Science.gov

Sample records for acyl-coa oxidase aco

  1. EXPRESSION OF TURKEY TRANSCRIPTION FACTORS AND ACYL COA OXIDASE IN DIFFERENT TISSUES AND GENETIC POPULATIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several transcription factors are involved in regulating lipid metabolism in various animal tissues. Peroxisome proliferator activated receptor (PPAR) gamma and PPAR alpha regulate both lipogenesis and fatty acid oxidation. Gene fragments for PPAR gamma, PPAR alpha, and acyl CoA oxidase (ACO) have b...

  2. 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

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

    PubMed Central

    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

    2014-01-01

    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. PMID:25001221

  4. An ACC Oxidase Gene Essential for Cucumber Carpel Development.

    PubMed

    Chen, Huiming; Sun, Jinjing; Li, Shuai; Cui, Qingzhi; Zhang, Huimin; Xin, Fengjiao; Wang, Huaisong; Lin, Tao; Gao, Dongli; Wang, Shenhao; Li, Xia; Wang, Donghui; Zhang, Zhonghua; Xu, Zhihong; Huang, Sanwen

    2016-09-01

    Sex determination in plants gives rise to unisexual flowers that facilitate outcrossing and enhance genetic diversity. In cucumber and melon, ethylene promotes carpel development and arrests stamen development. Five sex-determination genes have been identified, including four encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase that catalyzes the rate-limiting step in ethylene biosynthesis, and a transcription factor gene CmWIP1 that corresponds to the Mendelian locus gynoecious in melon and is a negative regulator of femaleness. ACC oxidase (ACO) converts ACC into ethylene; however, it remains elusive which ACO gene in the cucumber genome is critical for sex determination and how CmWIP1 represses development of female flowers. In this study, we discovered that mutation in an ACO gene, CsACO2, confers androecy in cucumber that bears only male flowers. The mutation disrupts the enzymatic activity of CsACO2, resulting in 50% less ethylene emission from shoot tips. CsACO2 was expressed in the carpel primordia and its expression overlapped with that of CsACS11 in female flowers at key stages for sex determination, presumably providing sufficient ethylene required for proper CsACS2 expression. CmACO3, the ortholog of CsACO2, showed a similar expression pattern in the carpel region, suggesting a conserved function of CsACO2/CmACO3. We demonstrated that CsWIP1, the ortholog of CmWIP1, could directly bind the promoter of CsACO2 and repress its expression. Taken together, we propose a presumably conserved regulatory module consisting of WIP1 transcription factor and ACO controls unisexual flower development in cucumber and melon. PMID:27403533

  5. Characteristics of Rural Accountable Care Organizations (ACOs) – A Survey of Medicare ACOs with Rural Presence.

    PubMed

    Salako, Abiodun; Zhu, Xi; MacKinney, A Clinton; Ullrich, Fred; Mueller, Keith

    2015-05-01

    Accountable Care Organizations (ACOs) are groups of health care providers, principally physicians and hospitals, who develop a new entity that contracts to provide coordinated care to assigned patients with the goal of improving quality of care while controlling costs. Section 3022 of the Patient Protection and Affordable Care Act of 2010 created the Medicare Shared Savings Program (SSP). The Centers for Medicare & Medicaid Services (CMS) implements this program and has approved SSP contracts in five cycles since 2011, including some that participated in a special demonstration project that provided advance payment (as a forgivable loan). A new ACO Investment Model (AIM) program starts in 2015 that provides initial investment capital and variable monthly payments to ACO participants in rural and underserved areas who may not have access to the capital needed for successful ACO formation and operation. CMS also contracted with 32 organizations under a special demonstration project, "Pioneer ACOs" (as of November 16, 2014, there were 19 remaining).8 At the time of the research reported in this brief, there were 455 Medicare ACOs (Pioneer and SSP). While there is growing literature about ACOs, much remains to be learned about ACO development in rural areas. A previous RUPRI Center policy brief 2 examined the formation of four rural ACOs. The authors found that prior experience with risk sharing and provider integration facilitated ACO formation. This brief expands on the earlier brief by describing the findings of a survey of 27 rural ACOs, focusing on characteristics important to their formation and operation. Prospective rural ACO participants can draw from the experiences of predecessors, and the survey findings can inform policy discussions about ACO formation and operation. Key Findings from 27 Respondents. (1) Sixteen rural ACOs were formed by pre-existing integrated delivery networks. (2) Physician groups played a more prominent role than other participant

  6. 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. PMID:27587453

  7. Postharvest Analysis of Lowland Transgenic Tomato Fruits Harboring hpRNAi-ACO1 Construct

    PubMed Central

    Behboodian, Bita; Mohd Ali, Zainon; Ismail, Ismanizan; Zainal, Zamri

    2012-01-01

    The plant hormone, ethylene, is an important regulator which involved in regulating fruit ripening and flower senescence. In this study, RNA interference (RNAi) technology was employed to silence the genes involved in ethylene biosynthetic pathway. This was achieved by blocking the expression of specific gene encoding the ACC oxidase. Initially, cDNA corresponding to ACO1 of lowland tomato cultivar (MT1), which has high identity with ACO1 of Solanum lycopersicum in GenBank, was cloned through RT-PCR. Using a partial coding region of ACO1, one hpRNAi transformation vector was constructed and expressed ectopically under the 35S promoter. Results showed that transgenic lines harboring the hpRNA-ACO1 construct had lower ethylene production and a longer shelf life of 32 days as compared to 10 days for wild-type fruits. Changes in cell wall degrading enzyme activities were also investigated in cases where the transgenic fruits exhibited reduced rates of firmness loss, which can be associated with a decrease in pectin methylesterase (PME) and polygalacturonase (PG) activities. However, no significant change was detected in both transgenic and wild-type fruits in terms of β-galactosidase (β-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid. PMID:22919320

  8. Super ACO FEL oscillation at 300 nm

    NASA Astrophysics Data System (ADS)

    Nutarelli, D.; Garzella, D.; Renault, E.; Nahon, L.; Couprie, M. E.

    2000-05-01

    Some recent improvements, involving both the optical cavity mirrors and the positron beam dynamics in the storage ring, have allowed us to achieve a laser oscillation at 300 nm on the Super ACO Storage Ring FEL. The Super ACO storage ring is operated at 800 MeV which is the nominal energy for the usual synchrotron radiation users, and the highest energy for a storage ring FEL. The lasing at 300 nm could be kept during 2 h per injection, with a stored current ranging between 30 and 60 mA. The FEL characteristics are presented here. The longitudinal stability and the FEL optics behaviour are also discussed.

  9. BROWN ADIPOSE TISSUE FUNCTION IN SHORT-CHAIN ACYL-COA DEHYDROGENASE DEFICIENT MICE

    PubMed Central

    Skilling, Helen; Coen, Paul M.; Fairfull, Liane; Ferrell, Robert E.; Goodpaster, Bret H.; Vockley, Jerry; Goetzman, Eric S.

    2010-01-01

    Brown adipose tissue is a highly specialized organ that uses mitochondrial fatty acid oxidation to fuel nonshivering thermogenesis. In mice, mutations in the acyl-CoA dehydrogenase family of fatty acid oxidation genes are associated with sensitivity to cold. Brown adipose tissue function has not previously been characterized in these knockout strains. Short-chain acyl-CoA dehydrogenase (SCAD) deficient mice were found to have increased brown adipose tissue mass as well as modest cardiac hypertrophy. Uncoupling protein-1 was reduced by 70% in brown adipose tissue and this was not due to a change in mitochondrial number, nor was it due to decreased signal transduction through protein kinase A which is known to be a major regulator of uncoupling protein-1 expression. PKA activity and in vitro lipolysis were normal in brown adipose tissue, although in white adipose tissue a modest increase in basal lipolysis was seen in SCAD−/ − mice. Finally, an in vivo norepinephrine challenge of brown adipose tissue thermogenesis revealed normal heat production in SCAD−/− mice. These results suggest that reduced brown adipose tissue function is not the major factor causing cold sensitivity in acyl-CoA dehydrogenase knockout strains. We speculate that other mechanisms such as shivering capacity, cardiac function, and reduced hepatic glycogen stores are involved. PMID:20727852

  10. 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...

  11. 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. PMID:26450153

  12. A Compilation of Redshifts and Velocity Dispersions for ACO Clusters

    NASA Astrophysics Data System (ADS)

    Struble, Mitchell F.; Rood, Herbert J.

    1999-11-01

    We present a compilation of redshifts for 1572 Abell, Corwin, & Olowin (ACO) clusters, referenced to both the heliocentric and cosmic background radiation reference frames, and 395 velocity dispersions corrected to the reference frame of the cluster, available from the literature as of 1998 December. We present an additional list of 81 ACO clusters with published redshifts which are probably those of galaxies or groups superimposed on, or near, the ACO cluster position.

  13. A fifth member of the tomato 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene family harbours a leucine zipper and is anaerobically induced.

    PubMed

    Sell, Simone; Hehl, Reinhard

    2005-02-01

    Using the leucine zipper domain of a small anaerobically induced bZIP transcription factor in a yeast two hybrid screen, anaerobically induced genes were identified. One peptide corresponds to an anaerobically induced IDS4-like protein that maybe involved in G-protein signaling. Surprisingly, another interacting peptide corresponds to a novel anaerobically induced 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, designated ACO5. ACO5 harbours a leucine zipper and transcription is mainly induced in fruits and to a lesser extend in leaves. The role of ACO5 in the low oxygen response of tomato is discussed. PMID:16040352

  14. Orsay free electron laser activities on the storage rings ACO and super-ACO

    NASA Astrophysics Data System (ADS)

    Couprie, M. E.; Bazin, C.; Bergher, M.; Billardon, M.; Elleaume, P.; Ortega, J. M.; Petroff, Y.; Prazeres, R.; Velghe, M.

    1989-10-01

    The main results obtained on the free electron laser and on optical harmonic generation with the storage ring ACO at Orsay in France are reviewed. A specific magnetic device, generally called an undulator, allows one to collect coherent light. In the free electron laser case, the undulator radiation is stored and amplified in an optical cavity. Tunable light was obtained in the visible range. For optical harmonic generation, external laser light is emitted in the undulator area, generating coherent harmonic radiation. Experimentally, 350, 177 and 107 nm wavelengths were obtained with a Nd-YAG laser. New experiments are planned with the Super-ACO storage ring, and preliminary experiments with the optical klystron are given.

  15. Cost-outcomes focus is essential for ACO success.

    PubMed

    Greenspun, Harry; Bercik, William

    2013-02-01

    To succeed under value-based payment, accountable care organizations (ACOs) must be able to link, analyze, and compare clinical and administrative data from across their constituent organizations. ACOs require a precise costing methodology, such as activity-based costing, to be able to manage costs effectively and gain critical insight into which service lines are delivering value from a clinical and financial standpoint. To support informed strategic decision-making, ACOs also require ready access to integrated patient encounter data to be able to perform the sophisticated modeling of predictive analytics. PMID:23413676

  16. Asthma and chronic obstructive pulmonary disease overlap syndrome (ACOS): structured literature review and physician insights

    PubMed Central

    Ding, B.; Enstone, A

    2016-01-01

    ABSTRACT Objectives: To understand the key characteristics of Asthma and Chronic Obstructive Pulmonary Disease Overlap Syndrome (ACOS) and to identify evidence gaps relating to the identification, treatment and management of ACOS patients. Methods: A structured literature review and 1-hour telephone interviews with specialist respiratory physicians were conducted (n=10; China, France, Germany, Japan and the USA). Results: All 10 physicians used the term ACOS in clinical practice. ACOS was not clearly defined in the literature. Prevalence of ACOS among adult patients with COPD or asthma ranged from 12–55%. ACOS patients had severe disease, with increased exacerbations and hospitalisations compared to some asthma and COPD patients. ACOS represented a clinical challenge due to a lack of evidence-based guidelines distinguishing between asthma, COPD and ACOS. Published data quantifying ACOS costs were limited. Conclusions: There is a need for consensus evidence-based guidance to facilitate earlier diagnosis and to optimise the management of ACOS patients. PMID:26789845

  17. Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus.

    PubMed Central

    Priefert, H; Steinbüchel, A

    1992-01-01

    The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC 6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in two transposon-induced mutants of A. eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE. The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source. An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54. Images PMID:1356967

  18. 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

  19. 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

  20. Exogenously induced expression of ethylene biosynthesis, ethylene perception, phospholipase D, and Rboh-oxidase genes in broccoli seedlings

    PubMed Central

    Jakubowicz, Małgorzata; Gałgańska, Hanna; Nowak, Witold; Sadowski, Jan

    2010-01-01

    In higher plants, copper ions, hydrogen peroxide, and cycloheximide have been recognized as very effective inducers of the transcriptional activity of genes encoding the enzymes of the ethylene biosynthesis pathway. In this report, the transcriptional patterns of genes encoding the 1-aminocyclopropane-1-carboxylate synthases (ACSs), 1-aminocyclopropane-1-carboxylate oxidases (ACOs), ETR1, ETR2, and ERS1 ethylene receptors, phospholipase D (PLD)-α1, -α2, -γ1, and -δ, and respiratory burst oxidase homologue (Rboh)-NADPH oxidase-D and -F in response to these inducers in Brassica oleracea etiolated seedlings are shown. ACS1, ACO1, ETR2, PLD-γ1, and RbohD represent genes whose expression was considerably affected by all of the inducers used. The investigations were performed on the seedlings with (i) ethylene insensitivity and (ii) a reduced level of the PLD-derived phosphatidic acid (PA). The general conclusion is that the expression of ACS1, -3, -4, -5, -7, and -11, ACO1, ETR1, ERS1, and ETR2, PLD-γ 1, and RbohD and F genes is undoubtedly under the reciprocal cross-talk of the ethylene and PAPLD signalling routes; both signals affect it in concerted or opposite ways depending on the gene or the type of stimuli. The results of these studies on broccoli seedlings are in agreement with the hypothesis that PA may directly affect the ethylene signal transduction pathway via an inhibitory effect on CTR1 (constitutive triple response 1) activity. PMID:20581125

  1. Characterization of transcriptional profiles of MA-ACS1 and MA-ACO1 genes in response to ethylene, auxin, wounding, cold and different photoperiods during ripening in banana fruit.

    PubMed

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2008-12-01

    The ripening-specific genes MA-ACS1 (Musa acuminata ACC synthase1) and MA-ACO1 (M. acuminata ACC oxidase 1) are regulated in response to a wide variety of factors. Here, we have studied the differential transcript accumulation pattern and protein levels of MA-ACS1 and MA-ACO1 genes in response to ethylene, auxin, wounding and low temperature in preclimacteric banana fruit. We have shown that exogenous application of ethylene and auxin induced the expression of MA-ACS1, while MA-ACO1 showed marginal expression following ethylene treatment in preclimacteric stage. Auxin did not induce MA-ACO1 expression. Thus, auxin-treated banana fruits showed lower ethylene production rate as compared to ethylene-treated fruits. Conversely, wounding and cold treatment down-regulated the expression of both the genes and thus inhibited ethylene production. Furthermore, we have detected a GCC-box putative ethylene-responsive element (ERE)- and an auxin-responsive element (ARE)-specific DNA-binding activity in the banana pulp and studied the ethylene and auxin responsive characteristics of the GCC-box and ARE (TGTCTC) containing synthetic promoter fragments. In addition, we have detected an enhanced ethylene production rate and expression level of MA-ACS1 and MA-ACO1 genes along with a strong GCC-box-specific DNA-binding activity following exposure to constant dark period for 8d at the preclimacteric stage. Together, our study provides interesting information about the regulation of expression of MA-ACS1 and MA-ACO1 genes in response to various factors during ripening in banana fruit, which may have physiological relevance concerning ethylene biosynthesis during post-harvest conditions. PMID:18554749

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

    PubMed Central

    Lewis, Valerie A.; Tierney, Emily; Muhlestein, David B.

