. The results of these studies support the safe use of PGO in food for human consumption.

  13. Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

    NASA Astrophysics Data System (ADS)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-03-01

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na2SeO3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant ( K m) values and maximal reaction velocity ( V max) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  14. The NADPH oxidase Nox4 has anti-atherosclerotic functions

    PubMed Central

    Schürmann, Christoph; Rezende, Flavia; Kruse, Christoph; Yasar, Yakub; Löwe, Oliver; Fork, Christian; van de Sluis, Bart; Bremer, Rolf; Weissmann, Norbert; Shah, Ajay M.; Jo, Hanjoong; Brandes, Ralf P.; Schröder, Katrin

    2015-01-01

    Aims Oxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than O2− . Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme. Methods and results Tamoxifen-induced Nox4-knockout mice were crossed with ApoE−/− mice and spontaneous atherosclerosis under regular chow as well as accelerated atherosclerosis in response to partial carotid artery ligation under high-fat diet were determined. Deletion of Nox4 resulted in increased atherosclerosis formation in both models. Mechanistically, pro-atherosclerotic and pro-inflammatory changes in gene expression were observed prior to plaque development. Moreover, inhibition of Nox4 or deletion of the enzyme in the endothelium but not in macrophages resulted in increased adhesion of macrophages to the endothelial surface. Conclusions The H2O2-producing NADPH oxidase Nox4 is an endogenous anti-atherosclerotic enzyme. Nox4 inhibitors, currently under clinical evaluation, should be carefully monitored for cardiovascular side-effects. PMID:26385958

  15. Xanthine oxidase inhibition attenuates ischemic-reperfusion lung injury

    SciTech Connect

    Lynch, M.J.; Grum, C.M.; Gallagher, K.P.; Bolling, S.F.; Deeb, G.M.; Morganroth, M.L.

    1988-05-01

    Ischemic-reperfusion lung injury is a factor potentially limiting the usefulness of distant organ procurement for heart-lung transplantation. Toxic oxygen metabolites are considered a major etiologic factor in reperfusion injury. Although oxygen-free radicals may be generated by many mechanisms, we investigated the role of xanthine oxidase in this injury process by using lodoxamide, a xanthine oxidase inhibitor, to inhibit ischemic-reperfusion injury in an isolated rat lung model. Isolated rat lungs were perfused with physiologic salt solution (PSS) osmotically stabilized with Ficoll until circulating blood elements were nondetectable in the pulmonary venous effluent. Lungs were rendered ischemic by interrupting ventilation and perfusion for 2 hr at 37/sup 0/C. After the ischemic interval, the lungs were reperfused with whole blood and lung injury was determined by measuring the accumulation of /sup 125/I-bovine serum albumin in lung parenchyma and alveolar lavage fluid as well as by gravimetric measurements. Lung effluent was collected immediately pre- and postischemia for analysis of uric acid by high-pressure liquid chromatography. Lodoxamide (1 mM) caused significant attenuation of postischemic lung injury. Uric acid levels in the lung effluent confirmed inhibition of xanthine oxidase. Protection from injury was not complete, however, implying that additional mechanisms may contribute to ischemic-reperfusion injury in the lung.

  16. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

    Knowles, P.F.; Brown, R.D. III; Koenig, S.H.

    1995-07-19

    X-ray absorption and EPR spectroscopy have been used to probe the copper site structure in galactose oxidase at pH 4.5 and 7.0. the results suggest that there are no major differences in the structure of the tetragonal Cu(II) site at these pH values. Analysis of the extended X-ray absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 {Angstrom}; these are presumably the equatorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore results obtained on the one-electron reduced enzyme, containing Cu(II) but not the tyrosine radical, probably also apply to the catalytically active Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor binding, and substrate binding have been probed via nuclear magnetic relaxation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a single, equatorial water ligand with a Cu(II)-O separation of about 2.4 {Angstrom}. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effects on the NMRD profile of galactose oxidase, indicating that substrates also bind to the active site Cu(II) in an equatorial position.

  17. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis.

    PubMed

    Kopacz, Malgorzata M; Heuts, Dominic P H M; Fraaije, Marco W

    2014-10-01

    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 stopped-flow spectroscopy and putrescine as a substrate. During the fast and irreversible reductive half-reaction no radical intermediates were observed, suggesting a direct hydride transfer from the substrate to the FAD. The rate constant of flavin reoxidation depends on the ligand binding; when the imine product was bound to the enzyme the rate constant was higher than with free enzyme species. Similar results were obtained with product-mimicking ligands and this indicates that a ternary complex is formed during catalysis. The obtained kinetic data were used together with steady-state rate equations derived for ping-pong, ordered sequential and bifurcated mechanisms to explore which mechanism is operative. The integrated analysis revealed that PuO employs a bifurcated mechanism due to comparable rate constants of product release from the reduced enzyme and reoxidation of the reduced enzyme-product complex.

  18. Multi-Copper Oxidases and Human Iron Metabolism

    PubMed Central

    Vashchenko, Ganna; MacGillivray, Ross T. A.

    2013-01-01

    Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis. PMID:23807651

  19. Essential role of lysyl oxidases in notochord development

    PubMed Central

    Gansner, John M.; Mendelsohn, Bryce A.; Hultman, Keith A.; Johnson, Stephen L.; Gitlin, Jonathan D.

    2007-01-01

    Recent studies reveal a critical role for copper in the development of the zebrafish notochord, suggesting that specific cuproenzymes are required for the structural integrity of the notochord sheath. We now demonstrate that β-aminopropionitrile, a known inhibitor of the copper-dependent lysyl oxidases, causes notochord distortion in the zebrafish embryo identical to that seen in copper deficiency. Characterization of the zebrafish lysyl oxidase genes reveals eight unique sequences, several of which are expressed in the developing notochord. Specific gene knockdown demonstrates that loss of loxl1 results in notochord distortion, and that loxl1 and loxl5b have overlapping roles in notochord formation. Interestingly, while notochord abnormalities are not observed following partial knockdown of loxl1 or loxl5b alone, in each case this markedly sensitizes developing embryos to notochord distortion if copper availability is diminished. Likewise, partial knockdown of the lysyl oxidase substrate col2a1 results in notochord distortion when combined with reduced copper availability or partial knockdown of loxl1 or loxl5b. These data reveal a complex interplay of gene expression and nutrient availability critical to notochord development. They also provide insight into specific genetic and nutritional factors that may play a role in the pathogenesis of structural birth defects of the axial skeleton. PMID:17543297

  20. Proton transfer in ba(3) cytochrome c oxidase from Thermus thermophilus.

    PubMed

    von Ballmoos, Christoph; Adelroth, Pia; Gennis, Robert B; Brzezinski, Peter

    2012-04-01

    The respiratory heme-copper oxidases catalyze reduction of O(2) to H(2)O, linking this process to transmembrane proton pumping. These oxidases have been classified according to the architecture, location and number of proton pathways. Most structural and functional studies to date have been performed on the A-class oxidases, which includes those that are found in the inner mitochondrial membrane and bacteria such as Rhodobacter sphaeroides and Paracoccus denitrificans (aa(3)-type oxidases in these bacteria). These oxidases pump protons with a stoichiometry of one proton per electron transferred to the catalytic site. The bacterial A-class oxidases use two proton pathways (denoted by letters D and K, respectively), for the transfer of protons to the catalytic site, and protons that are pumped across the membrane. The B-type oxidases such as, for example, the ba(3) oxidase from Thermus thermophilus, pump protons with a lower stoichiometry of 0.5 H(+)/electron and use only one proton pathway for the transfer of all protons. This pathway overlaps in space with the K pathway in the A class oxidases without showing any sequence homology though. Here, we review the functional properties of the A- and the B-class ba(3) oxidases with a focus on mechanisms of proton transfer and pumping.

  1. Decavanadate inhibits the cell-free activation of neutrophil NADPH oxidase without affecting tyrosine phosphorylation.

    PubMed

    Okamura, N; Sakai, T; Nishimura, Y; Sakai, M; Araki, S; Yamaguchi, M; Ishibashi, S

    1999-08-01

    NADPH oxidase was activated by arachidonate in a cell-free system consisting of membrane and cytosol fractions prepared from guinea pig neutrophils. Vanadate apparently inhibited the NADPH oxidase activity in the cell-free system (IC50=2 microM) without phosphotyrosine accumulation. The pH dependency and stability of the inhibitory effect observed for vanadate solution indicated that decavanadate, an isopolyanion of vanadate, was responsible for the inhibition. Pervanadate (vanadyl hydroperoxide) also inhibited the oxidase activity but at a higher concentration (IC50=0.2 mM). Decavanadate lowered the Vmax but did not affect the Km value of NADPH oxidase for NADPH. Decavanadate inhibited the activation process of NADPH oxidase but not the oxidase activity itself. Decavanadate-pretreatment of membrane and cytosol fractions irreversibly decreased the abilities of both fractions to activate NADPH oxidase in the cell-free system. Translocation of p47-phox, one of the cytosolic activation factors of NADPH oxidase, from cytosol to membrane, was little affected by decavanadate. These results suggest that decavanadate inhibits the activation of NADPH oxidase in the cell-free system without affecting the phosphotyrosine phosphatase, and that decavanadate can bind to both the membrane and cytosolic activation factors when they are in a dormant state, but not to the active oxidase complex.

  2. Two variants of the assembly factor Surf1 target specific terminal oxidases in Paracoccus denitrificans.

    PubMed

    Bundschuh, Freya A; Hoffmeier, Klaus; Ludwig, Bernd

    2008-10-01

    Biogenesis of cytochrome c oxidase (COX) relies on a large number of assembly proteins, one of them being Surf1. In humans, the loss of Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder. In the soil bacterium Paracoccus denitrificans, homologous genes specifying Surf1 have been identified and located in two operons of terminal oxidases: surf1q is the last gene of the qox operon (coding for a ba(3)-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa(3)-type cytochrome c oxidase). We introduced chromosomal single and double deletions for both surf1 genes, leading to significantly reduced oxidase activities in membrane. Our experiments on P. denitrificans surf1 single deletion strains show that both Surf1c and Surf1q are functional and act independently for the aa(3)-type cytochrome c oxidase and the ba(3)-type quinol oxidase, respectively. This is the first direct experimental evidence for the involvement of a Surf1 protein in the assembly of a quinol oxidase. Analyzing the heme content of purified cytochrome c oxidase, we conclude that Surf1, though not indispensable for oxidase assembly, is involved in an early step of cofactor insertion into subunit I.

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

    PubMed

    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.

  4. Inhibition of xanthine oxidase by the aldehyde oxidase inhibitor raloxifene: implications for identifying molybdopterin nitrite reductases.

    PubMed

    Weidert, E R; Schoenborn, S O; Cantu-Medellin, N; Choughule, K V; Jones, J P; Kelley, E E

    2014-02-15

    Sources of nitric oxide alternative to nitric oxide synthases are gaining significant traction as crucial mediators of vessel function under hypoxic inflammatory conditions. For example, capacity to catalyze the one electron reduction of nitrite (NO2-) to ·NO has been reported for hemoglobin, myoglobin and molybdopterin-containing enzymes including xanthine oxidoreductase (XOR) and aldehyde oxidase (AO). For XOR and AO, use of selective inhibition strategies is therefore crucial when attempting to assign relative contributions to nitrite-mediated ·NO formation in cells and tissue. To this end, XOR inhibition has been accomplished with application of classic pyrazolopyrimidine-based inhibitors allo/oxypurinol or the newly FDA-approved XOR-specific inhibitor, Uloric® (febuxostat). Likewise, raloxifene, an estrogen receptor antagonist, has been identified as a potent (Ki=1.0 nM) inhibitor of AO. Herein, we characterize the inhibition kinetics of raloxifene for XOR and describe the resultant effects on inhibiting XO-catalyzed ·NO formation. Exposure of purified XO to raloxifene (PBS, pH 7.4) resulted in a dose-dependent (12.5-100 μM) inhibition of xanthine oxidation to uric acid. Dixon plot analysis revealed a competitive inhibition process with a Ki=13 μM. This inhibitory process was more effective under acidic pH; similar to values encountered under hypoxic/inflammatory conditions. In addition, raloxifene also inhibited anoxic XO-catalyzed reduction of NO2- to NO (EC50=64 μM). In contrast to having no effect on XO-catalyzed uric acid production, the AO inhibitor menadione demonstrated potent inhibition of XO-catalyzed NO2- reduction (EC50=60 nM); somewhat similar to the XO-specific inhibitor, febuxostat (EC50=4 nM). Importantly, febuxostat was found to be a very poor inhibitor of human AO (EC50=613 μM) suggesting its usefulness for validating XO-dependent contributions to NO2- reduction in biological systems. Combined, these data indicate care should be taken

  5. Limitation of adipose tissue enlargement in rats chronically treated with semicarbazide-sensitive amine oxidase and monoamine oxidase inhibitors.

    PubMed

    Carpéné, C; Abello, V; Iffiú-Soltész, Z; Mercier, N; Fève, Bruno; Valet, P

    2008-06-01

    Inhibition of semicarbazide-sensitive amine oxidases (SSAO) and monoamine oxidases (MAO) reduces fat deposition in obese rodents: chronic administration of the SSAO-inhibitor semicarbazide (S) in combination with pargyline (MAO-inhibitor) has been shown to reduce body weight gain in obese Zucker rats, while (E)-2-(4-fluorophenethyl)-3-fluoroallylamine, an SSAO- and MAO-B inhibitor, has been reported to limit weight gain in obese and diabetic mice. Our aim was to state whether such weight gain limitation could occur in non-obese, non-diabetic rats and to extend these observations to other amine oxidase inhibitors. Prolonged treatment of non-obese rats with a high dose of S (900 micromol kg(-1) day(-1)) reduced body weight gain and limited white adipose tissue enlargement. When chronically administered at a threefold lower dose, S also inhibited SSAO activity but not fat depot enlargement, suggesting that effects other than SSAO inhibition were involved in adipose tissue growth retardation. However, combined treatment of this lower dose of S with pargyline inhibited SSAO, MAO, energy intake, weight gain and fat deposition. Adipocytes from treated rats exhibited unchanged insulin responsiveness but impaired antilipolytic responses to amine oxidase substrates. Phenelzine clearly inhibited both MAO and SSAO when tested on adipocytes. Obese rats receiving phenelzine i.p. at 17 micromol kg(-1) day(-1) for 3 weeks, exhibited blunted MAO and SSAO activities in any tested tissue, diminished body weight gain and reduced intra-abdominal adipose tissue. Their adipocytes were less responsive to lipogenesis activation by tyramine or benzylamine. These observations suggest that SSAO inhibition is not sufficient to impair fat deposition. However, combined MAO and SSAO inhibition limits adiposity in non-obese as well as in obese rats.

  6. Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability

    PubMed Central

    Vuong, Thu V.; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R.

    2016-01-01

    Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H2O2. By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H2O2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H2O2, the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H2O2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications. PMID:27869125

  7. Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.

    PubMed

    Vuong, Thu V; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R

    2016-11-21

    Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H2O2. By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H2O2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H2O2, the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H2O2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

  8. Evidence for cancer-associated expression of NADPH oxidase 1 (Nox1)-based oxidase system in the human stomach.

    PubMed

    Tominaga, Kumiko; Kawahara, Tsukasa; Sano, Toshiaki; Toida, Kazunori; Kuwano, Yuki; Sasaki, Hideyuki; Kawai, Tomoko; Teshima-Kondo, Shigetada; Rokutan, Kazuhito

    2007-12-15

    Helicobacter pylori infection has been suggested to stimulate expression of the NADPH oxidase 1 (Nox1)-based oxidase system in guinea pig gastric epithelium, whereas Nox1 mRNA expression has not yet been documented in the human stomach. PCR of human stomach cDNA libraries showed that Nox1 and Nox organizer 1 (NOXO1) messages were absent from normal stomachs, while they were specifically coexpressed in intestinal- and diffuse-type adenocarcinomas including signet-ring cell carcinoma. Immunohistochemistry showed that Nox1 and NOXO1 proteins were absent from chronic atrophic gastritis (15 cases), adenomas (4 cases), or surrounding tissues of adenocarcinomas (45 cases). In contrast, Nox1 and its partner proteins were expressed in intestinal-type adenocarcinomas (19/21 cases), diffuse-type adenocarcinomas (15/15 cases), and signet-ring cell carcinomas (9/9 cases). Confocal microscopy revealed that Nox1, NOXO1, Nox activator 1, and p22(phox) were predominantly associated with Golgi apparatus in these cancer cells, while diffuse-type adenocarcinomas also contained cancer cells having Nox1 and its partner proteins in their nuclei. Nox1-expressing cancer cells exhibited both gastric and intestinal phenotypes, as assessed by expression of mucin core polypeptides. Thus, the Nox1-base oxidase may be a potential marker of neoplastic transformation and play an important role in oxygen radical- and inflammation-dependent carcinogenesis in the human stomach.

  9. Comparing hydrazine-derived reactive groups as inhibitors of quinone-dependent amine oxidases.

    PubMed

    Burke, Ashley A; Severson, Elizabeth S; Mool, Shreya; Solares Bucaro, Maria J; Greenaway, Frederick T; Jakobsche, Charles E

    2017-12-01

    Lysyl oxidase has emerged as an important enzyme in cancer metastasis. Its activity has been reported to become upregulated in several types of cancer, and blocking its activity has been shown to limit the metastatic potential of various cancers. The small-molecules phenylhydrazine and β-aminopropionitrile are known to inhibit lysyl oxidase; however, issues of stability, toxicity, and poorly defined mechanisms limit their potential use in medical applications. The experiments presented herein evaluate three other families of hydrazine-derived compounds - hydrazides, alkyl hydrazines, and semicarbazides - as irreversible inhibitors of lysyl oxidase including determining the kinetic parameters and comparing the inhibition selectivities for lysyl oxidase against the topaquinone-containing diamine oxidase from lentil seedlings. The results suggest that the hydrazide group may be a useful core functionality that can be developed into potent and selective inhibitors of lysyl oxidase and eventually find application in cancer metastasis research.

  10. A method for colocating satellite XCO2 data to ground-based data and its application to ACOS-GOSAT and TCCON

    NASA Astrophysics Data System (ADS)

    Nguyen, H.; Osterman, G.; Wunch, D.; O'Dell, C.; Mandrake, L.; Wennberg, P.; Fisher, B.; Castano, R.

    2014-02-01

    Satellite measurements are often compared with higher-precision ground-based measurements as part of validation efforts. The satellite soundings are rarely perfectly coincident in space and time with the ground-based measurements, so a colocation methodology is needed to aggregate "nearby" soundings into what the instrument would have seen at the location and time of interest. We are particularly interested in validation efforts for satellite-retrieved total column carbon dioxide (XCO2), where XCO2 data from Greenhouse Gas Observing Satellite (GOSAT) retrievals (ACOS, NIES, RemoteC, PPDF, etc.) or SCanning Imaging Absorption SpectroMeter for Atmospheric CHartographY (SCHIACHY) are often colocated and compared to ground-based column XCO2 measurement from Total Carbon Column Observing Network (TCCON). Current colocation methodologies for comparing satellite measurements of total column dry-air mole fractions of CO2 (XCO2) with ground-based measurements typically involve locating and averaging the satellite measurements within some latitudinal, longitudinal, and temporal window. We examine a geostatistical colocation methodology that takes a weighted average of satellite observations depending on the "distance" of each observation from a ground-based location of interest. The "distance" function that we use is a modified Euclidian distance with respect to latitude, longitude, time, and mid-tropospheric temperature at 700 hPa. We apply this methodology to XCO2 retrieved from Greenhouse Gas Observing Satellite (GOSAT) spectra by the ACOS team, cross-validate the results to TCCON XCO2 ground-based data, and present some comparison between our methodology and standard existing colocation methods showing that in general geostatistical colocation produces smaller mean-squared error.

  11. A method for colocating satellite XCO2 data to ground-based data and its application to ACOS-GOSAT and TCCON

    NASA Astrophysics Data System (ADS)

    Nguyen, H.; Osterman, G.; Wunch, D.; O'Dell, C.; Mandrake, L.; Wennberg, P.; Fisher, B.; Castano, R.

    2014-08-01

    Satellite measurements are often compared with higher-precision ground-based measurements as part of validation efforts. The satellite soundings are rarely perfectly coincident in space and time with the ground-based measurements, so a colocation methodology is needed to aggregate "nearby" soundings into what the instrument would have seen at the location and time of interest. We are particularly interested in validation efforts for satellite-retrieved total column carbon dioxide (XCO2), where XCO2 data from Greenhouse Gas Observing Satellite (GOSAT) retrievals (ACOS, NIES, RemoteC, PPDF, etc.) or SCanning Imaging Absorption SpectroMeter for Atmospheric CHartographY (SCIAMACHY) are often colocated and compared to ground-based column XCO2 measurement from Total Carbon Column Observing Network (TCCON). Current colocation methodologies for comparing satellite measurements of total column dry-air mole fractions of CO2 (XCO2) with ground-based measurements typically involve locating and averaging the satellite measurements within a latitudinal, longitudinal, and temporal window. We examine a geostatistical colocation methodology that takes a weighted average of satellite observations depending on the "distance" of each observation from a ground-based location of interest. The "distance" function that we use is a modified Euclidian distance with respect to latitude, longitude, time, and midtropospheric temperature at 700 hPa. We apply this methodology to XCO2 retrieved from GOSAT spectra by the ACOS team, cross-validate the results to TCCON XCO2 ground-based data, and present some comparisons between our methodology and standard existing colocation methods showing that, in general, geostatistical colocation produces smaller mean-squared error.

  12. Quantitation of rat liver xanthine oxidase by radioimmunoassay. A mechanism for sex-specific differences

    SciTech Connect

    Decker, D.E.; Levinson, D.J.

    1982-03-01

    To further delineate the mechanism responsible for the differences in xanthine oxidase activity in male and female Sprague-Dawley rats, a sensitive and specific radioimmunoassay (RIA) was developed for the measurement of hepatic xanthine oxidase. The RIA could detect as little as 5 mg of liver enzyme. Specificity of the RIA was confirmed by 1) Ouchterlony double immuno-diffusion in which a single precipitin band exhibited xanthine oxidase activity, when crude liver homogenate and an enzyme-specific stain were used; 2) parallelism between purified 125I-labeled xanthine oxidase and serial dilutions of crude liver homogenate; 3) a linear correlation between xanthine oxidase activity and the level of enzyme protein; and 4) a single protein band coincident with purified xanthine oxidase, when an immunoprecipitate prepared from antisera and crude liver homogenate was analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gels. Whether xanthine oxidase activity was assayed in the absence of nicotinamide adenine dinucleotide (NAD+) (oxidase form) or in the presence of NAD+ (dehydrogenase), male values were consistently higher, and both forms of the enzyme correlated significantly with each other. When purified to homogeneity, neither form of the enzyme was appreciably affected by 17 beta-estradiol or testosterone propionate. When the RIA was employed, levels of hepatic xanthine oxidase were significantly greater in male than in female rats. We concluded from these data that increased xanthine oxidase activity in the male corresponds to a greater quantitative complement of xanthine oxidase protein. Furthermore, lower xanthine oxidase activity in the female cannot be explained by immunologically cross-reactive material without enzyme activity nor by a direct sex-steroid enzyme interaction.

  13. The Role of the RAS Tumor Suppressor, Lysyl Oxidase, in Breast Cancer Development and Progression.

    DTIC Science & Technology

    1997-11-01

    lymphocytes. In ductal carcinoma, lysyl oxidase expression was absent in some cases , patchy in others, and sometimes appeared unchanged. The expression of...in tumor tissue sections. Ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC). Several cases of DCIS, IDC and mixed DCIS-IDC...were examined by in situ RT-PCR. The expression of lysyl oxidase in cases of ductal carcinoma was variable. In some patients, lysyl oxidase message

  14. A biochemical approach to study the role of the terminal oxidases in aerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    Le Laz, Sébastien; Kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2014-01-01

    The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed.

  15. The Terminal Oxidase Cytochrome bd Promotes Sulfide-resistant Bacterial Respiration and Growth

    PubMed Central

    Forte, Elena; Borisov, Vitaliy B.; Falabella, Micol; Colaço, Henrique G.; Tinajero-Trejo, Mariana; Poole, Robert K.; Vicente, João B.; Sarti, Paolo; Giuffrè, Alessandro

    2016-01-01

    Hydrogen sulfide (H2S) impairs mitochondrial respiration by potently inhibiting the heme-copper cytochrome c oxidase. Since many prokaryotes, including Escherichia (E.) coli, generate H2S and encounter high H2S levels particularly in the human gut, herein we tested whether bacteria can sustain sulfide-resistant O2-dependent respiration. E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo3 enzyme and two bd oxidases much less sensitive to cyanide. Working on the isolated enzymes, we found that, whereas the bo3 oxidase is inhibited by sulfide with half-maximal inhibitory concentration IC50 = 1.1 ± 0.1 μM, under identical experimental conditions both bd oxidases are insensitive to sulfide up to 58 μM. In E. coli respiratory mutants, both O2-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected by ≤200 μM sulfide when either bd enzyme acted as the only terminal oxidase. Accordingly, wild-type E. coli showed sulfide-insensitive respiration and growth under conditions favouring the expression of bd oxidases. In all tested conditions, cyanide mimicked the functional effect of sulfide on bacterial respiration. We conclude that bd oxidases promote sulfide-resistant O2-consumption and growth in E. coli and possibly other bacteria. The impact of this discovery is discussed. PMID:27030302

  16. The Terminal Oxidase Cytochrome bd Promotes Sulfide-resistant Bacterial Respiration and Growth.

    PubMed

    Forte, Elena; Borisov, Vitaliy B; Falabella, Micol; Colaço, Henrique G; Tinajero-Trejo, Mariana; Poole, Robert K; Vicente, João B; Sarti, Paolo; Giuffrè, Alessandro

    2016-03-31

    Hydrogen sulfide (H2S) impairs mitochondrial respiration by potently inhibiting the heme-copper cytochrome c oxidase. Since many prokaryotes, including Escherichia (E.) coli, generate H2S and encounter high H2S levels particularly in the human gut, herein we tested whether bacteria can sustain sulfide-resistant O2-dependent respiration. E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo3 enzyme and two bd oxidases much less sensitive to cyanide. Working on the isolated enzymes, we found that, whereas the bo3 oxidase is inhibited by sulfide with half-maximal inhibitory concentration IC50 = 1.1 ± 0.1 μM, under identical experimental conditions both bd oxidases are insensitive to sulfide up to 58 μM. In E. coli respiratory mutants, both O2-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected by ≤200 μM sulfide when either bd enzyme acted as the only terminal oxidase. Accordingly, wild-type E. coli showed sulfide-insensitive respiration and growth under conditions favouring the expression of bd oxidases. In all tested conditions, cyanide mimicked the functional effect of sulfide on bacterial respiration. We conclude that bd oxidases promote sulfide-resistant O2-consumption and growth in E. coli and possibly other bacteria. The impact of this discovery is discussed.

  17. Pacific oyster polyamine oxidase: a protein missing link in invertebrate evolution.

    PubMed

    Cervelli, Manuela; Polticelli, Fabio; Angelucci, Emanuela; Di Muzio, Elena; Stano, Pasquale; Mariottini, Paolo

    2015-05-01

    Polyamine oxidases catalyse the oxidation of polyamines and acetylpolyamines and are responsible for the polyamine interconversion metabolism in animal cells. Polyamine oxidases from yeast can oxidize spermine, N(1)-acetylspermine, and N(1)-acetylspermidine, while in vertebrates two different enzymes, namely spermine oxidase and acetylpolyamine oxidase, specifically catalyse the oxidation of spermine, and N(1)-acetylspermine/N(1)-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylpolyamine oxidases have arisen from an ancestor invertebrate polyamine oxidase with lower specificity for polyamine substrates, as demonstrated by the enzymatic activity of the mollusc polyamine oxidase characterized here. This is the first report of an invertebrate polyamine oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to oxidase both N(1)-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.

  18. A method for highly efficient catalytic immobilisation of glucose oxidase on the surface of silica.

    PubMed

    Sim, Yong-Kyun; Park, Jung-Woo; Kim, Bo-Hyeong; Jun, Chul-Ho

    2013-12-11

    A simple, mild and convenient method has been developed for catalytic immobilisation of glucose oxidase (GOx), chemically modified to contain pendant methallylsilyl groups, on an untreated silica surface.

  19. Involvement of Polyamine Oxidase in Wound Healing12[W

    PubMed Central

    Angelini, Riccardo; Tisi, Alessandra; Rea, Giuseppina; Chen, Martha M.; Botta, Maurizio; Federico, Rodolfo; Cona, Alessandra

    2008-01-01

    Hydrogen peroxide (H2O2) is involved in plant defense responses that follow mechanical damage, such as those that occur during herbivore or insect attacks, as well as pathogen attack. H2O2 accumulation is induced during wound healing processes as well as by treatment with the wound signal jasmonic acid. Plant polyamine oxidases (PAOs) are H2O2 producing enzymes supposedly involved in cell wall differentiation processes and defense responses. Maize (Zea mays) PAO (ZmPAO) is a developmentally regulated flavoprotein abundant in primary and secondary cell walls of several tissues. In this study, we investigated the effect of wounding on ZmPAO gene expression in the outer tissues of the maize mesocotyl and provide evidence that ZmPAO enzyme activity, protein, and mRNA levels increased in response to wounding as well as jasmonic acid treatment. Histochemically detected ZmPAO activity especially intensified in the epidermis and in the wound periderm, suggesting a tissue-specific involvement of ZmPAO in wound healing. The role played by ZmPAO-derived H2O2 production in peroxidase-mediated wall stiffening events was further investigated by exploiting the in vivo use of N-prenylagmatine (G3), a selective and powerful ZmPAO inhibitor, representing a reliable diagnostic tool in discriminating ZmPAO-mediated H2O2 production from that generated by peroxidase, oxalate oxidase, or by NADPH oxidase activity. Here, we demonstrate that G3 inhibits wound-induced H2O2 production and strongly reduces lignin and suberin polyphenolic domain deposition along the wound, while it is ineffective in inhibiting the deposition of suberin aliphatic domain. Moreover, ZmPAO ectopic expression in the cell wall of transgenic tobacco (Nicotiana tabacum) plants strongly enhanced lignosuberization along the wound periderm, providing evidence for a causal relationship between PAO and peroxidase-mediated events during wound healing. PMID:17993545

  20. Alternative oxidase: distribution, induction, properties, structure, regulation, and functions.

    PubMed

    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.

  1. Oxidation of the flavonol fisetin by polyphenol oxidase.

    PubMed

    Jiménez, M; Escribano-Cebrián, J; García-Carmona, F

    1998-11-27

    The present study demonstrates the antiradical efficiency of fisetin, a flavonol widely distributed in fruits and vegetables, by its ability to react with two different free radicals, ABTS; and DPPH;. The polyphenolic nature of fisetin led us to consider whether it might be oxidised by polyphenol oxidase (PPO), and the results reported show that it can be oxidised by PPO extracted and partially purified from broad bean seeds. The reaction was followed by recording spectral changes with time, with maximal spectral changes being observed at 282 nm (increase in absorbance) and at 362 nm (decrease). The presence of two isosbectic points (at 265 and 304 nm) suggested that only one absorbent product was formed. These spectral changes were not observed in the absence of PPO. The oxidation rate varied with the pH, reaching its highest value at pH 5.5. The fisetin oxidation rate increased in the presence of sodium dodecyl sulfate, an activator of polyphenol oxidase. Maximal activity was obtained at 0.87 mM sodium dodecyl sulfate. The following kinetic parameters were determined: Vmax=49 microM/min, Km=0.6 mM, Vmax/Km=8.2x10-2 min-1. Flavonol oxidation was inhibited by selective PPO inhibitors such as cinnamic acid (a classical competitive inhibitor, Ki=1.4 mM) and 4-hexylresorcinol, which behaved as a slow-binding inhibitor. The results reported show that fisetin oxidation was strictly dependent on the presence of polyphenol oxidase.

  2. 9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors.

    PubMed

    Rodrigues, Marili V N; Barbosa, Alexandre F; da Silva, Júlia F; dos Santos, Deborah A; Vanzolini, Kenia L; de Moraes, Marcela C; Corrêa, Arlene G; Cass, Quezia B

    2016-01-15

    A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1±9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein.

  3. Bioactive compounds of inhibiting xanthine oxidase from Selaginella labordei.

    PubMed

    Tan, Wen-Jie; Xu, Jia-Cheng; Li, Li; Chen, Ke-Li

    2009-01-01

    Four flavone compounds were isolated from the effective fractions inhibiting xanthine oxidase (XOD) of the medicinal plant Selaginella labordei with anti-virus activity, and the structures were elucidated as 4'-methylether robustaflavone (1), robustaflavone (2), eriodictyol (3) and amentoflavone (4). The 50% inhibitory concentration (IC(50)) of the three compounds of inhibiting XOD were 61.0, 0.199, 16.0 and 32.0 mg L(-1), respectively. All of these compounds were isolated from the species for the first time, and eriodictyol was found from Selaginellaceae for the first time. Among these compounds, robustaflavone has been reported as an effective compound against the hepatitis B virus.

  4. Functions of the hydrophilic channels in protonmotive cytochrome c oxidase

    PubMed Central

    Rich, Peter R.; Maréchal, Amandine

    2013-01-01

    The structures and functions of hydrophilic channels in electron-transferring membrane proteins are discussed. A distinction is made between proton channels that can conduct protons and dielectric channels that are non-conducting but can dielectrically polarize in response to the introduction of charge changes in buried functional centres. Functions of the K, D and H channels found in A1-type cytochrome c oxidases are reviewed in relation to these ideas. Possible control of function by dielectric channels and their evolutionary relation to proton channels is explored. PMID:23864498

  5. A Simple Litmus Test for Aldehyde Oxidase Metabolism of Heteroarenes

    PubMed Central

    2015-01-01

    The bioavailability of aromatic azaheterocyclic drugs can be affected by the activity of aldehyde oxidase (AO). Susceptibility to AO metabolism is difficult to predict computationally and can be complicated in vivo by differences between species. Here we report the use of bis(((difluoromethyl)sulfinyl)oxy)zinc (DFMS) as a source of CF2H radical for a rapid and inexpensive chemical “litmus test” for the early identification of heteroaromatic drug candidates that have a high probability of metabolism by AO. PMID:24472070

  6. Differential expression of multiple terminal oxidases for aerobic respiration in Pseudomonas aeruginosa.

    PubMed

    Kawakami, Takuro; Kuroki, Miho; Ishii, Masaharu; Igarashi, Yasuo; Arai, Hiroyuki

    2010-06-01

    Pseudomonas aeruginosa has five terminal oxidases for aerobic respiration. Two of them, the bo(3) oxidase (Cyo) and the cyanide-insensitive oxidase (CIO), are quinol oxidases and the other three, the cbb(3)-1 oxidase (Cbb3-1), the cbb(3)-2 oxidase (Cbb3-2) and the aa(3) oxidase (Aa3), are cytochrome c oxidases. The expression pattern of the genes for these terminal oxidases under various growth conditions was investigated by using lacZ transcriptional fusions and some novel regulatory issues were found. The Aa3 genes were induced under starvation conditions. The Cyo genes were induced by exposure to the nitric oxide-generating reagent S-nitrosoglutathione. The CIO genes were induced by exposure to sodium nitroprusside as well as cyanide. The stationary phase sigma factor RpoS was found to be involved in the expression of the Aa3 and CIO genes. The role of two redox-responsive transcriptional regulators, ANR and RoxSR, was investigated using the anr and roxSR mutant strains. The ANR was involved in the repression of the CIO genes and induction of the Cbb3-2 genes. The other three terminal oxidase genes were not significantly regulated by ANR. On the other hand, all five terminal oxidase genes were shown to be directly or indirectly regulated by RoxSR. The Aa3 genes were repressed but the genes for the other four enzymes were induced by RoxSR. The transcriptome data also showed that some respiration-related genes were regulated by RoxSR, suggesting that this two-component regulatory system plays an important role in the regulation of respiration in P. aeruginosa.

  7. Identification and biochemical characterization of polyamine oxidases in amphioxus: Implications for emergence of vertebrate-specific spermine and acetylpolyamine oxidases.

    PubMed

    Wang, Huihui; Liu, Baobao; Li, Hongyan; Zhang, Shicui

    2016-01-10

    Polyamine oxidases (PAOs) have been identified in a wide variety of animals, as well as in fungi and plant. Generally, plant PAOs oxidize spermine (Spm), spermidine (Spd) and their acetylated derivatives, N(1)-acetylspermine (N(1)-Aspm) and N(1)-acetylspermidine (N(1)-Aspd), while yeast PAOs oxidize Spm, N(1)-Aspm and N(1)-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N(1)-Aspm/N(1)-Aspd, respectively. However, our knowledge on the biochemical and structural characterization of PAOs remains rather limited, and their evolutionary history is still enigmatic. In this study, two amphioxus (Branchiostoma japonicum) PAO genes, named Bjpao1 and Bjpao2, were cloned and characterized. Both Bjpao1 and Bjpao2 displayed distinct tissue-specific expression patterns. Notably, rBjPAO1 oxidized both spermine and spermidine, but not N(1)-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N(1)-acetylspermine, but not spermine. To understand structure-function relationship, the enzymatic activities of mutant BjPAOs that were generated by site-directed mutagenesis and expressed in E. coli were examined, The results indicate that the residues H64, K301 and T460 in rBjPAO1, and H69, K315 and T467 in rBjPAO2 were all involved in substrate binding and enzyme catalytic activity to some extent. Based on our results and those of others, a model depicting the divergent evolution and functional specialization of vertebrate SMO and APAO genes is proposed.

  8. Crystal Structure of the Deglycating Enzyme Fructosamine Oxidase (Amadoriase ll)

    SciTech Connect

    Collard, F.; Zhang, J; Nemet, I; Qanungo, K; Monnier, V; Yee, V

    2008-01-01

    Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6{angstrom} resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12{angstrom} deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.

  9. Crystal Structure of the Deglycating Enzyme Fructosamine Oxidase (Amadoriase II)

    SciTech Connect

    Collard, François; Zhang, Jianye; Nemet, Ina; Qanungo, Kaustubha R.; Monnier, Vincent M.; Yee, Vivien C.

    2009-01-12

    Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6{angstrom} resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12{angstrom} deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.

  10. Alternative oxidase in animals: unique characteristics and taxonomic distribution.

    PubMed

    McDonald, Allison E; Vanlerberghe, Greg C; Staples, James F

    2009-08-01

    Alternative oxidase (AOX), a ubiquinol oxidase, introduces a branch point into the respiratory electron transport chain, bypassing complexes III and IV and resulting in cyanide-resistant respiration. Previously, AOX was thought to be limited to plants and some fungi and protists but recent work has demonstrated the presence of AOX in most kingdoms of life, including animals. In the present study we identified AOX in 28 animal species representing nine phyla. This expands the known taxonomic distribution of AOX in animals by 10 species and two phyla. Using bioinformatics we found AOX gene sequences in members of the animal phyla Porifera, Placozoa, Cnidaria, Mollusca, Annelida, Nematoda, Echinodermata, Hemichordata and Chordata. Using reverse-transcriptase polymerase chain reaction (RT-PCR) with degenerate primers designed to recognize conserved regions of animal AOX, we demonstrated that AOX genes are transcribed in several animals from different phyla. An analysis of full-length AOX sequences revealed an amino acid motif in the C-terminal region of the protein that is unique to animal AOXs. Animal AOX also lacks an N-terminal cysteine residue that is known to be important for AOX enzyme regulation in plants. We conclude that the presence of AOX is the ancestral state in animals and hypothesize that its absence in some lineages, including vertebrates, is due to gene loss events.

  11. Partial purification and characterization of polyphenol oxidase from persimmon.

    PubMed

    Navarro, José L; Tárrega, Amparo; Sentandreu, Miguel A; Sentandreu, Enrique

    2014-08-15

    Activity of polyphenol oxidase (PPO) from "Rojo Brillante" persimmon (Diospyros kaki L.) fruits was characterized. Crude extracts were used for characterization of enzyme activity and stability at different temperatures (60, 70 and 80 °C), pHs (from 3.5 to 7.5) and substrate concentrations (catechol from 0 to 0.5M). Maximum enzyme activity was reached at pH 5.5 and 55 °C. Enzyme stability was higher than PPO activities found in other natural sources, since above pH 5.5 the minimum time needed to achieve an enzyme inactivation of 90% was 70 min at 80 °C. However, at pH 4.0 the enzyme stability decreased, reaching inactivation levels above 90% after 10 min even at 60 °C. Thus it was concluded that acidification can circumvent browning problems caused by PPO activity. Moreover, polyacrylamide gel electrophoresis of the enriched extract revealed the presence of at least four bands with strong oxidase activity, suggesting the existence of different PPO isoforms.

  12. Characterization of copper atoms in bilirubin oxidase by spectroscopic analyses.

    PubMed

    Gotoh, Y; Kondo, Y; Kaji, H; Takeda, A; Samejima, T

    1989-10-01

    Bilirubin oxidase [EC 1.3.3.5], purified from the culture medium of Myrothecium verrucaria, was found to contain two blue copper atoms per protein molecule with a molecular weight of ca. 52 kDa. The two copper atoms were estimated to be in the all cupric state by the cuproine colorimetric method and also atomic absorption analysis. We could remove the reduce cuprous ions from the holo enzyme by adding ascorbate, followed by a KCN solution, yielding an apo-enzyme with no activity. The apo-enzyme can be reconstituted with Cu or other divalent cations such as Co, Fe, and Cd, with accompanying recovery of the enzyme activity. The activity recovery depended upon the species of cation employed; Cu being most effective, an almost 100% recovery, and Cd the least, only a 25% recovery. We could obtain information on the copper ions and their coordination structure by spectroscopic analyses of the apo- and reconstituted enzymes, obtaining such as absorption, CD, MCD, and XPS spectra. The bilirubin oxidase catalyzed-reaction was a second order reaction with respect to copper bound with protein. The donor set was of the CuSS*N2 (S = Cys, S* = Met, N = His) type, i.e., the same as in the case of blue copper proteins. On studying the Co-substituted enzyme, it was revealed that the copper site of the enzyme had a 4-coordinated structure.

  13. Location and catalytic characteristics of a multipotent bacterial polyphenol oxidase.

    PubMed

    Fernández, E; Sanchez-Amat, A; Solano, F

    1999-10-01

    The melanogenic marine bacterium Marinomonas mediterranea contains a multipotent polyphenol oxidase (PPO) able to oxidize substrates characteristic for tyrosinase and laccase. Thus, this enzyme shows tyrosine hydroxylase activity and it catalyzes the oxidation of a wide variety of o-diphenol as well as o-methoxy-activated phenols. The study of its sensitivity to different inhibitors also revealed intermediate features between laccase and tyrosinase. It is similar to tyrosinases in its sensitivity to tropolone, but it resembles laccases in its resistance to cinnamic acid and phenylthiourea, and in its sensitivity to fluoride anion. This enzyme is mostly membrane-bound and can be solubilized either by non-ionic detergent or lipase treatments of the membrane. The expression of this enzymatic activity is growth-phase regulated, reaching a maximum in the stationary phase of bacterial growth, but L-tyrosine, Cu(II) ions, or 2,5-xylidine do not induce it. This enzyme can be separated from a second PPO form by gel permeation chromatography. The second PPO is located in the soluble fraction and shows a sodium dodecyl sulfate (SDS)-activated action on the characteristic substrates for tyrosinase, L-tyrosine, and L-dopa, but it does not show activity towards laccase-specific substrates. The involvement of the multipotent PPO in melanogenesis and its relationship with the SDS-activated form and with the alternative functions proposed for multicopper oxidases in other microorganisms are discussed.

  14. Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

    SciTech Connect

    Hurtado, Paola A.; Vora, Siddharth; Sume, Siddika Selva; Yang, Dan; Hilaire, Cynthia St.; Guo Ying; Palamakumbura, Amitha H.; Schreiber, Barbara M.; Ravid, Katya; Trackman, Philip C.

    2008-02-01

    Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-{alpha}-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.

  15. Purification and characterization of amine oxidase from pea seedlings.

    PubMed

    Vianello, F; Malek-Mirzayans, A; Di Paolo, M L; Stevanato, R; Rigo, A

    1999-03-01

    A novel, simple, and rapid procedure for the purification of pea seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation, was purified in two steps: the first one by anion-exchange chromatography and the second one by affinity chromatography. The first chromatography step was carried out on a diethylaminoethyl-cellulose column. By lowering the amount of protein loaded on the column and the buffer concentration it was possible to obtain an enzyme pure at 95% (sp act 1.2 microkat/mg). To achieve a higher degree of purification various affinity resins were prepared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best results were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1, 4-diaminocyclohexane. This last compound was found to be a relatively strong competitive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (Ki = 32 microM). According to this procedure an electrophoretically homogeneous enzyme, characterized by a specific activity of 1.63 microkat/mg, was obtained.

  16. Crystal Structure of the Deglycating Enzyme Fructosamine Oxidase (Amadoriase II)*

    PubMed Central

    Collard, François; Zhang, Jianye; Nemet, Ina; Qanungo, Kaustubha R.; Monnier, Vincent M.; Yee, Vivien C.

    2008-01-01

    Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6Å resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12Å deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates. PMID:18667417

  17. Differential Expression of the Three Multicopper Oxidases from Myxococcus xanthus▿

    PubMed Central

    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

  18. Inhibiting Cytochrome C Oxidase Leads to Alleviated Ischemia Reperfusion Injury

    PubMed Central

    Yang, Zhaoyun; Duan, Zhongxin; Yu, Tian; Xu, Junmei

    2017-01-01

    Background and Objectives The overall purpose of this study was to investigate the role of cytochrome C oxidase (CcO) in preventing ischemia reperfusion-induced cardiac injury through gaseous signaling molecule pathways. Materials and Methods We used CcO inhibitor, potassium cyanide (KCN) to mimic the pre-treatment of gaseous signaling molecules in a global ischemia/reperfusion (IR) injury model in rats. Intracellular reactive oxygen species (ROS) was determined by measuring mitochondrial H2O2 and mitochondrial complex activity. Results KCN pre-treatment led to decreased infarction area after IR injury and improved cardiac function. KCN pre-treated group challenged with IR injury was associated with reduced ROS production through inhibition of activity and not downregulation of CcO expression. In addition, KCN pre-treatment was associated with enhanced expression and activity of mitochondrial antioxidase, suggesting the role of CcO in regulating IR injury through oxidative stress. Conclusion KCN pre-treatment reduced the severity of IR injury. The potential mechanism could be increased endogenous anti-oxidase activity and consequently, the enhanced clearance of ROS. PMID:28382074

  19. Purification, characterization and decolorization of bilirubin oxidase from Myrothecium verrucaria 3.2190

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myrothecium verrucaria 3.2190 is a nonligninolytic fungus that produces bilirubin oxidase. Both Myrothecium verrucaria and the extracellular bilirubin oxidase were tested for their ability to decolorize indigo carmine. The biosorption and biodegradation of the dye were detected during the process of...

  20. Hydroxychavicol: a potent xanthine oxidase inhibitor obtained from the leaves of betel, Piper betle.

    PubMed

    Murata, Kazuya; Nakao, Kikuyo; Hirata, Noriko; Namba, Kensuke; Nomi, Takao; Kitamura, Yoshihisa; Moriyama, Kenzo; Shintani, Takahiro; Iinuma, Munekazu; Matsuda, Hideaki

    2009-07-01

    The screening of Piperaceous plants for xanthine oxidase inhibitory activity revealed that the extract of the leaves of Piper betle possesses potent activity. Activity-guided purification led us to obtain hydroxychavicol as an active principle. Hydroxychavicol is a more potent xanthine oxidase inhibitor than allopurinol, which is clinically used for the treatment of hyperuricemia.

  1. Vectorial nature of redox Bohr effects in bovine heart cytochrome c oxidase.

    PubMed

    Capitanio, N; Capitanio, G; De Nitto, E; Papa, S

    1997-09-08

    The vectorial nature of redox Bohr effects (redox-linked pK shifts) in cytochrome c oxidase from bovine heart incorporated in liposomes has been analyzed. The Bohr effects linked to oxido-reduction of heme a and CuB display membrane vectorial asymmetry. This provides evidence for involvement of redox Bohr effects in the proton pump of the oxidase.

  2. Purification and characterization of the cytochrome c oxidase from Rhodopseudomonas sphaeroides.

    PubMed

    Gennis, R B; Casey, R P; Azzi, A; Ludwig, B

    1982-06-15

    When grown aerobically in the dark, Rhodopseudomonas sphaeroides develops a respiratory chain similar to that in mitochondria and the photosynthetic apparatus is suppressed. The aa3-type cytochrome c oxidase from Rps. sphaeroides has been purified in Triton X-100 by affinity chromatography with Sepharose 4B coupled to yeast cytochrome c. The oxidase contains 14 nmol heme a/mg protein and is composed of three polypeptide subunits with relative molecular masses of 45000, 37000 and 35000. The enzyme is highly active in the presence of detergents, with a maximal velocity of 300 s-1/mol oxidase using either yeast or horse-heart cytochrome c. The Rps. sphaeroides oxidase is cross-reactive with antibodies directed against the oxidases from Paracoccus denitrificans and Saccharomyces cerevisiae. A particularly close relationship is indicated in the case of P. denitrificans. The Rps. sphaeroides oxidase has been incorporated into phospholipid vesicles. The resulting oxidase in these vesicles demonstrates high enzymatic activity and a respiratory control ratio of 5. Using these vesicles, no evidence for proton extrusion accompanying cytochrome c oxidation was observed. The data suggest that the Rps. sphaeroides oxidase does not function as a proton pump.

  3. A colorimetric method for lysyl oxidase activity in copper deficient rats fed a high fructose diet

    SciTech Connect

    Werman, M.J.; Bhathena, S.J. )

    1991-03-11

    Lysyl oxidase is involved in initiating cross link formation in collagen and elastin. The activity of lysyl oxidase is traditionally assessed by the tritium released assay. The authors describe a simplified and modified method for measuring lysyl oxidase activity in rats, based on measuring ammonia release according to the Bertholet colorimetric reaction. Lysyl oxidase activity was measured in copper deficient rats using this method. Sixteen weanling Sprague Dawley male rats were fed for four weeks either copper adequate or copper deficient diets containing 62% fructose. Copper deficiency was confirmed by significant low copper levels in heart, brain, liver and skin, and by nondetectable levels of ceruloplasmin. Lysyl oxidase activity was significantly lower in heart and skin of rats fed a copper deficient diet compared to those fed a copper adequate diet. No significant difference in activity was observed in brain tissue. A correlation was not observed between decreased tissue copper levels and decreased lysyl oxidase activity. Thus, the determination of ammonia liberated during lysyl oxidase activity may serve as an effective tool in assessing lysyl oxidase activity.

  4. The role of aldehyde oxidase in ethanol-induced hepatic lipid peroxidation in the rat.

    PubMed Central

    Shaw, S; Jayatilleke, E

    1990-01-01

    Hepatic lipid peroxidation has been implicated in the pathogenesis of alcohol-induced liver injury, but the mechanism(s) by which ethanol metabolism or resultant free radicals initiate lipid peroxidation is not fully defined. The role of the molybdenum-containing enzymes aldehyde oxidase and xanthine oxidase in the generation of such free radicals was investigated by measuring alkane production (lipoperoxidation products) in isolated rat hepatocytes during ethanol metabolism. Inhibition of aldehyde oxidase and xanthine oxidase (by feeding tungstate at 100 mg/day per kg) decreased alkane production (80-95%), whereas allopurinol (20 mg/kg by mouth), a marked inhibitor of xanthine oxidase, inhibited alkane production by only 35-50%. Addition of acetaldehyde (0-100 microM) (in the presence of 50 microM-4-methylpyrazole) increased alkane production in a dose-dependent manner (Km of aldehyde oxidase for acetaldehyde 1 mM); menadione, an inhibitor of aldehyde oxidase, virtually inhibited alkane production. Desferrioxamine (5-10 microM) completely abolished alkane production induced by both ethanol and acetaldehyde, indicating the importance of catalytic iron. Thus free radicals generated during the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism in the initiation of alcohol-induced liver injury. PMID:2363695

  5. Effect of rapid eye movement sleep deprivation on rat brain monoamine oxidases.

    PubMed

    Thakkar, M; Mallick, B N

    1993-08-01

    Monoamine oxidase, monoamine oxidase-A, and monoamine oxidase-B activities were compared in free moving, rapid eye movement sleep-deprived, recovered, and control rat brains. The activities were estimated in the whole brain, cerebrum, cerebellum, whole brainstem, medulla, pons, and midbrain. The flowerpot method was used for continuing deprivation for one, two, or four days. Monoamine oxidase activity decreased significantly in the cerebrum and the cerebellum of the sleep-deprived rats, whereas monoamine oxidase-A and monoamine oxidase-B were differentially affected. Medullary MAO-A was the first to be affected, showing an increase after just one day of rapid eye movement sleep deprivation, while longer deprivation decreased its activity. The activity of monoamine oxidase-B was not significantly affected in any brain areas of the deprived rats until after two days of rapid eye movement sleep deprivation. All the altered enzyme activities returned to control levels after recovery. Control experiments suggest that the decrease was primarily caused by the rapid eye movement sleep deprivation and was not due to nonspecific effects. These findings are consistent with past studies and may help to explain earlier observations. The results support the involvement of aminergic mechanisms in rapid eye movement sleep. The plausible reasons for the changes in the activities of monoamine oxidases, after rapid eye movement sleep deprivation, are discussed.

  6. Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase

    SciTech Connect

    Koland, J.G.; Miller, M.J.; Gennis, R.B.

    1984-01-31

    Pyruvate oxidase is a flavoprotein dehydrogenase located on the inner surface of the Escherichia coli cytoplasmic membrane and coupled to the E. coli aerobic respiratory chain. The role of quinones in the pyruvate oxidase system is investigated, and a minimal respiratory chain is described consisting of only two pure proteins plus ubiquinone 8 incorporated in phospholipid vesicles. The enzymes used in this reconstitution are the flavorprotein and the recently purified E. coli cytochrome d terminal oxidase. The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with E. coli membranes. It is also shown that electron transport from pyruvate to oxygen in the liposome system generates a transmembrane potential of at least 180 mV (negative inside), which is sensitive to the uncouplers carbonyl cyanide p-(trichloromethoxy)phenylhydrazone and valinomycin. A transmembrane potential is also generated by the oxidation of ubiquinol 1 by the terminal oxidase in the absence of the flavoprotein. It is concluded that: the flavoprotein can directly reduce ubiquinone 8 within the phospholipid bilayer; menaquinone 8 will not effectively substitute for ubiquinone 8 in this electron-transfer chain; and the cytochrome d terminal oxidase functions as a ubiquinol 8 oxidase and serves as a coupling site in the E. coli aerobic respiratory chain. These investigations suggest a relatively simple organization for the E. coli respiratory chain.

  7. Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase.

    PubMed

    Koland, J G; Miller, M J; Gennis, R B

    1984-01-31

    Pyruvate oxidase is a flavoprotein dehydrogenase located on the inner surface of the Escherichia coli cytoplasmic membrane and coupled to the E. coli aerobic respiratory chain. In this paper, the role of quinones in the pyruvate oxidase system is investigated, and a minimal respiratory chain is described consisting of only two pure proteins plus ubiquinone 8 incorporated in phospholipid vesicles. The enzymes used in this reconstitution are the flavoprotein and the recently purified E. coli cytochrome d terminal oxidase. The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with E. coli membranes. It is also shown that electron transport from pyruvate to oxygen in the liposome system generates a transmembrane potential of at least 180 mV (negative inside), which is sensitive to the uncouplers carbonyl cyanide p-(tri-chloromethoxy)phenylhydrazone and valinomycin. A trans-membrane potential is also generated by the oxidation of ubiquinol 1 by the terminal oxidase in the absence of the flavoprotein. It is concluded that (1) the flavoprotein can directly reduce ubiquinone 8 within the phospholipid bilayer, (2) menaquinone 8 will not effectively substitute for ubiquinone 8 in this electron-transfer chain, and (3) the cytochrome d terminal oxidase functions as a ubiquinol 8 oxidase and serves as a "coupling site" in the E. coli aerobic respiratory chain. These investigations suggest a relatively simple organization for the E. coli respiratory chain.

  8. The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function.

    PubMed

    Noonan, Thomas; Lukas, Susan; Peet, Gregory W; Pelletier, Josephine; Panzenbeck, Mark; Hanidu, Adedayo; Mazurek, Suzanne; Wasti, Ruby; Rybina, Irina; Roma, Teresa; Kronkaitis, Anthony; Shoultz, Alycia; Souza, Donald; Jiang, Huiping; Nabozny, Gerald; Modis, Louise Kelly

    2013-01-01

    Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1.

  9. The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function

    PubMed Central

    Noonan, Thomas; Lukas, Susan; Peet, Gregory W; Pelletier, Josephine; Panzenbeck, Mark; Hanidu, Adedayo; Mazurek, Suzanne; Wasti, Ruby; Rybina, Irina; Roma, Teresa; Kronkaitis, Anthony; Shoultz, Alycia; Souza, Donald; Jiang, Huiping; Nabozny, Gerald; Modis, Louise Kelly

    2013-01-01

    Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1. PMID:23885334

  10. Design, synthesis and molecular modeling of aloe-emodin derivatives as potent xanthine oxidase inhibitors.

    PubMed

    Shi, Da-Hua; Huang, Wei; Li, Chao; Liu, Yu-Wei; Wang, Shi-Fan

    2014-03-21

    A series of aloe-emodin derivatives were synthesized and evaluated as xanthine oxidase inhibitors. Among them, four aloe-emodin derivatives showed significant inhibitory activities against xanthine oxidase. The compound 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbaldehyde (A1) possessed the best xanthine oxidase inhibitory activity with IC50 of 2.79 μM. Lineweaver-Burk plot analysis revealed that A1 acted as a mixed-type inhibitor for xanthine oxidase. The docking study revealed that the molecule A1 had strong interactions with the active site of xanthine oxidase and this result was in agreement with kinetic study. Consequently, compound A1 is a new-type candidate for further development for the treatment of gout.

  11. Cox26 is a novel stoichiometric subunit of the yeast cytochrome c oxidase.

    PubMed

    Levchenko, Maria; Wuttke, Jan-Moritz; Römpler, Katharina; Schmidt, Bernhard; Neifer, Klaus; Juris, Lisa; Wissel, Mirjam; Rehling, Peter; Deckers, Markus

    2016-07-01

    The cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain. The complex accepts electrons from cytochrome c and passes them onto molecular oxygen. This process contributes to energy capture in the form of a membrane potential across the inner membrane. The enzyme complex assembles in a stepwise process from the three mitochondria-encoded core subunits Cox1, Cox2 and Cox3, which associate with nuclear-encoded subunits and cofactors. In the yeast Saccharomyces cerevisiae, the cytochrome c oxidase associates with the bc1-complex into supercomplexes, allowing efficient energy transduction. Here we report on Cox26 as a protein found in respiratory chain supercomplexes containing cytochrome c oxidase. Our analyses reveal Cox26 as a novel stoichiometric structural subunit of the cytochrome c oxidase. A loss of Cox26 affects cytochrome c oxidase activity and respirasome organization.

  12. Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

    PubMed Central

    Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

    2001-01-01

    Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

  13. Digenic inheritance of mutations in the coproporphyrinogen oxidase and protoporphyrinogen oxidase genes in a unique type of porphyria.

    PubMed

    van Tuyll van Serooskerken, Anne Moniek; de Rooij, Felix W; Edixhoven, Annie; Bladergroen, Reno S; Baron, Jens M; Joussen, Sylvia; Merk, Hans F; Steijlen, Peter M; Poblete-Gutiérrez, Pamela; te Velde, Kornelis; Wilson, J H Paul; Koole, Rita H; van Geel, Michel; Frank, Jorge

    2011-11-01

    The simultaneous dysfunction of two enzymes within the heme biosynthetic pathway in a single patient is rare. Not more than 15 cases have been reported. A woman with a transient episode of severe photosensitivity showed a biochemical porphyrin profile suggestive of hereditary coproporphyria (HCP), whereas some of her relatives had a profile that was suggestive of variegate porphyria (VP). HCP and VP result from a partial enzymatic deficiency of coproporphyrinogen oxidase (CPOX) and protoporphyrinogen oxidase (PPOX), respectively. DNA analysis in the index patient revealed mutations in both the CPOX and PPOX genes, designated as c.557-15C>G and c.1289dupT, respectively. The CPOX mutation leads to a cryptic splice site resulting in retention of 14 nucleotides from intron 1 in the mRNA transcript. Both mutations encode null alleles and were associated with nonsense-mediated mRNA decay. Given the digenic inheritance of these null mutations, coupled with the fact that both HCP and VP can manifest with life-threatening acute neurovisceral attacks, the unusual aspect of this case is a relatively mild clinical phenotype restricted to dermal photosensitivity.

  14. Enzymatic oxidation of phthalazine with guinea pig liver aldehyde oxidase and liver slices: inhibition by isovanillin.

    PubMed

    Panoutsopoulos, Georgios I; Beedham, Christine

    2004-01-01

    The enzymes aldehyde oxidase and xanthine oxidase catalyze the oxidation of a wide range of N-heterocycles and aldehydes. These enzymes are widely known for their role in the metabolism of N-heterocyclic xenobiotics where they provide a protective barrier by aiding in the detoxification of ingested nitrogen-containing heterocycles. Isovanillin has been shown to inhibit the metabolism of aromatic aldehydes by aldehyde oxidase, but its inhibition towards the heterocyclic compounds has not been studied. The present investigation examines the oxidation of phthalazine in the absence and in the presence of the inhibitor isovanillin by partially purified aldehyde oxidase from guinea pig liver. In addition, the interaction of phthalazine with freshly prepared guinea pig liver slices, both in the absence and presence of specific inhibitors of several liver oxidizing enzymes, was investigated. ldehyde oxidase rapidly converted phthalazine into 1-phthalazinone, which was completely inhibited in the presence of isovanillin (a specific inhibitor of aldehyde oxidase). In freshly prepared liver slices, phthalazine was also rapidly converted to 1-phthalazinone. The formation of 1-phthalazinone was completely inhibited by isovanillin, whereas disulfiram (a specific inhibitor of aldehyde dehydrogenase) only inhibited 1-phthalazinone formation by 24% and allopurinol (a specific inhibitor of xanthine oxidase) had little effect. Therefore, isovanillin has been proved as an inhibitor of the metabolism of heterocyclic substrates, such as phthalazine, by guinea pig liver aldehyde oxidase, since it had not been tested before. Thus it would appear from the inhibitor results that aldehyde oxidase is the predominant enzyme in the oxidation of phthalazine to 1-phthalazinone in freshly prepared guinea pig liver slices, whereas xanthine oxidase only contributes to a small extent and aldehyde dehydrogenase does not take any part.

  15. Expression of ACO1, ERS1 and ERF1 genes in harvested bananas in relation to heat-induced defense against Colletotrichum musae.

    PubMed

    Zhu, Xiangfei; Wang, Aiping; Zhu, Shijiang; Zhang, Lubin

    2011-09-15

    The aim of this study was to investigate the connection between heat-induced ethylene signal changes and enhanced disease resistance. Heat enhanced ripening and elevated MaACO1 expression in naturally ripened bananas (NRB), while it delayed ripening and reduced MaACO1expression in the ethephon-treated bananas (ETB). However, in both cases, heat reduced lesion sizes infected by Colletotrichum musae. This indicates that heat-induced disease resistance in bananas was independent of ripening rate. The expression of MaERS1 gene was inhibited by heat treatment in both NRB and ETB, implying that heat as a physical signal could be sensed by banana fruits through the inhibition of ethylene receptor gene expression. The intensity of MaERF1 transcript signals was elevated in heated bananas, suggesting that the enhanced accumulation of MaERF1 transcript following heat treatment could play an important role in activation of the defense system. In ETB, inhibition of JA biosynthesis by application of IBU down-regulated the expression of MaERF and significantly weakened disease resistance, suggesting involvement of endogenous JA in induction of the gene expression, which was reconfirmed by the fact that exposure to exogenous MeJA following the combination of heat plus IBU treatment restored part of the gene expression. On the other hand, in NRB, application of IBU elevated level of MaERF1 expression at 24h and enhanced disease resistance, suggesting that, when banana was not exposed to ethephon, the expression of MaERF1 gene was not JA dependent, which was verified by the fact that MeJA application did not enhance MaERF1 gene expression. In conclusion, heat-induced disease resistance in harvested bananas could involve down-regulation of MaERS1 expression and up-regulation of MaERF1 expression and JA pathway could be involved in heat activation of the defense system in bananas exposed to ethephon.

  16. Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

    PubMed Central

    Smesrud, Logan; Tebo, Bradley M.