    2016-01-01

    Relationships between physicians and hospitals have changed considerably over the past decade as hospitals and physician groups integrate and new public and private payment policies create 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 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 2015, and hospitals that were in urban areas, non-profit, or had a smaller share of Medicare patients were more likely to participate in ACOs compared to other ACOs. Qualitative data identified several 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 ACOs that include hospitals (63 percent of ACOs) offered more comprehensive services compared to ACOs without hospitals, we found no differences among ACOs in their ability to manage hospital-related aspects of patient care. PMID:26953297

  3. 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. PMID:24889945

  4. SamACO: variable sampling ant colony optimization algorithm for continuous optimization.

    PubMed

    Hu, Xiao-Min; Zhang, Jun; Chung, Henry Shu-Hung; Li, Yun; Liu, Ou

    2010-12-01

    An ant colony optimization (ACO) algorithm offers algorithmic techniques for optimization by simulating the foraging behavior of a group of ants to perform incremental solution constructions and to realize a pheromone laying-and-following mechanism. Although ACO is first designed for solving discrete (combinatorial) optimization problems, the ACO procedure is also applicable to continuous optimization. This paper presents a new way of extending ACO to solving continuous optimization problems by focusing on continuous variable sampling as a key to transforming ACO from discrete optimization to continuous optimization. The proposed SamACO algorithm consists of three major steps, i.e., the generation of candidate variable values for selection, the ants' solution construction, and the pheromone update process. The distinct characteristics of SamACO are the cooperation of a novel sampling method for discretizing the continuous search space and an efficient incremental solution construction method based on the sampled values. The performance of SamACO is tested using continuous numerical functions with unimodal and multimodal features. Compared with some state-of-the-art algorithms, including traditional ant-based algorithms and representative computational intelligence algorithms for continuous optimization, the performance of SamACO is seen competitive and promising. PMID:20371409

  5. On the molecular relationships between high-zinc tolerance and aconitase (Aco1) in Saccharomyces cerevisiae.

    PubMed

    Guirola, Maria; Jiménez-Martí, Elena; Atrian, Silvia

    2014-03-01

    Zinc is an essential metal for all organisms, as it participates in the structure and/or function of many proteins. However, zinc excess is as deleterious to cells as zinc deficiency. A genome-wide study of the transcriptomic response to high zinc in S. cerevisiae performed in our laboratory allowed the identification of a zinc hyper-tolerant deletion mutant (pif1Δ), which lacks the Pif1 DNA helicase. Further molecular characterization of this strain phenotype revealed that the lack of Pif1 leads to increased iron accumulation, redistribution of the aconitase protein to mitochondria, and also a loss of aconitase activity, despite normal Aco1 protein levels being present, probably due to the epistasis in protecting mtDNA between PIF1 and ACO1. The results presented in this work focus now on the characterization of different features related to the Aco1 protein and activity in yeast and the tolerance to high zinc. Hence, multiple phenotypic traits related to metal metabolism, namely Aco1 protein content and activity levels, succinate dehydrogenase activity, citrate levels, metal content, BPS influence in cultures, and the range of transcription of some iron metabolism related genes, have been analyzed for several strains, some of them constructed to this end, including BY4741, the deletants pif1Δ and aco1Δ, and the aco1 mutants aco1Δ-d4, aco1-C448S, aco1-R476S and aco1-R668S. Overall, lack of Aco1 enzymatic activity in mitochondria, citrate accumulation and lack of activity of [Fe-S] enzymes, e.g. succinate dehydrogenase, appear to be direct molecular indicators of increased zinc tolerance in S. cerevisiae. PMID:24503898

  6. Energy spread measurements on the ACO storage ring

    NASA Astrophysics Data System (ADS)

    Ortega, J. M.; Elleaume, P.; Billardon, M.; Deacon, D.; Girard, B.; Lapierre, Y.

    1985-06-01

    Absolute and relative energy spread measurements have been made on the ACO storage ring using two different methods. The first one consists of a spectral analysis of the electron bunch length based on the proportionality of the longitudinal bunch length versus energy spread. The second one constitutes a direct measurement using the on axis synchrotron radiation emitted by an optical klystron. The two measurement techniques present a very high signal/noise ratio allowing time resolved records. Examples are presented of the energy spread time evolution in the FEL oscillator and of harmonic generation experiments.

  7. 42 CFR 425.214 - Managing changes to the ACO during the agreement.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Managing changes to the ACO during the agreement. 425.214 Section 425.214 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH... Application Procedures and Participation Agreement § 425.214 Managing changes to the ACO during the...

  8. 42 CFR 425.214 - Managing changes to the ACO during the agreement.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 3 2014-10-01 2014-10-01 false Managing changes to the ACO during the agreement. 425.214 Section 425.214 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH... Application Procedures and Participation Agreement § 425.214 Managing changes to the ACO during the...

  9. 42 CFR 425.214 - Managing changes to the ACO during the agreement.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Managing changes to the ACO during the agreement. 425.214 Section 425.214 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH... Application Procedures and Participation Agreement § 425.214 Managing changes to the ACO during the...

  10. 42 CFR 425.220 - Termination of an agreement by the ACO.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Termination of an agreement by the ACO. 425.220 Section 425.220 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN...) Payment consequences of early termination. The ACO will not share in any savings for the performance...

  11. 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

  12. ACOs: time for ED managers to get involved, shape how their departments will add value.

    PubMed

    2011-06-01

    Even with a proposed rule outlining how accountable care organizations (ACO) will be structured under fee-for-service Medicare, emergency medicine experts are concerned about how ACOs will impact patient access to the ED. Further, some see a clear need to beef-up case management staff, while others say ED managers need to get involved with ACO-development now so their interests and concerns are represented. Under a proposed rule unveiled by the Department of Health and Human Services, a handful of quality indicators will require the tracking of ED visits related to certain ambulatory care-sensitive conditions; experts say it could be a challenge for ED staff to differentiate between appropriate and inappropriate use of the ED. EDs that figure out how to contribute value under the ACO mission will be better positioned to prosper under the new model. The ACO model is likely to add considerable complexity to billing and reimbursement. PMID:21675150

  13. CHARACTERISTICS OF POLYPHENOL OXIDASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO, EC 1.14.18.1 or EC 1.10.3.1) catalyzes the oxidation of o-diphenols to o-quinones. Highly reactive o-quinones couple with phenolics and specific amino acids on proteins to form the characteristic browning products in many wounded fruits, vegetables, and leaf tissues of plant...

  14. Physicians' Participation In ACOs Is Lower In Places With Vulnerable Populations Than In More Affluent Communities.

    PubMed

    Yasaitis, Laura C; Pajerowski, William; Polsky, Daniel; Werner, Rachel M

    2016-08-01

    Early evidence suggested that accountable care organizations (ACOs) could improve health care quality while constraining costs, and ACOs are expanding throughout the United States. However, if disadvantaged patients have unequal access to physicians who participate in ACOs, that expansion may exacerbate health care disparities. We examined the relationship between physicians' participation in both Medicare and commercial ACOs across the country and the sociodemographic characteristics of their likely patient populations. Physicians' participation in ACOs varied widely across hospital referral regions, from nearly 0 percent to over 85 percent. After we adjusted for individual physician and practice characteristics, we found that physicians who practiced in ZIP Code Tabulation Areas where a higher percentage of the population was black, living in poverty, uninsured, or disabled or had less than a high school education-compared to other areas-had significantly lower rates of ACO participation than other physicians. Our findings suggest that vulnerable populations' access to physicians participating in ACOs may not be as great as access for other groups, which could exacerbate existing disparities in health care quality. PMID:27503961

  15. 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. PMID:26953297

  16. 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

    2015-05-01

    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. PMID:25969323

  17. MOEA/D-ACO: a multiobjective evolutionary algorithm using decomposition and AntColony.

    PubMed

    Ke, Liangjun; Zhang, Qingfu; Battiti, Roberto

    2013-12-01

    Combining ant colony optimization (ACO) and the multiobjective evolutionary algorithm (EA) based on decomposition (MOEA/D), this paper proposes a multiobjective EA, i.e., MOEA/D-ACO. Following other MOEA/D-like algorithms, MOEA/D-ACO decomposes a multiobjective optimization problem into a number of single-objective optimization problems. Each ant (i.e., agent) is responsible for solving one subproblem. All the ants are divided into a few groups, and each ant has several neighboring ants. An ant group maintains a pheromone matrix, and an individual ant has a heuristic information matrix. During the search, each ant also records the best solution found so far for its subproblem. To construct a new solution, an ant combines information from its group's pheromone matrix, its own heuristic information matrix, and its current solution. An ant checks the new solutions constructed by itself and its neighbors, and updates its current solution if it has found a better one in terms of its own objective. Extensive experiments have been conducted in this paper to study and compare MOEA/D-ACO with other algorithms on two sets of test problems. On the multiobjective 0-1 knapsack problem,MOEA/D-ACO outperforms the MOEA/D with conventional genetic operators and local search on all the nine test instances. We also demonstrate that the heuristic information matrices in MOEA/D-ACO are crucial to the good performance of MOEA/D-ACO for the knapsack problem. On the biobjective traveling salesman problem, MOEA/D-ACO performs much better than the BicriterionAnt on all the 12 test instances. We also evaluate the effects of grouping, neighborhood, and the location information of current solutions on the performance of MOEA/D-ACO. The work in this paper shows that reactive search optimization scheme, i.e., the "learning while optimizing" principle, is effective in improving multiobjective optimization algorithms. PMID:23757576

  18. 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. PMID:26062323

  19. Asthma-COPD Overlap Syndrome (ACOS): Single disease entity or not? Could exhaled nitric oxide be a useful biomarker for the differentiation of ACOS, asthma and COPD?

    PubMed

    Karampitsakos, Theodoros; Gourgoulianis, Konstantinos I

    2016-06-01

    Asthma and chronic obstructive pulmonary disease (COPD) represent two major public health problems. However, there is a significant proportion of patients with a mixed asthma-COPD phenotype. This condition is defined as asthma-COPD overlap syndrome (ACOS). Since there are no internationally accepted criteria for the diagnosis of that syndrome, its management remains difficult. Given the fact that patients with ACOS have an increased risk of exacerbation and hospitalization, there is a pressing need for a more targeted approach and better management. We propose that fractional exhaled nitric oxide (FeNO), a marker of eosinophilic inflammation, could help clinicians differentiate ACOS from asthma and COPD. We evaluate this hypothesis, using data derived from the existing literature. PMID:27142135

  20. The Impact Of Medicare ACOs On Improving Integration And Coordination Of Physical And Behavioral Health Care.

    PubMed

    Fullerton, Catherine A; Henke, Rachel M; Crable, Erica; Hohlbauch, Andriana; Cummings, Nicholas

    2016-07-01

    The accountable care organization (ACO) model holds the promise of reducing costs and improving the quality of care by realigning payment incentives to focus on health outcomes instead of service volume. One key to managing the total cost of care is improving care coordination for and treatment of people with behavioral health disorders. We examined qualitative data from ninety organizations participating in Medicare ACO demonstration programs from 2012 through 2015 to determine whether and how they focused on behavioral health care. These ACOs had mixed degrees of engagement in improving behavioral health care for their populations. The biggest challenges included a lack of behavioral health care providers, data availability, and sustainable financing models. Nonetheless, we found substantial interest in integrating behavioral health care into primary care across a majority of the ACOs. PMID:27385242

  1. A Preliminary Validation of the ACOS/GOSAT Xco2 Product Using TCCON Data

    NASA Astrophysics Data System (ADS)

    Wunch, D.; Wennberg, P. O.; Keppel-Aleks, G.; Connor, B. J.; Fisher, B.; Osterman, G. B.; Thompson, D. R.; Oyafuso, F. A.; Toon, G. C.; Roehl, C. M.; Miller, C. E.; O'Dell, C.; Salawitch, R. J.; Crisp, D.; Castano, R.; Mandrake, L.; Sherlock, V.; Deutscher, N. M.; Griffith, D. W.; Macatangay, R.; Rettinger, M.; Sussmann, R.; Messerschmidt, J.; Notholt, J.; Warneke, T.

    2010-12-01

    The JAXA/NIES/MOE Greenhouse gas Observing Satellite (GOSAT) was launched in 2009, carrying its main instrument, the TANSO-FTS (Fourier transform spectrometer), to measure total columns of CO2 (Xco2). The Atmospheric CO2 Observations from Space (ACOS) group, which was formed out of the Orbiting Carbon Observatory (OCO) science team, has worked with the GOSAT Research Announcement (RA) office to retrieve Xco2 values from the GOSAT spectra. We describe a validation technique for the ACOS/GOSAT Xco2 data product with the spatially sparse but precise and accurate Total Carbon Column Observing Network (TCCON). To account for non-coincidence of the GOSAT and TCCON observations, we exploit the strong correlation between free-tropospheric temperature and Xco2. Systematic errors and biases in the ACOS/GOSAT product are described. After correcting for these biases, a more precise and accurate global picture of Xco2 is derived from the ACOS/GOSAT data.

  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. A pheromone-rate-based analysis on the convergence time of ACO algorithm.

    PubMed

    Huang, Han; Wu, Chun-Guo; Hao, Zhi-Feng

    2009-08-01

    Ant colony optimization (ACO) has widely been applied to solve combinatorial optimization problems in recent years. There are few studies, however, on its convergence time, which reflects how many iteration times ACO algorithms spend in converging to the optimal solution. Based on the absorbing Markov chain model, we analyze the ACO convergence time in this paper. First, we present a general result for the estimation of convergence time to reveal the relationship between convergence time and pheromone rate. This general result is then extended to a two-step analysis of the convergence time, which includes the following: 1) the iteration time that the pheromone rate spends on reaching the objective value and 2) the convergence time that is calculated with the objective pheromone rate in expectation. Furthermore, four brief ACO algorithms are investigated by using the proposed theoretical results as case studies. Finally, the conclusions of the case studies that the pheromone rate and its deviation determine the expected convergence time are numerically verified with the experiment results of four one-ant ACO algorithms and four ten-ant ACO algorithms. PMID:19380276

  4. 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

  5. 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. PMID:27149517

  6. Crystallization of Mitochondrial Cytochrome Oxidase

    NASA Astrophysics Data System (ADS)

    Ozawa, Takayuki; Tanaka, Masashi; Wakabayashi, Takashi

    1982-12-01

    Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria. By washing the oxidase with detergent on a hydrophobic interaction column, phospholipids were depleted to the level of 1 mol of cardiolipin per mol of heme a. Hydrophobic impurities and partially denatured oxidase were separated from the intact oxidase on an affinity column with cytochrome c as the specific ligand. The final preparation of the oxidase contained seven distinct polypeptides. The molecular weight of the oxidase was estimated to be 130,000 from its specific heme a and copper content and from the subunit composition. Crystals of the oxidase were obtained by slow removal of the detergent from the buffer in which the oxidase was dissolved. The needle-shaped crystals were 100 μ m in average length and 5 μ m in width, and they strongly polarized visible light. Electron diffraction patterns were obtained with an unstained glutaraldehyde-fixed single crystal by electron microscopy using 1,000-kV electrons. From electron micrographs and the diffraction patterns of the crystal, it was concluded that the crystal is monoclinic in the space group P21, with unit cell dimensions a = 92 angstrom, b = 84 angstrom, and c = 103 angstrom, and α =β 90 degrees, γ = 126 degrees.

  7. Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

    PubMed

    Qi, Jing; Dong, Zhen; Zhang, Yu-Xing

    2015-12-01

    The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti‑sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti‑sense RNA technology in the genetic improvement of pears and other fruit. PMID:26460204

  8. 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.