    2016-01-01

    ABSTRACT The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes. IMPORTANCE The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the

  17. Banana Transcription Factor MaERF11 Recruits Histone Deacetylase MaHDA1 and Represses the Expression of MaACO1 and Expansins during Fruit Ripening1[OPEN

    PubMed Central

    Han, Yan-Chao; Kuang, Jian-Fei; Xiao, Yun-Yi; Fu, Chang-Chun; Wang, Jun-Ning

    2016-01-01

    Phytohormone ethylene controls diverse developmental and physiological processes such as fruit ripening via modulation of ethylene signaling pathway. Our previous study identified that ETHYLENE RESPONSE FACTOR11 (MaERF11), a transcription factor in the ethylene signaling pathway, negatively regulates the ripening of banana, but the mechanism for the MaERF11-mediated transcriptional regulation remains largely unknown. Here we showed that MaERF11 has intrinsic transcriptional repression activity in planta. Electrophoretic mobility shift assay and chromatin immunoprecipitation analyses demonstrated that MaERF11 binds to promoters of three ripening-related Expansin genes, MaEXP2, MaEXP7 and MaEXP8, as well as an ethylene biosynthetic gene MaACO1, via the GCC-box motif. Furthermore, expression patterns of MaACO1, MaEXP2, MaEXP7, and MaEXP8 genes are correlated with the changes of histone H3 and H4 acetylation level during fruit ripening. Moreover, we found that MaERF11 physically interacts with a histone deacetylase, MaHDA1, which has histone deacetylase activity, and the interaction significantly strengthens the MaERF11-mediated transcriptional repression of MaACO1 and Expansins. Taken together, these findings suggest that MaERF11 may recruit MaHDA1 to its target genes and repress their expression via histone deacetylation. PMID:27208241

  18. Polyphenol Oxidases in Crops: Biochemical, Physiological and Genetic Aspects

    PubMed Central

    Taranto, Francesca; Pasqualone, Antonella; Mangini, Giacomo; Tripodi, Pasquale; Miazzi, Monica Marilena; Pavan, Stefano; Montemurro, Cinzia

    2017-01-01

    Enzymatic browning is a colour reaction occurring in plants, including cereals, fruit and horticultural crops, due to oxidation during postharvest processing and storage. This has a negative impact on the colour, flavour, nutritional properties and shelf life of food products. Browning is usually caused by polyphenol oxidases (PPOs), following cell damage caused by senescence, wounding and the attack of pests and pathogens. Several studies indicated that PPOs play a role in plant immunity, and emerging evidence suggested that PPOs might also be involved in other physiological processes. Genomic investigations ultimately led to the isolation of PPO homologs in several crops, which will be possibly characterized at the functional level in the near future. Here, focusing on the botanic families of Poaceae and Solanaceae, we provide an overview on available scientific literature on PPOs, resulting in useful information on biochemical, physiological and genetic aspects. PMID:28208645

  19. Amperometric catechol biosensor based on polyaniline-polyphenol oxidase.

    PubMed

    Tan, Yongyan; Guo, Xiaoxia; Zhang, Jinghui; Kan, Jinqing

    2010-03-15

    A novel catechol biosensor was described based on the immobilization of polyphenol oxidase (PPO) into polyaniline (PANI), which was easily constructed by direct electropolymerization of aniline in a solution containing ionic liquid, 1-ethyl-3-methylimidazolium ethyl sulfate (EMIES). The developed biosensor for the detection of catechol has a linear range of 1.25-150 micromol dm(-3). The maximum response current (I(max)) and the Michaelis-Menten constant (k'(m)) are 0.62 microA and 146 micromol dm(-3), respectively. The activation energy (E(a)) of the PPO catalytic reaction is 31.1 kJ mol(-1) in the B-R buffer. The biosensor shows good reproducibility (a relative standard deviation of 3.1% was obtained) and remarkable long-term stability (it retains 75% of the original activity after four months). The effects of potential and pH on the response current of the biosensor are also described.

  20. Studies of dimethylglycine oxidase isoenzymes in Arthrobacter globiformis cells.

    PubMed

    Casaitė, Vida; Povilonienė, Simona; Meškienė, Rita; Rutkienė, Rasa; Meškys, Rolandas

    2011-04-01

    Glycine betaine (GB) could be used by Arthrobacter globiformis cells as a sole carbon source. The cells took up this molecule in the low as well as in the high salinity medium. Addition of GB to the mineral medium with high salt concentration revealed that GB was also used as an osmoprotectant. Dimethylglycine oxidase (DMGO) was involved in the catabolism of GB. Two genes for DMGO were detected in a cloned 26267 bp fragment of A. globiformis DNA. The genes involved in the tetrahydrofolate-dependent assimilation of methyl groups were located nearby the two of DMGO genes. Both cloned A. globiformis DMGO were active. The activity of DMGO was detected in A. globiformis cells and it depended on the addition of GB and the salinity of the medium. Reverse transcription-PCR demonstrated that the addition of GB influenced the transcription of dmg genes.

  1. Potential role of NADPH oxidase in pathogenesis of pancreatitis.

    PubMed

    Cao, Wei-Li; Xiang, Xiao-Hui; Chen, Kai; Xu, Wei; Xia, Shi-Hai

    2014-08-15

    Studies have demonstrated that reactive oxygen species (ROS) are closely related to inflammatory disorders. Nicotinamide adenine dinucleotide phosphate oxidase (NOX), originally found in phagocytes, is the main source of ROS in nonphagocytic cells. Besides directly producing the detrimental highly reactive ROS to act on biomolecules (lipids, proteins, and nucleic acids), NOX can also activate multiple signal transduction pathways, which regulate cell growth, proliferation, differentiation and apoptosis by producing ROS. Recently, research on pancreatic NOX is no longer limited to inflammatory cells, but extends to the aspect of pancreatic acinar cells and pancreatic stellate cells, which are considered to be potentially associated with pancreatitis. In this review, we summarize the literature on NOX protein structure, activation, function and its role in the pathogenesis of pancreatitis.

  2. The NADPH oxidase Nox4 and aging in the heart.

    PubMed

    Ago, Tetsuro; Matsushima, Shouji; Kuroda, Junya; Zablocki, Daniela; Kitazono, Takanari; Sadoshima, Junichi

    2010-12-01

    Oxidative stress in mitochondria is believed to promote aging. Although passive leakage of electron from the mitochondrial electron transport chain has been considered as a major source of oxidative stress in the heart and the cardiomyocytes therein, enzymes actively producing reactive oxygen species may also exist in mitochondria. We have shown recently that Nox4, a member of the NADPH oxidase family, is localized on intracellular membranes, primarily at mitochondria, in cardiomyocytes. Mitochondrial expression of Nox4 is upregulated by cardiac stress and aging in the heart, where Nox4 could become a major source of oxidative stress. This raises an intriguing possibility that Nox4 may play an important role in mediating aging of the heart. Here we discuss the potential involvement of Nox4 in mitochondrial oxidative stress and aging in the heart.

  3. Brofaromine: a monoamine oxidase-A and serotonin uptake inhibitor.

    PubMed

    Waldmeier, P C; Glatt, A; Jaekel, J; Bittiger, H

    1993-01-01

    Brofaromine is a tight-binding, reversible inhibitor of monoamine oxidase-A (MAO-A), with concomitant serotonin (5-HT) uptake-inhibiting properties. In psychopharmacologic investigations, the compound shows the properties expected of an MAO inhibitor, antagonizing the effects of reserpine, tetrabenazine, and 5-hydroxytryptophan in rats and mice, and suppressing rapid eye movement sleep in cats. Brofaromine showed antidepressant-like activity in a rat social conflict test. In radioligand binding assays, brofaromine exhibited weak or no interaction with alpha 1- and alpha 2-noradrenergic, 5-HT1, 5-HT2, 5-HT3, cholinergic, histamine H1 and H2, mu-opiate, GABAA, benzodiazepine, adenosine, neurotensin, and substance P receptors. Comparison of in vitro and in vivo potencies to inhibit 5-HT uptake with those of reference drugs, and direct evidence in patients and volunteers suggest that 5-HT uptake inhibition plays a role in the clinical profile of brofaromine.

  4. Presence of a flavin semiquinone in methanol oxidase.

    PubMed Central

    Mincey, T; Tayrien, G; Mildvan, A S; Abeles, R H

    1980-01-01

    Methanol oxidase from Hansenula polymorpha contains five "red" flavin semiquinones and two oxidized flavins per octamer. Addition of substrate results in the reduction of the two oxidized flavins but does not affect the flavin semiquinones. Enhanced water proton relaxation rates indicate that the unpaired electron of the flavin semiquinones is accessible to the solvent and this accessibility is significantly decreased upon binding of the suicide inhibitor cyclopropanol. In the native enzyme, the semiquinones are not oxidizable by air. All flavins were resolved from the enzyme, and holoenzyme was reconstituted by addition of oxidized flavin. The reconstituted enzyme was catalytically active. The specific activity was 50% that of the original enzyme. It was concluded that the semiquinone is not required for the oxidation of methanol, although it may be present at an otherwise intact site. PMID:6261238

  5. Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy.

    PubMed

    Horvath, Rita; Kemp, John P; Tuppen, Helen A L; Hudson, Gavin; Oldfors, Anders; Marie, Suely K N; Moslemi, Ali-Reza; Servidei, Serenella; Holme, Elisabeth; Shanske, Sara; Kollberg, Gittan; Jayakar, Parul; Pyle, Angela; Marks, Harold M; Holinski-Feder, Elke; Scavina, Mena; Walter, Maggie C; Coku, Jorida; Günther-Scholz, Andrea; Smith, Paul M; McFarland, Robert; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N; Hirano, Michio; Lochmüller, Hanns; Taylor, Robert W; Chinnery, Patrick F; Tulinius, Mar; DiMauro, Salvatore

    2009-11-01

    Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as 'benign cytochrome c oxidase deficiency myopathy' is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

  6. Colocalization of Polyphenol Oxidase and Photosystem II Proteins.

    PubMed

    Lax, A R; Vaughn, K C

    1991-05-01

    Polyphenol oxidase (PPO) appears to be ubiquitous in higher plants but, as yet, no function has been ascribed to it. Herein, we report on the localization of PPO based upon biochemical fractionation of chloroplast membranes in Vicia faba (broad bean) into various complexes and immunocytochemical electron microscopic investigations. Sucrose density gradient fractionations of thylakoid membranes after detergent solubilization reveals that PPO protein (by reactivity with anti-PPO antibody) and activity (based upon ability to oxidize di-dihydroxyphenylalanine) are found only in fractions enriched in photosystem II (PSII). Furthermore, of the PSII particles isolated using three different protocols utilizing several plant species, all had PPO. Immunogold localization of PPO on thin sections reveals exclusive thylakoid labeling with a distribution pattern consistent with other PSII proteins (80% grana, 20% stroma). These data strongly indicate that PPO is at least peripherally associated with the PSII complex.

  7. Potential role of NADPH oxidase in pathogenesis of pancreatitis

    PubMed Central

    Cao, Wei-Li; Xiang, Xiao-Hui; Chen, Kai; Xu, Wei; Xia, Shi-Hai

    2014-01-01

    Studies have demonstrated that reactive oxygen species (ROS) are closely related to inflammatory disorders. Nicotinamide adenine dinucleotide phosphate oxidase (NOX), originally found in phagocytes, is the main source of ROS in nonphagocytic cells. Besides directly producing the detrimental highly reactive ROS to act on biomolecules (lipids, proteins, and nucleic acids), NOX can also activate multiple signal transduction pathways, which regulate cell growth, proliferation, differentiation and apoptosis by producing ROS. Recently, research on pancreatic NOX is no longer limited to inflammatory cells, but extends to the aspect of pancreatic acinar cells and pancreatic stellate cells, which are considered to be potentially associated with pancreatitis. In this review, we summarize the literature on NOX protein structure, activation, function and its role in the pathogenesis of pancreatitis. PMID:25133019

  8. Comparative studies in series of cytochrome c oxidase models.

    PubMed

    Melin, F; Trivella, A; Lo, M; Ruzié, C; Hijazi, I; Oueslati, N; Wytko, J A; Boitrel, B; Boudon, C; Hellwig, P; Weiss, J

    2012-03-01

    This study compares the behavior as cytochrome c oxidase (CcO) functional and structural models of a series of reported and unreported ligands that provide either a binding site for copper without a built-in proximal base, or both a flexible binding site for copper and a built-in proximal base, or a fixed binding site for copper with a built-in proximal base. The comparisons of the models show that the relative position of the two metal sites is not only a crucial parameter in the control of the catalytic behavior but also essential in mimicking other features of the enzyme such as CO exchange between the ferrous heme a(3) and the cuprous Cu(B) center.

  9. Resolution of thylakoid polyphenol oxidase and a protein kinase

    SciTech Connect

    Race, H.L.; Davenport, J.W.; Hind, G.

    1995-12-31

    The predominant protein kinase activity in octylglucoside (OG) extracts of spinach thylakoids has been attributed to a 64-kDa protein, tp64. Recent work calls into question the relation between tp64 and protein kinase activity, which were fractionated apart using fluid phase IEF and hydroxylapatite chromatography. Hind et al. sequenced tp64 from the cDNA and showed it to be a polyphenol oxidase (PPO) homolog. Its transit peptide indicates a location for the mature protein within the thylakoid lumen, where there is presumably no ATP and where it is remote from the presumed kinase substrates: the stromally exposed regions of integral PS-II membrane proteins. Here the authors suggest that the kinase is a 64-kDa protein distinct from tp64.

  10. Isolation and characterization of sulfite oxidase from Alligator mississipiensis

    SciTech Connect

    Robbins, A.; Neame, P.J.; Barber, M.J. )

    1991-03-11

    Sulfite oxidase has been isolated from fresh alligator liver using ammonium sulfate and acetone fractionation, DEAE chromatography and FPLC on Mono Q. The enzyme is dimeric and exhibits a subunit M. Wt. of approximately 58 kDa, larger than that of chicken SO. EPR spectroscopy of the partially-reduced enzyme revealed a single Mo(V) species while visible spectroscopy revealed the presence of cytochrome b{sub 557}. Maximal activities were obtained at pH 8 and 9, respectively. K{sub m}'s for SO{sub 3}{sup 2 {minus}}, cyt. c and Fe(CN){sub 6}{sup 3 {minus}} were 23.5 uM, 2.9 uM and 8.0 uM, respectively. Sequencing of peptides obtained by endoprotease K digestion indicated regions of extensive sequence similarity to chicken and rat enzymes in both heme and Mo-pterin domains. Regions of sequence dissimilarity were also found.

  11. Redox-mediated signal transduction by cardiovascular Nox NADPH oxidases.

    PubMed

    Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

    2014-08-01

    The only known function of the Nox family of NADPH oxidases is the production of reactive oxygen species (ROS). Some Nox enzymes show high tissue-specific expression and the ROS locally produced are required for synthesis of hormones or tissue components. In the cardiovascular system, Nox enzymes are low abundant and function as redox-modulators. By reacting with thiols, nitric oxide (NO) or trace metals, Nox-derived ROS elicit a plethora of cellular responses required for physiological growth factor signaling and the induction and adaptation to pathological processes. The interactions of Nox-derived ROS with signaling elements in the cardiovascular system are highly diverse and will be detailed in this article, which is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".

  12. Translational neuroimaging: positron emission tomography studies of monoamine oxidase.

    PubMed

    Fowler, Joanna S; Logan, Jean; Volkow, Nora D; Wang, Gene-Jack

    2005-01-01

    Positron emission tomography (PET) using radiotracers with high molecular specificity is an important scientific tool in studies of monoamine oxidase (MAO), an important enzyme in the regulation of the neurotransmitters dopamine, norepinephrine, and serotonin as well as the dietary amine, tyramine. MAO occurs in two different subtypes, MAO A and MAO B, which have different substrate and inhibitor specificity and which are different gene products. The highly variable subtype distribution with different species makes human studies of special value. MAO A and B can be imaged in the human brain and certain peripheral organs using PET and carbon-11 (half-life 20.4 minutes) labeled mechanism-based irreversible inhibitors, clorgyline and L -deprenyl, respectively. In this article we introduce MAO and describe the development of these radiotracers and their translation from preclinical studies to the investigation of variables affecting MAO in the human brain and peripheral organs.

  13. STUDIES ON A SARCOSINE OXIDASE OF BACTERIAL ORIGIN

    PubMed Central

    Kopper, Paul H.

    1950-01-01

    A "sarcosine oxidase" was prepared from a creatinine-decomposing strain of Pseudomonas aeruginosa. The enzyme is inactivated by drying, lyophilization, and dialysis against distilled water. No dialyzable cofactor was found. Optimal activity of the enzyme is reached at pH 7.8. Enzyme activity is directly proportional to enzyme concentration and also to substrate concentration up to the point of saturation of enzyme with substrate molecules. One molecule of enzyme combines with one molecule of substrate. Data concerning the effect of temperature and of a variety of chemical compounds on the enzyme are presented. Its inactivation by heat follows the course of a first order reaction, and the critical thermal increment between 48° and 52°C. was calculated to be 103,000 calories per mol. The relationship of enzyme concentration to heat inactivation rates is illustrated. PMID:14778974

  14. Optical characterization of porous silicon microcavities for glucose oxidase biosensing

    NASA Astrophysics Data System (ADS)

    Palestino, G.; Agarwal, V.; Garcia, D. B.; Legros, R.; Pérez, E.; Gergely, C.

    2008-04-01

    PSi microcavity (PSiMc) is characterized by a narrow resonance peak in the optical spectrum that is very sensitive to small changes in the refractive index. We report that the resonant optical cavities of PSi structures can be used to enhance the detection of labeled fluorescent biomolecules. Various PSi configurations were tested in order to compare the optical response of the PSi devices to the capture of organic molecules. Morphological and topographical analyses were performed on PSiMc using Atomic Force (AFM) and Scanning Electron (SEM) microscopies. The heterogeneity in pores lengths resulting from etching process assures a better penetration of larger molecules into the pores and sensor sensitivity depends on the pore size. Molecular detection is monitored by the successive red shifts in the reflectance spectra after the stabilization of PSiMc with 3-aminopropyltriethoxysilane (APTES). The glucose oxidase was cross linked into the PSiMc structures following a silane-glutaraldehyde (GTA) chemistry.

  15. Electrochemistry of xanthine oxidase and its interaction with nitric oxide.

    PubMed

    Zhou, Hui; Xu, Yi; Chen, Ting; Suzuki, Iwao; Li, Genxi

    2006-02-01

    With the help of nanocrystalline TiO2, the direct electrochemistry of xanthine oxidase (XOD) was achieved and two pairs of redox waves were observed. The interaction between XOD and nitric oxide (NO) was also investigated. The experimental results reveal that NO can be reduced at a XOD-nano TiO2 film modified electrode. When the NO concentration was low, the reduced product, HNO, would inactivate the protein. However, when the NO concentration was high, HNO would continue to react with NO to form N2O2- and N3O3-, which would not inhibit XOD, and thus the amount of active protein did not decrease any further.

  16. Physico-chemical properties of molten dimer ascorbate oxidase.

    PubMed

    Nicolai, Eleonora; Di Venere, Almerinda; Rosato, Nicola; Rossi, Antonello; Finazzi Agro', Alessandro; Mei, Giampiero

    2006-11-01

    The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.

  17. The Dynamic Disulfide Relay of Quiescin Sulfhydryl Oxidase

    PubMed Central

    Alon, Assaf; Grossman, Iris; Gat, Yair; Kodali, Vamsi K.; DiMaio, Frank; Mehlman, Tevie; Haran, Gilad; Baker, David; Thorpe, Colin; Fass, Deborah

    2012-01-01

    Protein stability, assembly, localization, and regulation often depend on formation of disulfide cross-links between cysteine side chains. Enzymes known as sulfhydryl oxidases catalyze de novo disulfide formation and initiate intra- and intermolecular dithiol/disulfide relays to deliver the disulfides to substrate proteins1,2. Quiescin sulfhydryl oxidase (QSOX) is a unique, multi-domain disulfide catalyst that is localized primarily to the Golgi apparatus and secreted fluids3 and has attracted attention due to its over-production in tumors4,5. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulfide formation pathways. How disulfides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. We determined the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulfide relay were found more than 40 Å apart in this structure, too far for direct disulfide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulfide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulfide bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented herein, reveals additional biochemical features that facilitate disulfide transfer in metazoan orthologs. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of novel catalytic relays. PMID:22801504

  18. Ionizing radiation induces tumor cell lysyl oxidase secretion

    PubMed Central

    2014-01-01

    Background Ionizing radiation (IR) is a mainstay of cancer therapy, but irradiation can at times also lead to stress responses, which counteract IR-induced cytotoxicity. IR also triggers cellular secretion of vascular endothelial growth factor, transforming growth factor β and matrix metalloproteinases, among others, to promote tumor progression. Lysyl oxidase is known to play an important role in hypoxia-dependent cancer cell dissemination and metastasis. Here, we investigated the effects of IR on the expression and secretion of lysyl oxidase (LOX) from tumor cells. Methods LOX-secretion along with enzymatic activity was investigated in multiple tumor cell lines in response to irradiation. Transwell migration assays were performed to evaluate invasive capacity of naïve tumor cells in response to IR-induced LOX. In vivo studies for confirming IR-enhanced LOX were performed employing immunohistochemistry of tumor tissues and ex vivo analysis of murine blood serum derived from locally irradiated A549-derived tumor xenografts. Results LOX was secreted in a dose dependent way from several tumor cell lines in response to irradiation. IR did not increase LOX-transcription but induced LOX-secretion. LOX-secretion could not be prevented by the microtubule stabilizing agent patupilone. In contrast, hypoxia induced LOX-transcription, and interestingly, hypoxia-dependent LOX-secretion could be counteracted by patupilone. Conditioned media from irradiated tumor cells promoted invasiveness of naïve tumor cells, while conditioned media from irradiated, LOX- siRNA-silenced cells did not stimulate their invasive capacity. Locally applied irradiation to tumor xenografts also increased LOX-secretion in vivo and resulted in enhanced LOX-levels in the murine blood serum. Conclusions These results indicate a differential regulation of LOX-expression and secretion in response to IR and hypoxia, and suggest that LOX may contribute towards an IR-induced migratory phenotype in

  19. Molecular Dynamic Studies of the Complex Polyethylenimine and Glucose Oxidase

    PubMed Central

    Szefler, Beata; Diudea, Mircea V.; Putz, Mihai V.; Grudzinski, Ireneusz P.

    2016-01-01

    Glucose oxidase (GOx) is an enzyme produced by Aspergillus, Penicillium and other fungi species. It catalyzes the oxidation of β-d-glucose (by the molecular oxygen or other molecules, like quinones, in a higher oxidation state) to form d-glucono-1,5-lactone, which hydrolyses spontaneously to produce gluconic acid. A coproduct of this enzymatic reaction is hydrogen peroxide (H2O2). GOx has found several commercial applications in chemical and pharmaceutical industries including novel biosensors that use the immobilized enzyme on different nanomaterials and/or polymers such as polyethylenimine (PEI). The problem of GOx immobilization on PEI is retaining the enzyme native activity despite its immobilization onto the polymer surface. Therefore, the molecular dynamic (MD) study of the PEI ligand (C14N8_07_B22) and the GOx enzyme (3QVR) was performed to examine the final complex PEI-GOx stabilization and the affinity of the PEI ligand to the docking sites of the GOx enzyme. The docking procedure showed two places/regions of major interaction of the protein with the polymer PEI: (LIG1) of −5.8 kcal/mol and (LIG2) of −4.5 kcal/mol located inside the enzyme and on its surface, respectively. The values of enthalpy for the PEI-enzyme complex, located inside of the protein (LIG1) and on its surface (LIG2) were computed. Docking also discovered domains of the GOx protein that exhibit no interactions with the ligand or have even repulsive characteristics. The structural data clearly indicate some differences in the ligand PEI behavior bound at the two places/regions of glucose oxidase. PMID:27801788

  20. Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells

    PubMed Central

    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. PMID:28386230

  1. Putrescine biosensor based on putrescine oxidase from Kocuria rosea.

    PubMed

    Bóka, Beáta; Adányi, Nóra; Szamos, Jenő; Virág, Diána; Kiss, Attila

    2012-10-10

    The novel putrescine oxidase based amperometric biosensor selectively measures putrescine, which can be considered as an indicator of microbial spoilage. Putrescine oxidase (PUOX, EC 1.4.3.10) was isolated from Kocuria rosea (Micrococcus rubens) by an improved and simplified purification process. Cells were grown on brain heart infusion medium supplemented with putrescine. Cell-free extract was prepared in Tris buffer (pH 8.0) by Bead-beater. A newly elaborated step based on three-phase partitioning (TPP) was applied in the purification protocol of PUOX. The purified enzyme was immobilized on the surface of a spectroscopic graphite electrode in redox hydrogel with horseradish peroxidase, Os mediator and poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) as crosslinking agent. This modified working electrode was used in wall-jet type amperometric cell together with the Ag/AgCl (0.1M KCl) reference electrode and a platinum wire as auxiliary electrode in flow injection analysis system (FIA). Hydrogel composition, pH and potential dependence were studied. Optimal working conditions were 0.45 mLmin(-1) flow rate of phosphate buffer (66 mM, pH 8.0) and +50 mV polarizing potential vs. Ag/AgCl. The linear measuring range of the method was 0.01-0.25 mM putrescine, while the detection limit was 5 μM. Beer samples were investigated by the putrescine biosensor and the results were compared by those of HPLC reference method.

  2. Proton exit channels in bovine cytochrome c oxidase.

    PubMed

    Popović, Dragan M; Stuchebrukhov, Alexei A

    2005-02-10

    Cytochrome c oxidase (CcO) is the terminal transmembrane enzyme of the respiratory electron transport chain in aerobic cells. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping, a process which results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping and proton exit pathways remain unknown. In our previous work, the continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine cytochrome c oxidase for different redox states of the enzyme. A possible mechanism of oxygen reduction and proton pumping via His291 was proposed. In this paper, using electrostatic calculations, we examine the proton exit pathways in the enzyme. By monitoring the changes of the protonation states, proton affinities, and energies of electrostatic interactions between the titratable groups in different redox states of CcO, we identified the clusters of strongly interacting residues. Using these data, we detected four possible proton exit points on the periplasmic side of the membrane (Lys171B/Asp173B, His24B/Asp25B, Asp51, and Asp300). We then were able to trace the proton exit pathways and to evaluate the energy profiles along the paths. On the basis of energetic considerations and the conservation of the residues in a protein sequence, the most likely exit pathway is one via the Lys171B/Asp173B site. The obtained results are fully consistent with our His291 model of proton pumping, and provide a rationale for the absence of proton leaking in CcO between the pumping strokes.

  3. Aspartate oxidase plays an important role in Arabidopsis stomatal immunity.

    PubMed

    Macho, Alberto P; Boutrot, Freddy; Rathjen, John P; Zipfel, Cyril

    2012-08-01

    Perception of pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin (or the peptide flg22), by surface-localized receptors activates defense responses and subsequent immunity. In a previous forward-genetic screen aimed at the identification of Arabidopsis (Arabidopsis thaliana) flagellin-insensitive (fin) mutants, we isolated fin4, which is severely affected in flg22-triggered reactive oxygen species (ROS) bursts. Here, we report that FIN4 encodes the chloroplastic enzyme ASPARTATE OXIDASE (AO), which catalyzes the first irreversible step in the de novo biosynthesis of NAD. Genetic studies on the role of NAD have been hindered so far by the lethality of null mutants in NAD biosynthetic enzymes. Using newly identified knockdown fin alleles, we found that AO is required for the ROS burst mediated by the NADPH oxidase RBOHD triggered by the perception of several unrelated PAMPs. AO is also required for RBOHD-dependent stomatal closure. However, full AO activity is not required for flg22-induced responses that are RBOHD independent. Interestingly, although the fin4 mutation dramatically affects RBOHD function, it does not affect functions carried out by other members of the RBOH family, such as RBOHC and RBOHF. Finally, we determined that AO is required for stomatal immunity against the bacterium Pseudomonas syringae. Altogether, our work reveals a novel specific requirement for AO activity in PAMP-triggered RBOHD-dependent ROS burst and stomatal immunity. In addition, the availability of viable mutants for the chloroplastic enzyme AO will enable future detailed studies on the role of NAD metabolism in different cellular processes, including immunity, in Arabidopsis.

  4. Molecular Dynamic Studies of the Complex Polyethylenimine and Glucose Oxidase.

    PubMed

    Szefler, Beata; Diudea, Mircea V; Putz, Mihai V; Grudzinski, Ireneusz P

    2016-10-27

    Glucose oxidase (GOx) is an enzyme produced by Aspergillus, Penicillium and other fungi species. It catalyzes the oxidation of β-d-glucose (by the molecular oxygen or other molecules, like quinones, in a higher oxidation state) to form d-glucono-1,5-lactone, which hydrolyses spontaneously to produce gluconic acid. A coproduct of this enzymatic reaction is hydrogen peroxide (H₂O₂). GOx has found several commercial applications in chemical and pharmaceutical industries including novel biosensors that use the immobilized enzyme on different nanomaterials and/or polymers such as polyethylenimine (PEI). The problem of GOx immobilization on PEI is retaining the enzyme native activity despite its immobilization onto the polymer surface. Therefore, the molecular dynamic (MD) study of the PEI ligand (C14N8_07_B22) and the GOx enzyme (3QVR) was performed to examine the final complex PEI-GOx stabilization and the affinity of the PEI ligand to the docking sites of the GOx enzyme. The docking procedure showed two places/regions of major interaction of the protein with the polymer PEI: (LIG1) of -5.8 kcal/mol and (LIG2) of -4.5 kcal/mol located inside the enzyme and on its surface, respectively. The values of enthalpy for the PEI-enzyme complex, located inside of the protein (LIG1) and on its surface (LIG2) were computed. Docking also discovered domains of the GOx protein that exhibit no interactions with the ligand or have even repulsive characteristics. The structural data clearly indicate some differences in the ligand PEI behavior bound at the two places/regions of glucose oxidase.

  5. Multicopper manganese oxidase accessory proteins bind Cu and heme.

    PubMed

    Butterfield, Cristina N; Tao, Lizhi; Chacón, Kelly N; Spiro, Thomas G; Blackburn, Ninian J; Casey, William H; Britt, R David; Tebo, Bradley M

    2015-12-01

    Multicopper oxidases (MCOs) catalyze the oxidation of a diverse group of metal ions and organic substrates by successive single-electron transfers to O2 via four bound Cu ions. MnxG, which catalyzes MnO2 mineralization by oxidizing both Mn(II) and Mn(III), is unique among multicopper oxidases in that it carries out two energetically distinct electron transfers and is tightly bound to accessory proteins. There are two of these, MnxE and MnxF, both approximately 12kDa. Although their sequences are similar to those found in the genomes of several Mn-oxidizing Bacillus species, they are dissimilar to those of proteins with known function. Here, MnxE and MnxF are co-expressed independent of MnxG and are found to oligomerize into a higher order stoichiometry, likely a hexamer. They bind copper and heme, which have been characterized by electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry. Cu is found in two distinct type 2 (T2) copper centers, one of which appears to be novel; heme is bound as a low-spin species, implying coordination by two axial ligands. MnxE and MnxF do not oxidize Mn in the absence of MnxG and are the first accessory proteins to be required by an MCO. This may indicate that Cu and heme play roles in electron transfer and/or Cu trafficking.

  6. Characterization of the multicopper oxidase gene family in Anopheles gambiae

    PubMed Central

    Gorman, Maureen J.; Dittmer, Neal T.; Marshall, Jeremy L.; Kanost, Michael R.

    2008-01-01

    The multicopper oxidase (MCO) family of enzymes includes laccases, which oxidize a broad range of substrates including diphenols, and several oxidases with specific substrates such as iron, copper or ascorbic acid. We have identified five putative MCO genes in the genome of Anopheles gambiae and have cloned cDNAs encompassing the full coding region for each gene. MCO1 mRNA was detected in all developmental stages and in all of the larval and adult tissues tested. We observed an increase in MCO1 transcript abundance in the midguts and Malphighian tubules of adult females following a blood meal and in adult abdominal carcasses in response to an immune challenge. Two alternatively spliced isoforms of MCO2 mRNA were identified. The A isoform of MCO2 was previously detected in larval and pupal cuticle where it probably catalyzes sclerotization reactions (He et al., 2007). The B isoform was transcriptionally upregulated in ovaries in response to a blood meal. MCO3 mRNA was detected in the adult midgut, Malpighian tubules, and male reproductive tissues; like MCO1, it was upregulated in response to an immune challenge or a blood meal. MCO4 and MCO5 were observed primarily in eggs and in the abdominal carcass of larvae. A phylogenetic analysis of insect MCO genes identified putative orthologs of MCO1 and MCO2 in all of the insect genomes tested, whereas MCO3, MCO4 and MCO5 were found only in the two mosquito species analyzed. MCO2 orthologs have especially high sequence similarity, suggesting that they are under strong purifying selection; the A isoforms are more conserved than the B isoforms. The mosquito specific group shares a common ancestor with MCO2. This initial study of mosquito MCOs suggests that MCO2 may be required for egg development or eggshell tanning in addition to cuticle tanning, while MCO1 and MCO3 may be involved in metal metabolism or immunity. PMID:18675911

  7. A Transition from Localized to Strongly Correlated Electron Behavior and Mixed Valence Driven by Physical or Chemical Pressure in ACo2As2 (A = Eu and Ca)

    SciTech Connect

    Tan, Xiaoyan; Fabbris, Gilberto; Haskel, Daniel; Yaroslavtsev, Alexander A.; Cao, H.; Thompson, Corey M.; Kovnir, Kirill; Menushenkov, Alexey P.; Chernikov, Roman V.; Garlea, V. Ovidiu; Shatruk, Michael

    2016-03-02

    We demonstrate that the action of physical pressure, chemical compression, or aliovalent substitution in ACo(2)As(2) (A = Eu and Ca) has a general consequence of causing these antiferromagnetic materials to become ferromagnets. In all cases, the mixed valence triggered at the electropositive A site results in the increase of the Co 3d density of states at the Fermi level. Remarkably, the dramatic alteration of magnetic behavior results from the very minor (<0.15 eleetron) change in the population of the 3d orbitals. The mixed valence state of En observed in the high-pressure (HP) form of EuCo2As2 exhibits a remarkable stability, achieving the average oxidation state of +2.25 at 12.6 GPa. In the case of CaCo2As2, substituting even 10% of Eu or La into the Ca site causes ferromagnetic ordering of Co moments. Similar to HP-EuCo2As2, the itinerant 3d ferromagnetism emerges from electronic doping into the Co layer because of chemical compression of Eu sites in Ca0.9Eu0.1Co1.91As2 or direct electron doping in Ca0.85La0.15Co1.89As2. The results reported herein demonstrate the general possibility of amplifying minor localized electronic effects to achieve major changes in material's properties via involvement of strongly correlated electrons.

  8. A Transition from Localized to Strongly Correlated Electron Behavior and Mixed Valence Driven by Physical or Chemical Pressure in ACo2As2 (A = Eu and Ca).

    PubMed

    Tan, Xiaoyan; Fabbris, Gilberto; Haskel, Daniel; Yaroslavtsev, Alexander A; Cao, Huibo; Thompson, Corey M; Kovnir, Kirill; Menushenkov, Alexey P; Chernikov, Roman V; Garlea, V Ovidiu; Shatruk, Michael

    2016-03-02

    We demonstrate that the action of physical pressure, chemical compression, or aliovalent substitution in ACo2As2 (A = Eu and Ca) has a general consequence of causing these antiferromagnetic materials to become ferromagnets. In all cases, the mixed valence triggered at the electropositive A site results in the increase of the Co 3d density of states at the Fermi level. Remarkably, the dramatic alteration of magnetic behavior results from the very minor (<0.15 electron) change in the population of the 3d orbitals. The mixed valence state of Eu observed in the high-pressure (HP) form of EuCo2As2 exhibits a remarkable stability, achieving the average oxidation state of +2.25 at 12.6 GPa. In the case of CaCo2As2, substituting even 10% of Eu or La into the Ca site causes ferromagnetic ordering of Co moments. Similar to HP-EuCo2As2, the itinerant 3d ferromagnetism emerges from electronic doping into the Co layer because of chemical compression of Eu sites in Ca0.9Eu0.1Co1.91As2 or direct electron doping in Ca0.85La0.15Co1.89As2. The results reported herein demonstrate the general possibility of amplifying minor localized electronic effects to achieve major changes in material's properties via involvement of strongly correlated electrons.

  9. Cytoplasmic CopZ-Like Protein and Periplasmic Rusticyanin and AcoP Proteins as Possible Copper Resistance Determinants in Acidithiobacillus ferrooxidans ATCC 23270.

    PubMed

    Navarro, Claudio A; von Bernath, Diego; Martínez-Bussenius, Cristóbal; Castillo, Rodrigo A; Jerez, Carlos A

    2015-12-04

    Acidophilic organisms, such as Acidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals. A. ferrooxidans contains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression of A. ferrooxidans genes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitive Escherichia coli strain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium.

  10. A Transition from Localized to Strongly Correlated Electron Behavior and Mixed Valence Driven by Physical or Chemical Pressure in ACo2As2 (A = Eu and Ca)

    DOE PAGES

    Tan, Xiaoyan; Fabbris, Gilberto; Haskel, Daniel; ...

    2016-02-03

    In this paper, we demonstrate that the action of physical pressure, chemical compression, or aliovalent substitution in ACo2As2 (A = Eu and Ca) has a general consequence of causing these antiferromagnetic materials to become ferromagnets. In all cases, the mixed valence triggered at the electropositive A site results in the increase of the Co 3d density of states at the Fermi level. Remarkably, the dramatic alteration of magnetic behavior results from the very minor (<0.15 electron) change in the population of the 3d orbitals. The mixed valence state of Eu observed in the high-pressure (HP) form of EuCo2As2 exhibits amore » remarkable stability, achieving the average oxidation state of +2.25 at 12.6 GPa. In the case of CaCo2As2, substituting even 10% of Eu or La into the Ca site causes ferromagnetic ordering of Co moments. Similar to HP-EuCo2As2, the itinerant 3d ferromagnetism emerges from electronic doping into the Co layer because of chemical compression of Eu sites in Ca0.9Eu0.1Co1.91As2 or direct electron doping in Ca0.85La0.15Co1.89As2. Finally, the results reported herein demonstrate the general possibility of amplifying minor localized electronic effects to achieve major changes in material’s properties via involvement of strongly correlated electrons.« less

  11. Processing optimization of probiotic yogurt containing glucose oxidase using response surface methodology.

    PubMed

    Cruz, A G; Faria, J A F; Walter, E H M; Andrade, R R; Cavalcanti, R N; Oliveira, C A F; Granato, D

    2010-11-01

    Exposure to oxygen may induce a lack of functionality of probiotic dairy foods because the anaerobic metabolism of probiotic bacteria compromises during storage the maintenance of their viability to provide benefits to consumer health. Glucose oxidase can constitute a potential alternative to increase the survival of probiotic bacteria in yogurt because it consumes the oxygen permeating to the inside of the pot during storage, thus making it possible to avoid the use of chemical additives. This research aimed to optimize the processing of probiotic yogurt supplemented with glucose oxidase using response surface methodology and to determine the levels of glucose and glucose oxidase that minimize the concentration of dissolved oxygen and maximize the Bifidobacterium longum count by the desirability function. Response surface methodology mathematical models adequately described the process, with adjusted determination coefficients of 83% for the oxygen and 94% for the B. longum. Linear and quadratic effects of the glucose oxidase were reported for the oxygen model, whereas for the B. longum count model an influence of the glucose oxidase at the linear level was observed followed by the quadratic influence of glucose and quadratic effect of glucose oxidase. The desirability function indicated that 62.32 ppm of glucose oxidase and 4.35 ppm of glucose was the best combination of these components for optimization of probiotic yogurt processing. An additional validation experiment was performed and results showed acceptable error between the predicted and experimental results.

  12. Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.) 1

    PubMed Central

    Lin, Liang-Shiou; Varner, Joseph E.

    1991-01-01

    The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall “loosening.” ImagesFigure 3Figure 4Figure 5 PMID:16668145

  13. The coordinate regulation of multiple terminal oxidases by the Pseudomonas putida ANR global regulator.

    PubMed

    Ugidos, Ana; Morales, Gracia; Rial, Eduardo; Williams, Huw D; Rojo, Fernando

    2008-07-01

    Pseudomonas putida KT2440 contains a branched aerobic respiratory chain with multiple terminal oxidases. Their relative proportion varies according to environmental conditions. The role of the oxygen-responsive ANR global regulator on expression of these terminal oxidases was analysed. During exponential growth in a highly aerated complete medium, ANR activated expression of the Cbb3-1 terminal oxidase (equivalent to Pseudomonas aeruginosa Cbb3-2), but had little role on expression of other terminal oxidases. In early stationary phase, or under oxygen limitation, inactivation of the anr gene led to increased expression of the bo(3)-type cytochrome (Cyo) and cyanide-insensitive (CIO) terminal oxidases, and to a much lower expression of Cbb3-1. DNase I footprints identified ANR binding sites at the promoters for these oxidases. Their location suggests that ANR is a transcriptional activator of Cbb3-1 genes and a repressor of CIO genes, consistent with expression data. ANR binding sites at the promoter for Cyo genes suggests a complex regulation in combination with other factors. Therefore, ANR coordinates expression of Cyo, CIO and Cbb3-1, but does not influence cytochrome aa3 and Cbb3-2 terminal oxidases under the conditions analysed. Functional assays showed that Cyo has a leading role during aerobic exponential growth, while Cbb3-1 becomes very important in stationary phase.

  14. Cytochrome c oxidase-intermediate fibres: importance in understanding the pathogenesis and treatment of mitochondrial myopathy.

    PubMed

    Murphy, Julie L; Ratnaike, Thiloka E; Shang, Ersong; Falkous, Gavin; Blakely, Emma L; Alston, Charlotte L; Taivassalo, Tanja; Haller, Ronald G; Taylor, Robert W; Turnbull, Doug M

    2012-08-01

    An important diagnostic muscle biopsy finding in patients with mitochondrial DNA disease is the presence of respiratory-chain deficient fibres. These fibres are detected as cytochrome c oxidase-deficient following a sequential cytochrome c oxidase-succinate dehydrogenase reaction, often in a mosaic pattern within a population of cytochrome c oxidase-normal fibres. Detailed analysis of muscle biopsies from patients with various mitochondrial DNA defects shows that a spectrum of deficiency exists, as there are a large number of fibres which do not correspond to being either completely cytochrome c oxidase-normal (brown staining) or cytochrome c oxidase-deficient (blue staining). We have used a combination of histochemical and immunocytochemical techniques to show that a population of cytochrome c oxidase-intermediate reacting fibres are a gradation between normal and deficient fibres. We show that cytochrome c oxidase-intermediate fibres also have different genetic characteristics in terms of amount of mutated and wild-type mtDNA, and as such, may represent an important transition between respiratory normal and deficient fibres. Assessing changes in intermediate fibres will be crucial to evaluating the responses to treatment and in particular to exercise training regimes in patients with mitochondrial DNA disease.

  15. Phenol oxidase is a necessary enzyme for the silkworm molting which is regulated by molting hormone.

    PubMed

    Wang, Mei-xian; Lu, Yan; Cai, Zi-zheng; Liang, Shuang; Niu, Yan-shan; Miao, Yun-gen

    2013-05-01

    Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. The molting process includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis excoriation etc. Polyphenol oxidases (PPOs), dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the phenol oxidase was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that polyphenol oxidases are one set of the key enzymes in molting, which closely related with the role of ecdysone in regulation of molting processes. The data showed that the expression peak of phenol oxidase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the phenol oxidase expression was significantly elevated compared to the control. PPO1 RNAi induced phenol oxidase expression obviously declined in the silkworm larvae, and caused the pupae incomplete pupation. Overall, the results described that the phenol oxidase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.

  16. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis

    PubMed Central

    Bataller, Ramón; Schwabe, Robert F.; Choi, Youkyung H.; Yang, Liu; Paik, Yong Han; Lindquist, Jeffrey; Qian, Ting; Schoonhoven, Robert; Hagedorn, Curt H.; Lemasters, John J.; Brenner, David A.

    2003-01-01

    Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II–induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox–/– mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox–/– mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle α-actin and expression of TGF-β1 were reduced in p47phox–/– mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis. PMID:14597764

  17. Sequence analysis of the cbb3 oxidases and an atomic model for the Rhodobacter sphaeroides enzyme.

    PubMed

    Sharma, Vivek; Puustinen, Anne; Wikström, Mårten; Laakkonen, Liisa

    2006-05-09

    The cbb3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. To understand the minimal common properties of the superfamily, and to learn about cbb3-type oxidases specifically, we have analyzed a wide set of heme-copper oxidase sequences and built a homology model of the catalytic subunit of the cbb3 oxidase from Rhodobacter sphaeroides. We conclude that with regard to the active site surroundings, the cbb3 oxidases greatly resemble the structurally known oxidases, while major differences are found in three segments: the additional N-terminal stretch of ca. 60 amino acids, the segment following helix 3 to the end of helix 5, and the C-terminus from helix 11 onward. The conserved core contains the active site tyrosine and also an analogue of the K-channel of proton transfer, but centered on a well-conserved histidine in the lower part of helix 7. Modeling the variant parts of the enzyme suggests that two periplasmic loops (between helices 3 and 4 and between helices 11 and 12) could interact with each other as a part of the active site structure and might have an important role in proton pumping. An analogue of the D-channel is not found, but an alternative channel might form around helix 9. A preliminary packing model of the trimeric enzyme is also presented.

  18. Partial purification of gibberellin oxidases from spinach leaves. [Spinacia oleracea L

    SciTech Connect

    Gilmour, S.J.; Bleecker, A.B.; Zeevaart, J.A.D.

    1987-09-01

    Four enzyme activities catalyzing the following oxidative steps in the gibberellin (GA) biosynthetic pathway have been extracted from spinach (Spinacia oleracea L.) leaves after exposure to 8 long days: GA/sub 12/ ..-->.. GA/sub 53/ ..-->.. GA/sub 44/ ..-->.. GA/sub 19/ ..-->.. GA/sub 20/. Two of these, GA/sub 53/ oxidase and GA/sup 19/ oxidase, were separable from the other two, GA/sub 44/ oxidase and GA/sub 12/ 13-hydroxylase, by anion exchange high performance liquid chromatography (HPLC). Apparent molecular weights of the four enzymes as determined by gel filtration HLPL are: GA/sub 12/ 13-hydroxylase, 28,400; GA/sub 43/ oxidase, 42,500; GA/sub 44/ oxidase, 38,100; GA/sub 19/ oxidase, 39,500. GA/sub 44/ oxidase was purified approximately 100-fold in 0.3% yield by a combination of ammonium sulfate fractionation, anion exchange HPLC, phenyl-Sepharose chromatography and gel filtration HLPC.

  19. Synthesis and Biological Evaluation of Novel Aryl-2H-pyrazole Derivatives as Potent Non-purine Xanthine Oxidase Inhibitors.

    PubMed

    Sun, Zhi-Gang; Zhou, Xiao-Jing; Zhu, Ming-Li; Ding, Wen-Ze; Li, Zhen; Zhu, Hai-Liang

    2015-01-01

    A series of aryl-2H-pyrazole derivatives were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro as potent xanthine oxidase inhibitors. Among them, 2 aryl-2H-pyrazole derivatives showed significant inhibitory activities against xanthine oxidase. Compound 19 emerged as the most potent xanthine oxidase inhibitor (IC50=9.8 µM) in comparison with allopurinol (IC50=9.5 µM). The docking study revealed that compound 19 might have strong interactions with the active site of xanthine oxidase. This compound is thus a new candidate for further development for the treatment of gout.

  20. Involvement of monoamine oxidase and diamine oxidase in the metabolism of the cell differentiating agent hexamethylene bisacetamide (HMBA).

    PubMed

    Conley, B A; Callery, P S; Egorin, M J; Subramanyam, B; Geelhaar, L A; Pan, S S

    1988-01-01

    We have previously demonstrated a number of metabolites of hexamethylene bisacetamide (HMBA) in the urine of patients treated with HMBA. These include N-acetyl-1,6-diaminohexane (NADAH), 6-acetamidohexanoic acid (6AcHA), 1,6-diaminohexane (DAH) and 6-aminohexanoic acid (6AmHA). Because these compounds have potential roles in the dose-limiting metabolic acidosis and neurotoxicity associated with HMBA therapy, and are similar in structure to known substrates of monoamine oxidase (MAO) and diamine oxidase (DAO), we investigated the activities of these enzymes in the metabolic interconversion of HMBA metabolites. NADAH (5 mM) was incubated with MAO and aldehyde dehydrogenase. 6AcHA production was verified by gas chromatography-mass spectrometry and quantified by gas chromatography. 6AcHA production was linear for up to 4 hr. Complete inhibition of MAO activity was observed with 2 mM tranyl-cypromine or pargyline. Mouse liver microsomes, which do not contain MAO, did not convert NADAH to 6AcHA and, in control experiments, did not degrade 6AcHA. The HMBA metabolite, DAH, was a substrate for DAO, producing 3,4,5,6-tetrahydro-2H-azepine. Participation of DAO in the metabolism of HMBA implies potential interaction of HMBA and metabolites with polyamine metabolism and may represent a mechanism for HMBA's effects on cellular growth and differentiation. Metabolism of NADAH, also a differentiator, by MAO implies that concurrent use of HMBA and an MAO inhibitor may be clinically useful.

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

    PubMed

    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.

  2. Cloning and sequencing of two Ceriporiopsis subvermispora bicupin oxalate oxidase allelic isoforms: implications for the reaction specificity of oxalate oxidases and decarboxylases.

    PubMed

    Escutia, Marta R; Bowater, Laura; Edwards, Anne; Bottrill, Andrew R; Burrell, Matthew R; Polanco, Rubén; Vicuña, Rafael; Bornemann, Stephen

    2005-07-01

    Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess < or =0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.

  3. Inhibition and oxygen activation in copper amine oxidases.

    PubMed

    Shepard, Eric M; Dooley, David M

    2015-05-19

    Copper-containing amine oxidases (CuAOs) use both copper and 2,4,5-trihydroxyphenylalanine quinone (TPQ) to catalyze the oxidative deamination of primary amines. The CuAO active site is highly conserved and comprised of TPQ and a mononuclear type II copper center that exhibits five-coordinate, distorted square pyramidal coordination geometry with histidine ligands and equatorially and axially bound water in the oxidized, resting state. The active site is buried within the protein, and CuAOs from various sources display remarkable diversity with respect to the composition of the active site channel and cofactor accessibility. Structural and mechanistic factors that influence substrate preference and inhibitor sensitivity and selectivity have been defined. This Account summarizes the strategies used to design selective CuAO inhibitors based on active site channel characteristics, leading to either enhanced steric fits or the trapping of reactive electrophilic products. These findings provide a framework to support the future development of candidate molecules aimed at minimizing the negative side effects associated with drugs containing amine functionalities. This is vital given the existence of human diamine oxidase and vascular adhesion protein-1, which have distinct amine substrate preferences and are associated with different metabolic processes. Inhibition of these enzymes by antifungal or antiprotozoal agents, as well as classic monoamine oxidase (MAO) inhibitors, may contribute to the adverse side effects associated with drug treatment. These observations provide a rationale for the limited clinical value associated with certain amine-containing pharmaceuticals and emphasize the need for more selective AO inhibitors. This Account also discusses the novel roles of copper and TPQ in the chemistry of O2 activation and substrate oxidation. Reduced CuAOs exist in a redox equilibrium between the Cu(II)-TPQAMQ (aminoquinol) and Cu(I)-TPQSQ (semiquinone). Elucidating

  4. Cyclic voltammetry at TCNQ and TTF-TCNQ modified platinum electrodes: A study of the glucose oxidase/glucose and galactose oxidase/galactose systems

    SciTech Connect

    Hale, P.D.; Skotheim, T.A.

    1988-01-01

    Recent work has shown that the synthetic metal TTF-TCNQ can be used as an electrode material for the oxidation of enzymes containing the prosthetic group flavin adenine dinucleotide (FAD). This direct electron transfer (direct in the sense that oxygen is not a mediator) between reduced enzyme and electrode, a process which does not occur to any measurable extent at a typical metal electrode, is not very well understood. In the present work, electron transfer between reduced glucose oxidase and TTF-TCNQ is investigated using cyclic voltammetry, and it is also shown that TCNQ itself can mediate this electron transfer between the enzyme and a platinum electrode. In addition to the glucose oxidase studies, cyclic voltammetric experiments have been performed on the galactose oxidase system, which contains a copper redox center rather than FAD. The results of these experiments demonstrate that the catalytic ability of TTF-TCNQ in enzyme-based electrochemical sensors is quite general. 15 refs., 4 figs.

  5. Optimization of catechol production by membrane-immobilized polyphenol oxidase: a modeling approach.

    PubMed

    Boshoff, A; Burton, M H; Burton, S G

    2003-07-05

    Although previous research has focused on phenol removal efficiencies using polyphenol oxidase in nonimmobilized and immobilized forms, there has been little consideration of the use of polyphenol oxidase in a biotransformation system for the production of catechols. In this study, polyphenol oxidase was successfully immobilized on various synthetic membranes and used to convert phenolic substrates to catechol products. A neural network model was developed and used to model the rates of substrate utilization and catechol production for both nonimmobilized and immobilized polyphenol oxidase. The results indicate that the biotransformation of the phenols to their corresponding catechols was strongly influenced by the immobilization support, resulting in differing yields of catechols. Hydrophilic membranes were found to be the most suitable immobilization supports for catechol production. The successful biocatalytic production of 3-methylcatechol, 4-methylcatechol, catechol, and 4-chlorocatechol is demonstrated.

  6. Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase.

    PubMed

    Lehninger, A L; Reynafarje, B; Costa, L

    1985-01-01

    The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.

  7. Effect of polymerization on antioxidant and xanthine oxidase inhibitory potential of sea buckthorn (H. rhamnoides) proanthocyanidins.

    PubMed

    Arimboor, Ranjith; Arumughan, C

    2012-10-01

    Inhibitory potential of sea buckthorn (Hippophae rhamnoides L) seed proanthocyanidins against oxidative stress and xanthine oxidase activity was evaluated. Composition of antioxidant proanthocyanidins was profiled by analyzing the cleavage products obtained by the acid catalyzed hydrolysis in the presence of phloroglucinol. Catechin, epicatechin, gallocatechin, and epigallocatechin were found as the extension and terminal subunits of proanthocyanidins with an average degree of polymerization (ADP) of 14.7. Seed proanthocyanidins showed considerably high antioxidant and xanthine oxidase inhibitory potentials. Antioxidant and xanthine oxidase inhibitory capacity evaluation of proanthocyanidin fractions with varying ADP showed that proanthocyanidins with lower molecular size were more effective as superoxide anion (ADP ≤ 4.2) and hydroxyl radical (ADP ≤ 5.9) scavengers and xanthine oxidase (ADP ≤ 3.1) inhibitors. ADP of the studied proanthocyanidin fractions did not show significant influence on their DPPH and ABTS radical scavenging and ferric reduction capacities.

  8. Polyphenol oxidase expression in potato (Solanum tuberosum) tubers inhibited to sprouting by treatment with iodine atmosphere.

    PubMed

    Eolini, Francesco; Hochkoeppler, Alejandro; Credi, Andrea; Rodríguez, Antonio Gonzàlez Vara Y; Poggi, Valeria

    2004-08-01

    Iodine-saturated atmosphere was found to inhibit the sprouting of potato (Solanum tuberosum L.) tubers. The iodine concentration in tuber tissues increased as a function of exposure length, and the onset of inhibition of sprouting was found to depend on tubers genotype. During the time-course of the treatment, the transcription of polyphenol oxidases (EC 1.10.3.1 and EC 1.14.18.1) was undetectable in tuber peel, whereas in bud tissues featured an increase, followed by a decrease occurring simultaneously with the suppression of sprouting. The treatment of tubers with iodine strongly affected the expression of polyphenol oxidases at the transcriptional level. Polyphenol oxidase activity in buds poorly reflected the corresponding level of transcription; similarly, little differences were found among the enzyme isoforms expressed in buds as a function of length of exposure to iodine. These findings suggest that the induction of polyphenol oxidases mRNAs transcription could probe the inhibition of sprouting by iodine.

  9. NADPH oxidases are critical targets for prevention of ethanol-induced bone loss

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanisms through which chronic alcohol consumption induce bone loss and osteoporosis are largely unknown. Ethanol increases expression and activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase enzymes (Nox) in osteoblasts leading to accumulation of reactive oxygen spe...

  10. Electron-microscopic cytochemical localization of diamine and polyamine oxidases in pea and maize tissues

    NASA Technical Reports Server (NTRS)

    Slocum, R. D.; Furey MJ, 3. d.

    1991-01-01

    An electron-microscopic cytochemical method was used to localize diamine oxidase (DAO) in pea and polyamine oxidase (PAO) in maize (Zea mays L.). The method, based on the precipitation of amine-oxidase-generated H2O2 by CeCl3, was shown to be specific for DAO and PAO and permitted their localization in plant tissues with a high degree of resolution. Both enzymes are localized exclusively in the cell wall. Both DAO- and PAO-activity staining is most intense in the middle lamellar region of the wall and in cells exhibiting highly lignified walls. The oxidases could provide H2O2 for peroxidase-mediated cross-linking reactions in the cell wall and may, in this capacity, play a role in the regulation of plant growth.

  11. Isolation of a gene encoding a glycosylated cytokinin oxidase from maize.

    PubMed

    Morris, R O; Bilyeu, K D; Laskey, J G; Cheikh, N N

    1999-02-16

    The major cytokinin oxidase in immature maize kernels was purified to homogeneity. Selected tryptic peptides were used to design degenerate oligonucleotide primers for PCR isolation of a fragment of the oxidase gene. Hybridization of the PCR fragment to a maize genomic library allowed isolation of a full-length cytokinin oxidase gene (ckx1). The gene encodes a protein of approximately 57 kDa that possesses a signal peptide, eight consensus N-glycosylation sequences and a consensus FAD binding sequence. Expression of ckx1 in Pichia caused secretion of active glycosylated cytokinin oxidase that contains a substrate-reducible FAD. The gene displays sequence homology with a putative oxidoreductase from Arabidopsis thaliana and with the fas5 gene from Rhodococcus fascians.

  12. NADH oxidase and alkyl hydroperoxide reductase subunit C (peroxiredoxin) from Amphibacillus xylanus form an oligomeric assembly.

    PubMed

    Arai, Toshiaki; Kimata, Shinya; Mochizuki, Daichi; Hara, Keita; Zako, Tamotsu; Odaka, Masafumi; Yohda, Masafumi; Arisaka, Fumio; Kanamaru, Shuji; Matsumoto, Takashi; Yajima, Shunsuke; Sato, Junichi; Kawasaki, Shinji; Niimura, Youichi

    2015-01-01

    The NADH oxidase-peroxiredoxin (Prx) system of Amphibacillus xylanus reduces hydroperoxides with the highest turnover rate among the known hydroperoxide-scavenging enzymes. The high electron transfer rate suggests that there exists close interaction between NADH oxidase and Prx. Variant enzyme experiments indicated that the electrons from β-NADH passed through the secondary disulfide, Cys128-Cys131, of NADH oxidase to finally reduce Prx. We previously reported that ionic strength is essential for a system to reduce hydroperoxides. In this study, we analyzed the effects of ammonium sulfate (AS) on the interaction between NADH oxidase and Prx by surface plasmon resonance analysis. The interaction between NADH oxidase and Prx was observed in the presence of AS. Dynamic light scattering assays were conducted while altering the concentration of AS and the ratio of NADH oxidase to Prx in the solutions. The results revealed that the two proteins formed a large oligomeric assembly, the size of which depended on the ionic strength of AS. The molecular mass of the assembly converged at approximately 300 kDa above 240 mM AS. The observed reduction rate of hydrogen peroxide also converged at the same concentration of AS, indicating that a complex formation is required for activation of the enzyme system. That the complex generation is dependent on ionic strength was confirmed by ultracentrifugal analysis, which resulted in a signal peak derived from a complex of NADH oxidase and Prx (300 mM AS, NADH oxidase: Prx = 1:10). The complex formation under this condition was also confirmed structurally by small-angle X-ray scattering.

  13. Role of Polyamine Oxidase (PAOh1/SMO) in Human Breast Cancer

    DTIC Science & Technology

    2006-04-01

    Huang Y, Hacker A, Woster PM, Casero RA, and Davidson NE. Proc AACR. Page 1224, 2004 Publications: Spermine oxidase SMO(PAOh1), not N1...Hacker A, Zhang Z, Woster PM, Davidson NE, and Casero RA Jr. J Biol Chem, 280: 39843-41, 2005. Page 8 Presentations: The role of spermine oxidase...perspective of polyamine metabolism. Biochem J. 376: 1-14, 2003. 2. Huang Y, Pledgie A, Casero RA, and Davidson NE. Molecular mechanisms of polyamine

  14. Enzymatic characterization and in vivo function of five terminal oxidases in Pseudomonas aeruginosa.

    PubMed

    Arai, Hiroyuki; Kawakami, Takuro; Osamura, Tatsuya; Hirai, Takehiro; Sakai, Yoshiaki; Ishii, Masaharu

    2014-12-01

    The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo(3)-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa3-type cytochrome c oxidase (aa3), and two cbb(3)-type cytochrome c oxidases (cbb(3)-1and cbb(3)-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 strains of P. aeruginosa that express only one aerobic terminal oxidase to investigate the enzymatic characteristics and in vivo function of each enzyme. The Km values of Cyo, CIO, and aa3 for oxygen were similar and were 1 order of magnitude higher than those of cbb(3)-1 and cbb(3)-2, indicating that Cyo, CIO, and aa3 are low-affinity enzymes and that cbb(3)-1 and cbb(3)-2 are high-affinity enzymes. Although cbb(3)-1 and cbb(3)-2 exhibited different expression patterns in response to oxygen concentration, they had similar Km values for oxygen. Both cbb(3)-1 and cbb(3)-2 utilized cytochrome c4 as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb(3)-1 and cbb(3)-2 generate a proton gradient across the cell membrane with similar efficiencies. The electron transport chain of aa3 had the highest proton translocation efficiency, whereas that of CIO had the lowest efficiency. The enzymatic properties of the terminal oxidases reported here are partially in agreement with their regulatory patterns and may explain the environmental adaptability and versatility of P. aeruginosa.

  15. Low Cytochrome Oxidase 1 Links Mitochondrial Dysfunction to Atherosclerosis in Mice and Pigs

    PubMed Central

    Vanhaverbeke, Maarten; Geeraert, Benjamine; De Keyzer, Dieuwke; Hulsmans, Maarten; Janssens, Stefan

    2017-01-01

    Background Cytochrome oxidase IV complex regulates energy production in mitochondria. Therefore, we determined the relation of COX genes with atherosclerosis in mice and pigs. Methods and results First, we compared atherosclerosis in the aortic arch of age-matched (24 weeks) C57BL/6J control (n = 10), LDL-receptor deficient (n = 8), leptin-deficient ob/ob (n = 10), and double knock-out (lacking LDL-receptor and leptin) mice (n = 12). Low aortic mitochondria-encoded cytochrome oxidase 1 in obese diabetic double knock-out mice was associated with a larger plaque area and higher propensity of M1 macrophages and oxidized LDL. Caloric restriction increased mitochondria-encoded cytochrome oxidase 1 and reduced plaque area and oxidized LDL. This was associated with a reduction of titer of anti-oxidized LDL antibodies, a proxy of systemic oxidative stress. Low of mitochondria-encoded cytochrome oxidase 1 was related to low expression of peroxisome proliferative activated receptors α, δ, and γ and of peroxisome proliferative activated receptor, gamma, co-activator 1 alpha reflecting mitochondrial dysfunction. Caloric restriction increased them. To investigate if there was a diabetic/obesity requirement for mitochondria-encoded cytochrome oxidase 1 to be down-regulated, we then studied atherosclerosis in LAD of hypercholesterolemic pigs (n = 37). Pigs at the end of the study were divided in three groups based on increasing LAD plaque complexity according to Stary (Stary I: n = 12; Stary II: n = 13; Stary III: n = 12). Low mitochondria-encoded cytochrome oxidase 1 in isolated plaque macrophages was associated with more complex coronary plaques and oxidized LDL. Nucleus-encoded cytochrome oxidase 4I1 and cytochrome oxidase 10 did not correlate with plaque complexity and oxidative stress. In mice and pigs, MT-COI was inversely related to insulin resistance. Conclusions Low MT-COI is related to mitochondrial dysfunction, oxidative stress and atherosclerosis and plaque

  16. Catalase activity of cytochrome C oxidase assayed with hydrogen peroxide-sensitive electrode microsensor.

    PubMed

    Bolshakov, I A; Vygodina, T V; Gennis, R; Karyakin, A A; Konstantinov, A A

    2010-11-01

    An iron-hexacyanide-covered microelectrode sensor has been used to continuously monitor the kinetics of hydrogen peroxide decomposition catalyzed by oxidized cytochrome oxidase. At cytochrome oxidase concentration ~1 µM, the catalase activity behaves as a first order process with respect to peroxide at concentrations up to ~300-400 µM and is fully blocked by heat inactivation of the enzyme. The catalase (or, rather, pseudocatalase) activity of bovine cytochrome oxidase is characterized by a second order rate constant of ~2·10(2) M(-1)·sec(-1) at pH 7.0 and room temperature, which, when divided by the number of H2O2 molecules disappearing in one catalytic turnover (between 2 and 3), agrees reasonably well with the second order rate constant for H2O2-dependent conversion of the oxidase intermediate F(I)-607 to F(II)-580. Accordingly, the catalase activity of bovine oxidase may be explained by H2O2 procession in the oxygen-reducing center of the enzyme yielding superoxide radicals. Much higher specific rates of H2O2 decomposition are observed with preparations of the bacterial cytochrome c oxidase from Rhodobacter sphaeroides. The observed second order rate constants (up to ~3000 M(-1)·sec(-1)) exceed the rate constant of peroxide binding with the oxygen-reducing center of the oxidized enzyme (~500 M(-1)·sec(-1)) several-fold and therefore cannot be explained by catalytic reaction in the a(3)/Cu(B) site of the enzyme. It is proposed that in the bacterial oxidase, H2O2 can be decomposed by reacting with the adventitious transition metal ions bound by the polyhistidine-tag present in the enzyme, or by virtue of reaction with the tightly-bound Mn2+, which in the bacterial enzyme substitutes for Mg2+ present in the mitochondrial oxidase.