  9. Effectiveness analysis of ACOS-Xco2 bias correction method with GEOS-Chem model results

    NASA Astrophysics Data System (ADS)

    Liu, Da; Lei, Liping; Liu, Min; Guo, Lijie; Wang, Qian; Bie, Nian

    2015-08-01

    Satellite observations and model simulations are of two important data sources to study atmospheric carbon dioxide concentration. For analyzing and evaluating the bias correction method of ACOS dry-air column averaged CO2 (Xco2) product, the GEOS-Chem Xco2 simulations are selected according to observing time and locations of the ACOS product. The GEOS-Chem simulations of CO2 profiles are transformed to Xco2 data by convolving with satellite averaging kernels and pressure weighting functions. The GEOS-Chem Xco2 data are then compared with both bias uncorrected and bias corrected satellite retrievals of ACOS. The comparisons show that the bias uncorrected ACOS retrievals are on average 1.12ppm higher than the model Xco2 data, while the corrected ACOS retrievals are only on average 0.06ppm lower than the model Xco2 data. By assuming consistency between model Xco2 simulations and true atmospheric Xco2, this study indicates that the bias can be obvious decreased through the bias correction method, and the correction is effective and necessary for satellite Xco2 retrievals.

  10. NADPH Oxidase and Neurodegeneration

    PubMed Central

    Hernandes, Marina S; Britto, Luiz R G

    2012-01-01

    NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases. PMID:23730256

  11. Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

    PubMed Central

    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. PMID:23762027

  12. DISTINCT TRANSCRIPTIONAL REGULATION OF LONG-CHAIN ACYL-COA SYNTHETASE ISOFORMS AND CYTOSOLIC THIOESTERASE 1 IN THE RODENT HEART BY FATTY ACIDS AND INSULIN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanism(s) responsible for channeling long-chain fatty acids (LCFAs) into oxidative versus nonoxidative pathways is (are) poorly understood in the heart. Intracellular LCFAs are converted to long-chain fatty acyl-CoAs (LCFA-CoAs) by a family of long-chain acyl-CoA synthetases (ACSLs)...

  13. A NOVEL 78-KDA FATTY ACYL-COA SYNTHETASE (ACS1) OF BABESIA BOVIS STIMULATES MEMORY CD4+ T LYMPHOCYTE RESPONSES IN B. BOVIS-IMMUNE CATTLE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antigen-specific CD4+ T lymphocyte responses contribute to protective immunity against Babesia bovis, however the antigens that induce these responses remain largely unknown. A proteomic approach was used to identify novel B. bovis antigens recognized by memory CD4+ T cells from immune cattle. Fract...

  14. Production of VUV coherent light by harmonic generation with the optical klystron of super-ACO

    NASA Astrophysics Data System (ADS)

    Prazeres, R.; Guyot-Sionnest, P.; Ortega, J. M.; Billardon, M.; Jaroszynski, D.

    1991-04-01

    The aim of the experiment presented was to develop a usable source of coherent light in the vacuum ultraviolet (VUV) range (200-50 nm) using the Super-ACO storage ring. A presentation is made of the most recent results where coherent photons have been produced at the 3rd (177 nm) and 5th (106 nm) harmonics of the incident laser. The better performance of the new machine and the laser, compared to the older ACO, is largely offset by experimental difficulties. The number of photons is, however. larger than that obtained with the ACO, but it is approximately one order of magnitude below the expected rate. This seems to be due to positron beam instabilities. The authors believe that the extremely critical alignment of the whole experiment, from the injection laser to the monochromator, does not appear to be compatible with a user facility.

  15. Asthma and COPD Overlap Syndrome (ACOS): A Systematic Review and Meta Analysis

    PubMed Central

    Alshabanat, A.

    2015-01-01

    Background The combination of asthma and chronic obstructive pulmonary disease (COPD), or ACOS is a recently defined syndrome. The epidemiology of the condition is poorly described and previous research has suggested ACOS is associated with worse outcomes than either condition alone. We therefore decided to complete a systematic review of the published literature. Methods This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta- Analyses guidelines. A structured search was performed in the PubMed, Embase, and Medline databases up to Feb 2015 to identify studies reporting incidence, prevalence, health care utilization, morbidity, or mortality in COPD and asthma. Results A total of 19 studies were included in the present study. The pooled prevalence of overlap among COPD was 27% (95% CI: 0.16–0.38, p<0.0001) and 28% (95% CI: 0.09–0.47, p = 0.0032) in the population and hospital-based studies, respectively. We found no significant difference between ACOS and COPD in terms of gender, smoking status, lung function and 6mWD. However, in comparison to subject with only COPD, ACOS subjects were significantly younger, had higher BMI, healthcare utilization, and lower HRQoL. Conclusion ACOS is a common condition that exists in a substantial proportion of subjects with COPD. ACOS represents a distinct clinical phenotype with more frequent exacerbations, hospitalization, worse health-related quality of life, and higher healthcare costs than either disease alone. There is a critical need to better define the management and treatment of this syndrome. PMID:26336076

  16. Few ACOs pursue innovative models that integrate care for mental illness and substance abuse with primary care.

    PubMed

    Lewis, Valerie A; Colla, Carrie H; Tierney, Katherine; Van Citters, Arica D; Fisher, Elliott S; Meara, Ellen

    2014-10-01

    Accountable care organizations (ACOs) may be well positioned to increase the focus on managing behavioral health conditions (mental health and substance abuse) through the integration of behavioral health treatment and primary care. We used a mixed-methods research design to examine the extent to which ACOs are clinically, organizationally, and financially integrating behavioral health care and primary care. We used data from 257 respondents to the National Survey of Accountable Care Organizations, a nationally representative survey of ACOs. The data were supplemented with semistructured, in-depth interviews with clinical leaders at sixteen ACOs purposively sampled to represent the spectrum of behavioral health integration. We found that most ACOs hold responsibility for some behavioral health care costs, and 42 percent include behavioral health specialists among their providers. However, integration of behavioral health care and primary care remains low, with most ACOs pursuing traditional fragmented approaches to physical and behavioral health care and only a minority implementing innovative models. Contract design and contextual factors appear to influence the extent to which ACOs integrate behavioral health care. Nevertheless, the ACO model has the potential to create opportunities for improving behavioral health care and integrating it with primary care. PMID:25288427

  17. An improved adaptive ACO meta heuristic for scheduling multimedia traffic across the 802.11e EDCA

    NASA Astrophysics Data System (ADS)

    Ditze, Michael; Becker, Markus

    2008-01-01

    This paper presents an adaptive near-optimal scheduler for multimedia traffic for the 802.11e Enhanced Distributed Channel Access (EDCA) medium access control scheme. The scheduler exploits the ant colony optimization (ACO) meta heuristic to tackle the challenge of packet scheduling. ACO is a biologically inspired algorithm that is known to find near-optimal solutions for combinatorial optimization problems. Thus, we expect that ACO scheduling produces more efficient schedules than comparable deterministic scheduling approaches at the expenses of a computational overhead it introduces. We compare ACO scheduling relevant deterministic scheduling approaches, and in particular the MLLF scheduler that is specifically designed for the needs of compressed multimedia applications. The purpose of the evaluation is twofold. It allows to draw conclusions on the feasibility of ACO scheduling for multimedia traffic while it serves as a benchmark to determine to what extent deterministic schedulers fall short of a near-optimal solution.

  18. 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. PMID:26463088

  19. Analysis of Carotenoid Isomerase Activity in a Prototypical Carotenoid Cleavage Enzyme, Apocarotenoid Oxygenase (ACO)*

    PubMed Central

    Sui, Xuewu; Kiser, Philip D.; Che, Tao; Carey, Paul R.; Golczak, Marcin; Shi, Wuxian; von Lintig, Johannes; Palczewski, Krzysztof

    2014-01-01

    Carotenoid cleavage enzymes (CCEs) constitute a group of evolutionarily related proteins that metabolize a variety of carotenoid and non-carotenoid substrates. Typically, these enzymes utilize a non-heme iron center to oxidatively cleave a carbon-carbon double bond of a carotenoid substrate. Some members also isomerize specific double bonds in their substrates to yield cis-apocarotenoid products. The apocarotenoid oxygenase from Synechocystis has been hypothesized to represent one such member of this latter category of CCEs. Here, we developed a novel expression and purification protocol that enabled production of soluble, native ACO in quantities sufficient for high resolution structural and spectroscopic investigation of its catalytic mechanism. High performance liquid chromatography and Raman spectroscopy revealed that ACO exclusively formed all-trans products. We also found that linear polyoxyethylene detergents previously used for ACO crystallization strongly inhibited the apocarotenoid oxygenase activity of the enzyme. We crystallized the native enzyme in the absence of apocarotenoid substrate and found electron density in the active site that was similar in appearance to the density previously attributed to a di-cis-apocarotenoid intermediate. Our results clearly demonstrated that ACO is in fact a non-isomerizing member of the CCE family. These results indicate that careful selection of detergent is critical for the success of structural studies aimed at elucidating structures of CCE-carotenoid/retinoid complexes. PMID:24648526

  20. Results of the first phase of the ACO storage ring laser experiment

    NASA Astrophysics Data System (ADS)

    Bazin, C.; Velghe, M.; Billardon, M.; Ortega, J. M.; Deacon, D. A. G.; Madey, J. M. J.; Robinson, K. E.; Elleaume, P.; Farge, Y.; Petroff, Y.

    A Superconducting undulator has been installed on the ACO storage ring and operated as a synchrotron radiation source, and as a free electron amplifier. Measurements of spontaneous emission spectrum, the gain as a function of the electron energy, and the laser induced lengthening of the electron bunch are reported.

  1. 42 CFR 425.500 - Measures to assess the quality of care furnished by an ACO.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 3 2012-10-01 2012-10-01 false Measures to assess the quality of care furnished by an ACO. 425.500 Section 425.500 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) MEDICARE SHARED SAVINGS PROGRAM Quality Performance Standards and Reporting...

  2. 42 CFR 425.500 - Measures to assess the quality of care furnished by an ACO.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 3 2014-10-01 2014-10-01 false Measures to assess the quality of care furnished by an ACO. 425.500 Section 425.500 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) MEDICARE SHARED SAVINGS PROGRAM Quality Performance Standards and Reporting...

  3. 42 CFR 425.502 - Calculating the ACO quality performance score.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... four domains: (i) Patient/care giver experience. (ii) Care coordination/Patient safety. (iii) Preventative health. (iv) At-risk population. (2) To satisfy quality performance requirements for a domain: (i... 42 Public Health 3 2014-10-01 2014-10-01 false Calculating the ACO quality performance score....

  4. 42 CFR 425.502 - Calculating the ACO quality performance score.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...) Patient/care giver experience. (ii) Care coordination/Patient safety. (iii) Preventative health. (iv) At... 42 Public Health 3 2013-10-01 2013-10-01 false Calculating the ACO quality performance score. 425... HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) MEDICARE SHARED SAVINGS PROGRAM...

  5. 42 CFR 425.500 - Measures to assess the quality of care furnished by an ACO.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 3 2013-10-01 2013-10-01 false Measures to assess the quality of care furnished by an ACO. 425.500 Section 425.500 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM (CONTINUED) MEDICARE SHARED SAVINGS PROGRAM Quality Performance Standards and Reporting...

  6. Coupling in cytochrome c oxidase

    PubMed Central

    Kessler, R. J.; Blondin, G. A.; Zande, H. Vande; Haworth, R. A.; Green, D. E.

    1977-01-01

    Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K+ when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome c and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid. Images PMID:198794

  7. 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.

  8. Protoporphyrinogen Oxidase-Inhibiting Herbicides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protoporphyrinogen oxidase-inhibiting herbicides (also referred to as Protox- or PPO-inhibiting herbicides) were commercialized in the 1960s and their market share reached approximately 10% (total herbicide active ingredient output) in the late 1990’s. The wide-spread adoption of glyphosate-resista...

  9. Betting on change: Tenet deal with Vanguard shows it's primed to try ACO effort, new payment model.

    PubMed

    Kutscher, Beth

    2013-07-01

    Tenet Healthcare Corp.'s acquisition of Vanguard Health Systems is a sign the investor-owned chain is willing to take a chance on alternative payment models such as accountable care organizations. There's no certainty that ACOs will deliver the improvements on quality or cost savings, but Vanguard Vice Chairman Keith Pitts, left, says his system's Pioneer ACO in Detroit has already achieved some cost savings. PMID:23882720

  10. Super-ACO: Results on a positron low emittance ring (invited)

    NASA Astrophysics Data System (ADS)

    Besson, J. C.; Certain, P.; Dael, A.; Damany, A.; Juan, P.; Labeque, A.; Level, M. P.; Marin, P. C.; Michaut, J.; Monet-Descombey, C.; Nghiem, P.; Sommer, M.; Souchet, R.; Zyngier, H.

    1989-07-01

    The dedicated VUV radiation source Super-ACO is part of the Orsay synchrotron radiation complex. The construction of this 800-MeV positron storage ring was started in 1982, and the first beam was stored on March 18, 1987. Super-ACO is expected to accommodate up to six undulators. The first, a 3.2-m-long optical klystron has already been operated between 500 and 800 MeV. A 1.3-m-long undulator has been recently installed. The experience gained during the three periods of operation is presented. From March to October 1987 measurements were taken of the machine properties in the low emittance regime, as well as performances in single and multibunch operation. In March 1988 the optical klystron was tested together with the commissioning of two beamlines out of a bending magnet. Starting from May 1988 two undulators and six beamlines progressively operated.

  11. Spontaneous emission of the Super-ACO fel optical klystron domino

    NASA Astrophysics Data System (ADS)

    Couprie, M. E.; Bazin, C.; Billardon, M.; Velghe, M.

    1989-12-01

    New free electron laser experiments are planned on the Orsay storage ring Super-ACO, which has been commisioned in 1987 at LURE (Laboratoire d'Utilisation du Rayonnement Electromagnétique). For this purpose, a new insertion device, DOMINO, was set up on Super-ACO in January 1988; it consists of a permanent magnet optical klystron composed of two undulators separated by a dispersive section; the magnetic gaps can be changed independently. Here, the parameters of the optical klystron are specified, and the main steps of the optimization are described. Then, the effect of the insertion device on the stored beam is discussed. The spontaneous emission of the optical klystron has been observed and analysed. Consequently, with the ring parameters measurements, the free electron laser gain versus the current can be evaluated.

  12. Applications of UV-storage ring free electron lasers: the case of super-ACO

    NASA Astrophysics Data System (ADS)

    Nahon, L.; Renault, E.; Couprie, M. E.; Mérola, F.; Dumas, P.; Marsi, M.; Taleb-Ibrahimi, A.; Nutarelli, D.; Roux, R.; Billardon, M.

    1999-06-01

    The potential of UV-storage ring free electron lasers (SRFELs) for the performance of original application experiments is shown with a special emphasis concerning their combination with the naturally synchronized synchrotron radiation (SR). The first two-color FEL+SR experiment, performed in surface science at Super-ACO is reported. The experimental parameters found to be the most important as gathered from the acquired experience, are underlined and discussed. Finally, future prospects for the scientific program of the Super-ACO FEL are presented with two-color experiments combining the FEL with SR undulator-based XUV and VUV beamlines as well as with a SR white light bending magnet beamline emiting in the IR-UV ( 20 μm- 0.25 μm) .

  13. Chromate reduction by rabbit liver aldehyde oxidase

    SciTech Connect

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

    1986-05-29

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

  14. Acetate utilization is inhibited by benzoate in Alcaligenes eutrophus: evidence for transcriptional control of the expression of acoE coding for acetyl coenzyme A synthetase.