  17. A comparative study on the archives of xanthine oxidase and aldehyde oxidase in different fish species from two rivers in the Western Niger-Delta.

    PubMed

    Isamah, Gabriel K; Asagba, Samuel O

    2004-02-01

    Glycaemia, a classical indicator of stress, xanthine oxidase and aldehyde oxidase which are involved in phase I detoxication were investigated in two different fish species from two rivers with different pollution levels in the Western Niger-Delta. Four sampling zones covering the entire lengths of Warri and Ethiope Rivers respectively were used in this study. For each species of fish five were obtained from a sampling zone in a river. Blood glucose was significantly higher (P < 0.001) in M. electricus from Warri River (82.13 +/- 5.50 mg cm(-3)) compared to the same species from Ethiope River (36.47 +/- 1.49 mg cm(-3)). With the same parameter a similar profile was observed for C. gariepinus; Warri River (56.92 +/- 10.31 mg cm(-3)); Ethiope River (37.65 +/- 0.90 mg cm(-3)) which was also significant (P < 0.01). The activity of xanthine oxidase in M. electricus from Warri River (255.80 +/- 41 it mol cm(-3)) was significantly higher (P < 0.001) compared to the value obtained for the same species (108 +/- 22.36 micro mol cm(-3)) from Ethiope River. Also the activity of xanthine oxidase in C. gariepinus from Warri River (197 +/- 34.65 micro mol cm(-3)) was significantly higher (P < 0.001) when matched with the value obtained for the same species (78.40 +/- 26.84 micro mol cm(-3)) from Ethiope River. That blood glucose level was related to xanthine oxidase activity in the two fish species from Warri River was supported by the high positive correlation between these two parameters (M. electricus. r = 1: C. gariepinus, r = 0.71). The activity of aldehyde oxidase in C. gariepinus from Warri River (143.80 +/- 28.45 micro mol cm(-3)) was significantly higher (P < 0.001) compared to the value obtained for the same species (61.20 +/- 15.21 micro mol cm(-3)) from Ethiope River. A similar profile in aldehyde oxidase activity observed for M. electricus; Warri River (130 +/- 28.39 micro mol cm(-3)); Ethiope River (89 +/- 19.70 micro mol cm(-3)) but an inferior statistical

  18. Correlation between electrical properties and thermodynamic stability of ACoO{sub 3-{delta}} perovskites (A= La, Pr, Nd, Sm, Gd)

    SciTech Connect

    Scherrer, Barbara; Harvey, Ashley S.; Martynczuk, Julia; Gauckler, Ludwig J.; Tanasescu, Speranta; Teodorescu, Florina; Botea, Alina; Conder, Kazimierz; Grundy, A. Nicholas

    2011-08-15

    For perovskites with the general formula ACoO{sub 3-{delta}} (A = La, Pr, Nd, Sm, and Gd) the influence of the A-site cation on the electrical conductivity, electronic structure, thermodynamic stability, and oxygen stoichiometry was studied. The perovskite oxide powders were produced by a combined citric acid and ethylenediaminetetraacetic acid complexing method. Ceramic specimens sintered at 1100 deg. C in air were single-phase perovskites. With increasing temperature, the electrical conductivity shows three discrete regimes. All compositions show semiconductivity up to a transition temperature of {approx}300 deg. C-450 deg. C and then behave like metallic conductors. The activation energies for the semiconductivity, as well as the transition temperatures to the metallic-like conduction, decrease monotonically with increasing pseudocubic lattice parameters, i.e., with increasing ionic radii of the A cation. This behavior correlates with decreasing oxygen nonstoichiometry and increased thermodynamic stability. The highest conductivity and the lowest activation energy of 0.66 eV were found for LaCoO{sub 3-{delta},} which also had the lowest semiconductor-metal transition temperature at 269 deg. C, the lowest oxygen nonstoichiometry of {delta}= 0.008, and the highest Gibbs free energy change for the decomposition reaction of 42.37 kJ/mol at 850 deg. C. GdCoO{sub 3-{delta}} had the highest oxygen nonstoichiometry with {delta}0.032, a high activation energy of 1.19 eV for the semiconductivity with a high transition temperature at 452 deg. C, and the lowest Gibbs free energy change of 26.54 kJ/mol at 850 deg. C. X-ray absorption spectroscopy data imply an increasing Co low-spin character with decreasing cation radius from La to Gd, while an increase in temperature increases the number of holes or Co 3d bandwidth. This correlates well with the electrical conductivity data.

  19. Impact of admission blood glucose on outcomes of nondiabetic patients with acute ST-elevation myocardial infarction (from the German Acute Coronary Syndromes [ACOS] Registry).

    PubMed

    Naber, Christoph K; Mehta, Rajendra H; Jünger, Claus; Zeymer, Uwe; Wienbergen, Harm; Sabin, Georg V; Erbel, Raimund; Senges, Jochen; Gitt, Anselm

    2009-03-01

    High blood glucose in patients with acute coronary syndromes have been associated with adverse short-term outcomes in patients without diabetes. However, the relation of admission glucose to long-term outcomes in these patients was less well established. Accordingly, consecutive patients with ST-elevation myocardial infarction (STEMI) without diabetes enrolled at 155 sites from July 2000 to November 2002 in the ACOS Registry were evaluated. Patients were categorized into tertiles based on admission blood glucose. Clinical end points of interest were 1-year mortality and composite of death, reinfarction, stroke, or rehospitalization (major adverse cardiac clinical events [MACCEs]) in the hospital and after discharge. Of 5,866 patients with STEMI, 36.9% had blood glucose <120 mg/dl; 33.1%, 120 to 150 mg/dl; and 30.0%, >150 mg/dl. Admission blood glucose was significantly related to increased risk of not only in-hospital events (death, glucose >150 vs <120 mg/dl, adjusted odds ratio [OR] 2.86, 95% confidence interval [CI] 2.13 to 3.82, p <0.0001; and MACCE, >150 vs <120 mg/dl, adjusted OR 1.88, 95% CI 1.52 to 2.33; p <0.0001), but this increased risk persisted beyond the acute phase during 1-year follow-up of a mean 380 days (median 387; death, glucose >150 vs <120 mg/dl, adjusted OR 1.46, 95% CI 1.04 to 2.03, p <0.0001; and MACCE, >150 vs <120 mg/dl, adjusted OR 1.31, 95% CI 1.00 to 1.71, p <0.0001). In conclusion, high blood glucose at admission to the hospital independently correlated with short- and midterm mortality in patients with STEMI.

  20. Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade.

    PubMed

    Kalachova, Tetiana; Iakovenko, Oksana; Kretinin, Sergii; Kravets, Volodymyr

    2013-05-01

    Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway.

  1. Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern 1

    PubMed Central

    Chatfield, J. Mark; Armstrong, Donald J.

    1986-01-01

    The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N6-(Δ2-isopentenyl)adenine-8-14C (i6 Ade-8-14C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i6 Ade-8-14C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded. PMID:16664650

  2. Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization.

    PubMed

    Doukyu, Noriyuki; Nihei, Shyou

    2015-07-01

    An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6β-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 °C and 30 °C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 °C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 °C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 °C. The enzyme oxidized 3β-hydroxysteroids such as cholesterol, β-cholestanol, and β-sitosterol at high rates. The Km value and Vmax value for the cholesterol were 92.6 μM and 15.9 μmol/min/mg of protein, respectively. The Vmax value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa.

  3. Ferricytochrome c protects mitochondrial cytochrome c oxidase against hydrogen peroxide-induced oxidative damage.

    PubMed

    Sedlák, Erik; Fabian, Marian; Robinson, Neal C; Musatov, Andrej

    2010-11-30

    An excess of ferricytochrome c protects purified mitochondrial cytochrome c oxidase and bound cardiolipin from hydrogen peroxide-induced oxidative modification. All of the peroxide-induced changes within cytochrome c oxidase, such as oxidation of Trp(19,IV) and Trp(48,VIIc), partial dissociation of subunits VIa and VIIa, and generation of cardiolipin hydroperoxide, no longer take place in the presence of ferricytochrome c. Furthermore, ferricytochrome c suppresses the yield of H(2)O(2)-induced free radical detectable by electron paramagnetic resonance spectroscopy within cytochrome c oxidase. These protective effects are based on two mechanisms. The first involves the peroxidase/catalase-like activity of ferricytochrome c, which results in the decomposition of H(2)O(2), with the apparent bimolecular rate constant of 5.1±1.0M(-1)s(-1). Although this value is lower than the rate constant of a specialized peroxidase, the activity is sufficient to eliminate H(2)O(2)-induced damage to cytochrome c oxidase in the presence of an excess of ferricytochrome c. The second mechanism involves ferricytochrome c-induced quenching of free radicals generated within cytochrome c oxidase. These results suggest that ferricytochrome c may have an important role in protection of cytochrome c oxidase and consequently the mitochondrion against oxidative damage.

  4. Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions.

    PubMed

    Unitt, David C; Hollis, Veronica S; Palacios-Callender, Miriam; Frakich, Nanci; Moncada, Salvador

    2010-03-01

    We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O(2)) conditions. The system measures the concentrations of O(2) and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O(2) concentration and electron turnover of the enzyme. At a high O(2) concentration (70 microM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O(2). At low O(2) (15 microM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O(2) consumption. At both high and low O(2) concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.

  5. Early Effects of Boron Deficiency on Indoleacetic Acid Oxidase Levels of Squash Root Tips

    PubMed Central

    Bohnsack, Charles W.; Albert, Luke S.

    1977-01-01

    The indoleacetic acid (IAA) oxidase activity of root tips of boron-sufficient, -deficient, recovering, and IAA-treated boron-sufficient squash plants (Cucurbita pepo L.) was determined. Apical and subapical root sections displayed an increase in IAA oxidase activity between 6 and 9 hours after boron was withheld, and after 24 hours the activity of the apical sections showed a 20-fold increase over +B controls. Root elongation of -B plants was inhibited before an increase in oxidase activity could be detected. Roots of plants subjected to 12 hours of -B treatment and then transferred to +B treatment for recovery regained normal elongation rates and oxidase activity within 18 to 20 hours. IAA treatment of +B plants increased IAA oxidase activity of apical and subapical root sections and also inhibited root elongation and caused symptoms similar to -B treatments. These results have demonstrated the earliest enzymic change for intact boron-deficient plants. The results are in agreement with the theory that boron deficiency symptoms may be the result of supraoptimal endogenous levels of IAA. These high levels of IAA may inhibit cell division and lead to an induction of the IAA oxidase enzyme. PMID:16659990

  6. NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

    PubMed Central

    Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

    2013-01-01

    Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

  7. Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products.

    PubMed

    Le Bourvellec, Carine; Le Quéré, Jean-Michel; Sanoner, Philippe; Drilleau, Jean-François; Guyot, Sylvain

    2004-01-14

    The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.

  8. The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

    NASA Astrophysics Data System (ADS)

    DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.

    2003-04-01

    The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

  9. Hyper-responsive Toll-like receptor 7 and 9 activation in NADPH oxidase-deficient B lymphoblasts.

    PubMed

    McLetchie, Shawna; Volpp, Bryan D; Dinauer, Mary C; Blum, Janice S

    2015-12-01

    Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40(phox) or p47(phox) subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients.

  10. 3-Coumaranone derivatives as inhibitors of monoamine oxidase

    PubMed Central

    Van Dyk, Adriaan S; Petzer, Jacobus P; Petzer, Anél; Legoabe, Lesetja J

    2015-01-01

    The present study examines the monoamine oxidase (MAO) inhibitory properties of a series of 20 3-coumaranone [benzofuran-3(2H)-one] derivatives. The 3-coumaranone derivatives are structurally related to series of α-tetralone and 1-indanone derivatives, which have recently been shown to potently inhibit MAO, with selectivity for MAO-B (in preference to the MAO-A isoform). 3-Coumaranones are similarly found to selectively inhibit human MAO-B with half-maximal inhibitory concentration (IC50) values of 0.004–1.05 µM. Nine compounds exhibited IC50<0.05 µM for the inhibition of MAO-B. For the inhibition of human MAO-A, IC50 values ranged from 0.586 to >100 µM, with only one compound possessing an IC50<1 µM. For selected 3-coumaranone derivatives, it is established that MAO-A and MAO-B inhibition are reversible since dialysis of enzyme–inhibitor mixtures almost completely restores enzyme activity. On the basis of the selectivity profiles and potent action, it may be concluded that the 3-coumaranone derivatives are suitable leads for the development of selective MAO-B inhibitors as potential treatment for disorders such as Parkinson’s disease and Alzheimer’s disease. PMID:26491258

  11. Purification and characterization of polyphenol oxidase from corn tassel.

    PubMed

    Gul Guven, R; Aslan, N; Guven, K; Matpan Bekler, F; Acer, O

    2016-11-30

    In this study, polyphenol oxidase (PPO) from corn tassel  was extracted and partially purified through  (NH4)2SO4 precipitation and gel filtration chromatography. Optimal temperatures for subsrates catechol and 4-methyl catechol were 40 °C and 30 °C, respectively. The optimal pH values were 8.0 for catechol and 6.0 for 4-methyl catechol. Catechol was the most suitible substrate (Km: 3.48 mM, Vmax: 1.0 Abs./ min.). The moleculer mass of PPO was determined as 158 kDa. In this work, sodium azide, ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) were found to inhibit the enzyme activity as 26.6 %,  22.2 % and 12.2 % ratio, respectively. Besides, the effects of carbohydrates such as sucrose, fructose, ribose and glucose on PPO activity were investigated. The enzyme was found to be activated 17 % by fructose and ribose, 16 % by glucose and 4 % by sucrose.

  12. Substrate inhibition competes with halide inhibition in polyphenol oxidase.

    PubMed

    Lim, Giselle Grace Fernando; Imura, Yuki; Yoshimura, Etsuro

    2012-10-01

    Polyphenol oxidase (PPO) is a ubiquitous enzyme important in the food industry. Although PPO activity followed Michaelis-Menten kinetics at catechol concentrations of up to 1 mM, it slowly decreased at catechol concentrations above 2 mM. This result indicated that in addition to the active site (site A), the enzyme possesses a second catechol-binding site (site B) that exerts an inhibitory effect on PPO activity. Halides inhibit PPO activity in such a way that substrate inhibition is lessened when halide concentration is increased. Furthermore, elevated concentrations of catechol diminished the degree of inhibition by halides. These findings suggest that halides also bind to site B to inhibit PPO activity. A steady-state kinetic analysis demonstrated that the dissociation constant between catechol and PPO depended on the binding of halides to site B. The dissociation constants were greatest when chloride bound to the site. Bromide and iodide yielded lower dissociation constants, in that order. These data indicate that the binding of halide to site B modulated the structure of site A, thereby exerting an inhibitory effect.

  13. Maladaptive defensive behaviours in monoamine oxidase A-deficient mice

    PubMed Central

    Godar, Sean C.; Bortolato, Marco; Frau, Roberto; Dousti, Mona; Chen, Kevin; Shih, Jean C.

    2012-01-01

    Rich evidence indicates that monoamine oxidase (MAO) A, the major enzyme catalysing the degradation of monoamine neurotransmitters, plays a key role in emotional regulation. Although MAOA deficiency is associated with reactive aggression in humans and mice, the involvement of this enzyme in defensive behaviour remains controversial and poorly understood. To address this issue, we tested MAOA knockout (KO) mice in a spectrum of paradigms and settings associated with variable degrees of threat. The presentation of novel inanimate objects induced a significant reduction in exploratory approaches and increase in defensive behaviours, such as tail-rattling, biting and digging. These neophobic responses were context-dependent and particularly marked in the home cage. In the elevated plus- and T-mazes, MAOA KO mice and wild-type (WT) littermates displayed equivalent locomotor activity and time in closed and open arms; however, MAOA KO mice featured significant reductions in risk assessment, as well as unconditioned avoidance and escape. No differences between genotypes were observed in the defensive withdrawal and emergence test. Conversely, MAOA KO mice exhibited a dramatic reduction of defensive and fear-related behaviours in the presence of predator-related cues, such as predator urine or an anaesthetized rat, in comparison with those observed in their WT littermates. The behavioural abnormalities in MAOA KO mice were not paralleled by overt alterations in sensory and microvibrissal functions. Collectively, these results suggest that MAOA deficiency leads to a general inability to appropriately assess contextual risk and attune defensive and emotional responses to environmental cues. PMID:21156093

  14. Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.).

    PubMed

    Siddiq, M; Dolan, K D

    2017-03-01

    Polyphenol oxidase (PPO) was extracted and characterized from high-bush blueberries. PPO showed an optimum activity at pH 6.1-6.3 and 35°C, with the enzyme showing significant activity over a wide temperature range (25-60°C). Catechol was the most readily oxidized substrate followed by 4-methylcatechol, DL-DOPA, and dopamine. Blueberry PPO showed a Km of 15mM and Vmax of 2.57 ΔA420nm/min×10(-1), determined with catechol. PPO was completely inactivated in 20min at 85°C, however, after 30minat 75°C it showed about 10% residual activity. Thermal treatment at 55 and 65°C for 30min resulted in the partial inactivation of PPO. Ascorbic acid, sodium diethyldithiocarbamic acid, L-cysteine, and sodium metabisulfite were effective inhibitors of PPO at 1.0mM. Benzoic acid and cinnamic acid series inhibitors showed relatively weak inhibition of PPO (21.8-27.6%), even at as high as 2.0mM concentration.

  15. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%.

  16. Lysyl Oxidase, a Targetable Secreted Molecule Involved in Cancer Metastasis.

    PubMed

    Cox, Thomas R; Gartland, Alison; Erler, Janine T

    2016-01-15

    Secondary metastatic cancer remains the single biggest cause of mortality and morbidity across most solid tumors. In breast cancer, 100% of deaths are attributed to metastasis. At present, there are no "cures" for secondary metastatic cancer of any form and there is an urgent unmet clinical need to improve the tools available in our arsenal against this disease, both in terms of treatment, but also prevention. Recently, we showed that hypoxic induction of the extracellular matrix modifying enzyme lysyl oxidase (LOX) correlates with metastatic dissemination to the bone in estrogen receptor negative breast cancer and is essential for the formation of premetastatic osteolytic lesions. We showed that in models of breast cancer metastasis, targeting LOX, or its downstream effects, significantly inhibited premetastatic niche formation and the resulting metastatic burden, offering preclinical validation of this enzyme as a therapeutic target for metastatic breast cancer. Our work is the latest in an emerging body of work supporting the targeting of LOX and calls for greater efforts in developing therapeutics against this extracellular secreted factor in the prevention of cancer progression across multiple solid tumor types.

  17. Lysyl oxidase is essential for hypoxia-induced metastasis.

    PubMed

    Erler, Janine T; Bennewith, Kevin L; Nicolau, Monica; Dornhöfer, Nadja; Kong, Christina; Le, Quynh-Thu; Chi, Jen-Tsan Ashley; Jeffrey, Stefanie S; Giaccia, Amato J

    2006-04-27

    Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell-cell or cell-matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.

  18. Structural Characterization of Glucooligosaccharide Oxidase from Acremonium strictum

    PubMed Central

    Lee, Meng-Hwan; Lai, Wen-Lin; Lin, Shuen-Fuh; Hsu, Cheng-Sheng; Liaw, Shwu-Huey; Tsai, Ying-Chieh

    2005-01-01

    Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the Km. Instead, the variants displayed kcat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin. PMID:16332885

  19. Hypoxia inhibits semicarbazide-sensitive amine oxidase activity in adipocytes.

    PubMed

    Repessé, Xavier; Moldes, Marthe; Muscat, Adeline; Vatier, Camille; Chetrite, Gérard; Gille, Thomas; Planes, Carole; Filip, Anna; Mercier, Nathalie; Duranteau, Jacques; Fève, Bruno

    2015-08-15

    Semicarbazide-sensitive amine oxidase (SSAO), an enzyme highly expressed on adipocyte plasma membranes, converts primary amines into aldehydes, ammonium and hydrogen peroxide, and is likely involved in endothelial damage during the course of diabetes and obesity. We investigated whether in vitro, adipocyte SSAO was modulated under hypoxic conditions that is present in adipose tissue from obese or intensive care unit. Physical or pharmacological hypoxia decreased SSAO activity in murine adipocytes and human adipose tissue explants, while enzyme expression was preserved. This effect was time-, dose-dependent and reversible. This down-regulation was confirmed in vivo in subcutaneous adipose tissue from a rat model of hypoxia. Hypoxia-induced suppression in SSAO activity was independent of the HIF-1-α pathway or of oxidative stress, but was partially antagonized by medium acidification. Hypoxia-induced down-regulation of SSAO activity could represent an adaptive mechanism to lower toxic molecules production, and may thus protect from tissue injury during these harmful conditions.

  20. A codon deletion confers resistance to herbicides inhibiting protoporphyrinogen oxidase

    PubMed Central

    Patzoldt, William L.; Hager, Aaron G.; McCormick, Joel S.; Tranel, Patrick J.

    2006-01-01

    Herbicides that act by inhibiting protoporphyrinogen oxidase (PPO) are widely used to control weeds in a variety of crops. The first weed to evolve resistance to PPO-inhibiting herbicides was Amaranthus tuberculatus, a problematic weed in the midwestern United States that previously had evolved multiple resistances to herbicides inhibiting two other target sites. Evaluation of a PPO-inhibitor-resistant A. tuberculatus biotype revealed that resistance was a (incompletely) dominant trait conferred by a single, nuclear gene. Three genes predicted to encode PPO were identified in A. tuberculatus. One gene from the resistant biotype, designated PPX2L, contained a codon deletion that was shown to confer resistance by complementation of a hemG mutant strain of Escherichia coli grown in the presence and absence of the PPO inhibitor lactofen. PPX2L is predicted to encode both plastid- and mitochondria-targeted PPO isoforms, allowing a mutation in a single gene to confer resistance to two herbicide target sites. Unique aspects of the resistance mechanism include an amino acid deletion, rather than a substitution, and the dual-targeting nature of the gene, which may explain why resistance to PPO inhibitors has been rare. PMID:16894159

  1. A codon deletion confers resistance to herbicides inhibiting protoporphyrinogen oxidase.

    PubMed

    Patzoldt, William L; Hager, Aaron G; McCormick, Joel S; Tranel, Patrick J

    2006-08-15

    Herbicides that act by inhibiting protoporphyrinogen oxidase (PPO) are widely used to control weeds in a variety of crops. The first weed to evolve resistance to PPO-inhibiting herbicides was Amaranthus tuberculatus, a problematic weed in the midwestern United States that previously had evolved multiple resistances to herbicides inhibiting two other target sites. Evaluation of a PPO-inhibitor-resistant A. tuberculatus biotype revealed that resistance was a (incompletely) dominant trait conferred by a single, nuclear gene. Three genes predicted to encode PPO were identified in A. tuberculatus. One gene from the resistant biotype, designated PPX2L, contained a codon deletion that was shown to confer resistance by complementation of a hemG mutant strain of Escherichia coli grown in the presence and absence of the PPO inhibitor lactofen. PPX2L is predicted to encode both plastid- and mitochondria-targeted PPO isoforms, allowing a mutation in a single gene to confer resistance to two herbicide target sites. Unique aspects of the resistance mechanism include an amino acid deletion, rather than a substitution, and the dual-targeting nature of the gene, which may explain why resistance to PPO inhibitors has been rare.

  2. Reye's syndrome: salicylate and mitochondrial monoamine oxidase function

    SciTech Connect

    Faraj, B.A.; Caplan, D.; Lolies, P.

    1986-03-01

    It has been suggested that aspirin is somehow linked with the onset of Reye's syndrome (RS). A general feature of Reye's syndrome is severe impairment of mitochondrial monoamine oxidase (MAO) function. The main objective of this investigation was to study the effect of salicylate on platelet mitochondrial MAO activity in three groups: group A (healthy children, n = 21) and group C (healthy adults, n = 10). Platelet MAO was measured by radio-enzymatic technique with /sup 14/C-tyramine as a substrate. The results showed that salicyclate (10 mM) had a 20 to 60 percent inhibitory effect on platelet MAO function in only 1, 3 and 2 of the subjects in group A, B and C. Furthermore, there was an association between low enzyme activity and salicylate MAO inhibitory effect in these subjects. These preliminary findings suggest that salicylate may induce deterioration in mitochondrial function in susceptible individuals and that the assessment of salicylate MAO inhibitory effect may identify those who may be at risk to develop aspirin poisoning and Reye's syndrome.

  3. BEHAVIORAL OUTCOMES OF MONOAMINE OXIDASE DEFICIENCY: PRECLINICAL AND CLINICAL EVIDENCE

    PubMed Central

    Bortolato, Marco; Shih, Jean C.

    2012-01-01

    Monoamine oxidase (MAO) isoenzymes A and B are mitochondrial-bound proteins, catalyzing the oxidative deamination of monoamine neurotransmitters as well as xenobiotic amines. Although they derive from a common ancestral progenitor gene, are located at X-chromosome and display 70% structural identity, their substrate preference, regional distribution, and physiological role are divergent. In fact, while MAO-A has high affinity for serotonin and norepinephrine, MAO-B primarily serves the catabolism of 2-phenylethylamine (PEA) and contributes to the degradation of other trace amines and dopamine. Convergent lines of preclinical and clinical evidence indicate that variations in MAO enzymatic activity—due to either genetic or environmental factors—can exert a profound influence on behavioral regulation and play a role in the pathophysiology of a large spectrum of mental and neurodegenerative disorders, ranging from antisocial personality disorder to Parkinson’s disease. Over the past few years, numerous advances have been made in our understanding of the phenotypical variations associated with genetic polymorphisms and mutations of the genes encoding for both isoenzymes. In particular, novel findings on the phenotypes of MAO-deficient mice are highlighting novel potential implications of both isoenzymes in a broad spectrum of mental disorders, ranging from autism and anxiety to impulse-control disorders and ADHD. These studies will lay the foundation for future research on the neurobiological and neurochemical bases of these pathological conditions, as well as the role of gene × environment interactions in the vulnerability to several mental disorders. PMID:21971001

  4. Electrical communication between glucose oxidase and different ferrocenylalkanethiol chain lengths

    SciTech Connect

    Rubin, S.; Bar, G.; Cutts, R.W.; Zawodzinski, T.A. Jr.; Chow, J.T.; Ferraris, J.P.

    1995-12-31

    We describe the factors affecting the electron transfer process between the different components of a self-assembled mixed monolayer. The system is comprised of mixed monolayers containing aminoalkanethiols (AMATs) and ferrocenylakanethiols (FATs) of variable chain lengths. We study the effects of different ratio of the two mixed monolayer components on the permeability of the monolayer toward a Ru(NH{sub 3}{sub 6}Cl{sub 3} redox probe. In order to study the electrical communication between the enzyme and the mediator molecules, the enzyme glucose oxidase (GOx) was attached to the AMAT sites to create a biosensor device. The relative efficiency of a biosensor of each chain-length combination of FAT and AMAT was examined. In light of this comparison, we consider the critical factors for efficient electron transfer between the ferrocene mediator and the GOx redox active site immobilized as part of the surface-confined system. We find that the biosensor response is greatest when the enzyme and the FATs are attached to the surface with different alkane chain lengths. We also find strong evidence for the existence of domains of FAT and AMAT in the mixed monolayer system.

  5. The Pea Gene LH Encodes ent-Kaurene Oxidase1

    PubMed Central

    Davidson, Sandra E.; Smith, Jennifer J.; Helliwell, Chris A.; Poole, Andrew T.; Reid, James B.

    2004-01-01

    The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants. PMID:14988475

  6. Lysyl oxidase is associated with increased thrombosis and platelet reactivity.

    PubMed

    Matsuura, Shinobu; Mi, Rongjuan; Koupenova, Milka; Eliades, Alexia; Patterson, Shenia; Toselli, Paul; Thon, Jonathan; Italiano, Joseph E; Trackman, Philip C; Papadantonakis, Nikolaos; Ravid, Katya

    2016-03-17

    Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Lox(tg/tg)) were generated. Pf4-Lox(tg/tg) mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox(tg/tg) mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Lox(tg/tg) platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox(tg/tg) platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2β1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.

  7. 2-acetylphenol analogs as potent reversible monoamine oxidase inhibitors

    PubMed Central

    Legoabe, Lesetja J; Petzer, Anél; Petzer, Jacobus P

    2015-01-01

    Based on a previous report that substituted 2-acetylphenols may be promising leads for the design of novel monoamine oxidase (MAO) inhibitors, a series of C5-substituted 2-acetylphenol analogs (15) and related compounds (two) were synthesized and evaluated as inhibitors of human MAO-A and MAO-B. Generally, the study compounds exhibited inhibitory activities against both MAO-A and MAO-B, with selectivity for the B isoform. Among the compounds evaluated, seven compounds exhibited IC50 values <0.01 µM for MAO-B inhibition, with the most selective compound being 17,000-fold selective for MAO-B over the MAO-A isoform. Analyses of the structure–activity relationships for MAO inhibition show that substitution on the C5 position of the 2-acetylphenol moiety is a requirement for MAO-B inhibition, and the benzyloxy substituent is particularly favorable in this regard. This study concludes that C5-substituted 2-acetylphenol analogs are potent and selective MAO-B inhibitors, appropriate for the design of therapies for neurodegenerative disorders such as Parkinson’s disease. PMID:26203229

  8. Microwave radiation effects on the thermally driven oxidase of erythrocytes

    SciTech Connect

    Keil, J.L.; Erwin, D.N.

    1986-01-01

    Sheep red blood cells (SRBCs) were labelled with a concanavalin A-luminol-bovine serum albumin conjugate specific for the transmembrane anion transport protein (Band 3) and exposed to 2450-MHz continuous-wave microwave radiation at an average specific absorption rate of 91W/kg for 10 min. The temperature was held constant at 25, 37, 40, 42, or 45C with an airflow heat-exchange system. Following exposure to microwave or air heating, the decrease in residual base-activated chemiluminescence (CL) of the SRBCs was measured as an indication of infield oxidase activity. Air heating resulted in a significant decrease in residual CL at temperatures above 37C (74% decrease at 45C). Microwave radiation inhibited the decline in residual CL above 37C. At 45C the inhibition was 40%. The results suggest microwave radiation either reversibly altered the thermodynamics of oxygen binding to haemoglobin or failed to energize a significant portion of the haemoglobin molecules in each sample to the thermal threshold of haemoglobin autoxidation.

  9. Upregulation of Mitochondrial Content in Cytochrome c Oxidase Deficient Fibroblasts.

    PubMed

    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.

  10. Monomolecular films of cholesterol oxidase and S-Layer proteins

    NASA Astrophysics Data System (ADS)

    Ferraz, Helen Conceição; Guimarães, Juliana Aguilar; Alves, Tito Livio Moitinho; Constantino, Carlos José Leopoldo

    2011-05-01

    Cholesterol oxidase (ChOx) is a flavoenzyme that catalyzes the oxidation of cholesterol to cholest-5-en-3-one and subsequently the isomerization to cholest-4-en-3-one. ChOx has been very commonly studied as the detection element in cholesterol biosensors. In the biosensor development field, a relatively new approach is the use of crystalline bacterial cell surface layers, known as S-Layer proteins. These proteins exhibit the ability of self-assembling at surfaces, opening a vast spectrum of applications, both in basic and applied researches. In our study, monomolecular films of ChOx and mixed films of ChOx/S-Layer proteins and DPPC/S-Layer proteins were produced using the Langmuir technique. Characterization of the films was performed by means of surface pressure-molecular area ( π- A) isotherms. Stable monolayers were obtained, which means that they can be transferred to solid substrates by Langmuir-Blodgett technique. Mixed monolayers showed an ideal like behavior.

  11. Study of umbelliferone hydroxylation to esculetin catalyzed by polyphenol oxidase.

    PubMed

    Garcia-Molina, Mary Of The Sea; Munoz-Munoz, Joseph Louis; Garcia-Molina, Francis; Rodriguez-Lopez, Joseph Neptune; Garcia-Canovas, Francis

    2013-01-01

    We characterize umbelliferone, a derivative of 2,4-dihydroxycoumaric acid, as a substrate of polyphenol oxidase. This enzyme hydroxylates umbelliferone to esculetin, its o-diphenol, and then oxidizes it to o-quinone. The findings show that umbelliferone, an intermediate in one of the coumarin biosynthesis pathways, may be transformed into its o-diphenol, esculetin, which is also an intermediate in the same pathway. The activity of the enzyme on umbelliferone was followed by measuring the consumption of oxygen, spectrophotometrically and by HPLC. Kinetic constants characterizing the hydroxylation process were: kcat=0.09±0.02 s(-1) and Km=0.17±0.06 mM. The o-diphenol, esculetin, was a better substrate and when its oxidation was followed spectrophotometrically, the kinetic constants were: kcat=1.31±0.25 s(-1) and Km=0.035±0.002 mM. Both compounds therefore can be considered as alternative substrates to L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA), since both indirectly inhibit melanogenesis.

  12. Polyphenol oxidases from latex of Hevea brasiliensis: purification and characterization.

    PubMed

    Wititsuwannakul, Dhirayos; Chareonthiphakorn, Nopphakaew; Pace, Mario; Wititsuwannakul, Rapepun

    2002-09-01

    Polyphenol oxidase (PPO) was isolated from the B-serum obtained after repetitive freeze-thawing of the bottom fraction isolated from ultracentrifuged fresh latex. The B-serum was subjected to acetone precipitation and CM-Sepharose chromatography, affording two PPOs, PPO-I and PPO-II, which, upon SDS-PAGE, were 32 and 34 kDa, respectively. Both PPOs possessed the same pI (9.2), optimum pH (7) and optimum temperature (35-45 degrees C). They are stable up to 60 degrees C and active at broad pH ranges from 4-9. The K(m) values of PPO-I for dopamine, L-dopa and catechol as substrates are 2.08, 8.33 and 9.09 mM, while those for PPO-II are 2.12, 4.76 and 7.14 mM, respectively. Among various PPO inhibitors tested, 4-hexylresorcinol was the most potent. Anionic detergents were among the most effective activators of the enzymes, while cationic and nonionic detergents showed little and no effect on the PPO activities, respectively.

  13. Partial purification of latent persimmon fruit polyphenol oxidase.

    PubMed

    Núñez-Delicado, Estrella; Sojo, M Mar; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

    2003-03-26

    Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).

  14. Some biochemical properties of polyphenol oxidase from celery.

    PubMed

    Yagar, Hülya

    2004-11-01

    Polyphenol oxidase (PPO, EC 1.14.18.1) was extracted from celery roots (Apium graveolens L.) with 0.1 M phosphate buffer, pH 7.0. The PPO was partially purified by (NH4)2SO4 and dialysis. Substrate specificity experiments were carried out with catechol, pyrogallol, L-DOPA, p-cresol, resorcinol, and tyrosine. The Km for pyrogallol, catechol, and L-DOPA were 4.5, 8.3, and 6.2mM, respectively, at 25 degrees C. Data for Vmax/Km values, which represent catalytic efficiency, show that pyrogallol has the highest value. The optimum pH and temperature were determined with catechol, pyrogallol, and L-DOPA. Optimum pH was 7.0 for catechol and L-DOPA, and 7.5 for pyrogallol. Optimum temperatures for maximum PPO activity were 25 degrees C for pyrogallol, 40 degrees C for catechol, and 45 degrees C for L-DOPA. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 60 degrees C. The order of inhibitor effectiveness was: L-cysteine > ascorbic acid > glycine > resorcinol > NaCl.

  15. Reexamining Michaelis-Menten enzyme kinetics for xanthine oxidase.

    PubMed

    Bassingthwaighte, James B; Chinn, Tamara M

    2013-03-01

    Abbreviated expressions for enzyme kinetic expressions, such as the Michaelis-Menten (M-M) equations, are based on the premise that enzyme concentrations are low compared with those of the substrate and product. When one does progress experiments, where the solute is consumed during conversion to form a series of products, the idealized conditions are violated. Here, we analyzed data of xanthine oxidase in vitro from Escribano et al. (Biochem J 254: 829, 1988) on two conversions in series, hypoxanthine to xanthine to uric acid. Analyses were done using four models: standard irreversible M-M reactions (model 1), Escribano et al.'s M-M forward reaction expressions with product inhibition (model 2), fully reversible M-M equations (model 3), and standard differential equations allowing forward and backward reactions with mass balance accounting for binding (model 4). The results showed that the need for invoking product inhibition vanishes with more complete analyses. The reactions were not quite irreversible, so the backward reaction had a small effect. Even though the enzyme concentration was only 1-2% of the initial substrate concentrations, accounting for the fraction of solutes bound to the enzyme did influence the parameter estimates, but in this case, the M-M model overestimated Michaelis constant values by only about one-third. This article also presents the research and models in a reproducible and publicly available form.

  16. The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress

    SciTech Connect

    Riganti, Chiara . E-mail: dario.ghigo@unito.it

    2006-05-01

    Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H{sub 2}O{sub 2} concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution.

  17. Reducing peanut allergens by high pressure combined with polyphenol oxidase

    NASA Astrophysics Data System (ADS)

    Chung, Si-Yin; Houska, Milan; Reed, Shawndrika

    2013-12-01

    Polyphenol oxidase (PPO) has been shown to reduce major peanut allergens. Since high pressure (HP) can increase enzyme activity, we postulated that further reduction of peanut allergens can be achieved through HP combined with PPO. Peanut extracts containing caffeic acid were treated with each of the following: (1) HP; (2) HP+PPO; (3) PPO; and (4) none. HP was conducted at 300 and 500 MPa, each for 3 and 10 min, 37 °C. After treatment, SDS-PAGE was performed and allergenic capacity (IgE binding) was determined colorimetrically in inhibition enzyme-linked immunosorbent assay and Western blots, using a pooled plasma from peanut-allergic patients. Data showed that HP alone had no effect on major peanut allergens. However, HP at 500 MPa combined with PPO (HP500/PPO) induced a higher (approximately twofold) reduction of major peanut allergens and IgE binding than PPO alone or HP300/PPO. There was no difference between treatment times. We concluded that HP500/PPO at 3-min enhanced a twofold reduction of the allergenic capacity of peanut extracts, as compared to PPO itself.

  18. Optimization of polyphenol oxidase immobilization in copper alginate beads.

    PubMed

    Kocaturk, Selin; Yagar, Hulya

    2010-05-01

    Polyphenol oxidase (PPO, EC 1.14.18.1) was isolated from artichoke head (Cynara scolymus L.) by using 0.1 M Tris-HCl buffer (pH 7.0), concentrated by (NH4)2SO4 precipitation, and immobilized in copper-alginate beads. Immobilization yield was determined to be 70%. The cresolase and catecholase activities of enzyme immobilized at optimum immobilization conditions were found to be 13.3 and 670 U g beads min(-1), respectively. Effects of immobilization conditions such as alginate concentration, CaCl2 concentration, amount of loading enzyme, bead size, and amount of beads on enzymatic activity were investigated. Optimum alginate and CuCl2 concentration were found to be 2 % and 3 % (w/v), respectively. Using bead (diameter 3 mm) amount of 0.25 g maximum enzyme activities were observed for both polyphenol activities. The initial concentrations of loading free enzyme were 6.5 U mL(-1) and 5815 U mL(-1) for cresolase activity and catecholase activities, respectively. Beads prepared at optimum immobilization conditions were suitable for up to 8 repeated uses.

  19. Characterization of polyphenol oxidase activity in Ataulfo mango.

    PubMed

    Cheema, Summervir; Sommerhalter, Monika

    2015-03-15

    Crude extracts of Ataulfo exhibited polyphenol oxidase (PPO) activity with pyrogallol, 3-methylcatechol, catechol, gallic acid, and protocatechuic acid. The substrate dependent pH optima ranged from pH 5.4 to 6.4 with Michaelis-Menten constants between 0.84 ± 0.09 and 4.6 ± 0.7 mM measured in MES or phosphate buffers. The use of acetate buffers resulted in larger Michaelis-Menten constants, up to 14.62 ± 2.03 mM. Sodium ascorbate, glutathione, and kojic acid are promising inhibitors to prevent enzymatic browning in Ataulfo. PPO activity increased with ripeness and was always higher in the skin compared to the pulp. Sodium dodecyl sulphate (SDS) enhanced PPO activity, with pulp showing a stronger increase than skin. SDS-PAGE gels stained for catecholase activity showed multiple bands, with the most prominent bands at apparent molecular weights of 53, 112, and 144 kDa.

  20. Alteration of drug metabolizing enzymes in sulphite oxidase deficiency

    PubMed Central

    Tutuncu, Begum; Kuçukatay, Vural; Arslan, Sevki; Sahin, Barbaros; Semiz, Asli; Sen, Alaattin

    2012-01-01

    The aim of this study was to investigate the possible effects of sulphite oxidase (SOX, E.C. 1.8.3.1) deficiency on xenobiotic metabolism. For this purpose, SOX deficiency was produced in rats by the administration of a low molybdenum diet with concurrent addition of 200 ppm tungsten to their drinking water. First, hepatic SOX activity in deficient groups was measured to confirm SOX deficiency. Then, aminopyrine N-demethylase, aniline 4-hydroxylase, aromatase, caffeine N-demethylase, cytochrome b5 reductase, erythromycin N-demethylase, ethoxyresorufin O-deethylase, glutathione S-transferase, N-nitrosodimethylamine N-demethylase and penthoxyresorufin O-deethylase activities were determined to follow changes in the activity of drug metabolizing enzymes in SOX-deficient rats. Our results clearly demonstrated that SOX deficiency significantly elevated A4H, caffeine N-demethylase, erythromycin N-demethylase and N-nitrosodimethylamine N-demethylase activities while decreasing ethoxyresorufin O-deethylase and aromatase activities. These alterations in drug metabolizing enzymes can contribute to the varying susceptibility and response of sulphite-sensitive individuals to different drugs and/or therapeutics used for treatments. PMID:22798713

  1. Enzymatic reactivity of glucose oxidase confined in nanochannels.

    PubMed

    Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin

    2014-05-15

    The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency.

  2. Inhibition of Monoamine Oxidase by Stilbenes from Rheum palmatum

    PubMed Central

    Wei, Bo; Yang, Zhong-Duo; Shi, Dan-Feng; Yao, Xiao-Jun; Wang, Ming-Gang

    2016-01-01

    Seven stilbenes and one catechin were bioactivity-guidedly isolated from the rhizomes of Rheum palmatem. Their structures were identified as piceatannol (1), resveratrol (2), piceid (3), rhapontigenin (4), piceatannol-3´-O-β-D-glucopyranoside (5), rhaponticin (6), catechin (7) and desoxyrhapontigenin (8). Anti-monoamine oxidase (MAO) activities of compounds 1–8 were tested. Compounds 1 and 8 showed significant MAO inhibitory activities with IC50 values 16.4 ± 1.5 μM and 11.5 ± 1.1, respectively, when the IC50 value of iproniazid as a standard was 6.5 ± 0.5 μM. The selectivity of compounds 1-8 against MAO-A and MAO-B were also evaluated. The results showed that compounds 4˴6˴8 preferred to inhibit MAO-A rather than MAO-B with selectivity values ([IC50 of MAO-B]/ [IC50 of MAO-A]) of 4.74, 10.01 and 9.42, respectively. The preliminary structure–activity relationships (SARs) of these compounds were discussed and the molecular modeling was also performed to explore the binding mode of inhibitors at the active site of MAO-A and MAO-B. PMID:28243286

  3. A tomato alternative oxidase protein with altered regulatory properties.

    PubMed

    Holtzapffel, Ruth C; Castelli, Joanne; Finnegan, Patrick M; Millar, A Harvey; Whelan, Jim; Day, David A

    2003-09-30

    We have investigated the expression and regulatory properties of the two alternative oxidase (Aox) proteins that are expressed in tomato (Lycopersicon esculentum L. Mill cv. Sweetie) after storage of green fruit at 4 degrees C. Four Aox genes were identified in the tomato genome, of which two (LeAox1a and LeAox1b) were demonstrated to be expressed in cold-treated fruit. The activity and regulatory properties of LeAox1a and LeAox1b were assayed after expression of each protein in yeast cells (Saccharomyces cerevisiae), proving that each is an active Aox protein. The LeAox1b protein was shown to have altered regulatory properties due to the substitution of a Ser for the highly conserved Cys(I) residue. LeAox1b could not form inactive disulfide-linked dimers and was activated by succinate instead of pyruvate. This is the first example of a dicot species expressing a natural Cys(I)/Ser isoform. The implications of the existence and expression of such Aox isoforms is discussed in the light of the hypothesised role for Aox in plant metabolism.

  4. Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis

    PubMed Central

    Wu, Jason Boyang; Shao, Chen; Li, Xiangyan; Li, Qinlong; Hu, Peizhen; Shi, Changhong; Li, Yang; Chen, Yi-Ting; Yin, Fei; Liao, Chun-Peng; Stiles, Bangyan L.; Zhau, Haiyen E.; Shih, Jean C.; Chung, Leland W.K.

    2014-01-01

    Tumors from patients with high-grade aggressive prostate cancer (PCa) exhibit increased expression of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines. Despite the association between MAOA and aggressive PCa, it is unclear how MAOA promotes PCa progression. Here, we found that MAOA functions to induce epithelial-to-mesenchymal transition (EMT) and stabilize the transcription factor HIF1α, which mediates hypoxia through an elevation of ROS, thus enhancing growth, invasiveness, and metastasis of PCa cells. Knockdown and overexpression of MAOA in human PCa cell lines indicated that MAOA induces EMT through activation of VEGF and its coreceptor neuropilin-1. MAOA-dependent activation of neuropilin-1 promoted AKT/FOXO1/TWIST1 signaling, allowing FOXO1 binding at the TWIST1 promoter. Importantly, the MAOA-dependent HIF1α/VEGF-A/FOXO1/TWIST1 pathway was activated in high-grade PCa specimens, and knockdown of MAOA reduced or even eliminated prostate tumor growth and metastasis in PCa xenograft mouse models. Pharmacological inhibition of MAOA activity also reduced PCa xenograft growth in mice. Moreover, high MAOA expression in PCa tissues correlated with worse clinical outcomes in PCa patients. These findings collectively characterize the contribution of MAOA in PCa pathogenesis and suggest that MAOA has potential as a therapeutic target in PCa. PMID:24865426

  5. Electrophoretic analysis of coniferyl alcohol oxidase and related laccases.

    PubMed

    Udagama-Randeniya, P; Savidge, R

    1994-01-01

    Gradient gel electrophoretic methods enabled a distinction to be made between coniferyl alcohol oxidase (CAO) of lignifying cell walls and a pI approximately 9 pine "laccase" recently implicated in lignification (Science 1993 260, 672). Following treatment of a partially purified protein mixture from developing xylem of Pinus strobus with 2-[N-morpholine]ethanesulfonic acid (MES) buffer, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that CAO had been selectively precipitated by MES and thereby purified to electrophoretic homogeneity. Purified CAO was determined to be a cell-wall-bound glycoprotein (38% glycan), M(r) 107,500, pI 7.6, pH and temperature optima 6.3 and 30 degrees C, respectively. By graphite-furnace atomic-absorption analysis, CAO contained one copper atom per protein molecule. Proteins obtained from lignifying cambial derivatives of conifers (family Pinaceae) and from Rhus typhina bark were compared with CAO and the pI approximately 9 pine "laccase" following electrophoresis and Western blotting. For Abies balsamea, Larix laricina, Picea rubens, Pinus banksiana, Pinus taeda, and R. typhina, the isoelectric points of oxidatively active bands were identical to those of purified CAO. In addition, for all species only the pI 7.6 band was immunoreactive with antibodies against periodate-deglycosylated CAO.

  6. Regulation and Function of Proline Oxidase under Nutrient Stress

    PubMed Central

    Pandhare, Jui; Cooper, Sandra K.; Donald, Steven P.; Phang, James M.

    2009-01-01

    Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of proline through the proline cycle initiated by proline oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and α-ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP-activated protein kinase (AMPK), the cellular energy sensor, by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX-dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels. PMID:19415679

  7. Monoamine oxidases as sources of oxidants in the heart

    PubMed Central

    Kaludercic, Nina; Mialet-Perez, Jeanne; Paolocci, Nazareno; Parini, Angelo; Di Lisa, Fabio

    2014-01-01

    Oxidative stress can be generated at several sites within the mitochondria. Among these, monoamine oxidases (MAO) have been described as a prominent source. MAO are mitochondrial flavoenzymes responsible for the oxidative deamination of catecholamines, serotonin and biogenic amines, and during this process they generate H2O2 and aldehyde intermediates. The role of MAO in cardiovascular pathophysiology has only recently gathered some attention since it has been demonstrated that both H2O2 and aldehydes may target mitochondrial function and consequently affect function and viability of the myocardium. In the present review, we will discuss the role of MAO in catecholamine and serotonin clearance and cycling in relation to cardiac structure and function. The relevant contribution of each MAO isoform (MAO-A or -B) will be discussed in relation to mitochondrial dysfunction and myocardial injury. Finally, we will examine both beneficial effects of their pharmacological or genetic inhibition along with potential adverse effects observed at baseline in MAO knockout mice, as well as the deleterious effects following their over-expression specifically at cardiomyocyte level. PMID:24412580

  8. Monoamine oxidases and alcoholism. II. Studies in alcoholic families

    SciTech Connect

    Suarez, B.K.; Hampe, C.L.; Parsian, A.; Cloninger, C.R.

    1995-10-09

    Thirty-five alcoholic families have been studied to investigate the relationship between DNA markers at the monoamine oxidase (MAO) loci and (1) platelet activity levels and (2) alcoholism. A quantitative linkage analysis failed to reveal any evidence that the variation in activity levels cosegregates with the DNA markers. A sib-pair analysis did not reveal a significant excess of MAO haplotype sharing among alcoholic sibs, although the deviation from random sharing was in the direction consistent with an X-linked component. A reanalysis of platelet MAO activity levels in a subset of these families revealed that the lower levels previously found in alcoholics is more likely due to the differences between males and females. Only among males and only when a {open_quotes}broad{close_quotes} definition of alcoholism is used (and MAO activity levels are transformed to normality) does it appear that alcoholics have depressed activities compared to nonalcoholics. Finally, when the confounding due to gender difference is removed, no differences between type I and type II alcoholics are found in these families. 63 refs., 6 tabs.

  9. Functional Heterogeneity of Nadph Oxidases in Atherosclerotic and Aneurysmal Diseases

    PubMed Central

    Kigawa, Yasuyoshi; Lei, Xiao-Feng; Kim-Kaneyama, Joo-ri; Miyazaki, Akira

    2017-01-01

    NADPH oxidases (NOX) are enzymes that catalyze the production of reactive oxygen species (ROS). Four species of NOX catalytic homologs (NOX1, NOX2, NOX4, and NOX5) are reportedly expressed in vascular tissues. The pro-atherogenic roles of NOX1, NOX2, and their organizer protein p47phox were manifested, and it was noted that the hydrogen peroxide-generating enzyme NOX4 possesses atheroprotective effects. Loss of NOX1 or p47phox appears to ameliorate murine aortic dissection and subsequent aneurysmal diseases; in contrast, the ablation of NOX2 exacerbates the aneurysmal diseases. It is possible that the loss of NOX2 activates inflammatory cascades in macrophages in the lesions. Roles of NOX5 in vascular functions are currently undetermined, owing to the absence of this enzyme in rodents and the limitation of the experimental procedure. Thus, it is possible that the NOX family of enzymes exhibits heterogeneity in the atherosclerotic diseases. In this aspect, subtype-selective NOX inhibitor may be promising when NOX systems serve as a molecular target for atherosclerotic and aneurysmal diseases. PMID:27476665

  10. Monoamine oxidase A (MAO A) inhibitors decrease glioma progression

    PubMed Central

    Vaikari, Vijaya Pooja; Kota, Rajesh; Chen, Kevin; Yeh, Tzu-Shao; Jhaveri, Niyati; Groshen, Susan L.; Olenyuk, Bogdan Z.; Chen, Thomas C.; Hofman, Florence M.; Shih, Jean C.

    2016-01-01

    Glioblastoma (GBM) is an aggressive brain tumor which is currently treated with temozolomide (TMZ). Tumors usually become resistant to TMZ and recur; no effective therapy is then available. Monoamine Oxidase A (MAO A) oxidizes monoamine neurotransmitters resulting in reactive oxygen species which cause cancer. This study shows that MAO A expression is increased in human glioma tissues and cell lines. MAO A inhibitors, clorgyline or the near-infrared-dye MHI-148 conjugated to clorgyline (NMI), were cytotoxic for glioma and decreased invasion in vitro. Using the intracranial TMZ-resistant glioma model, clorgyline or NMI alone or in combination with low-dose TMZ reduced tumor growth and increased animal survival. NMI was localized specifically to the tumor. Immunocytochemistry studies showed that the MAO A inhibitor reduced proliferation, microvessel density and invasion, and increased macrophage infiltration. In conclusion, we have identified MAO A inhibitors as potential novel stand-alone drugs or as combination therapy with low dose TMZ for drug-resistant gliomas. NMI can also be used as a non-invasive imaging tool. Thus has a dual function for both therapy and diagnosis. PMID:26871599

  11. Xanthine oxidase inhibiting effects of noni (Morinda citrifolia) fruit juice.

    PubMed

    Palu, Afa; Deng, Shixin; West, Brett; Jensen, Jarakae

    2009-12-01

    Morinda citrifolia L. (noni), family Rubiaceae, has been used in Polynesia for over 2000 years for its reputed health benefits, one of which is its therapeutic effects on gout (langa e hokotanga hui). However, its healing mechanism has not been elucidated. This study showed that in an in vitro bioassay that Tahitian Noni Juice (TNJ) inhibited xanthine oxidase (XO) concentration dependently. Concentrations of 1, 5 and 10 mg/mL of TNJ inhibited XO by 11%, 113% and 148%, respectively, with an IC50 of 3.8 mg compared with an IC50 of 2.4 microm for allopurinol. Noni fruit juice concentrate (NFJC) also inhibited XO concentration dependently. Concentrations of 1 and 5 mg/mL NFJC inhibited XO in vitro by 184% and 159%, respectively. A 0.1 mg/mL methanol extract (NFJME) from the fractionation of noni fruit puree inhibited XO by 64%. It was elucidated that the noni fruit juice inhibitory effect on XO enzymes is the mechanism by which noni ameliorates gout and gout-like diseases. Further, the results also support the traditional usage of noni in the treatment of gout.

  12. Cytochrome c Oxidase Inhibition by Anesthetics: Thermodynamic Analysis

    NASA Astrophysics Data System (ADS)

    Vanderkooi, Garret; Chazotte, Brad

    1982-06-01

    The thermodynamic parameters that characterize the inhibition of cytochrome c oxidase activity, in rat liver submitochondrial particles, by n-butanol, tetracaine, and dibucaine were obtained. Three equilibria were assumed in order to account for the data: for the interaction of inhibitor with the native state of the enzyme, for the interaction of inhibitor with the thermally (reversibly) denatured state, and for the change between the native and thermally denatured states. Inhibition results from interaction with both the native and denatured states but, because the interaction is stronger with the denatured than with the native state, the native/denatured equilibrium is shifted to the right by the anesthetics. The enthalpies of interaction are -2.3, -4.7, and 3.7 kcal/mol (1 cal = 4.18 J) for the native state and -10, -6, and -14 kcal/mol for the denatured state, for n-butanol, tetracaine, and dibucaine, respectively. These values are much smaller than the previous estimates obtained by using the assumption that anesthetics interact only with the thermally denatured state of enzymes (e.g., -81 kcal/mol for tetracaine inhibition of luciferase). Our results suggest that local anesthetics inhibit enzyme activity by causing a reversible perturbation of protein conformation. The magnitude of the perturbation is much smaller (in energetic terms) than that which accompanies thermal denaturation.

  13. Lysyl oxidase is associated with increased thrombosis and platelet reactivity

    PubMed Central

    Matsuura, Shinobu; Mi, Rongjuan; Koupenova, Milka; Eliades, Alexia; Patterson, Shenia; Toselli, Paul; Thon, Jonathan; Italiano, Joseph E.; Trackman, Philip C.; Papadantonakis, Nikolaos

    2016-01-01

    Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Loxtg/tg) were generated. Pf4-Loxtg/tg mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Loxtg/tg mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Loxtg/tg platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Loxtg/tg platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2β1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis. PMID:26755713

  14. A rational approach to elucidate human monoamine oxidase molecular selectivity.

    PubMed

    Mangiatordi, Giuseppe Felice; Alberga, Domenico; Pisani, Leonardo; Gadaleta, Domenico; Trisciuzzi, Daniela; Farina, Roberta; Carotti, Andrea; Lattanzi, Gianluca; Catto, Marco; Nicolotti, Orazio

    2017-04-01

    Designing highly selective human monoamine oxidase (hMAO) inhibitors is a challenging goal on the road to a more effective treatment of depression and anxiety (inhibition of hMAO-A isoform) as well as neurodegenerative diseases (inhibition of hMAO-B isoform). To uncover the molecular rationale of hMAOs selectivity, two recently prepared 2H-chromene-2-ones, namely compounds 1 and 2, were herein chosen as molecular probes being highly selective toward hMAO-A and hMAO-B, respectively. We performed molecular dynamics (MD) studies on four different complexes, cross-simulating one at a time the two hMAO-isoforms (dimer embedded in a lipid bilayer) with the two considered probes. Our comparative analysis on the obtained 100ns trajectories discloses a stable H-bond interaction between 1 and Gln215 as crucial for ligand selectivity toward hMAO-A whereas a water-mediated interaction might explain the observed hMAO-B selectivity of compound 2. Such hypotheses are further supported by binding free energy calculations carried out applying the molecular mechanics generalized Born surface area (MM-GBSA) method and allowing us to evaluate the contribution of each residue to the observed isoform selectivity. Taken as whole, this study represents the first attempt to explain at molecular level hMAO isoform selectivity and a valuable yardstick for better addressing the design of new and highly selective MAO inhibitors.

  15. NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs

    PubMed Central

    Wen, Zhenke; Shimojima, Yasuhiro; Shirai, Tsuyoshi; Li, Yinyin; Ju, Jihang; Yang, Zhen; Tian, Lu; Goronzy, Jörg J.

    2016-01-01

    Immune aging results in progressive loss of both protective immunity and T cell–mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo. We identified deficiency of NADPH oxidase 2 (NOX2) as the molecular underpinning of CD8 Treg failure in the older individuals and in patients with giant cell arteritis. CD8 Tregs suppress by releasing exosomes that carry preassembled NOX2 membrane clusters and are taken up by CD4 T cells. Overexpression of NOX2 in aged CD8 Tregs promptly restored suppressive function. Together, our data support NOX2 as a critical component of the suppressive machinery of CD8 Tregs and suggest that repairing NOX2 deficiency in these cells may protect older individuals from tissue-destructive inflammatory disease, such as large-vessel vasculitis. PMID:27088800

  16. NADPH Oxidase-Dependent Superoxide Production in Plant Reproductive Tissues.

    PubMed

    Jiménez-Quesada, María J; Traverso, José Á; Alché, Juan de Dios

    2016-01-01

    In the life cycle of a flowering plant, the male gametophyte (pollen grain) produced in the anther reaches the stigmatic surface and initiates the pollen-pistil interaction, an important step in plant reproduction, which ultimately leads to the delivery of two sperm cells to the female gametophyte (embryo sac) inside the ovule. The pollen tube undergoes a strictly apical expansion characterized by a high growth rate, whose targeting should be tightly regulated. A continuous exchange of signals therefore takes place between the haploid pollen and diploid tissue of the pistil until fertilization. In compatible interactions, theses processes result in double fertilization to form a zygote (2n) and the triploid endosperm. Among the large number of signaling mechanisms involved, the redox network appears to be particularly important. Respiratory burst oxidase homologs (Rbohs) are superoxide-producing enzymes involved in a broad range of processes in plant physiology. In this study, we review the latest findings on understanding Rboh activity in sexual plant reproduction, with a particular focus on the male gametophyte from the anther development stages to the crowning point of fertilization. Rboh isoforms have been identified in both the male and female gametophyte and have proven to be tightly regulated. Their role at crucial points such as proper growth of pollen tube, self-incompatibility response and eventual fertilization is discussed.

  17. Oxygen dependent pyruvate oxidase expression and production in Streptococcus sanguinis

    PubMed Central

    Zheng, Lan-yan; Itzek, Andreas; Chen, Zhi-yun; Kreth, Jens

    2011-01-01

    The objective of this study was to characterize the oxygen dependent regulation of pyruvate oxidase (SpxB) gene expression and protein production in Streptococcus sanguinis (S. sanguinis). SpxB is responsible for the generation of growth-inhibiting amounts of hydrogen peroxide (H2O2) able to antagonize cariogenic Streptococcus mutans (S. mutans). Furthermore, the ecological consequence of H2O2 production was investigated in its self-inhibiting ability towards the producing strain. Expression of spxB was determined with quantitative Real-Time RT-PCR and a fluorescent expression reporter strain. Protein abundance was investigated with FLAG epitope engineered in frame on the C-terminal end of SpxB. Self inhibition was tested with an antagonism plate assay. The expression and protein abundance decreased in cells grown under anaerobic conditions. S. sanguinis was resistant against its own produced H2O2, while cariogenic S. mutans was inhibited in its growth. The results suggest that S. sanguinis produces H2O2 as antimicrobial substance to inhibit susceptible niche competing species like S. mutans during initial biofilm formation, when oxygen availability allows for spxB expression and Spx production. PMID:21485312

  18. Monoamine oxidases as sources of oxidants in the heart.

    PubMed

    Kaludercic, Nina; Mialet-Perez, Jeanne; Paolocci, Nazareno; Parini, Angelo; Di Lisa, Fabio

    2014-08-01

    Oxidative stress can be generated at several sites within the mitochondria. Among these, monoamine oxidase (MAO) has been described as a prominent source. MAOs are mitochondrial flavoenzymes responsible for the oxidative deamination of catecholamines, serotonin and biogenic amines, and during this process they generate H2O2 and aldehyde intermediates. The role of MAO in cardiovascular pathophysiology has only recently gathered some attention since it has been demonstrated that both H2O2 and aldehydes may target mitochondrial function and consequently affect function and viability of the myocardium. In the present review, we will discuss the role of MAO in catecholamine and serotonin clearance and cycling in relation to cardiac structure and function. The relevant contribution of each MAO isoform (MAO-A or -B) will be discussed in relation to mitochondrial dysfunction and myocardial injury. Finally, we will examine both beneficial effects of their pharmacological or genetic inhibition along with potential adverse effects observed at baseline in MAO knockout mice, as well as the deleterious effects following their over-expression specifically at cardiomyocyte level. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".

  19. The pea gene LH encodes ent-kaurene oxidase.

    PubMed

    Davidson, Sandra E; Smith, Jennifer J; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2004-03-01

    The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants.

  20. Glucose oxidase characterization for the fabrication of hybrid microelectronic devices

    NASA Astrophysics Data System (ADS)

    Fichera, Manuela; Libertino, Sebania; Aiello, Venera; Scandurra, Antonino; Sinatra, Fulvia; Renis, Marcella; Lombardo, Salvatore

    2007-05-01

    We studied the enzyme glucose oxidase (GOx) immobilization on silicon oxide surfaces. In particular, we optimized the immobilization protocol and verified that it fulfills both requirements of enzyme preservation (measured by enzymatic activity) and VLSI compatibility. The immobilization consists of four steps: oxide activation, silanization, linker molecule deposition and GOx immobilization. It is crucial to form an uniform linker layer on the sample surface in order to maximize the sites available for enzyme bonding and achieving the best enzyme deposition. In this study, utilizing glutaraldehyde as bifunctional reagent, we monitored its uniformity on the surface through X-ray Photoelectron Spectroscopy (XPS). Once optimized, the same protocol was used to anchor the enzyme in a porous silicon dioxide matrix. Gold labeled GOx molecules were monitored by electron diffraction X-ray (EDX) measurements coupled with scanning electron microscopy (SEM). The enzymatic activity was also monitored to confirm the goodness of the proposed immobilization method. Finally, the electrical characterization of MOS capacitors, showing a shift of about 1 V in the flat band voltage, demonstrated the possibility to use this approach for electrical detection.