    PubMed Central

    Ampe, F; Lindley, N D

    1995-01-01

    During batch growth of Alcaligenes eutrophus on benzoate-acetate mixtures, benzoate was the preferred substrate, with acetate consumption being delayed until the rate of benzoate consumption had diminished. This effect was attributed to a transcriptional control of the synthesis of acetyl coenzyme A (acetyl-CoA) synthetase, an enzyme necessary for the entry of acetate into the central metabolic pathways, rather than to a biochemical modulation of the activity of this enzyme. Analysis of a 2.4-kb mRNA transcript hybridizing with the A. eutrophus acoE gene confirmed this repression effect. In a benzoate-limited chemostat culture, derepression was observed, with no increase in the level of expression following an acetate pulse. Benzoate itself was not the signal triggering the repression of acetyl-CoA synthetase. This role was played by catechol, which transiently accumulated in the medium when high specific rates of benzoate consumption were reached. The lack of rapid inactivation of the functional acetyl-CoA synthetase after synthesis has been stopped enables A. eutrophus to retain the capacity to metabolize acetate for prolonged periods while conserving minimal protein expenditure. PMID:7592330

  15. Dynamic filling factor in the Super-ACO free electron laser

    NASA Astrophysics Data System (ADS)

    Nutarelli, D.; Garzella, D.; Couprie, M.-E.; Billardon, M.

    1997-02-01

    Here, the Free Electron Laser evolution before the start-up from the spontaneous radiation of an undulator, is considered. This analysis is carried on through the dynamical behaviour of the Filling-factor, representing the transverse overlap between the light pulse and the electron bunch. In such a framework, the gain optimization before the laser start-up is presented and more generally applied to low-gain storage-ring free electron lasers, such as on Super-ACO.

  16. 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.

  17. Urate oxidase: primary structure and evolutionary implications.

    PubMed Central

    Wu, X W; Lee, C C; Muzny, D M; Caskey, C T

    1989-01-01

    Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyzes the oxidation of uric acid to allantoin in most mammals. In humans and certain other primates, however, the enzyme has been lost by some unknown mechanism. To identify the molecular basis for this loss, urate oxidase cDNA clones were isolated from pig, mouse, and baboon, and their DNA sequences were determined. The mouse urate oxidase open reading frame encodes a 303-amino acid polypeptide, while the pig and baboon urate oxidase cDNAs encode a 304-amino acid polypeptide due to a single codon deletion/insertion event. The authenticity of this single additional codon was confirmed by sequencing the mouse and pig genomic copies of the gene. The urate oxidase sequence contains a domain similar to the type 2 copper binding motif found in other copper binding proteins, suggesting that the copper ion in urate oxidase is coordinated as a type 2 structure. Based upon a comparison of the NH2-terminal peptide and deduced sequences, we propose that the maturation of pig urate oxidase involves the posttranslational cleavage of a six-amino acid peptide. Two nonsense mutations were found in the human urate oxidase gene, which confirms, at the molecular level, that the urate oxidase gene in humans is nonfunctional. The sequence comparisons favor the hypothesis that the loss of urate oxidase in humans is due to a sudden mutational event rather than a progressive mutational process. Images PMID:2594778

  18. 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

  19. Incorporation of copper into lysyl oxidase.

    PubMed

    Kosonen, T; Uriu-Hare, J Y; Clegg, M S; Keen, C L; Rucker, R B

    1997-10-01

    Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation. PMID:9355764

  20. Arsenite Oxidase Also Functions as an Antimonite Oxidase

    PubMed Central

    Wang, Qian; Warelow, Thomas P.; Kang, Yoon-Suk; Romano, Christine; Osborne, Thomas H.; Lehr, Corinne R.; Bothner, Brian; McDermott, Timothy R.

    2015-01-01

    Arsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work with Agrobacterium tumefaciens strain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show with in vivo experiments that a mutation in aioA [encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further, in vitro studies with the purified As(III) oxidase from Rhizobium sp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA of A. tumefaciens) provide direct evidence of Sb(III) oxidation but also show a significantly decreased Vmax compared to that of As(III) oxidation. The aioBA genes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereas arsR gene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium. PMID:25576601

  1. Characterization of free-electron laser bunch lengthening on the ACO storage ring

    NASA Astrophysics Data System (ADS)

    Robinson, K. E.; Deacon, D. A. G.; Madey, J. M. J.; Velghe, M. F.; Bazin, C.; Bergher, M.; Petroff, Y.; Billardon, M.; Ortega, J. M.; Elleaume, P.

    1985-01-01

    This paper summarizes the basic low-current, laser-induced bunch-lengthening measurements that have been made on the ACO Storage-Ring Free-Electron Laser (SRFEL). The measurements provide verification of both the functional dependence and absolute magnitude of SRFEL theoretical models. The method of measurement, which is explained, exploits frequency-domain techiques and is capable of accuracies comparable to those of a streak camera. The measurements are in good agreement with existing SRFEL theory and provide an important base for future work.

  2. Applications in biology with the super-ACO FEL and future prospects

    NASA Astrophysics Data System (ADS)

    Couprie, Marie-Emmanuelle; Renault, Eric; Garzella, David; Nutarelli, Daniele; de Ninno, G.; Merola, F.; Nahon, Laurent

    2002-04-01

    The first applications of a storage ring Free Electron Laser started in 1993 on the Super-ACO FEL with the study of the anisotropy decay of a coenzyme, NADH, allowing to understand the thermodynamical equilibrium of the different conformational states of the molecule and their hydrodynamical volume in solution. After this first one- color experiment using the time-resolved fluorescence technique, a transient absorption experiment was developed in which the system is excited with the UV FEL and is probed by Visible-UV absorption using synchrotron radiation. First results on the dynamical behavior were obtained for the acrinide molecule.

  3. Storage ring free electrons laser on super-ACO, ELETTRA and SOLEIL

    NASA Astrophysics Data System (ADS)

    Couprie, M. E.; Brunie, C.; Garzella, D.; Nahon, L.; de Ninno, G.; Nutarelli, D.; Marsi, M.

    2002-06-01

    We present here the state of the art of Storage Ring Free Electron Lasers under operation in Europe. Free electrons Lasers (FELs) are coherent, high power and tuneable radiation sources, obtained by the interaction of an optical wave with a bunch of ultrarelavistic electrons wiggling in a periodical permanent magnetic structure. The developments and the results obtained recently in the UV domain by the FELs operating in Europe on Synchrotron Facilities, Super ACO and ELETTRA, offer new opportunities for using these sources in several scientific domains, and foresee uniques performances for the proposed FEL on the third generation french Synchrotron SOLEIL.

  4. A Comparison of Aerosol Parameterizations in the ACOS XCO2 Retrieval Algorithm

    NASA Astrophysics Data System (ADS)

    Nelson, R. R.; O'Dell, C.; Crisp, D.; Eldering, A.; Frankenberg, C.; Gunson, M. R.; Natraj, V.; Fu, D.

    2014-12-01

    An effective parameterization of clouds and aerosols in retrieval algorithms is essential for reducing measurement errors and biases in estimates of the column-averaged dry-air mole fraction of carbon dioxide (XCO2) from space-based measurements of near-infrared reflected sunlight. The NASA Atmospheric CO2 Observations from Space (ACOS) XCO2 retrieval algorithm has evolved significantly over the past several years in an effort to more accurately represent the impact of clouds and aerosols on XCO2. Recent ACOS algorithm versions up to build 3.4 used a water cloud type, ice cloud type, and two generic aerosol types for each sounding. ACOS build 3.5 uses the same cloud parameterization, but was modified to replace the "one-size-fits-all" aerosol scheme. Build 3.5 uses a monthly aerosol climatology based on the Modern-Era Retrospective Analysis for Research and Applications (MERRA) reanalysis to choose the two most likely aerosol types for a given measurement location, along with typical optical depths. The five MERRA types available for selection are sulfate, dust, sea salt, organic carbon, and black carbon. The algorithm then uses a pre-assigned Gaussian width and height and fits for the aerosol amount and peak height based on information from the 760 nm O2 A-band and the CO2 bands centered near 1610 and 2060 nm. Here we compare ACOS builds 3.4 and build 3.5 to quantify the impact of the aerosol scheme update. Two types of tests were performed. Simulated Orbiting Carbon Observatory 2 (OCO-2) retrievals and their associated aerosol and cloud profiles were compared to the "true" aerosol and cloud profiles used to create the simulated environment for a given measurement. The retrieval algorithms were also run on Greenhouse gases Observing SATellite (GOSAT) observations and compared to AErosol RObotic NETwork (AERONET) aerosol optical depth measurements in order to quantify the ability of the algorithms to retrieve information about aerosol optical depths. XCO2 errors

  5. Studies on the Mechanism of Aldehyde Oxidase and Xanthine Oxidase

    PubMed Central

    Alfaro, Joshua F.

    2009-01-01

    DFT calculations support a concerted mechanism for xanthine oxidase and aldehyde oxidase hydride displacement from the sp2 carbon of 6-substituted 4-quinazolinones. The variations in transition state structure show that C-O bond formation is nearly complete in the transition state and the transition state changes are anti-Hammond with the C-H and C-O bond lengths being more product-like for the faster reactions. The C-O bond length in the transition state is around 90% formed. However, the C-H bond is only about 80% broken. This leads to a very tetrahedral transition state with an O-C-N angle of 109 degrees. Thus, while the mechanism is concerted, the anti-bonding orbital of the C-H bond that is broken is not directly attacked by the nucleophile and instead hydride displacement occurs after almost complete tetrahedral transition state formation. In support of this the C=N bond is lengthened in the transition state indicating that attack on the electrophilic carbon occurs by addition to the C=N bond with negative charge increasing on the nitrogen. Differences in experimental reaction rates are accurately reproduced by these calculations, and tend to support this mechanism. PMID:18998731

  6. Studies on the mechanism of aldehyde oxidase and xanthine oxidase.

    PubMed

    Alfaro, Joshua F; Jones, Jeffrey P

    2008-12-01

    DFT calculations support a concerted mechanism for xanthine oxidase and aldehyde oxidase hydride displacement from the sp(2) carbon of 6-substituted 4-quinazolinones. The variations in transition state structure show that C-O bond formation is nearly complete in the transition state and the transition state changes are anti-Hammond with the C-H and C-O bond lengths being more product-like for the faster reactions. The C-O bond length in the transition state is around 90% formed. However, the C-H bond is only about 80% broken. This leads to a very tetrahedral transition state with an O-C-N angle of 109 degrees. Thus, while the mechanism is concerted, the antibonding orbital of the C-H bond that is broken is not directly attacked by the nucleophile and instead hydride displacement occurs after almost complete tetrahedral transition state formation. In support of this the C=N bond is lengthened in the transition state indicating that attack on the electrophilic carbon occurs by addition to the C=N bond with negative charge increasing on the nitrogen. Differences in experimental reaction rates are accurately reproduced by these calculations and tend to support this mechanism. PMID:18998731

  7. 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

  8. 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.

  9. 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

  10. Implementing storage rings free electron lasers for users on synchrotron radiation facilities: from Super-ACO to SOLEIL

    NASA Astrophysics Data System (ADS)

    Couprie, M. E.; Nutarelli, D.; Billardon, M.

    1998-09-01

    Storage Ring Free Electron Laser (SRFEL) sources can be implemented on synchrotron radiation facilities. Although in the beginning an additional experiment on the accelerator requires specific operating conditions as on Super-ACO at Orsay (France), they can now be conceived as an integral part of the project, providing coherent picosecond tunable light in the UV-VUV range, synchronized with synchrotron radiation for the scientific community, as on the SOLEIL project. Third generation storage ring beam characteristics are discussed in terms of synchrotron radiation and FEL optimization. FEL performances are presented, showing the improvement between the Super-ACO and the SOLEIL cases, including stability issues.

  11. Transient absorption spectroscopy using the super-ACO storage ring FEL

    NASA Astrophysics Data System (ADS)

    Renault, Eric; Nahon, Laurent; Nutarelli, Daniele; Garzella, David; Merola, F.; Couprie, Marie-Emmanuelle

    2000-04-01

    The Super-ACO storage ring FEL is operating with a high average power in the UV range (300 mW at 350 nm), and recently at wavelengths down to 300 nm. In addition this source exhibits high stability and long lifetime which makes it a unique tool for user applications. The coupling of the FEL with other synchrotron based sources (bending magnet and undulator) opens many unexplored possibilities for various types of two-color time-resolved spectroscopies. Presently, we are developing a two-color experiment where we study the sub-nanosecond time-resolved absorption of different chromophoric compounds. In this type of pump-probe experiments, the intense UV pulse of the Super-ACO FEL is used to prepare a high initial concentration of chromophores in their first singlet electronic excited state. The nearby bending magnet synchrotron radiation provides on the other hand a pulsed, white light continuum ranging from UV to IR, which is naturally synchronized with the FEL pulses and can be used to probe the photochemical subsequent events and the transient species. With a dye molecule (POPOP), we have obtained a two-color effect which demonstrates the feasibility of the experiment in terms of flux. Applications on various chromophores of biological interest are planned.

  12. The super-ACO free electron laser source in the UV and its applications

    NASA Astrophysics Data System (ADS)

    De Ninno, G.; Nutarelli, D.; Garzella, D.; Nahon, L.; Hirsch, M.; Renault, E.; Couprie, M. E.

    2001-06-01

    Free electron lasers (FELs) are new sources of tuneable coherent radiation, based on the interaction of a relativistic electron beam with a permanent magnetic field. The Super-ACO FEL operates in the UV (down to 300 nm) at 800 MeV, the nominal energy of the electron beam, with a high repetition rate (8 MHz) . It presents a high average extracted power (up to 300 mW), short pulses (15-50 ps FWHM) and small bandwidth (3×10 -2 nm) . Taking advantage of these characteristics, we demonstrated for the first time the possibility of using a storage ring FEL as a coherent source of radiation for scientific applications. In particular, the tuneable Super-ACO FEL source, combined with synchrotron radiation covering the X-ray to infra-red range, is a unique tool for the time-dependent studies of excited states. Such analysis benefits from the natural synchronisation of both sources at a high repetition rate, their mutual tunability, high intensity and coherence. Several experimental set-ups are now under operation.

  13. RRM analysis of protoporphyrinogen oxidase.

    PubMed

    Sauren, M; Pirogova, E; Cosic, I

    2004-12-01

    Enzymes are crucial in accelerating metabolic reactions in living organisms. Protoporphyrinogen oxidase (PpOI) is an enzyme that catalyses the production of protoporphyrin IX (PpIX), a protein used in a cancer treatment known as photodynamic therapy (PDT). In this study, a structure-function analysis of PpOI was carried out using the Resonant Recognition Model (RRM), a physico-mathematical approach for analysis of proteins interactions. This method is based on the finding that the distribution of delocalised electron energies along the protein plays a crucial role in determining the protein's biological activity. Two digital signal processing (DSP) methods were used: Fourier Transform (FT) and Continuous Wavelet Transform (CWT). Here we have determined the characteristic frequencies and the "hot spot" amino acids, and predicted the location of proteins' active site(s). Several proteins that potentially belong to the PpOI functional group were also analysed to distinguish their viability in this role. PMID:15712584

  14. Crosstalk between mitochondria and NADPH oxidases

    PubMed Central

    Dikalov, Sergey

    2011-01-01

    Reactive oxygen species (ROS) play an important role in physiological and pathological processes. In recent years, a feed-forward regulation of the ROS sources has been reported. The interaction between main cellular sources of ROS, such as mitochondria and NADPH oxidases, however, remain obscure. This work summarizes the latest findings on the role of crosstalk between mitochondria and NADPH oxidases in pathophysiological processes. Mitochondria have the highest levels of antioxidants in the cell and play an important role in the maintenance of cellular redox status, thereby acting as an ROS and redox sink and limiting NADPH oxidase activity. Mitochondria, however, are not only a target for ROS produced by NADPH oxidase but also a significant source of ROS, which under certain condition may stimulate NADPH oxidases. This crosstalk between mitochondria and NADPH oxidases, therefore, may represent a feed-forward vicious cycle of ROS production which can be pharmacologically targeted under conditions of oxidative stress. It has been demonstrated that mitochondria-targeted antioxidants break this vicious cycle, inhibiting ROS production by mitochondria and reducing NADPH oxidase activity. This may provide a novel strategy for treatment of many pathological conditions including aging, atherosclerosis, diabetes, hypertension and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role. It is conceivable that the use of mitochondria-targeted treatments would be effective in these conditions. PMID:21777669

  15. Structural Insights into Sulfite Oxidase Deficiency

    SciTech Connect

    Karakas,E.; Wilson, H.; Graf, T.; Xiang, S.; Jaramillo-Busquets, S.; Rajagopalan, K.; Kisker, C.

    2005-01-01

    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. 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

  17. Optimization of the permanent magnet optical klystron for the SUPER-ACO storage ring free electron laser

    NASA Astrophysics Data System (ADS)

    Couprie, M. E.; Bazin, C.; Billardon, M.

    1989-06-01

    A permanent magnet optical klystron has been optimized for free electron laser experiments and optical harmonic generation on the new storage ring SUPER-ACO at Orsay. The conditions of the optimization and the different steps of the field characterization measurements of this insertion device are discussed. Its effects on the stored beam and the undulator radiation measurements are described.

  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 beginning April 1 or July 1, 2012. 425.608 Section 425.608 Public Health CENTERS FOR MEDICARE & MEDICAID... April 1 or July 1, 2012. (a) For April 1 and July 1, 2012 starters, first year (defined as 21 and...

  19. Human lysyl oxidase-like 2.

    PubMed

    Moon, Hee-Jung; Finney, Joel; Ronnebaum, Trey; Mure, Minae

    2014-12-01

    Lysyl oxidase like-2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises Cu(2+)- and lysine tyrosylquinone (LTQ)-dependent amine oxidases. LOXL2 is proposed to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 has also been proposed to regulate extracellular and intracellular cell signaling pathways. Dysregulation of LOXL2 has been linked to many diseases, including cancer, pro-oncogenic angiogenesis, fibrosis and heart diseases. In this review, we will give an overview of the current understandings and hypotheses regarding the molecular functions of LOXL2. PMID:25146937

  20. NADPH Oxidases and Angiotensin II Receptor Signaling

    PubMed Central

    Garrido, Abel Martin; Griendling, Kathy K.

    2010-01-01

    Over the last decade many studies have demonstrated the importance of reactive oxygen species (ROS) production by NADPH oxidases in angiotensin II (Ang II) signaling, as well as a role for ROS in the development of different diseases in which Ang II is a central component. In this review, we summarize the mechanism of activation of NADPH oxidases by Ang II and describe the molecular targets of ROS in Ang II signaling in the vasculature, kidney and brain. We also discuss the effects of genetic manipulation of NADPH oxidase function on the physiology and pathophysiology of the renin angiotensin system. PMID:19059306

  1. Contributions to GOSAT Data Analysis by the NASA Atmospheric Carbon Observations from Space (ACOS) Team (Invited)

    NASA Astrophysics Data System (ADS)

    Crisp, D.; Atmospheric Carbon Observations From Space (Acos) Team

    2010-12-01

    Ground-based greenhouse gas measurements provide the accuracy and coverage needed to quantify the abundance and rate of change of CO2 on global scales, but still lack the resolution and spatial extent needed to identify and quantify CO2 fluxes on regional scales. One way to address this need is to collect precise, spatially-resolved, global measurements of the column-averaged CO2 dry air mole fraction, XCO2, from space. High precision is essential because CO2 sources and sinks must be inferred from spatial and temporal variations in XCO2, and this quantity typically varies by no more than 1% on regional scales. The NASA Orbiting Carbon Observatory (OCO) and Japanese Greenhouse Gases Observing Satellite (GOSAT) were the first two satellites designed specifically to measure of XCO2. The OCO and GOSAT science teams formed a close collaboration prior to the launch of these two missions to cross calibrate their instruments and cross validate their CO2 retrievals against common standards. Immediately after the loss of the OCO spacecraft, the GOSAT team invited the OCO science team to join their efforts to analyze GOSAT data. NASA responded by reformulating the OCO science team as the Atmospheric Carbon Observations from Space (ACOS) team to (i) meet the NASA’s obligations to its GOSAT partners, (ii) recover some of the science knowledge expected from OCO, and (iii) validate the OCO retrieval algorithms in a realistic environment. The third goal has taken on greater significance since the U.S. Congress authorized a restart of the OCO project, and the President’s 2010 budget proposal included funding to produce a “carbon copy” of OCO that could be ready for launch by February 2013. To address these objectives, the ACOS team has hosted a series of GOSAT vicarious calibration campaigns over Railroad Valley, NV, retrieved global estimates of XCO2 from GOSAT soundings, and validated these XCO2 retrievals against ground-based remote sensing observations by Fourier

  2. Changes in Health Care Spending and Quality for Medicare Beneficiaries Associated with a Commercial ACO Contract

    PubMed Central

    McWilliams, J. Michael; Landon, Bruce E.; Chernew, Michael E.

    2013-01-01

    Importance In a multi-payer system, new payment incentives implemented by one insurer for an accountable care organization (ACO) may affect spending and quality of care for another insurer’s enrollees served by the ACO. Such “spillover” effects reflect the extent of organizational efforts to reform care delivery and can contribute to the total impact of ACOs. Objective We examined whether the Blue Cross Blue Shield (BCBS) of Massachusetts’ Alternative Quality Contract (AQC), an early commercial ACO initiative associated with reduced spending and improved quality for BCBS enrollees, was also associated with changes in spending and quality for Medicare beneficiaries, who were not covered by the AQC. Design and Exposure Quasi-experimental comparisons from 2007–2010 of Medicare beneficiaries served by 11 provider organizations entering the AQC in 2009 or 2010 (intervention group) vs. beneficiaries served by other providers (control group). Using a difference-in-differences approach, we estimated changes in spending and quality for the intervention group in the first and second years of exposure to the AQC relative to concurrent changes for the control group. Regression and propensity-score methods were used to adjust for differences in sociodemographic and clinical characteristics. Participants and Setting Elderly fee-for-service Medicare beneficiaries in Massachusetts (1,761,325 person-years). Main Outcome Measures The primary outcome was total quarterly medical spending per beneficiary. Secondary outcomes included spending by setting and type of service, 5 process measures of quality, potentially avoidable hospitalizations, and 30-day readmissions. Results Before entering the AQC, total quarterly spending for the intervention group was $150 (95% CI, $25–$274) higher than for the control group and rose at a similar rate. In year 2 of the intervention group’s exposure to the AQC, this difference was reduced to $51 (95% CI, −$109–$210; P=0

  3. Regulation of NADPH oxidases in skeletal muscle.

    PubMed

    Ferreira, Leonardo F; Laitano, Orlando

    2016-09-01

    The only known function of NAD(P)H oxidases is to produce reactive oxygen species (ROS). Skeletal muscles express three isoforms of NAD(P)H oxidases (Nox1, Nox2, and Nox4) that have been identified as critical modulators of redox homeostasis. Nox2 acts as the main source of skeletal muscle ROS during contractions, participates in insulin signaling and glucose transport, and mediates the myocyte response to osmotic stress. Nox2 and Nox4 contribute to skeletal muscle abnormalities elicited by angiotensin II, muscular dystrophy, heart failure, and high fat diet. Our review addresses the expression and regulation of NAD(P)H oxidases with emphasis on aspects that are relevant to skeletal muscle. We also summarize: i) the most widely used NAD(P)H oxidases activity assays and inhibitors, and ii) studies that have defined Nox enzymes as protagonists of skeletal muscle redox homeostasis in a variety of health and disease conditions. PMID:27184955

  4. Activation of Polyphenol Oxidase of Chloroplasts 1

    PubMed Central

    Tolbert, N. E.

    1973-01-01

    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 × mg−1 chlorophyll × 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

  5. Heme/copper terminal oxidases

    SciTech Connect

    Ferguson-Miller, S.; Babcock, G.T.

    1996-11-01

    Spatially well-organized electron-transfer reactions in a series of membrane-bound redox proteins form the basis for energy conservation in both photosynthesis and respiration. The membrane-bound nature of the electron-transfer processes is critical, as the free energy made available in exergonic redox chemistry is used to generate transmembrane proton concentration and electrostatic potential gradients. These gradients are subsequently used to drive ATP formation, which provides the immediate energy source for constructive cellular processes. The terminal heme/copper oxidases in respiratory electron-transfer chains illustrate a number of the thermodynamic and structural principles that have driven the development of respiration. This class of enzyme reduces dioxygen to water, thus clearing the respiratory system of low-energy electrons so that sustained electron transfer and free-energy transduction can occur. By using dioxygen as the oxidizing substrate, free-energy production per electron through the chain is substantial, owing to the high reduction potential of O{sub 2} (0.815 V at pH 7). 122 refs.

  6. Proline dehydrogenase (oxidase) in cancer.

    PubMed

    Liu, Wei; Phang, James M

    2012-01-01

    Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the proline degradative pathway, plays a special role in tumorigenesis and tumor development. Proline metabolism catalyzed by PRODH/POX is closely linked with the tricarboxylic acid (TCA) cycle and urea cycle. The proline cycle formed by the interconversion of proline and Δ(1) -pyrroline-5-carboxylate (P5C) between mitochondria and cytosol interlocks with pentose phosphate pathway. Importantly, by catalyzing proline to P5C, PRODH/POX donates electrons into the electron transport chain to generate ROS or ATP. In earlier studies, we found that PRODH/POX functions as a tumor suppressor to initiate apoptosis, inhibit tumor growth, and block the cell cycle, all by ROS signaling. It also suppresses hypoxia inducible factor signaling by increasing α-ketoglutarate. During tumor progression, PRODH/POX is under the control of various tumor-associated factors, such as tumor suppressor p53, inflammatory factor peroxisome proliferator-activated receptor gamma (PPARγ), onco-miRNA miR-23b*, and oncogenic transcription factor c-MYC. Recent studies revealed the two-sided features of PRODH/POX-mediated regulation. Under metabolic stress such as oxygen and glucose deprivation, PRODH/POX can be induced to serve as a tumor survival factor through ATP production or ROS-induced autophagy. The paradoxical roles of PRODH/POX can be understood considering the temporal and spatial context of the tumor. Further studies will provide additional insights into this protein and on its metabolic effects in tumors, which may lead to new therapeutic strategies. PMID:22886911

  7. The composition of milk xanthine oxidase

    PubMed Central

    Hart, L. I.; McGartoll, Mary A.; Chapman, Helen R.; Bray, R. C.

    1970-01-01

    The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed `de-molybdo-xanthine oxidase', and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in `milk microsomes' (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed `inactivated xanthine oxidase', causes variability in activity relative to E450 or to Mo content and formation of it decreases these ratios

  8. NADPH oxidases: new actors in thyroid cancer?

    PubMed

    Ameziane-El-Hassani, Rabii; Schlumberger, Martin; Dupuy, Corinne

    2016-08-01

    Hydrogen peroxide (H2O2) is a crucial substrate for thyroid peroxidase, a key enzyme involved in thyroid hormone synthesis. However, as a potent oxidant, H2O2 might also be responsible for the high level of oxidative DNA damage observed in thyroid tissues, such as DNA base lesions and strand breakages, which promote chromosomal instability and contribute to the development of tumours. Although the role of H2O2 in thyroid hormone synthesis is well established, its precise mechanisms of action in pathological processes are still under investigation. The NADPH oxidase/dual oxidase family are the only oxidoreductases whose primary function is to produce reactive oxygen species. As such, the function and expression of these enzymes are tightly regulated. Thyrocytes express dual oxidase 2, which produces most of the H2O2 for thyroid hormone synthesis. Thyrocytes also express dual oxidase 1 and NADPH oxidase 4, but the roles of these enzymes are still unknown. Here, we review the structure, expression, localization and function of these enzymes. We focus on their potential role in thyroid cancer, which is characterized by increased expression of these enzymes. PMID:27174022

  9. No ROI in ACO. Risk, expense of Medicare program has most for-profit groups shying away.

    PubMed

    Kutscher, Beth

    2012-04-23

    For-profit healthcare companies aren't beating down the door to get in on Medicare ACOs. Several of the big chains say there is too much uncertainty about the experiment, and they're waiting to see how the program evolves. One company on the sideline is Capella Healthcare. Chairman and CEO Daniel Slipkovich, left, says there is a potential for high patient dropout rates and risks in population management. PMID:22571000

  10. 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. PMID:22352172

  11. CYTOCHROME OXIDASE IN NORMAL AND REGENERATING NEURONS

    PubMed Central

    Howe, Howard A.; Mellors, Robert C.

    1945-01-01

    Manometric determinations of cytochrome oxidase activity were carried out on grey matter from the thalamus and anterior horn of cats and monkeys under various experimental conditions. The thalamus of the cat was studied following the degeneration of virtually all the thalamic neurons secondary to decortication. In comparing the deneuronated thalamus with the normal one, it was found that approximately 34 per cent of the cytochrome oxidase activity was contributed by the neurons and the balance by neuroglia and mesodermal tissues which on the operated side remained comparable to that of the normal side. Total activity of the normal thalamus averaged 5.52 units per mg. of dry weight where I unit is defined as the amount of cytochrome oxidase required to produce a net oxygen consumption of 10 c.mm. per hour under the specified conditions of the experiment. The grey matter of the anterior horns of the spinal cord was isolated by a special technique and its cytochrome oxidase activity was compared with anterior horns in which motoneurons had been stimulated to regenerative activity by section of peripheral nerves. Each animal was studied in relation to an anterior horn which was normal and one in which only the functional state of the motoneurons had been changed. Average normal levels of 2.23 units were found for cat anterior horn and 0.69 units for the monkey. Reductions of cytochrome oxidase activity in the range of 22 to 23 per cent were observed for both cat and monkey following nerve section. In the latter the time sequence was carefully studied in relation to the cytological cycle known as chromatolysis and a virus refractory state previously described by us. It was found that maximal reduction of cytochrome oxidase activity coincided with maximal refractoriness of the cells to poliomyelitis virus (30 to 70 days following nerve section). Neither of these states could be correlated in time with maximal chromatolysis (10 to 15 days). PMID:19871471

  12. 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.

  13. 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

  14. Oxidative stress, NADPH oxidases, and arteries.

    PubMed

    Sun, Qi-An; Runge, Marschall S; Madamanchi, Nageswara R

    2016-05-10

    Atherosclerosis and its major complications - myocardial infarction and stroke - remain major causes of death and disability in the United States and world-wide. Indeed, with dramatic increases in obesity and diabetes mellitus, the prevalence and public health impact of cardiovascular diseases (CVD) will likely remain high. Major advances have been made in development of new therapies to reduce the incidence of atherosclerosis and CVD, in particular for treatment of hypercholesterolemia and hypertension. Oxidative stress is the common mechanistic link for many CVD risk factors. However, only recently have the tools existed to study the interface between oxidative stress and CVD in animal models. The most important source of reactive oxygen species (and hence oxidative stress) in vascular cells are the multiple forms of enzymes nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Recently published and emerging studies now clearly establish that: 1) NADPH oxidases are of critical importance in atherosclerosis and hypertension in animal models; 2) given the tissue-specific expression of key components of NADPH oxidase, it may be possible to target vascular oxidative stress for prevention of CVD. PMID:25649240

  15. The substrate tolerance of alcohol oxidases.

    PubMed

    Pickl, Mathias; Fuchs, Michael; Glueck, Silvia M; Faber, Kurt

    2015-08-01

    Alcohols are a rich source of compounds from renewable sources, but they have to be activated in order to allow the modification of their carbon backbone. The latter can be achieved via oxidation to the corresponding aldehydes or ketones. As an alternative to (thermodynamically disfavoured) nicotinamide-dependent alcohol dehydrogenases, alcohol oxidases make use of molecular oxygen but their application is under-represented in synthetic biotransformations. In this review, the mechanism of copper-containing and flavoprotein alcohol oxidases is discussed in view of their ability to accept electronically activated or non-activated alcohols and their propensity towards over-oxidation of aldehydes yielding carboxylic acids. In order to facilitate the selection of the optimal enzyme for a given biocatalytic application, the substrate tolerance of alcohol oxidases is compiled and discussed: Substrates are classified into groups (non-activated prim- and sec-alcohols; activated allylic, cinnamic and benzylic alcohols; hydroxy acids; sugar alcohols; nucleotide alcohols; sterols) together with suitable alcohol oxidases, their microbial source, relative activities and (stereo)selectivities. PMID:26153139

  16. Structure-function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family.

    PubMed

    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

  17. 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

  18. 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.