  1. Monoamine Oxidase-A Occupancy by Moclobemide and Phenelzine: Implications for the Development of Monoamine Oxidase Inhibitors

    PubMed Central

    Chiuccariello, Lina; Cooke, Robert G; Miler, Laura; Levitan, Robert D; Baker, Glen B; Kish, Stephen J; Kolla, Nathan J; Rusjan, Pablo M; Houle, Sylvain; Wilson, Alan A

    2016-01-01

    Background: Monoamine oxidase inhibitors (MAOIs) are being developed for major depressive disorder, Alzheimer’s, and Parkinson’s Disease. Newer MAOIs have minimal sensitivity to tyramine, but a key limitation for optimizing their development is that standards for in vivo monoamine oxidase-A (MAO-A) occupancy in humans are not well established. The objectives were to determine the dose-occupancy relationship of moclobemide and the occupancy of phenelzine at typical clinical dosing. Methods: Major depressive episode (MDE) subjects underwent [11C]harmine positron emission tomography scanning prior to and following 6 weeks of treatment with moclobemide or phenelzine. Results: Mean brain MAO-A occupancies were 74.23±8.32% for moclobemide at 300–600mg daily (n = 11), 83.75±5.52% for moclobemide at 900–1200mg daily (n = 9), and 86.82±6.89% for phenelzine at 45–60mg daily (n = 4). The regional dose-occupancy relationship of moclobemide fit a hyperbolic function [F(x) = a(x/[b + x]); F(1,18) = 5.57 to 13.32, p = 0.002 to 0.03, mean ‘a’: 88.62±2.38%, mean ‘b’: 69.88±4.36 mg]. Multivariate analyses of variance showed significantly greater occupancy of phenelzine (45–60mg) and higher-dose moclobemide (900–1200mg) compared to lower-dose moclobemide [300–600mg; F(7,16) = 3.94, p = 0.01]. Conclusions: These findings suggest that for first-line MDE treatment, daily moclobemide doses of 300–600mg correspond to a MAO-A occupancy of 74%, whereas for treatment-resistant MDE, either phenelzine or higher doses of moclobemide correspond to a MAO-A occupancy of at least 84%. Therefore, novel MAO inhibitor development should aim for similar thresholds. The findings provide a rationale in treatment algorithm design to raise moclobemide doses to inhibit more MAO-A sites, but suggest switching from high-dose moclobemide to phenelzine is best justified by binding to additional targets. PMID:26316187

  2. NADPH Oxidase 1 and NADPH Oxidase 4 Have Opposite Prognostic Effects for Patients with Hepatocellular Carcinoma after Hepatectomy

    PubMed Central

    Ha, Sang Yun; Paik, Yong-Han; Yang, Jung Wook; Lee, Min Ju; Bae, Hyunsik; Park, Cheol-Keun

    2016-01-01

    Background/Aims Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-mediated reactive oxygen species contribute to various liver diseases, including hepatocellular carcinoma (HCC). Uncertainties remain regarding the prognostic relevance of NOX1 and NOX4 protein expression in HCC. Methods NOX1 and NOX4 protein expression was examined by using immunohistochemistry in tumor tissue from 227 HCC patients who underwent hepatectomy. Results High immunoreactivity for NOX1 was observed in 197 (86.8%) of the 227 HCC cases and low immunoreactivity for NOX4 in 112 (49.3%). NOX1 and NOX4 proteins had opposite prognostic effects. High NOX1 expression was an independent predictor of both shorter recurrence-free survival (RFS) (p<0.01) and shorter overall survival (OS) (p=0.01). Low NOX4 expression was an independent predictor of both shorter RFS (p<0.01) and shorter OS (p=0.01). Subgroup analysis showed that, among patients with normal α-fetoprotein levels, patients with tumor size ≤5.0 cm and patients in Barcelona Clinic Liver Cancer stage A, high NOX1 expression had unfavorable effects on RFS, whereas low NOX4 expression had unfavorable effects on both RFS and OS. Conclusions These findings demonstrated that NOX1 and NOX4 protein expression had opposite prognostic effects for HCC patients. Moreover, both proteins had prognostic value in HCC patients with normal α-fetoprotein levels or with early-stage HCC. PMID:27282266

  3. On the oxygen reactivity of flavoprotein oxidases: an oxygen access tunnel and gate in brevibacterium sterolicum cholesterol oxidase.

    PubMed

    Piubelli, Luciano; Pedotti, Mattia; Molla, Gianluca; Feindler-Boeckh, Susanne; Ghisla, Sandro; Pilone, Mirella S; Pollegioni, Loredano

    2008-09-05

    The flavoprotein cholesterol oxidase from Brevibacterium sterolicum (BCO) possesses a narrow channel that links the active center containing the flavin to the outside solvent. This channel has been proposed to serve for the access of dioxygen; it contains at its "bottom" a Glu-Arg pair (Glu-475-Arg-477) that was found by crystallographic studies to exist in two forms named "open" and "closed," which in turn was suggested to constitute a gate functioning in the control of oxygen access. Most mutations of residues that flank the channel have minor effects on the oxygen reactivity. Mutations of Glu-311, however, cause a switch in the basic kinetic mechanism of the reaction of reduced BCO with dioxygen; wild-type BCO and most mutants show a saturation behavior with increasing oxygen concentration, whereas for Glu-311 mutants a linear dependence is found that is assumed to reflect a "simple" second order process. This is taken as support for the assumption that residue Glu-311 finely tunes the Glu-475-Arg-477 pair, forming a gate that functions in modulating the access/reactivity of dioxygen.

  4. Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A/sub 2/

    SciTech Connect

    Gamache, D.A.; Kornberg, L.J.; Bartolf, M.; Franson, R.C.

    1986-05-01

    Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1-/sup 14/C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A/sub 2/ (PLA/sub 2/) activity (64.3-545.6 nmols/min/mg). The PLA/sub 2/ was maximally active in the neutral-alkaline pH range, was Ca/sup 2 +/-dependent, and was unaffected by the addition of xanthine. PLA/sub 2/ activity was totally inhibited by 1mM EDTA whereas radical production by optimal concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA/sub 2/ activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca/sup 2 +/-dependent PLA/sub 2/ measured in various tissue homogenates (less than or equal to 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA/sub 2/ may have influenced previously published reports, and such studies should be interpreted cautiously.

  5. Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate.

    PubMed

    Stonehuerner, J; O'Brien, P; Kendrick, L; Hall, J; Millett, F

    1985-09-25

    Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.

  6. Purification and characterization of a novel caffeine oxidase from Alcaligenes species.

    PubMed

    Mohapatra, B R; Harris, N; Nordin, R; Mazumder, A

    2006-09-18

    Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.

  7. NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells.

    PubMed

    Masamune, Atsushi; Watanabe, Takashi; Kikuta, Kazuhiro; Satoh, Kennichi; Shimosegawa, Tooru

    2008-01-01

    Activated pancreatic stellate cells (PSCs) play an important role in pancreatic fibrosis and inflammation, where oxidative stress is implicated in the pathogenesis. NADPH oxidase might be a source of reactive oxygen species (ROS) in the injured pancreas. This study aimed to clarify the expression and regulation of cell functions by NADPH oxidase in PSCs. PSCs were isolated from rat and human pancreas tissues. Expression of NADPH oxidase was assessed by reverse transcription-PCR and immunostaining. Intracellular ROS production was assessed using 2',7'-dichlorofluorescin diacetate. The effects of diphenylene iodonium (DPI) and apocynin, inhibitors of NADPH oxidase, on key parameters of PSC activation were evaluated in vitro. In vivo, DPI (at 1 mg.kg body wt(-1).day(-1)) was administered in drinking water to 10-wk-old male Wistar Bonn/Kobori rats for 10 wk and to rats with chronic pancreatitis induced by dibutyltin dichloride (DBTC). PSCs expressed key components of NADPH oxidase (p22(phox), p47(phox), NOX1, gp91(phox)/NOX2, NOX4, and NOX activator 1). PDGF-BB, IL-1beta, and angiotensin II induced ROS production, which was abolished by DPI and apocynin. DPI inhibited PDGF-induced proliferation, IL-1beta-induced chemokine production, and expression of alpha-smooth muscle actin and collagen. DPI inhibited transformation of freshly isolated cells to a myofibroblast-like phenotype. In addition, DPI inhibited the development of pancreatic fibrosis in Wistar Bonn/Kobori rats and in rats with DBTC-induced chronic pancreatitis. In conclusion, PSCs express NADPH oxidase to generate ROS, which mediates key cell functions and activation of PSCs. NADPH oxidase might be a potential target for the treatment of pancreatic fibrosis.

  8. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    SciTech Connect

    Saad, Fawzy A.; Torres, Marie; Wang, Hao; Graham, Lila

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  9. Expression of ascorbic acid oxidase in zucchini squash (Cucurbita pepo L. )

    SciTech Connect

    Lin, Liangshiou; Varner, J.E. )

    1991-05-01

    The expression of ascorbic acid oxidase was studied in zuchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, the authors have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall loosening.

  10. Comparative resonance Raman study of cytochrome c oxidase from beef heart and Paracoccus denitrificans.

    PubMed

    Heibel, G E; Hildebrandt, P; Ludwig, B; Steinrücke, P; Soulimane, T; Buse, G

    1993-10-12

    Well-resolved, Soret band excited resonance Raman spectra were measured from the fully oxidized and fully reduced cytochrome c oxidase from beef heart and Paracoccus denitrificans. The vibrational patterns in the marker band region (1450-1700 cm-1) were analyzed, and a complete assignment of heme a and heme a3 vibrational modes is presented, permitting a detailed structural comparison of the mammalian and bacterial enzymes. Similar frequencies of the porphyrin modes for the reduced heme a and the reduced and oxidized heme a3 are found, indicating a close relationship of the ground-state conformations in all oxidase species studied. In oxidized heme a, however, significant frequency differences are observed and interpreted in terms of a ruffled porphyrin structure in the three- and two-subunit forms of the Paracoccus enzyme compared to the planar heme a of beef heart oxidase. The structural distortions, which also perturb the conformation of the formyl substituent and its electronic coupling with the porphyrin, reflect the specific heme-protein interactions at heme a. Since in the fully reduced state heme a appears to be largely planar in all oxidase species, the redox-linked conformational transition requires a more drastic rearrangement of the heme a-protein interactions in the bacterial than in the mammalian oxidase. For both heme a and heme a3 in the reduced state and for heme a3 in the oxidize state, frequency, intensity, and bandwidth differences of the formyl stretching vibration and intensity differences of some porphyrin modes are noted between the three oxidase forms. The same modes are also affected by quaternary structure changes in the bovine oxidase caused by different detergents and isolation procedures. These effects are attributed to differences of the dielectric properties of the heme environment, due to subtle structural changes in the heme pockets, induced by protein-protein interactions of subunit III with subunits I and/or II.

  11. NADPH oxidases, reactive oxygen species, and hypertension: clinical implications and therapeutic possibilities.

    PubMed

    Paravicini, Tamara M; Touyz, Rhian M

    2008-02-01

    Reactive oxygen species (ROS) influence many physiological processes including host defense, hormone biosynthesis, fertilization, and cellular signaling. Increased ROS production (termed "oxidative stress") has been implicated in various pathologies, including hypertension, atherosclerosis, diabetes, and chronic kidney disease. A major source for vascular and renal ROS is a family of nonphagocytic NAD(P)H oxidases, including the prototypic Nox2 homolog-based NAD(P)H oxidase, as well as other NAD(P)H oxidases, such as Nox1 and Nox4. Other possible sources include mitochondrial electron transport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase, and uncoupled nitric oxide synthase. NAD(P)H oxidase-derived ROS plays a physiological role in the regulation of endothelial function and vascular tone and a pathophysiological role in endothelial dysfunction, inflammation, hypertrophy, apoptosis, migration, fibrosis, angiogenesis, and rarefaction, important processes underlying cardiovascular and renal remodeling in hypertension and diabetes. These findings have evoked considerable interest because of the possibilities that therapies against nonphagocytic NAD(P)H oxidase to decrease ROS generation and/or strategies to increase nitric oxide (NO) availability and antioxidants may be useful in minimizing vascular injury and renal dysfunction and thereby prevent or regress target organ damage associated with hypertension and diabetes. Here we highlight current developments in the field of reactive oxygen species and cardiovascular disease, focusing specifically on the recently identified novel Nox family of NAD(P)H oxidases in hypertension. We also discuss the potential role of targeting ROS as a therapeutic possibility in the management of hypertension and cardiovascular disease.

  12. Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role.

    PubMed

    Sluse, F E; Jarmuszkiewicz, W

    1998-06-01

    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.

  13. Zinc ions as cytochrome C oxidase inhibitors: two sites of action.

    PubMed

    Kuznetsova, S S; Azarkina, N V; Vygodina, T V; Siletsky, S A; Konstantinov, A A

    2005-02-01

    Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome-reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix. This interaction is fully reversed by EDTA and results in a partial inhibition of the enzyme activity (50-90%, depending on preparation) with an effective K(i) of approximately 10 microM. The rapid effect of zinc is observed with the solubilized enzyme, it vanishes upon incorporation of cytochrome oxidase in liposomes, and it re-appears when proteoliposomes are supplied with alamethicin that makes the membrane permeable to low molecular weight substances. Zinc presumably blocks the entrance of the D-protonic channel opening into the inner aqueous phase. Second, zinc interacts slowly (tens of minutes, hours) with a site of cytochrome oxidase accessible from the outer aqueous phase bringing about complete inhibition of the enzymatic activity. The slow phase is characterized by high affinity of the inhibitor for the enzyme: full inhibition can be achieved upon incubation of the solubilized oxidase for 24 h with zinc concentration as low as 2 microM. The rate of zinc inhibitory action in the slow phase is proportional to Zn(2+) concentration. The slow interaction of zinc with the outer surface of liposome-reconstituted cytochrome oxidase is observed only with the enzyme turning over or in the presence of weak reductants, whereas incubation of zinc with the fully oxidized proteoliposomes does not induce the inhibition. It is shown that zinc ions added to cytochrome oxidase proteoliposomes from the outside inhibit specifically the slow electrogenic phase of proton transfer, coupled to a transition of cytochrome oxidase from the oxo-ferryl to the oxidized state (the F --> O step corresponding to transfer of the 4th electron in the catalytic

  14. Fine-tuning of NADH oxidase decreases byproduct accumulation in respiration deficient xylose metabolic Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    Background Efficiently utilizing all available carbon from lignocellulosic feedstock presents a major barrier to the production of economically feasible biofuel. Previously, to enable xylose utilization, we introduced a cofactor-dependent xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway, or a cofactor-independent xylose isomerase (XI) pathway, into Saccharomyces cerevisiae. The resulting strains metabolized xylose with high efficiency. However, in both pathway recombinant strains, the cofactor imbalance caused accumulation of the byproducts glycerol and/or xylitol and reduced the ethanol production efficiency. Results In this study, we introduced NADH oxidase from Lactococcus lactis into both XI and XR-XDH pathway recombinant strains. To reduce byproduct accumulation while maintaining xylose metabolism, we optimized the expression level of NADH oxidase by comparing its expression under the control of different promoters and plasmids. In recombinant XI strains, NADH oxidase was expressed at different levels, regulated by the GPD2 promoter or TEF1 promoter in the 2 μ plasmid. The expression under the control of GPD2 promoter decreased glycerol production by 84% and increased the ethanol yield and specific growth rate by 8% and 12%, respectively. In contrast, in the recombinant XR-XDH strains, such expression level was not efficient enough to decrease the byproduct accumulation. Therefore, higher NADH oxidase expression levels were tested. In the strain expressing NADH oxidase under the control of the TEF1 promoter in the centromeric plasmids, xylitol and glycerol production were reduced by 60% and 83%, respectively, without significantly affecting xylose consumption. Conclusions By fine-tuning NADH oxidase expression, we decreased the glycerol or/and xylitol production in both recombinant XI and XR-XDH xylose-metabolizing yeast strains. The optimal NADH oxidase expression levels depend on metabolic pathways. Similar cofactor engineering strategies

  15. Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes.

    PubMed

    Berry, E A; Trumpower, B L

    1985-02-25

    An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.

  16. Characterizing the proton loading site in cytochrome c oxidase.

    PubMed

    Lu, Jianxun; Gunner, M R

    2014-08-26

    Cytochrome c oxidase (CcO) uses the energy released by reduction of O2 to H2O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a3, Cu(B), Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1-4 residues load and release protons. However, the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded.

  17. How hydrogen peroxide is metabolized by oxidized cytochrome c oxidase.

    PubMed

    Jancura, Daniel; Stanicova, Jana; Palmer, Graham; Fabian, Marian

    2014-06-10

    In the absence of external electron donors, oxidized bovine cytochrome c oxidase (CcO) exhibits the ability to decompose excess H2O2. Depending on the concentration of peroxide, two mechanisms of degradation were identified. At submillimolar peroxide concentrations, decomposition proceeds with virtually no production of superoxide and oxygen. In contrast, in the millimolar H2O2 concentration range, CcO generates superoxide from peroxide. At submillimolar concentrations, the decomposition of H2O2 occurs at least at two sites. One is the catalytic heme a3-CuB center where H2O2 is reduced to water. During the interaction of the enzyme with H2O2, this center cycles back to oxidized CcO via the intermediate presence of two oxoferryl states. We show that at pH 8.0 two molecules of H2O2 react with the catalytic center accomplishing one cycle. In addition, the reactions at the heme a3-CuB center generate the surface-exposed lipid-based radical(s) that participates in the decomposition of peroxide. It is also found that the irreversible decline of the catalytic activity of the enzyme treated with submillimolar H2O2 concentrations results specifically from the decrease in the rate of electron transfer from heme a to the heme a3-CuB center during the reductive phase of the catalytic cycle. The rates of electron transfer from ferrocytochrome c to heme a and the kinetics of the oxidation of the fully reduced CcO with O2 were not affected in the peroxide-modified CcO.

  18. Regulation of NADPH Oxidase 5 by Protein Kinase C Isoforms

    PubMed Central

    Chen, Feng; Yu, Yanfang; Haigh, Steven; Johnson, John; Lucas, Rudolf; Stepp, David W.; Fulton, David J. R.

    2014-01-01

    NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes

  19. Purification and characterization of Ocimum basilicum L. polyphenol oxidase.

    PubMed

    Dogan, Serap; Turan, Pinar; Dogan, Mehmet; Arslan, Oktay; Alkan, Mahir

    2005-12-28

    A partial characterization of polyphenol oxidase (PPO) activity in Ocimum basilicum L. is described. PPO in O. basilicum L. was extracted and purified through (NH4)2SO4 precipitation, dialysis, and a Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. The samples obtained from (NH4)2SO4 precipitation and dialysis were used for the characterization of PPO. At the end of purification by affinity chromatography, 11.5-fold purification was achived. The purified enzyme exhibited a clear single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be approximately 54 kDa. The contents of total phenolic and protein of O. basilicum L. extracts were determined. The total phenolic content of O. basilicum L. was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 280 mg 100 g(-1) on a fresh weight basis. The protein content was determined according to the Bradford method. The enzyme showed activity to 4-methylcatechol, catechol, and pyrogallol substrates, but not to tyrosine. Therefore, of these three substrates, 4-methylcatecol was the best substrate due to the highest V(max)/K(m) value, followed by pyrogallol and catechol. The optimum pH was at 6, 8, and 9 for 4-methylcatechol, catechol, and pyrogallol, respectively. The enzyme had an optimum temperature of 20, 40, and 50 degrees C for 4-methylcatechol, catechol, and pyrogallol, respectively. It was found that optimum temperature and pH were dependent on the substrates studied. The enzyme activity with increasing temperature and inactivation time for 4-methylcatechol, catechol, and pyrogallol substrates decreased due to heat denaturation of the enzyme.

  20. Targeting monoamine oxidase A in advanced prostate cancer

    PubMed Central

    Flamand, Vincent; Zhao, Hongjuan

    2010-01-01

    Purpose Inhibitors of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades neurotransmitters including serotonin and norepinephrine, are commonly used to treat neurological conditions including depression. Recently, we and others identified high expression of MAOA in normal basal prostatic epithelium and high-grade primary prostate cancer (PCa). In contrast, MAOA is low in normal secretory prostatic epithelium and low-grade PCa. An irreversible inhibitor of MAOA, clorgyline, induced secretory differentiation in primary cultures of normal basal epithelial cells and high-grade PCa. Furthermore, clorgyline inhibited several oncogenic pathways in PCa cells, suggesting clinical value of MAOA inhibitors as a pro-differentiation and anti-oncogenic therapy for high-risk PCa. Here, we extended our studies to a model of advanced PCa, VCaP cells, which were derived from castration-resistant metastatic PCa and express a high level of MAOA. Methods Growth of VCaP cells in the presence or absence of clorgyline was evaluated in vitro and in vivo. Gene expression changes in response to clorgyline were determined by microarray and validated by quantitative real-time polymerase chain reaction. Results Treatment with clorgyline in vitro inhibited growth and altered the transcriptional pattern of VCaP cells in a manner consistent with the pro-differentiation and anti-oncogenic effects seen in treated primary PCa cells. Src, beta-catenin, and MAPK oncogenic pathways, implicated in androgen-independent growth and metastasis, were significantly downregulated. Clorgyline treatment of mice bearing VCaP xenografts slowed tumor growth and induced transcriptome changes similar to those noted in vitro. Conclusion Our results support the possibility that anti-depressant drugs that target MAOA might find a new application in treating PCa. PMID:20204405

  1. Import, targeting, and processing of a plant polyphenol oxidase.

    PubMed Central

    Sommer, A; Ne'eman, E; Steffens, J C; Mayer, A M; Harel, E

    1994-01-01

    A tomato (Lycopersicon esculentum L.) gene encoding a precursor of polyphenol oxidase (PPO) was transcribed and translated in vitro. The import, targeting, and processing of the [35S]methionine-labeled precursor protein (pPPO) were studied in isolated chloroplasts. The protein was routed to the thylakoid lumen in two steps. The 67-kD precursor was first imported into the stroma in an ATP-dependent step. It was processed to a 62-kD intermediate by a stromal peptidase. Translocation into the lumen was light dependent and involved processing of the 62-kD to the 59-kD mature form. The mature polypeptide was soluble in the lumen and not bound to thylakoids. This two-step targeting pattern was observed in plastids from a variety of plants including pea (Pisum sativum L.), tomato, and maize (Zea mays L.). The ratio between the intermediate and mature forms observed depended on the plant species, leaf age, growth conditions, and illumination regime to which the plants had been subjected. Cu2+ was not required for pPPO import or processing. Furthermore, low concentrations of Cu2+ (1-5 microM) markedly inhibited the first import step. Tentoxin specifically inhibited pPPO import, leaving the precursor bound to the envelope membrane. The two-step routing of pPPO into chloroplasts, typical of thylakoid lumen proteins, is consistent with the two-domain structure of the transit peptide and appears to be a feature of all plant PPO genes isolated so far. No evidence was found for unorthodox routing mechanisms, which have been suggested to be involved in the import of plant PPOs. The two-step routing may account for some of the multiplicity of PPO observed in vivo. PMID:7972497

  2. [Covalent immobilization of glucose oxidase within organic media].

    PubMed

    Zhou, Tao; Zhu, Xiongjun; Su, Jianhua; Yao, Dongsheng; Liu, Daling

    2012-04-01

    Activity losing during the covalent immobilization of enzyme is a serious problem. Here we studied organic phase immobilization by using glucose oxidase (GOD) as a model. After lyophilized at optimum pH, GOD is covalently immobilized onto glutaraldhyde-activated chitosan microsphere carrier under the condition of water, 1, 4-dioxane, ether and ethanol separately. The special activities, enzyme characterization and kinetic parameters are determined. Results show that all of the organic phase immobilized GODs have higher special activities and larger K(cat) than that of aqueous phase. Under the conditions of 0.1% of glutaraldehyde, 1.6% moisture content with 80 mg of GOD added to per gram of carrier, 2.9-fold of the special activity and 3-fold of the effective activity recovery ratio were obtained, and 3-fold of the residue activity was demonstrated after 7 runs when compares 1, 4-dioxane phase immobilized GOD with water phase immobilized one. In addition, kinetic study shows that 1,4-dioxane immobilized GOD (Km(app) = 5.63 mmol/L, V(max) = 1.70 micromol/(min x mg GOD), K(cat) = 0.304 s(-1) was superior to water immobilized GOD (Km(app) = 7.33 mmol/L, V(max) = 1.02 micromol/(min x mg GOD), K(cat) = 0.221 s(-1)). All above indicated GOD immobilized in proper organic media presented a better activity with improved catalytic performance. Organic phase immobilization might be one of the ways to overcome the conformational denature of enzyme protein during covalent modification.

  3. Alternative oxidase involvement in Daucus carota somatic embryogenesis.

    PubMed

    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.

  4. Predicting Monoamine Oxidase Inhibitory Activity through Ligand-Based Models

    PubMed Central

    Vilar, Santiago; Ferino, Giulio; Quezada, Elias; Santana, Lourdes; Friedman, Carol

    2013-01-01

    The evolution of bio- and cheminformatics associated with the development of specialized software and increasing computer power has produced a great interest in theoretical in silico methods applied in drug rational design. These techniques apply the concept that “similar molecules have similar biological properties” that has been exploited in Medicinal Chemistry for years to design new molecules with desirable pharmacological profiles. Ligand-based methods are not dependent on receptor structural data and take into account two and three-dimensional molecular properties to assess similarity of new compounds in regards to the set of molecules with the biological property under study. Depending on the complexity of the calculation, there are different types of ligand-based methods, such as QSAR (Quantitative Structure-Activity Relationship) with 2D and 3D descriptors, CoMFA (Comparative Molecular Field Analysis) or pharmacophoric approaches. This work provides a description of a series of ligand-based models applied in the prediction of the inhibitory activity of monoamine oxidase (MAO) enzymes. The controlled regulation of the enzymes’ function through the use of MAO inhibitors is used as a treatment in many psychiatric and neurological disorders, such as depression, anxiety, Alzheimer’s and Parkinson’s disease. For this reason, multiple scaffolds, such as substituted coumarins, indolylmethylamine or pyridazine derivatives were synthesized and assayed toward MAO-A and MAO-B inhibition. Our intention is to focus on the description of ligand-based models to provide new insights in the relationship between the MAO inhibitory activity and the molecular structure of the different inhibitors, and further study enzyme selectivity and possible mechanisms of action. PMID:23231398

  5. The subunit composition and function of mammalian cytochrome c oxidase.

    PubMed

    Kadenbach, Bernhard; Hüttemann, Maik

    2015-09-01

    Cytochrome c oxidase (COX) from mammals and birds is composed of 13 subunits. The three catalytic subunits I-III are encoded by mitochondrial DNA, the ten nuclear-coded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIII) by nuclear DNA. The nuclear-coded subunits are essentially involved in the regulation of oxygen consumption and proton translocation by COX, since their removal or modification changes the activity and their mutation causes mitochondrial diseases. Respiration, the basis for ATP synthesis in mitochondria, is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits IV, VIa, VIb, VIIa, VIIb, and VIII, but the holoenzyme in mammals is always composed of 13 subunits. Various proteins and enzymes were shown, e.g., by co-immunoprecipitation, to bind to specific COX subunits and modify its activity, but these interactions are reversible, in contrast to the tightly bound 13 subunits. In addition, the formation of supercomplexes with other oxidative phosphorylation complexes has been shown to be largely variable. The regulatory complexity of COX is increased by protein phosphorylation. Up to now 18 phosphorylation sites have been identified under in vivo conditions in mammals. However, only for a few phosphorylation sites and four nuclear-coded subunits could a specific function be identified. Research on the signaling pathways leading to specific COX phosphorylations remains a great challenge for understanding the regulation of respiration and ATP synthesis in mammalian organisms. This article reviews the function of the individual COX subunits and their isoforms, as well as proteins and small molecules interacting and regulating the enzyme.

  6. Molecular cloning and characterisation of banana fruit polyphenol oxidase.

    PubMed

    Gooding, P S; Bird, C; Robinson, S P

    2001-09-01

    Polyphenol oxidase (PPO; EC 1.10.3.2) is the enzyme thought to be responsible for browning in banana [Musa cavendishii (AAA group, Cavendish subgroup) cv. Williams] fruit. Banana flesh was high in PPO activity throughout growth and ripening. Peel showed high levels of activity early in development but activity declined until ripening started and then remained constant. PPO activity in fruit was not substantially induced after wounding or treatment with 5-methyl jasmonate. Banana flowers and unexpanded leaf roll had high PPO activities with lower activities observed in mature leaves, roots and stem. Four different PPO cDNA clones were amplified from banana fruit (BPO1, BPO11, BPO34 and BPO35). Full-length cDNA and genomic clones were isolated for the most abundant sequence (BPO1) and the genomic clone was found to contain an 85-bp intron. Introns have not been previously found in PPO genes. Northern analysis revealed the presence of BPO1 mRNA in banana flesh early in development but little BPO1 mRNA was detected at the same stage in banana peel. BPO11 transcript was only detected in very young flesh and there was no detectable expression of BPO34 or BPO35 in developing fruit samples. PPO transcripts were also low throughout ripening in both flesh and peel. BPO1 transcripts were readily detected in flowers, stem, roots and leaf roll samples but were not detected in mature leaves. BPO11 showed a similar pattern of expression to BPO1 in these tissues but transcript levels were much lower. BPO34 and BPO35 mRNAs were only detected at a low level in flowers and roots and BPO34 transcript was detected in mature leaves, the only clone to do so. The results suggest that browning of banana fruit during ripening results from release of pre-existing PPO enzyme, which is synthesised very early in fruit development.

  7. NADPH oxidase 4 is an oncoprotein localized to mitochondria.

    PubMed

    Graham, Kelly A; Kulawiec, Mariola; Owens, Kjerstin M; Li, Xiurong; Desouki, Mohamed Mokhtar; Chandra, Dhyan; Singh, Keshav K

    2010-08-01

    Reactive oxygen species (ROS) are known to be involved in many physiological and pathological processes. Initially ROS-producing NADPH oxidase (NOX) proteins were thought to be present in phagocytes. However, recent studies have demonstrated that NOX proteins are expressed in many other cell types and tissues. NOX family members' expression and function seems to vary from tissue to tissue. We determined the expression of the NOX family of proteins (NOX1-5) in normal breast tissue and breast tumors. Our study revealed that normal breast tissues express NOX1, 4 and 5 genes. Similar pattern of expression was revealed in a breast epithelial cell line. We found that NOX4 was overexpressed in the majority of breast cancer cell lines and primary breast tumors. NOX4 was also overexpressed in ovarian tumors. Overexpression of NOX4 in normal breast epithelial cells resulted in cellular senescence, resistance to apoptosis, and tumorigenic transformation. Overexpression of NOX4 in already transformed breast tumor cells also showed increased tumorigenicity. Strong evidence suggests that regulation of these processes occurs through NOX4 generation of ROS in the mitochondria. We demonstrate that the NOX4 protein contains a 73 amino acid long mitochondrial localization signal at the N-terminus that is capable of transporting a passenger protein GFP into the mitochondria. Treatment of NOX4 overexpressing cells with catalase resulted in decreased tumorigenic characteristics. Together, this study provides evidence for an oncogenic function for NOX4 protein localized to mitochondria and suggests that NOX4 is a novel source of ROS produced in the mitochondria. This study also identifies a possible treatment of NOX4-induced breast cancer by antioxidant treatment.