  19. Monoclonal antibodies to the alternative oxidase of higher plant mitochondria

    SciTech Connect

    Elthon, T.E.; Nickels, R.L.; McIntosh, L. )

    1989-04-01

    The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M{sub r} of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states. AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase.

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

    NASA Astrophysics Data System (ADS)

    Couprie, M. E.; Bakker, R.; Garzella, D.; Nahn, L.; Marsi, M.; Mérola, F.; Hara, T.; Billardon, M.

    1996-02-01

    On the Super-ACO free electron laser were developed the first applications of a storage ring based FEL and of a UV FEL. The feasibility of use was demonstrated in 1993 with a time resolved polarised fluorescence experiment in biology. After these experiments, the stability of the laser source was significantly improved, with the installation of a longitudinal feedback. A particular advantage of a storage ring FEL source is to provide as well naturally synchronized synchrotron radiation. A first pump-probe time-resolved experiment coupling such two tunable, synchronized light sources was then performed in surface physics.

  1. NADPH oxidases in the arbuscular mycorrhizal symbiosis.

    PubMed

    Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

    2016-04-01

    Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules. PMID:27018627

  2. Human copper-dependent amine oxidases.

    PubMed

    Finney, Joel; Moon, Hee-Jung; Ronnebaum, Trey; Lantz, Mason; Mure, Minae

    2014-03-15

    Copper amine oxidases (CAOs) are a class of enzymes that contain Cu(2+) and a tyrosine-derived quinone cofactor, catalyze the conversion of a primary amine functional group to an aldehyde, and generate hydrogen peroxide and ammonia as byproducts. These enzymes can be classified into two non-homologous families: 2,4,5-trihydroxyphenylalanine quinone (TPQ)-dependent CAOs and the lysine tyrosylquinone (LTQ)-dependent lysyl oxidase (LOX) family of proteins. In this review, we will focus on recent developments in the field of research concerning human CAOs and the LOX family of proteins. The aberrant expression of these enzymes is linked to inflammation, fibrosis, tumor metastasis/invasion and other diseases. Consequently, there is a critical need to understand the functions of these proteins at the molecular level, so that strategies targeting these enzymes can be developed to combat human diseases. PMID:24407025

  3. 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.

  4. INCREASED XANTHINE OXIDASE IN THE SKIN OF PREECLAMPTIC WOMEN

    PubMed Central

    Bainbridge, Shannon A.; Deng, Jau-Shyong; Roberts, James M.

    2010-01-01

    Xanthine oxioreductase is the holoenzyme responsible for terminal purine catabolism. Under conditions of metabolic stress or heightened pro-inflammatory cytokine production this enzyme is preferentially in it’s oxidized form, xanthine oxidase, with catalytic action that generates uric acid and the free radical superoxide. As preeclampsia is characterized by heightened inflammation, oxidative stress and hyperuricemia it has been proposed that xanthine oxidase plays a pivotal role in this hypertensive disorder of pregnancy. We sought to determine whether xanthine oxidase protein content was higher in maternal tissue of preeclamptic mothers, compared to healthy pregnant controls, using immunohistochemical analysis of skin biopsies. We further compared xanthine oxidase immunoreactivity in skin biopsies from preeclamptic women and patients with several inflammatory conditions. In preeclamptic women, intense xanthine oxidase immunoreactivity was present within the epidermis. By contrast, only very faint xanthine oxidase staining was observed in skin biopsies from healthy pregnant controls. Further, a role for inflammation in the increase of xanthine oxidase was suggested by similar findings of heightened xanthine oxidase immunoreactivity in the skin biopsies from non-pregnant individuals diagnosed with conditions of systemic inflammation. The finding of increased xanthine oxidase in maternal tissue, most likely as the result of heightened maternal inflammation, suggest maternal xanthine oxidase as a source of free radical and uric acid generation in preeclampsia. PMID:19196876

  5. Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases.

    PubMed

    Zajoncová, L; Frébort, I; Luhová, L; Sebela, M; Galuszka, P; Pec, P

    1999-01-01

    Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed. PMID:10092944

  6. Transient absorption spectroscopy in biology using the Super-ACO storage ring FEL and the synchrotron radiation combination

    NASA Astrophysics Data System (ADS)

    Renault, Eric; Nahon, Laurent; Garzella, David; Nutarelli, Daniele; De Ninno, Giovanni; Hirsch, Matthias; Couprie, Marie Emmanuelle

    2001-12-01

    The Super-ACO storage ring FEL, covering the UV range down to 300 nm with a high average power (300 mW at 350 nm) together with a high stability and long lifetime, is a unique tool for the performance of users applications. We present here the first pump-probe two color experiments on biological species using a storage ring FEL coupled to the synchrotron radiation. The intense UV pulse of the Super-ACO FEL is used to prepare a high initial concentration of chromophores in their first singlet electronic excited state. The nearby bending magnet synchrotron radiation provides, on the other hand a pulsed, white light continuum (UV-IR), naturally synchronized with the FEL pulses and used to probe the photochemical subsequent events and the associated transient species. We have demonstrated the feasibility with a dye molecule (POPOP) observing a two-color effect, signature of excited state absorption and a temporal signature with Acridine. Applications on various chromophores of biological interest are carried out, such as the time-resolved absorption study of the first excited state of Acridine.

  7. Spatial Analyses of a Complete Sample of Rich Abell/ACO Clusters to z=0.14

    NASA Astrophysics Data System (ADS)

    Miller, C. J.

    1999-12-01

    Galaxy clusters act as excellent mass tracers in our local Universe. They are bright, contain hundreds of galaxies, and are easy to observe. The most recognized galaxy cluster catalogs are those of Abell (1958) and Abell, Corwin and Olowin (1989-ACO). I will present the results of a new survey of Abell/ACO cluster redshifts that extends our knowledge of the large-scale mass distribution to a redshift of z = 0.14. The volume of the Universe traced by this survey is four times larger than any of its predecessors. The large number of clusters within the survey boundaries ( 560) as well as the large volume traced, allow for statistical analyses on scales never before probed. I will present the cluster power spectrum to scales approaching 1000h-1Mpc. I will also demonstrate the high-level of completeness for this survey, which allows one to study the effects of large-scale structure on internal cluster properties. I will then summarize some recent collaborative efforts which show that (1) Wide-Angle Tail radio jets are ``blown'' in the direction of the local large-scale axis; (2) High dM/dt cooling flow clusters reside in crowded environments; (3) Clusters with close near-neighbors are also close to the fundamental plane created from Lo, Lx and Ro. CM was funded in part by the National Aeronautics and Space Administration and the Maine Science and Technology Foundation.

  8. Pathological changes in platelet histamine oxidases in atopic eczema

    PubMed Central

    Ionescu, Gruia

    1993-01-01

    Increased plasma histamine levels were associated with significantly lowered diamine and type B monoamine oxidase activities in platelet-rich plasma of atopic eczema (AE) patients. The diamine oxidase has almost normal cofactor levels (pyridoxal phosphate and Cu2+) but the cofactor levels for type B monoamine oxidase (flavin adenine dinucleotide and Fe2+) are lowered. The biogenic amines putrescine, cadaverine, spermidine, spermine, tyramine and serotonin in the sera, as well as dopamine and epinephrine in EDTA-plasma were found to be normal. It is unlikely, therefore, that these amines are responsible for the decreased activities of monoamine and diamine oxidase in these patients. The most likely causative factors for the inhibition of the diamine oxidase are nicotine, alcohol, food additives and other environmental chemicals, or perhaps a genetic defect of the diamine oxidase. PMID:18475554

  9. Gravity Responsive NADH Oxidase of the Plasma Membrane

    NASA Technical Reports Server (NTRS)

    Morre, D. James (Inventor)

    2002-01-01

    A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

  10. An overview on alcohol oxidases and their potential applications.

    PubMed

    Goswami, Pranab; Chinnadayyala, Soma Sekhar R; Chakraborty, Mitun; Kumar, Adepu Kiran; Kakoti, Ankana

    2013-05-01

    Alcohol oxidases (Alcohol: O₂ Oxidoreductase; EC 1.1.3.x) are flavoenzymes that catalyze the oxidation of alcohols to the corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. Based on substrate specificity, alcohol oxidases may be categorized broadly into four different groups namely, (a) short chain alcohol oxidase (SCAO), (b) long chain alcohol oxidase (LCAO), (c) aromatic alcohol oxidase (AAO), and (d) secondary alcohol oxidase (SAO). The sources reported for these enzymes are mostly limited to bacteria, yeast, fungi, plant, insect, and mollusks. However, the quantum of reports for each category of enzymes considerably varies across these sources. The enzymes belonging to SCAO and LCAO are intracellular in nature, whereas AAO and SAO are mostly secreted to the medium. SCAO and LCAO are invariably reported as multimeric proteins with very high holoenzyme molecular masses, but the molecular characteristics of these enzymes are yet to be clearly elucidated. One of the striking features of the alcohol oxidases that make them distinct from the widely known alcohol dehydrogenase is the avidly bound cofactor to the redox center of these enzymes that obviate the need to supplement cofactor during the catalytic reaction. These flavin-based redox enzymes have gained enormous importance in the development of various industrial processes and products primarily for developing biosensors and production of various industrially useful carbonyl compounds. The present review provides an overview on alcohol oxidases from different categories focusing research on these oxidases during the last decade along with their potential industrial applications. PMID:23525937

  11. Xanthine oxidase status in ethanol-intoxicated rat liver.

    PubMed

    Abbondanza, A; Battelli, M G; Soffritti, M; Cessi, C

    1989-12-01

    The status of xanthine oxidase in ethanol-induced liver injury has been investigated in the rat, by acute and chronic ethanol treatments. A 38% increase of the enzyme O-form was observed after repeated ethanol administration. Chronic intoxication caused a significant decrease of total xanthine oxidase activity after both prolonged ethanol feeding and life span ethanol ingestion. The intermediate D/O-form of xanthine oxidase (that can act either as an oxidase or as a dehydrogenase, being able to react with O2 as well as with NAD+ as electron acceptor) increased 5.5-fold after prolonged ethanol feeding. PMID:2690670

  12. 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

  13. NADPH oxidase promotes neutrophil extracellular trap formation in pulmonary aspergillosis.

    PubMed

    Röhm, Marc; Grimm, Melissa J; D'Auria, Anthony C; Almyroudis, Nikolaos G; Segal, Brahm H; Urban, Constantin F

    2014-05-01

    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 (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) 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

  14. 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.

  15. 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

  16. 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

  17. Effect of contraceptive steroids on monoamine oxidase activity

    PubMed Central

    Southgate, Jennifer; Collins, G. G. S.; Pryse-Davies, J.; Sandler, M.

    1969-01-01

    Cyclical variations in monoamine oxidase activity during the human menstrual cycle, specific to the endometrium and modified in women undergoing contraceptive steroid treatment, may reflect changes in hormonal environment. Treatment of rats with individual constituents of the contraceptive pill causes analogous changes: oestrogens inhibit and progestogens potentiate uterine monoamine oxidase activity. ImagesFig. 2Fig. 3

  18. 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. PMID:25340468

  19. Activation of antibacterial autophagy by NADPH oxidases

    PubMed Central

    Huang, Ju; Canadien, Veronica; Lam, Grace Y.; Steinberg, Benjamin E.; Dinauer, Mary C.; Magalhaes, Marco A. O.; Glogauer, Michael; Grinstein, Sergio; Brumell, John H.

    2009-01-01

    Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcγ receptors (FcγRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy. PMID:19339495

  20. CO2 Measurements from Space: Lessons Learned from the Collaboration between the ACOS/OCO-2 and GOSAT Teams

    NASA Astrophysics Data System (ADS)

    Crisp, D.; Eldering, A.; Gunson, M. R.

    2012-12-01

    The NASA Orbiting Carbon Observatory (OCO) and the Japanese Greenhouse gases Observing SATellite (GOSAT) were the first two missions designed to collect space-based observations of the column-averaged CO2 dry air mole fraction, XCO2, with the sensitivity, coverage, and resolution needed to quantify CO2 fluxes on regional scales over the globe. The OCO and GOSAT teams formed a close collaboration during the development phases of these missions. After the loss of OCO, the GOSAT project team invited the OCO team to contribute to the analysis of measurements collected by the GOSAT Thermal And Near infrared Sensor for carbon Observations-Fourier Transform Spectrometer (TANSO-FTS). NASA responded by reformulating the OCO science team under the Atmospheric CO2 Observations from Space (ACOS) task to exploit this opportunity. This collaboration is providing an independent GOSAT XCO2 product, and valuable insights into the retrieval algorithms, calibration methods, and validation techniques that are being developed to analyze data anticipated the NASA Orbiting Carbon Observatory-2 (OCO-2). The ACOS/OCO-2 and GOSAT teams have conducted four, joint, vicarious calibration campaigns at Railroad Valley, Nevada to track the long-term radiometric performance of the TANSO-FTS instrument. The methods used in these campaigns evolved from those used to characterize the radiometric performance of high spatial resolution, imaging spectroradiometers. For TANSO-FTS, the conventional, surface based radiometric measurements have been augmented with surface and aircraft measurements of atmospheric temperature and trace gas profiles, as well as surface observations from MODIS and ASTER to characterize spatial variations of the surface reflectance within the (relatively large) sounding footprints. Similar methods will be needed for OCO-2. The ACOS/OCO-2 retrieval algorithm and associated data screening methods have been modified to estimate XCO2 from TANSO-FTS observations. Comparisons of TANSO

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

    PubMed

    Li, Xiaogang; Zhang, Dong; Li, Jing

    2014-06-01

    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. PMID:24632149

  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. Ascorbic acid and L-gulonolactone oxidase in lagomorphs.

    PubMed

    Jenness, R; Birney, E C; Ayaz, K L

    1978-01-01

    1. The activity of L-gulonolactone oxidase (EC 1.1.3.8) in the liver of eastern cottontail rabbits (Sylvilagus floridanus) is about 10-fold greater in winter than in summer. 2. L-gulonolactone oxidase activity is low and tissue ascorbate high during all seasons in snowshoe hares (Lepus americanus). 3. Liver contents of ascorbate fall to low levels in L. americanus fed on rabbit chow in the laboratory. 4. The activity of L-gulonolactone oxidase in liver of Sylvilagus and Oryctolagus is depressed by feeding high levels of L-ascorbic acid. 5. The New Zealand White breed of domestic rabbit (Oryctolagus cuniculus) has considerably higher levels of L-gulonolactone oxidase and liver ascorbate than does the Dutch breed. 6. In a wild population of Oryctolagus sampled in Australia L-gulonolactone oxidase levels were intermediate between those of the two domestic breeds and more variable than either. PMID:318384

  4. Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus.

    PubMed

    Sakamoto, J; Koga, E; Mizuta, T; Sato, C; Noguchi, S; Sone, N

    1999-04-21

    Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151-158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of alpha-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I (cbdA) encodes 448 amino acid residues with a molecular weight of 50195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II (cbdB) encodes 342 residues with a molecular weight of 38992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a

  5. 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

  6. In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

    PubMed

    Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

    2016-01-01

    In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors. PMID:27409598

  7. NADPH Oxidases in Chronic Liver Diseases

    PubMed Central

    Jiang, Joy X.; Török, Natalie J.

    2015-01-01

    Oxidative stress is a common feature observed in a wide spectrum of chronic liver diseases including viral hepatitis, alcoholic, and nonalcoholic steatohepatitis. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are emerging as major sources of reactive oxygen species (ROS). Several major isoforms are expressed in the liver, including NOX1, NOX2, and NOX4. While the phagocytic NOX2 has been known to play an important role in Kupffer cell and neutrophil phagocytic activity and inflammation, the nonphagocytic NOX homologues are increasingly recognized as key enzymes in oxidative injury and wound healing. In this review, we will summarize the current advances in knowledge on the regulatory pathways of NOX activation, their cellular distribution, and their role in the modulation of redox signaling in liver diseases. PMID:26436133

  8. Degradation of pentachlorophenol by potato polyphenol oxidase.

    PubMed

    Hou, Mei-Fang; Tang, Xiao-Yan; Zhang, Wei-De; Liao, Lin; Wan, Hong-Fu

    2011-11-01

    In this study, polyphenol oxidase (PPO) was extracted from commercial potatoes. Degradation of pentachlorophenol by potato PPO was investigated. The experimental results show that potato PPO is more active in weak acid than in basic condition and that the optimum pH for the reaction is 5.0. The degradation of pentachlorophenol by potato PPO reaches a maximum at 298 K. After reaction for 1 h, the removal of both pentachlorophenol and total organic carbon is >70% with 6.0 units/mL potato PPO at pH 5.0 and 298 K. Pentachlorophenol can be degraded through dechlorination and ring-opening by potato PPO. The work demonstrates that pentachlorophenol can be effectively eliminated by crude potato PPO. PMID:21967325

  9. 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.

  10. Drugs related to monoamine oxidase activity.

    PubMed

    Fišar, Zdeněk

    2016-08-01

    Progress in understanding the role of monoamine neurotransmission in pathophysiology of neuropsychiatric disorders was made after the discovery of the mechanisms of action of psychoactive drugs, including monoamine oxidase (MAO) inhibitors. The increase in monoamine neurotransmitter availability, decrease in hydrogen peroxide production, and neuroprotective effects evoked by MAO inhibitors represent an important approach in the development of new drugs for the treatment of mental disorders and neurodegenerative diseases. New drugs are synthesized by acting as multitarget-directed ligands, with MAO, acetylcholinesterase, and iron chelation as targets. Basic information is summarized in this paper about the drug-induced regulation of monoaminergic systems in the brain, with a focus on MAO inhibition. Desirable effects of MAO inhibition include increased availability of monoamine neurotransmitters, decreased oxidative stress, decreased formation of neurotoxins, induction of pro-survival genes and antiapoptotic factors, and improved mitochondrial functions. PMID:26944656

  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. Modeling dioxygen reduction at multicopper oxidase cathodes.

    PubMed

    Agbo, Peter; Heath, James R; Gray, Harry B

    2014-10-01

    We report a general kinetics model for catalytic dioxygen reduction on multicopper oxidase (MCO) cathodes. Our rate equation combines Butler-Volmer (BV) electrode kinetics and the Michaelis-Menten (MM) formalism for enzymatic catalysis, with the BV model accounting for interfacial electron transfer (ET) between the electrode surface and the MCO type 1 copper site. Extending the principles of MM kinetics to this system produced an analytical expression incorporating the effects of subsequent intramolecular ET and dioxygen binding to the trinuclear copper cluster into the cumulative model. We employed experimental electrochemical data on Thermus thermophilus laccase as benchmarks to validate our model, which we suggest will aid in the design of more efficient MCO cathodes. In addition, we demonstrate the model's utility in determining estimates for both the electronic coupling and average distance between the laccase type-1 active site and the cathode substrate. PMID:25188422

  13. POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5

    PubMed Central

    Liu, Taibo; Wook Kim, Dong; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

    2014-01-01

    POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5–2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5–2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5–2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5. PMID:25763711

  14. POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5.

    PubMed

    Liu, Taibo; Wook Kim, Dong; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

    2014-07-25

    POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5-2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5-2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5-2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5. PMID:25061821

  15. Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity.

    PubMed

    Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Kaku, Masaru; Fong, Keith S K; Csiszar, Katalin; Yamauchi, Mitsuo

    2008-12-01

    Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling. PMID:18835815

  16. Phagocyte NADPH oxidase and specific immunity.

    PubMed

    Cachat, Julien; Deffert, Christine; Hugues, Stephanie; Krause, Karl-Heinz

    2015-05-01

    The phagocyte NADPH oxidase NOX2 produces reactive oxygen species (ROS) and is a well-known player in host defence. However, there is also increasing evidence for a regulatory role of NOX2 in adaptive immunity. Deficiency in phagocyte NADPH oxidase causes chronic granulomatous disease (CGD) in humans, a condition that can also be studied in CGD mice. Clinical observations in CGD patients suggest a higher susceptibility to autoimmune diseases, in particular lupus, idiopathic thrombocytopenic purpura and rheumatoid arthritis. In mice, a strong correlation exists between a polymorphism in a NOX2 subunit and the development of autoimmune arthritis. NOX2 deficiency in mice also favours lupus development. Both CGD patients and CGD mice exhibit increased levels of immunoglobulins, including autoantibodies. Despite these phenotypes suggesting a role for NOX2 in specific immunity, mechanistic explanations for the typical increase of CGD in autoimmune disease and antibody levels are still preliminary. NOX2-dependent ROS generation is well documented for dendritic cells and B-lymphocytes. It is unclear whether T-lymphocytes produce ROS themselves or whether they are exposed to ROS derived from dendritic cells during the process of antigen presentation. ROS are signalling molecules in virtually any cell type, including T- and B-lymphocytes. However, knowledge about the impact of ROS-dependent signalling on T- and B-lymphocyte phenotype and response is still limited. ROS might contribute to Th1/Th2/Th17 cell fate decisions during T-lymphocyte activation and might enhance immunoglobulin production by B-lymphocytes. In dendritic cells, NOX2-derived ROS might be important for antigen processing and cell activation. PMID:25760962

  17. 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

  18. Evaluation of oxalate decarboxylase and oxalate oxidase for industrial applications.

    PubMed

    Cassland, Pierre; Sjöde, Anders; Winestrand, Sandra; Jönsson, Leif J; Nilvebrant, Nils-Olof

    2010-05-01

    Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching. PMID:19763895

  19. 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.

  20. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  1. CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

    PubMed Central

    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

  2. The terminal oxidase in the marine bacterium Pseudomonas nautica 617.

    PubMed

    Simpson, H; Denis, M; Malatesta, F

    1997-06-01

    The molecular properties of a novel membrane quinol oxidase from the marine bacterium Pseudomonas nautica 617 are presented. The protein contains 2b hemes/mole which may be distinguished by EPR spectroscopy but not by optical spectroscopy and electrochemistry. Respiration, though being cyanide insensitive, is not inhibited by carbon monoxide and oxygen reduction is carried out only half-way with production of hydrogen peroxide. The terminal oxidase represents, therefore, a unique example in the large family of terminal oxidases known up to date. PMID:9337488

  3. Identification of yeasts from clinical specimens by oxidase test.

    PubMed

    Kumar, S; Arora, B S; Mathur, M D

    2000-10-01

    A total of 100 yeasts and yeast like fungi isolates from clinical specimens were negative for oxidase production on Sabouraud dextrose agar. When grown on Columbia agar, chocolate agar, tryptose agar, Mueller-Hinton agar, brain heart infusion and a medium resembling Sabouraud's dextrose agar but with starch instead of dextrose, all the isolate of Candida albicans (55), C. guilliermondii (6), C. parapsilosis (14), C. tropicalis (6), C. pseudotropicalis (6) and Crytococcus neoformans (2) were positive for oxidase producation. Torulopsis glabrata (2), Saccharomyces cervisiae (2) and two out of seven isolates of C. krusei were negative for oxidase test. PMID:11344606

  4. Aiding and abetting roles of NOX oxidases in cellular transformation

    PubMed Central

    Block, Karen; Gorin, Yves

    2013-01-01

    NADPH oxidases of the NADPH oxidase (NOX) family are dedicated reactive oxygen species-generating enzymes that broadly and specifically regulate redox-sensitive signalling pathways that are involved in cancer development and progression. They act at specific cellular membranes and microdomains through the activation of oncogenes and the inactivation of tumour suppressor proteins. In this Review, we discuss primary targets and redox-linked signalling systems that are influenced by NOX-derived ROS, and the biological role of NOX oxidases in the aetiology of cancer. PMID:22918415

  5. Multilayered Polyelectrolyte Microcapsules: Interaction with the Enzyme Cytochrome C Oxidase

    PubMed Central

    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. PMID:25372607

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

    PubMed Central

    Sullivan, Michael L.

    2015-01-01

    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. PMID:25642234

  7. Inhibition of plant and mammalian diamine oxidase by substrate analogues.

    PubMed

    Biegański, T; Osińska, Z; Masliński, C

    1982-04-01

    Imidazoles, aliphatic substrate analogues and the natural dipeptides, carnosine and anserine, were investigated as inhibitors of diamine oxidase from the pig kidney, human pregnancy plasma and pea seedlings. Imidazole, methylimidazoles, N-acetylimidazole, histamine and N tau-methylhistamine are relatively potent inhibitors of mammalian diamine oxidase showing no influence on plant enzymes. Anserine and carnosine are inhibitors of pig kidney and pea seedling enzymes. Ki values are 2 microM and 10 microM respectively. Investigated natural derivatives of putrescine and cadaverine have no influence on diamine oxidase of different origin. In conclusion, we present some evidence to suggest that mammalian diamine oxidase, despite a high reaction rate with putrescine, is better adapted to histamine oxidation, whereas for plant enzymes the diamines are preferred substrates. PMID:6805264

  8. [Heterogeneity of molecular forms of phenol oxidase from grape leaves].

    PubMed

    Pruidze, G N; Zaprometov, M N; Durmishidze, S V; Kintsurashvili, D F

    1983-07-01

    The substrate specificity and some kinetic properties of the monomeric (Mr = 26 000--35 000) and dimeric (Mr = 55 000--70 000) forms of phenol oxidase from vine leaves were studied. These forms possess different hydroxylating and o-diphenol oxidase activities. A kinetic analysis demonstrated that the monomeric form of the enzyme possesses a higher affinity for monophenols and can more effectively accomplish the hydroxylation reaction as compared to the dimeric one. During vine vegetation the ratio of molecular forms of phenol oxidase is altered manifesting itself in quantitative and qualitative changes of enzymatic activity. During plant maturation the dimeric fraction is predominant. The maturation process is associated with a sharp rise of the o-phenol oxidase activity, a disappearance of the hydroxylating activity and a substantial deceleration of phenol compounds production. PMID:6412775

  9. Design and performance of a beam line with a 1 m focal length toroidal grating monochromator at the ACO storage ring

    NASA Astrophysics Data System (ADS)

    Gudat, W.; Kisker, E.; Rothberg, G. M.; Depautex, C.

    We describe a new beam line at the ACO storage ring for solid state photoelectron spectorscopy incorporating a 1 m focal length toroidal grating monochromator for the photon energy range 6 eV to 120 eV custom-built by Jobin-Yvon Instruments S.A. Test results for the TGM with laboratory line light sources are reported as well as performance data with synchrotron radiation.

  10. 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.

  11. Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis.

    PubMed

    Wiel, C; Augert, A; Vincent, D F; Gitenay, D; Vindrieux, D; Le Calvé, B; Arfi, V; Lallet-Daher, H; Reynaud, C; Treilleux, I; Bartholin, L; Lelievre, E; Bernard, D

    2013-01-01

    Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability. PMID:24113189

  12. 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.

  13. Molecular aspects of monoamine oxidase B.

    PubMed

    Ramsay, Rona R

    2016-08-01

    Monoamine oxidases (MAO) influence the monoamine levels in brain by virtue of their role in neurotransmitter breakdown. MAO B is the predominant form in glial cells and in platelets. MAO B structure, function and kinetics are described as a background for the effect of alterations in its activity on behavior. The need to inhibit MAO B to combat decreased brain amines continues to drive the search for new drugs. Reversible and irreversible inhibitors are now designed using data-mining, computational screening, docking and molecular dynamics. Multi-target ligands designed to combat the elevated activity of MAO B in Alzheimer's and Parkinson's Diseases incorporate MAO inhibition (usually irreversible) as well as iron chelation, antioxidant or neuroprotective properties. The main focus of drug design is the catalytic activity of MAO, but the imidazoline I2 site in the entrance cavity of MAO B is also a pharmacological target. Endogenous regulation of MAO B expression is discussed briefly in light of new studies measuring mRNA, protein, or activity in healthy and degenerative samples, including the effect of DNA methylation on the expression. Overall, this review focuses on examples of recent research on the molecular aspects of the expression, activity, and inhibition of MAO B. PMID:26891670

  14. 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. PMID:17316020

  15. 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

  16. Origin and evolution of lysyl oxidases.

    PubMed

    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

  17. 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.

  18. Photodynamic therapy using a protoporphyrinogen oxidase inhibitor.

    PubMed

    Fingar, V H; Wieman, T J; McMahon, K S; Haydon, P S; Halling, B P; Yuhas, D A; Winkelman, J W

    1997-10-15

    The use of endogenously created porphyrins as an alternative to photosensitizer injection for photodynamic therapy is a rapidly evolving area of study. One common method to induce porphyrin synthesis and accumulation in cells is the topical, oral, or parenteral administration of 5-aminolevulinic acid, a precursor for heme biosynthesis. Porphyrin accumulation may also be elicited by the use of enzyme inhibitors of the heme biosynthetic pathway. Groups of DBA/2 mice bearing SMT-F mammary tumors were placed on a diet containing 0-4000 ppm of a protoporphyrinogen oxidase inhibitor, FP-846. This agent blocks a critical step in porphyrin metabolism and results in elevated intracellular levels of protoporphyrin IX. Light treatment of tumors produced both initial and long-term regression that was dependent on the amount of inhibitor, the duration of inhibitor exposure to animals, and the amount of light used in PDT. Tumor regression occurred without significant destruction of normal tissues in the treatment field and without initial vascular constriction or blood flow stasis. Tumor cure in animals given 4000 ppm FP-846 in feed for 3 days and 300 J/cm2 602-670 nm light (23% cure) was similar to the response in animals given 10 mg/kg Photofrin and the same light dose (20%). PMID:9377568

  19. Analyzing the electrogenicity of cytochrome c oxidase.

    PubMed

    Kim, Ilsoo; Warshel, Arieh

    2016-07-12

    Measurements of voltage changes in response to charge separation within membrane proteins can offer fundamental information on spectroscopically "invisible" steps. For example, results from studies of voltage changes associated with electron and proton transfer in cytochrome c oxidase could, in principle, be used to discriminate between different theoretical models describing the molecular mechanism of proton pumping. Earlier analyses of data from these measurements have been based on macroscopic considerations that may not allow for exploring the actual molecular mechanisms. Here, we have used a coarse-grained model describing the relation between observed voltage changes and specific charge-transfer reactions, which includes an explicit description of the membrane, the electrolytes, and the electrodes. The results from these calculations offer mechanistic insights at the molecular level. Our main conclusion is that previously assumed mechanistic evidence that was based on electrogenic measurements is not unique. However, the ability of our calculations to obtain reliable voltage changes means that we have a tool that can be used to describe a wide range of electrogenic charge transfers in channels and transporters, by combining voltage measurements with other experiments and simulations to analyze new mechanistic proposals. PMID:27357681