  8. Redox-controlled proton gating in bovine cytochrome c oxidase

    NASA Astrophysics Data System (ADS)

    Rousseau, Denis

    2015-03-01

    Cytochrome c oxidase is the terminal enzyme in the electron transfer chain of essentially all organisms that utilize oxygen to generate energy. It reduces oxygen to water and harnesses the energy to pump protons across the mitochondrial membrane in eukaryotes and the plasma membrane in prokaryotes. The mechanism by which proton pumping is coupled to the oxygen reduction reaction remains unresolved, owing to the difficulty of visualizing proton movement within the massive membrane-associated protein matrix. Here, with a novel hydrogen/deuterium exchange resonance Raman spectroscopy method, we have identified two critical elements of the proton pump: a proton loading site near the propionate groups of heme a, which is capable of transiently storing protons uploaded from the negative-side of the membrane prior to their release into the positive-side of the membrane and a conformational gate that controls proton translocation in response to the change in the redox state of heme a. These findings form the basis for a postulated molecular model describing a detailed mechanism by which unidirectional proton translocation is coupled to electron transfer from heme a to heme a3, associated with oxygen chemistry occurring in the heme a3 site, during enzymatic turnover. Each time heme a undergoes an oxidation-reduction transition a proton is translocated across the membrane accounting for the observation that two protons are translocated during the oxidative phase of the enzymatic cycle and two more are translocated during the reductive phase. This work was done in collaboration with Drs. Tsuyoshi Egawa and Syun-Ru Yeh. This work was supported the National Institutes of Health Grant GM098799 to D.L.R and National Science Foundation Grant NSF 0956358 to S.-R.Y.

  9. Polyphenol oxidase from wheat bran is a serpin.

    PubMed

    Yamasaki, Yoshiki; Konno, Haruyoshi; Noda, Kazuhiko

    2008-01-01

    Polyphenol oxidase (PPO; EC 1.10.3.2) was isolated from wheat bran by a procedure that included ammonium sulfate fractionation, batch adsorption by DEAE-cellulofine, CM-cellulofine column chromatography, DEAE-cellulofine column chromatography, preparative isoelectric focusing, adsorption on the membrane of a Vivapure Q Maxi H spin column, and heat treatment. These procedures led to 150-fold purification with 4.2% recovery. The PPO was homogeneous by SDS/PAGE. The relative molecular weight of the PPO was estimated to be 37,000 based on its mobility in SDS/PAGE. The isoelectric point of the PPO was 4.4. The K(m) values of the PPO for caffeic acid, chlorogenic acid, pyrocatechol, 4-methyl catechol and l-DOPA as substrates were 0.077, 0.198, 1.176, 1.667 and 4.545 mM. The PPO was strongly inhibited by tropolone. The K(i) value for tropolone is 2.2 x 10(-7) M. The sequence of the 15 N-terminal amino-acid residues was determined to be ATDVRLSIAHQTRFA, which was identical to those of serpin from Triticum aestivum and protein Z from Hordeum vulgare. The PPO strongly inhibited the activity of trypsin, which is an enzyme of serine proteases; 50% inhibition was observed with 1.5 x 10(-7) M PPO. The K(i) value for PPO is 2.3 x 10(-8) M. The wheat bran PPO should be a very important protein for protecting wheat against disease, virus, insect and herbivore damages by both the activities of PPO and protease inhibitor.

  10. Inhibition of polyphenol oxidases activity by various dipeptides.

    PubMed

    Girelli, Anna M; Mattei, Enrico; Messina, Antonella; Tarola, Anna M

    2004-05-19

    In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%.

  11. Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase.

    PubMed

    Ferreira, Patricia; Hernández-Ortega, Aitor; Lucas, Fátima; Carro, Juan; Herguedas, Beatriz; Borrelli, Kenneth W; Guallar, Victor; Martínez, Angel T; Medina, Milagros

    2015-08-01

    Aryl-alcohol oxidase (AAO, EC 1.1.3.7) generates H2 O2 for lignin degradation at the expense of benzylic and other π system-containing primary alcohols, which are oxidized to the corresponding aldehydes. Ligand diffusion studies on Pleurotus eryngii AAO showed a T-shaped stacking interaction between the Tyr92 side chain and the alcohol substrate at the catalytically competent position for concerted hydride and proton transfers. Bi-substrate kinetics analysis revealed that reactions with 3-chloro- or 3-fluorobenzyl alcohols (halogen substituents) proceed via a ping-pong mechanism. However, mono- and dimethoxylated substituents (in 4-methoxybenzyl and 3,4-dimethoxybenzyl alcohols) altered the mechanism and a ternary complex was formed. Electron-withdrawing substituents resulted in lower quantum mechanics stacking energies between aldehyde and the tyrosine side chain, contributing to product release, in agreement with the ping-pong mechanism observed in 3-chloro- and 3-fluorobenzyl alcohol kinetics analysis. In contrast, the higher stacking energies when electron donor substituents are present result in reaction of O2 with the flavin through a ternary complex, in agreement with the kinetics of methoxylated alcohols. The contribution of Tyr92 to the AAO reaction mechanism was investigated by calculation of stacking interaction energies and site-directed mutagenesis. Replacement of Tyr92 by phenylalanine does not alter the AAO kinetic constants (on 4-methoxybenzyl alcohol), most probably because the stacking interaction is still possible. However, introduction of a tryptophan residue at this position strongly reduced the affinity for the substrate (i.e. the pre-steady state Kd and steady-state Km increase by 150-fold and 75-fold, respectively), and therefore the steady-state catalytic efficiency, suggesting that proper stacking is impossible with this bulky residue. The above results confirm the role of Tyr92 in substrate binding, thus governing the kinetic mechanism

  12. Quiescin-sulfhydryl oxidase inhibits prion formation in vitro

    PubMed Central

    Yuan, Jue; Zeng, Liang; Dang, Johnny; Camacho Martinez, Manuel; Wang, Zerui; Mikol, Jacqueline; Lehmann, Sylvain; Bu, Shizhong; Steyaert, Jan; Cui, Li; Petersen, Robert B.; Kong, Qingzhong; Wang, Gong-Xiang; Wohlkonig, Alexandre; Zou, Wen-Quan

    2016-01-01

    Prions are infectious proteins that cause a group of fatal transmissible diseases in animals and humans. The scrapie isoform (PrPSc) of the cellular prion protein (PrPC) is the only known component of the prion. Several lines of evidence have suggested that the formation and molecular features of PrPSc are associated with an abnormal unfolding/refolding process. Quiescin-sulfhydryl oxidase (QSOX) plays a role in protein folding by introducing disulfides into unfolded reduced proteins. Here we report that QSOX inhibits human prion propagation in protein misfolding cyclic amplification reactions and murine prion propagation in scrapie-infected neuroblastoma cells. Moreover, QSOX preferentially binds PrPSc from prion-infected human or animal brains, but not PrPC from uninfected brains. Surface plasmon resonance of the recombinant mouse PrP (moPrP) demonstrates that the affinity of QSOX for monomer is significantly lower than that for octamer (312 nM vs 1.7 nM). QSOX exhibits much lower affinity for N-terminally truncated moPrP (PrP89-230) than for the full-length moPrP (PrP23-231) (312 nM vs 2 nM), suggesting that the N-terminal region of PrP is critical for the interaction of PrP with QSOX. Our study indicates that QSOX may play a role in prion formation, which may open new therapeutic avenues for treating prion diseases. PMID:27959866

  13. Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana.

    PubMed

    Takahashi, Yoshihiro; Cong, Runzi; Sagor, G H M; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

    2010-09-01

    The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed.

  14. Oxidative phenols in forage crops containing polyphenol oxidase enzymes.

    PubMed

    Parveen, Ifat; Threadgill, Michael D; Moorby, Jon M; Winters, Ana

    2010-02-10

    Polyphenol oxidases (PPOs) are copper-containing enzymes that catalyze oxidation of endogenous monophenols to ortho-dihydroxyaryl compounds and of ortho-dihydroxyaryl compounds to ortho-quinones. Subsequent nucleophilic addition reactions of phenols, amino acids, and proteins with the electrophilic ortho-quinones form brown-, black-, or red-colored secondary products associated with the undesired discolouration of fruit and vegetables. Several important forage plants also exhibit significant PPO activity, and a link with improved efficiency of ruminant production has been established. In ruminant animals, extensive degradation of forage proteins, following consumption, can result in high rates of excretion of nitrogen, which contributes to point-source and diffuse pollution. Reaction of quinones with forage proteins leads to the formation of protein-phenol complexes that are resistant to proteolytic activity during ensilage and during rumen fermentation. Thus, PPO in red clover (Trifolium pratense) has been shown to improve protein utilization by ruminants. While PPO activity has been demonstrated in a number of forage crops, little work has been carried out to identify substrates of PPO, knowledge of which would be beneficial for characterizing this trait in these forages. In general, a wide range of 1,2-dihydroxyarenes can serve as PPO substrates because these are readily oxidized because of the ortho positioning of the hydroxy groups. Naturally occurring phenols isolated from forage crops with PPO activity are reviewed. A large number of phenols, which may be directly or indirectly oxidized as a consequence of PPO activity, have been identified in several forage grass, legume, cereal, and brassica species; these include hydroxybenzoic acids, hydroxycinnamates, and flavonoids. In conclusion, a number of compounds are known or postulated to enable PPO activity in important PPO-expressing forage crops. Targeting the matching of these compounds with PPO activity

  15. Detection, diversity and expression of aerobic bacterial arsenite oxidase genes.

    PubMed

    Inskeep, William P; Macur, Richard E; Hamamura, Natsuko; Warelow, Thomas P; Ward, Seamus A; Santini, Joanne M

    2007-04-01

    The arsenic (As) drinking water crisis in south and south-east Asia has stimulated intense study of the microbial processes controlling the redox cycling of As in soil-water systems. Microbial oxidation of arsenite is a critical link in the global As cycle, and phylogenetically diverse arsenite-oxidizing microorganisms have been isolated from various aquatic and soil environments. However, despite progress characterizing the metabolism of As in various pure cultures, no functional gene approaches have been developed to determine the importance and distribution of arsenite-oxidizing genes in soil-water-sediment systems. Here we report for the first time the successful amplification of arsenite oxidase-like genes (aroA/asoA/aoxB) from a variety of soil-sediment and geothermal environments where arsenite is known to be oxidized. Prior to the current work, only 16 aroA/asoA/aoxB-like gene sequences were available in GenBank, most of these being putative assignments from homology searches of whole genomes. Although aroA/asoA/aoxB gene sequences are not highly conserved across disparate phyla, degenerate primers were used successfully to characterize over 160 diverse aroA-like sequences from 10 geographically isolated, arsenic-contaminated sites and from 13 arsenite-oxidizing organisms. The primer sets were also useful for confirming the expression of aroA-like genes in an arsenite-oxidizing organism and in geothermal environments where arsenite is oxidized to arsenate. The phylogenetic and ecological diversity of aroA-like sequences obtained from this study suggests that genes for aerobic arsenite oxidation are widely distributed in the bacterial domain, are widespread in soil-water systems containing As, and play a critical role in the biogeochemical cycling of As.

  16. Oxalate oxidases and differentiating surface structure in wheat: germins.

    PubMed Central

    Lane, B G

    2000-01-01

    Oxalate oxidases (OXOs) have been found to be concentrated in the surface tissues of wheat embryos and grains: germin is concentrated in root and leaf sheaths that surround germinated embryos; pseudogermin (OXO-psi) is concentrated in the epidermis and bracts that 'encircle' mature grains. Most strikingly, the epidermal accumulation of OXO-psi was found to presage the transition of a delicate 'skin', similar to the fragile epidermis of human skin, into the tough shell (the miller's 'beeswing') that is typical of mature wheat grains. A narrow range of oxalate concentration (1--2 mM) in the hydrated tissues of major crop cereals (barley, maize, oat, rice, rye and wheat) contrasted with wide variations in their OXO expression, e.g. cold-tolerant and cold-sensitive varieties of maize have similar oxalate contents but the former was found to contain approx. 20-fold more germin than did the latter. Well-known OXOs in sorghum, a minor cereal, and beet, a dicotyledon, were found to have little antigenic relatedness to the germins, but the beet enzyme did share some of the unique stability properties that are peculiar to the germin-like OXOs that are found only in the major crop cereals. Their concentration in surface structures of domesticated wheat suggests a biochemical role for germin-like OXOs: programmed cell death in surface tissues might be a constitutive as well as an adaptive form of differentiation that helps to produce refractory barriers against tissue invasion by predators. Incidental to the principal investigation, and using an OXO assay (oxalate-dependent release of CO(2)) that did not rely on detecting H(2)O(2), which is often fully degraded in cell extracts, it was found that OXO activity in soluble extracts of wheat was manifested only in standard solution assays if the extract was pretreated in a variety of ways, which included preincubation with pepsin or highly substituted glucuronogalactoarabinoxylans (cell-wall polysaccharides). PMID:10861243

  17. Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes.

    PubMed

    Hutzler, J Matthew; Yang, Young-Sun; Albaugh, Daniel; Fullenwider, Cody L; Schmenk, Jennifer; Fisher, Michael B

    2012-02-01

    Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

  18. Crystal Structure of a Two-domain Multicopper Oxidase: Implications for the Evolution of Multicooper Blue Proteins

    SciTech Connect

    Lawton, Thomas J.; Sayavedra-Soto, Luis A.; Arp, Daniel J.; Rosenzweig, Amy C.

    2009-06-01

    The two-domain multicopper oxidases are proposed to be key intermediates in the evolution of three-domain multicopper oxidases. A number of two-domain multicopper oxidases have been identified from genome sequences and are classified as type A, type B, or type C on the basis of the predicted location of the type 1 copper center. The crystal structure of blue copper oxidase, a type C two-domain multicopper oxidase from Nitrosomonas europaea, has been determined to 1.9 A resolution. Blue copper oxidase is a trimer, of which each subunit comprises two cupredoxin domains. Each subunit houses a type 1 copper site in domain 1 and a type 2/type 3 trinuclear copper cluster at the subunit-subunit interface. The coordination geometry at the trinuclear copper site is consistent with reduction of the copper ions. Although the overall architecture of blue copper oxidase is similar to nitrite reductases, detailed structural alignments show that the fold and domain orientation more closely resemble the three-domain multicopper oxidases. These observations have important implications for the evolution of nitrite reductases and multicopper oxidases.

  19. Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

    SciTech Connect

    Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

    2012-04-18

    Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.

  20. Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis[C][W

    PubMed Central

    Grienenberger, Etienne; Kim, Sung Soo; Lallemand, Benjamin; Geoffroy, Pierrette; Heintz, Dimitri; Souza, Clarice de Azevedo; Heitz, Thierry; Douglas, Carl J.; Legrand, Michel

    2010-01-01

    The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors. PMID:21193572

  1. Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells.

    PubMed

    Wu, Feng; Schuster, David P; Tyml, Karel; Wilson, John X

    2007-01-01

    The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.

  2. Three-dimensional organization of three-domain copper oxidases: A review

    NASA Astrophysics Data System (ADS)

    Zhukhlistova, N. E.; Zhukova, Yu. N.; Lyashenko, A. V.; Zaĭtsev, V. N.; Mikhaĭlov, A. M.

    2008-01-01

    “Blue” copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrena maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.

  3. Effect of dietary protein and iron on the fractional turnover rate of rat liver xanthine oxidase

    SciTech Connect

    Cherry, D.M.; Amy, N.K.

    1987-12-01

    Rat liver xanthine oxidase activity is regulated in response to dietary protein and iron. To investigate whether the change in activity was mediated by a change in the rate of protein degradation, we measured the fractional turnover rate using the double-isotope technique with (/sup 3/H)- and (/sup 14/C)leucine and calculated the apparent half-life of xanthine oxidase in rats fed diets containing either 20 or 5% casein with either 35 or 5 mg iron/kg diet. Under control conditions, xanthine oxidase had an apparent half-life of 4.8 d and approximately 65% of the enzyme subunits were active. Rats fed diets with low dietary protein had lower xanthine oxidase activity, but the enzyme had a slower fractional turnover rate, resulting in an apparent half-life of 6.4 d, and only 15-20% of the enzyme was active. The apparent half-life of xanthine oxidase increased to 7.5 d in rats fed diets with low dietary iron, but dietary iron did not affect the specific activity of the enzyme or the percentage of active subunits. These results suggest that the loss of enzyme activity is not due to loss of enzyme protein by increased degradation, but rather to inactivation of the enzyme.

  4. Single mutations that redirect internal proton transfer in the ba3 oxidase from Thermus thermophilus.

    PubMed

    Smirnova, Irina; Chang, Hsin-Yang; von Ballmoos, Christoph; Ädelroth, Pia; Gennis, Robert B; Brzezinski, Peter

    2013-10-08

    The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa3-type oxidases proton uptake to the catalytic site and "pump site" occurs simultaneously. However, with ba3 oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than that with the aa3 oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba3 CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though ba3 CytcO uses only a single proton pathway for transfer of the substrate and "pumped" protons, the amino-acid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site.

  5. Cloning and Analysis of the Alternative Oxidase Gene of Neurospora Crassa

    PubMed Central

    Li, Q.; Ritzel, R. G.; McLean, LLT.; McIntosh, L.; Ko, T.; Bertrand, H.; Nargang, F. E.

    1996-01-01

    Mitochondria of Neurospora crassa contain a cyanide-resistant alternative respiratory pathway in addition to the cytochrome pathway. The alternative oxidase is present only when electron flow through the cytochrome chain is restricted. Both genomic and cDNA copies for the alternative oxidase gene have been isolated and analyzed. The sequence of the predicted protein is homologous to that of other species. The mRNA for the alternative oxidase is scarce in wild-type cultures grown under normal conditions, but it is abundant in cultures grown in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis, or in mutants deficient in mitochondrial cytochromes. Thus, induction of alternative oxidase appears to be at the transcriptional level. Restriction fragment length polymorphism mapping of the isolated gene demonstrated that it is located in a position corresponding to the aod-1 locus. Sequence analysis of mutant aod-1 alleles reveals mutations affecting the coding sequence of the alternative oxidase. The level of aod-1 mRNA in an aod-2 mutant strain that had been grown in the presence of chloramphenicol was reduced several fold relative to wild-type, supporting the hypothesis that the product of aod-2 is required for optimal expression of aod-1. PMID:8770590

  6. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  7. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    PubMed

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device.

  8. The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa.

    PubMed

    Parr, S R; Barber, D; Greenwood, C; Brunori, M

    1977-11-01

    A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.

  9. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding.

    PubMed

    Liko, Idlir; Degiacomi, Matteo T; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V

    2016-07-19

    Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance.

  10. Amyloid-β peptide binds to cytochrome C oxidase subunit 1.

    PubMed

    Hernandez-Zimbron, Luis Fernando; Luna-Muñoz, Jose; Mena, Raul; Vazquez-Ramirez, Ricardo; Kubli-Garfias, Carlos; Cribbs, David H; Manoutcharian, Karen; Gevorkian, Goar

    2012-01-01

    Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1-42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.

  11. A purification procedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa.

    PubMed

    Parr, S R; Barber, D; Greenwood, C

    1976-08-01

    The production of the soluble cytochrome oxidase/nitrite reductase in the bacterium Pseudomonas aeruginosa is favoured by anaerobic conditions and the presence of KNO3(20g/l) in the culture medium. Of three methods commonly used for the disruption of bacterial suspensions (ultrasonication, liquid-shear homogenization and glass-bead grinding), sonication proved the most efficient in releasing the Pseudomonas cytochrome oxidase. A polarographic assay of Pseudomonas cytochrome oxidase activity with sodium ascorbate as substrate and NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride as electron mediator is described. A purification procedure was developed which can be used on the small scale (40-litre cultures) or the large scale (400-litre cultures) and provides high yields of three respiratory-chain proteins, Pseudomonas cytochrome oxidase, cytochrome c551 and azurin, in a pure state. A typical preparation of 250g of Ps.aeruginosa cell paste yielded 180mg of Pseudomonas cytochrome oxidase, 81 mg of Pseudomonas cytochrome c551 and 275mg of Pseudomonas azurin.

  12. Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME.

    PubMed

    Golbeck, J H; Cammarata, K V

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14.18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.Sonication releases polyphenol oxidase from the membrane largely in the latent state. C(18) fatty acids, especially linolenic acid, are potent activators of the enzymic activity. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time.Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. The K(m) values for 3,4-dihydroxyphenylalanine and O(2) are 6.5 and 0.065 millimolar, respectively. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K(m) A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  13. Excess boron reduces polyphenol oxidase activities in embryo and endosperm of maize seed during germination.

    PubMed

    Olçer, Hillya; Kocaçaliskan, Ismail

    2007-01-01

    The effects of increasing concentrations of boron (0, 0.1, 1, 10 and 20 mM) as boric acid on the rate of germination and polyphenol oxidase activities in embryo and endosperm tissues of maize seeds (Zea mays L. cv. Arifiye) were studied. The germination percentage of maize seeds was not affected by boron concentrations up to 10 mM, and decreased by 20 mM. Distilled water and lower boron concentrations (0.1 and 1 mM) increased polyphenol oxidase activities at the beginning of germination up to 12 h whereas its excess levels (10 and 20 mM) decreased polyphenol oxidase activities in embryos and endosperm during germination. Polyphenol oxidase activities with o-diphenolic substrates (caffeic acid, catechol and dopa) were found to be higher than with a monophenolic substrat (tyrosine) in both embryos and endosperms. Further, caffeic acid oxidizing polyphenol oxidase was found to show more activity in embryos of the seeds germinating in distilled water when compared to other substrates.

  14. In vitro evaluation of selected benzimidazole derivatives as an antioxidant and xanthine oxidase inhibitors.

    PubMed

    Nile, Shivraj H; Kumar, Brajesh; Park, Se W

    2013-09-01

    2-Aryl-1-arylmethyl-1H-benzimidazole derivatives having different side chains on the structure were examined in vitro for their antioxidant abilities by 2,2-diphenyl-1-picryl hydrazine radical scavenging activity, reducing ability, OH radical scavenging activity, inhibition of polyphenol oxidase and xanthine oxidase. Overall, with few exceptions, all the 2-aryl-1-arylmethyl-1H-benzimidazoles showed moderate biological activity with all parameters examined. The 2-aryl-1-arylmethyl-1H-benzimidazoles were found to be reactive toward 2,2-diphenyl-1-picryl hydrazine radical and had considerable reducing ability, with significant xanthine oxidase inhibition. With few exceptions, all the compounds under study were found to possess moderate-to-poor OH radical scavenging activity and inhibited polyphenol oxidase significantly. These findings suggest that these 2-aryl-1-arylmethyl-1H-benzimidazoles can be considered as potential antioxidant and xanthine oxidase inhibitory agents, those might be further, explored for the design of lead antioxidant and antigout drug candidates using in vivo trials.

  15. Three-dimensional organization of three-domain copper oxidases: A review

    SciTech Connect

    Zhukhlistova, N. E. Zhukova, Yu. N.; Lyashenko, A. V.; Zaitsev, V. N.; Mikhailov, A. M.

    2008-01-15

    'Blue' copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrena maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.

  16. NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys

    PubMed Central

    Peck, Ammon B.; Khan, Saeed R.

    2013-01-01

    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. PMID:23840917

  17. Timing of electron and proton transfer in the ba(3) cytochrome c oxidase from Thermus thermophilus.

    PubMed

    von Ballmoos, Christoph; Lachmann, Peter; Gennis, Robert B; Ädelroth, Pia; Brzezinski, Peter

    2012-06-05

    Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O(2) to H(2)O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba(3) oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O(2) reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H(+)/electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1 H(+)/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba(3) oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a "proton-loading site" (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases.

  18. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-08

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  19. Shall we dance? How a multicopper oxidase chooses its electron transfer partner.

    PubMed

    Quintanar, Liliana; Stoj, Christopher; Taylor, Alexander B; Hart, P John; Kosman, Daniel J; Solomon, Edward I

    2007-06-01

    Multicopper oxidases (MCOs) are encoded in the genomes of Eukarya, Bacteria, and Archea. These proteins are unique in that they contain at least four Cu atom prosthetic groups organized into one each of the three spectral classifications of copper sites in biology: type 1 (T1), type 2 (T2), and binuclear type 3 (T3), where the T2 and T3 sites form a trinuclear Cu cluster. With these four redox-active copper sites, the multicopper oxidases catalyze the four-electron (4e(-)) reduction of dioxygen to 2H2O, an activity that they alone share with the terminal heme-containing oxidases. Most MCOs exhibit broad specificity towards organic reductants, while a relatively small number of family members exhibit equally robust activity towards metal ions like Fe(II), Cu(I), and Mn(II) and, thus, are considered metallo-oxidases. This Account analyzes the structure-activity features of multicopper oxidases that determine their relative substrate specificity. Since the substrate oxidation step involves an outer-sphere electron transfer from the reductant to the T1Cu site in the protein, the concepts of Marcus theory are applied to unravel the origin of the substrate specificity of the multicopper ferroxidases.

  20. Fluorescence quenching study of quercetin interaction with bovine milk xanthine oxidase

    NASA Astrophysics Data System (ADS)

    Rasoulzadeh, Farzaneh; Jabary, Hamideh Nadjarpour; Naseri, Abdolhossein; Rashidi, Mohammad-Reza

    2009-02-01

    Quercetin is a natural flavonoid with many important therapeutic properties. The interaction of this polyphenolic compound bovine milk xanthine oxidase as one of its major target proteins was studied using fluorescence quenching method for the first time. It was found that the fluorescence quenching of xanthine oxidase occurs through a static mechanism. The results revealed the presence of a single binding site on xanthine oxidase with the binding constant value equals to 1.153 × 10 4 l mol -1 at 310 K and pH 7.4. The thermodynamic parameters were also calculated at different temperatures. The enthalpy and entropy changes were found as -10.661 kJ mol -1 and +43.321 J mol -1 K -1 indicating that both hydrogen binding and hydrophobic are involved in the interaction of this polyphenolic natural compound with xanthine oxidase. The results may provide a ground for further studies with different flavonoids to find a safe alternative for allopurinol, the only xanthine oxidase inhibitor with clinical application.

  1. Spinach thylakoid polyphenol oxidase isolation, activation, and properties of the native chloroplast enzyme

    SciTech Connect

    Golbeck, J.H.; Cammarata, K.V.

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  2. Induction of the d-Amino Acid Oxidase from Trigonopsis variabilis

    PubMed Central

    Horner, R.; Wagner, F.; Fischer, L.

    1996-01-01

    Induction of the d-amino acid oxidase (EC. 1.4.3.3) from the yeast Trigonopsis variabilis was investigated by using a minimal medium containing glucose as the carbon and energy source, (NH(inf4))(inf2)SO(inf4) as the nitrogen source, and various d- and dl-amino acid derivatives as inducers. The best new inducers found were N-carbamoyl-d-alanine, N-acetyl-d-tryptophan, and N-chloroacetyl-d-(alpha)-aminobutyric acid; when the induction effects of these compounds were compared with the effects of d-alanine as the nitrogen source and inducer, the resulting activities of d-amino acid oxidase per gram of dried yeast were 4.2, 2.1, and 1.5 times higher, respectively. The optimum concentration of the best inducer, N-carbamoyl-d-alanine, was 5 mM. This inducer could also be used in its racemic form. The induction was pH dependent. After cultivation of the yeast in a 50-liter bioreactor, d-amino acid oxidase activity of about 3,850 (mu)kat (231,000 U) was obtained. In addition, production of the d-amino acid oxidase was found to be significantly dependent on the metal salt composition of the medium. Addition of zinc ions was required to obtain high d-amino acid oxidase levels in the cells. The optimum concentration of ZnSO(inf4) was about 140 (mu)M. PMID:16535339

  3. Amyloid-β Peptide Binds to Cytochrome C Oxidase Subunit 1

    PubMed Central

    Hernandez-Zimbron, Luis Fernando; Luna-Muñoz, Jose; Mena, Raul; Vazquez-Ramirez, Ricardo; Kubli-Garfias, Carlos; Cribbs, David H.; Manoutcharian, Karen; Gevorkian, Goar

    2012-01-01

    Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1–42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1–42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1–42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1–42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1–42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD. PMID:22927926

  4. Targeting and Regulation of Reactive Oxygen Species Generation by Nox Family NADPH Oxidases

    PubMed Central

    Morand, Stanislas; Hurt, Darrell; Ueyama, Takehiko

    2009-01-01

    Abstract Nox family NADPH oxidases serve a variety of functions requiring reactive oxygen species (ROS) generation, including antimicrobial defense, biosynthetic processes, oxygen sensing, and redox-based cellular signaling. We explored targeting, assembly, and activation of several Nox family oxidases, since ROS production appears to be regulated both spatially and temporally. Nox1 and Nox3 are similar to the phagocytic (Nox2-based) oxidase, functioning as multicomponent superoxide-generating enzymes. Factors regulating their activities include cytosolic activator and organizer proteins and GTP-Rac. Their regulation varies, with the following rank order: Nox2 > Nox1 > Nox3. Determinants of subcellular targeting include: (a) formation of Nox-p22phox heterodimeric complexes allowing plasma membrane translocation, (b) phospholipids-binding specificities of PX domain-containing organizer proteins (p47phox or Nox organizer 1 (Noxo1 and p40phox), and (c) variably splicing of Noxo1 PX domains directing them to nuclear or plasma membranes. Dual oxidases (Duox1 and Duox2) are targeted by different mechanisms. Plasma membrane targeting results in H2O2 release, not superoxide, to support extracellular peroxidases. Human Duox1 and Duox2 have no demonstrable peroxidase activity, despite their extensive homology with heme peroxidases. The dual oxidases were reconstituted by Duox activator 2 (Duoxa2) or two Duoxa1 variants, which dictate maturation, subcellular localization, and the type of ROS generated by forming stable complexes with Duox. Antioxid Redox Signal. 11, 2607–2619. PMID:19438290

  5. Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility.

    PubMed

    Thipyapong, Piyada; Hunt, Michelle D; Steffens, John C

    2004-11-01

    Polyphenol oxidases (PPOs; EC 1.14.18.1 or EC 1.10.3.2) catalyze the oxidation of phenolics to quinones, highly reactive intermediates whose secondary reactions are responsible for much of the oxidative browning that accompanies plant senescence, wounding, and responses to pathogens. To assess the impact of PPO expression on resistance to Pseudomonas syringae pv. tomato we introduced a chimeric antisense potato PPO cDNA into tomato (Lycopersicon esculentum L.). Oxidation of caffeic acid, the dominant o-diphenolic aglycone of tomato foliage, was decreased ca. 40-fold by antisense expression of PPO. All members of the PPO gene family were downregulated: neither immunoreactive PPO nor PPO-specific mRNA were detectable in the transgenic plants. In addition, the antisense PPO construct suppressed inducible increases in PPO activity. Downregulation of PPO in antisense plants did not affect growth, development, or reproduction of greenhouse-grown plants. However, antisense PPO expression dramatically increased susceptibility to P. syringae expressing the avirulence gene avrPto in both Pto and pto backgrounds. In a compatible (pto) interaction, plants constitutively expressing an antisense PPO construct exhibited a 55-fold increase in bacterial growth, three times larger lesion area, and ten times more lesions cm(-2) than nontransformed plants. In an incompatible (Pto) interaction, antisense PPO plants exhibited 100-fold increases in bacterial growth and ten times more lesions cm(-2) than nontransformed plants. Although it is not clear whether hypersusceptibility of antisense plants is due to low constitutive PPO levels or failure to induce PPO upon infection, these findings suggest a critical role for PPO-catalyzed phenolic oxidation in limiting disease development. As a preliminary effort to understand the role of induced PPO in limiting disease development, we also examined the response of PPO promoter::beta-glucuronidase constructs when plants are challenged with P

  6. Functional analysis of polyphenol oxidases by antisense/sense technology.

    PubMed

    Thipyapong, Piyada; Stout, Michael J; Attajarusit, Jutharat

    2007-07-27

    Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting

  7. Xanthine oxidase and xanthine dehydrogenase from an estivating land snail.

    PubMed

    Hermes-Lima, M; Storey, K B

    1995-01-01

    During arousal from estivation in land snails, Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transition from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (XO) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and some properties of XO and XO+XDH (sum of XO and xanthine dehydrogenase activities) were measured in hepatopancreas extracts. Km values for pterin for XO and XO+XDH were 9 and 6 microM, respectively, and the Km of XDH for methylene blue was 5 microM. Both XO+XDH and XO activities were inhibited by allopurinol (I50 = 2 microM), pre-incubation at 40 degrees C, and by 5 min H2O2 pre-exposure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H2O2 due to inhibition of high endogenous catalase activity. The I50 for H2O2 of XO+XDH and XO activities in the presence of azide was 0.04 and 0.11 mM, respectively. Unlike the situation for mammalian XO, a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H2O2 effects. Interestingly, methylene blue partially prevented both heat- and H2O2-induced inactivation of XO+XDH activity. These data indicate that the formation of an enzyme-methylene blue complex induces protection against heat and oxidative damage at the FAD-active site. Both XO and XO+XDH activites were significantly higher in snails after 35 days of estivation compared with active snails 24 h after arousal from dormancy. The ratio of XO/(XO+XDH) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nmol.min-1.mg protein-1 in estivating snails. Compared with hepatopancreas catalase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and

  8. Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst.

    PubMed

    Akard, L P; English, D; Gabig, T G

    1988-07-01

    The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N-ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N-ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was