  20. Molecularly "wired" cholesterol oxidase for biosensing.

    PubMed

    Leonida, Mihaela D; Aurian-Blajeni, Benedict

    2015-02-01

    The influence of several factors on the activity of cholesterol oxidase (ChOx) transiently exposed to a room temperature ionic liquid (RTIL) was studied. Presence of flavin adenine dinucleotide (FAD, prosthetic group of ChOx) during exposure to RTIL makes the procedure enzyme-friendly, while the use of RTIL (green reagent) makes it environmentally-friendly. Following exposure to RTIL and its subsequent removal, FAD becomes part of the molecular structure of the refolded protein (a molecular "wire"). This makes the procedure used here a molecular one. The factors studied were: FAD presence in RTIL during modification, water presence during exposure to RTIL, and ratio FAD:RTIL during "wiring". Performance parameters monitored were: enzyme activity before and after "wiring" (expressed as (dA/dt)/mg enzyme, and measured spectrophotometrically), peak current in an amperometric biosensor for cholesterol detection, and linearity of the biosensor response depending on cholesterol concentration. After RTIL removal, the modified enzyme (ME) retained a high percentage of the added FAD, which supplemented that of the native enzyme (functioning as a "wire" and enhancing electron transfer kinetics), and a fraction of the initial activity. Used in an amperometric biosensor, ME showed catalytic activity, linear behavior as a function of cholesterol concentration, and stability. PMID:25579496

  1. Crystallization of carbohydrate oxidase from Microdochium nivale.

    PubMed

    Dusková, Jarmila; Dohnálek, Jan; Skálová, Tereza; Østergaard, Lars Henrik; Fuglsang, Claus Crone; Kolenko, Petr; Stepánková, Andrea; Hasek, Jindrich

    2009-06-01

    Microdochium nivale carbohydrate oxidase was produced by heterologous recombinant expression in Aspergillus oryzae, purified and crystallized. The enzyme crystallizes with varying crystal morphologies depending on the crystallization conditions. Several different crystal forms were obtained using the hanging-drop vapour-diffusion method, two of which were used for diffraction measurements. Hexagon-shaped crystals (form I) diffracted to 2.66 A resolution, with unit-cell parameters a = b = 55.7, c = 610.4 A and apparent space group P6(2)22. Analysis of the data quality showed almost perfect twinning of the crystals. Attempts to solve the structure by molecular replacement did not give satisfactory results. Recently, clusters of rod-shaped crystals (form II) were grown in a solution containing PEG MME 550. These crystals belonged to the monoclinic system C2, with unit-cell parameters a = 132.9, b = 56.6, c = 86.5 A, beta = 95.7 degrees . Data sets were collected to a resolution of 2.4 A. The structure was solved by the molecular-replacement method. Model refinement is currently in progress. PMID:19478452

  2. Crystallization of carbohydrate oxidase from Microdochium nivale

    PubMed Central

    Dušková, Jarmila; Dohnálek, Jan; Skálová, Tereza; Østergaard, Lars Henrik; Fuglsang, Claus Crone; Kolenko, Petr; Štěpánková, Andrea; Hašek, Jindřich

    2009-01-01

    Microdochium nivale carbohydrate oxidase was produced by heterologous recombinant expression in Aspergillus oryzae, purified and crystallized. The enzyme crystallizes with varying crystal morphologies depending on the crystallization conditions. Several different crystal forms were obtained using the hanging-drop vapour-diffusion method, two of which were used for diffraction measurements. Hexagon-shaped crystals (form I) diffracted to 2.66 Å resolution, with unit-cell parameters a = b = 55.7, c = 610.4 Å and apparent space group P6222. Analysis of the data quality showed almost perfect twinning of the crystals. Attempts to solve the structure by molecular replacement did not give satisfactory results. Recently, clusters of rod-shaped crystals (form II) were grown in a solution containing PEG MME 550. These crystals belonged to the monoclinic system C2, with unit-cell parameters a = 132.9, b = 56.6, c = 86.5 Å, β = 95.7°. Data sets were collected to a resolution of 2.4 Å. The structure was solved by the molecular-replacement method. Model refinement is currently in progress. PMID:19478452

  3. Monoamine oxidase: Radiotracer chemistry and human studies

    DOE PAGESBeta

    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 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

  4. 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.

  5. An ultrafiltration assay for lysyl oxidase.

    PubMed

    Shackleton, D R; Hulmes, D J

    1990-03-01

    A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware. PMID:1971160

  6. Better Rooting Procedure to Enhance Survival Rate of Field Grown Malaysian Eksotika Papaya Transformed with 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Gene

    PubMed Central

    Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom

    2013-01-01

    A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets. PMID:25969786

  7. Better rooting procedure to enhance survival rate of field grown malaysian eksotika papaya transformed with 1-aminocyclopropane-1-carboxylic Acid oxidase gene.

    PubMed

    Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom

    2013-01-01

    A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets. PMID:25969786

  8. An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase activity.

    PubMed

    Linke, Diana; Lehnert, Nicole; Nimtz, Manfred; Berger, Ralf G

    2014-01-01

    An intracellular alcohol oxidase (AOX) was isolated from the white-rot basidiomycete Phanerochaete chrysosporium (Pch), grown on l-lactate induction medium, and purified to electrophoretic homogeneity. The dimeric protein consisted of two identical 75kDa subunits. The open reading frame of 1,956bp resulted in a monomer consisting of 651 amino acids. The enzyme showed a pI at 5.4, a pH optimum of 9, a temperature optimum at 50°C, possessed putative conserved domains of the GMC superfamily, a FAD binding domain, and showed up to 86% homology to alcohol oxidase sequences of Gloeophyllum trabeum and Coprinopsis cinerea. As was shown for the first time for an AOX from a basidiomycete, not only methanol, but also lower primary alcohols and glycerol were accepted as substrates. An assay based on aldehyde dehydrogenase confirmed d-glyceraldehyde as the product of the reaction. A bioprocess based on this enzyme could alleviate the problems associated with the huge side-stream of glycerol occurring during the manufacture of biodiesel, yielding the green oxidant hydrogen peroxide. PMID:24910330

  9. Monoamine oxidase and agitation in psychiatric patients.

    PubMed

    Nikolac Perkovic, Matea; Svob Strac, Dubravka; Nedic Erjavec, Gordana; Uzun, Suzana; Podobnik, Josip; Kozumplik, Oliver; Vlatkovic, Suzana; Pivac, Nela

    2016-08-01

    Subjects with schizophrenia or conduct disorder display a lifelong pattern of antisocial, aggressive and violent behavior and agitation. Monoamine oxidase (MAO) is an enzyme involved in the degradation of various monoamine neurotransmitters and neuromodulators and therefore has a role in various psychiatric and neurodegenerative disorders and pathological behaviors. Platelet MAO-B activity has been associated with psychopathy- and aggression-related personality traits, while variants of the MAOA and MAOB genes have been associated with diverse clinical phenotypes, including aggressiveness, antisocial problems and violent delinquency. The aim of the study was to evaluate the association of platelet MAO-B activity, MAOB rs1799836 polymorphism and MAOA uVNTR polymorphism with severe agitation in 363 subjects with schizophrenia and conduct disorder. The results demonstrated significant association of severe agitation and smoking, but not diagnosis or age, with platelet MAO-B activity. Higher platelet MAO-B activity was found in subjects with severe agitation compared to non-agitated subjects. Platelet MAO-B activity was not associated with MAOB rs1799836 polymorphism. These results suggested the association between increased platelet MAO-B activity and severe agitation. No significant association was found between severe agitation and MAOA uVNTR or MAOB rs1799836 polymorphism, revealing that these individual polymorphisms in MAO genes are not related to severe agitation in subjects with schizophrenia and conduct disorder. As our study included 363 homogenous Caucasian male subjects, our data showing this negative genetic association will be a useful addition to future meta-analyses. PMID:26851573

  10. Structure and function of mammalian aldehyde oxidases.

    PubMed

    Terao, Mineko; Romão, Maria João; Leimkühler, Silke; Bolis, Marco; Fratelli, Maddalena; Coelho, Catarina; Santos-Silva, Teresa; Garattini, Enrico

    2016-04-01

    Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX. PMID:26920149

  11. Monoamine oxidase inhibitory activities of heterocyclic chalcones.

    PubMed

    Minders, Corné; Petzer, Jacobus P; Petzer, Anél; Lourens, Anna C U

    2015-11-15

    Studies have shown that natural and synthetic chalcones (1,3-diphenyl-2-propen-1-ones) possess monoamine oxidase (MAO) inhibition activities. Of particular importance to the present study is a report that a series of furanochalcones acts as MAO-B selective inhibitors. Since the effect of heterocyclic substitution, other than furan (and more recently thiophene, piperidine and quinoline) on the MAO inhibitory properties of the chalcone scaffold remains unexplored, the aim of this study was to synthesise and evaluate further heterocyclic chalcone analogues as inhibitors of the human MAOs. For this purpose, heterocyclic chalcone analogues that incorporate pyrrole, 5-methylthiophene, 5-chlorothiophene and 6-methoxypyridine substitution were examined. Seven of the nine synthesised compounds exhibited IC50 values <1 μM for the inhibition of MAO-B, with all compounds exhibiting higher affinities for MAO-B compared to the MAO-A isoform. The most potent MAO-B inhibitor (4h) displays an IC50 value of 0.067 μM while the most potent MAO-A inhibitor (4e) exhibits an IC50 value of 3.81 μM. It was further established that selected heterocyclic chalcones are reversible and competitive MAO inhibitors. 4h, however, may exhibit tight-binding to MAO-B, a property linked to its thiophene moiety. We conclude that high potency chalcones such as 4h represent suitable leads for the development of MAO-B inhibitors for the treatment of Parkinson's disease and possibly other neurodegenerative disorders. PMID:26432037

  12. Role of NADPH Oxidases in Liver Fibrosis

    PubMed Central

    Paik, Yong-Han; Kim, Jonghwa; Aoyama, Tomonori; De Minicis, Samuele; Bataller, Ramon

    2014-01-01

    Abstract Significance: Hepatic fibrosis is the common pathophysiologic process resulting from chronic liver injury, characterized by the accumulation of an excessive extracellular matrix. Multiple lines of evidence indicate that oxidative stress plays a pivotal role in the pathogenesis of liver fibrosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. In addition to phagocytic NOX2, there are six nonphagocytic NOX proteins. Recent Advances: In the liver, NOX is functionally expressed both in the phagocytic form and in the nonphagocytic form. NOX-derived ROS contributes to various kinds of liver disease caused by alcohol, hepatitis C virus, and toxic bile acids. Recent evidence indicates that both phagocytic NOX2 and nonphagocytic NOX isoforms, including NOX1 and NOX4, mediate distinct profibrogenic actions in hepatic stellate cells, the main fibrogenic cell type in the liver. The critical role of NOX in hepatic fibrogenesis provides a rationale to assess pharmacological NOX inhibitors that treat hepatic fibrosis in patients with chronic liver disease. Critical Issues: Although there is compelling evidence indicating a crucial role for NOX-mediated ROS generation in hepatic fibrogenesis, little is known about the expression, subcellular localization, regulation, and redox signaling of NOX isoforms in specific cell types in the liver. Moreover, the exact mechanism of NOX-mediated fibrogenic signaling is still largely unknown. Future Directions: A better understanding through further research about NOX-mediated fibrogenic signaling may enable the development of novel anti-fibrotic therapy using NOX inhibition strategy. Antioxid. Redox Signal. 20, 2854–2872. PMID:24040957

  13. Terminal Oxidases of Bacillus subtilis Strain 168: One Quinol Oxidase, Cytochrome aa3 or Cytochrome bd, Is Required for Aerobic Growth

    PubMed Central

    Winstedt, Lena; von Wachenfeldt, Claes

    2000-01-01

    The gram-positive endospore-forming bacterium Bacillus subtilis has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome caa3 is a cytochrome c oxidase, whereas cytochrome bd and cytochrome aa3 are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochrome aa3 or cytochrome bd) is required for aerobic growth of B. subtilis strain 168. Data indicating that cytochrome aa3 is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome caa3 or cytochrome aa3, is required for efficient sporulation of B. subtilis strain 168 and that deletion of YthAB in a strain lacking cytochrome aa3 makes the strain sporulation deficient. PMID:11073895

  14. 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

  15. 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. PMID:27125069

  16. Sulfide inhibition of and metabolism by cytochrome c oxidase.

    PubMed

    Nicholls, Peter; Marshall, Doug C; Cooper, Chris E; Wilson, Mike T

    2013-10-01

    Hydrogen sulfide (H2S), a classic cytochrome c oxidase inhibitor, is also an in vitro oxidase substrate and an in vivo candidate hormonal ('gasotransmitter') species affecting sleep and hibernation. H2S, nitric oxide (NO) and carbon monoxide (CO) share some common features. All are low-molecular-mass physiological effectors and also oxidase inhibitors, capable of binding more than one enzyme site, and each is an oxidizable 'substrate'. The oxidase oxidizes CO to CO2, NO to nitrite and sulfide to probable persulfide species. Mitochondrial cytochrome c oxidase in an aerobic steady state with ascorbate and cytochrome c is rapidly inhibited by sulfide in a biphasic manner. At least two successive inhibited species are involved, probably partially reduced. The oxidized enzyme, in the absence of turnover, occurs in at least two forms: the 'pulsed' and 'resting' states. The pulsed form reacts aerobically with sulfide to form two intermediates, 'P' and 'F', otherwise involved in the reaction of oxygen with reduced enzyme. Sulfide can directly reduce the oxygen-reactive a3CuB binuclear centre in the pulsed state. The resting enzyme does not undergo such a step, but only a very slow one-electron reduction of the electron-transferring haem a. In final reactivation phases, both the steady-state inhibition of catalysis and the accumulation of P and F states are reversed by slow sulfide oxidation. A model for this complex reaction pattern is presented. PMID:24059525

  17. Crystal Structure of Alcohol Oxidase from Pichia pastoris.

    PubMed

    Koch, Christian; Neumann, Piotr; 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

  18. Bone marrow purging by a xanthine oxidase-antibody conjugate.

    PubMed

    Dinota, A; Tazzari, P L; Abbondanza, A; Battelli, M G; Gobbi, M; Stirpe, F

    1990-07-01

    The selective cytotoxicity of the xanthine oxidase conjugated to an 8A monoclonal antibody recognizing a human plasma cell-associated antigen has been described. The selectivity and the toxicity of the hypoxanthine/conjugated xanthine oxidase system was increased by removing the excess of conjugate and by adding chelated iron. Under these experimental conditions the cytotoxicity of the conjugate exceeded that of free xanthine oxidase by one order of magnitude. The conjugate effectively purged bone marrow from infiltrating neoplastic plasma cells and added target Raji cells, provided blood was removed and bone marrow peroxidases were exhausted. In conditions of purging effectiveness the conjugate had no toxicity to CFU-GM. No toxicity to mice was observed after i.v. injection of xanthine oxidase-antibody conjugate up to 2.9 U/kg body weight. Thus the hypoxanthine/conjugated xanthine oxidase system could be an effective and nontoxic tool for the ex vivo bone marrow purging in multiple myeloma patients for autologous transplantation. PMID:2390631

  19. Regulation of Ascorbate Oxidase Expression in Pumpkin by Auxin and Copper 1

    PubMed Central

    Esaka, Muneharu; Fujisawa, Kouichi; Goto, Miwa; Kisu, Yasutomo

    1992-01-01

    Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:16652952

  20. Regulation of ascorbate oxidase expression in pumpkin by auxin and copper.

    PubMed

    Esaka, M; Fujisawa, K; Goto, M; Kisu, Y

    1992-09-01

    Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation. PMID:16652952

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

    PubMed Central

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

    2014-01-01

    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. PMID:24293544

  2. Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity1[C][W

    PubMed Central

    Kim, Dong Wook; Watanabe, Kanako; Murayama, Chihiro; Izawa, Sho; Niitsu, Masaru; Michael, Anthony J.; Berberich, Thomas; Kusano, Tomonobu

    2014-01-01

    The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development. PMID:24906355

  3. 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.

  4. X-ray absorption spectroscopy of chicken sulfite oxidase crystals

    SciTech Connect

    George, G.N.; Pickering, I.J.; Kisker, C.

    1999-05-17

    Sulfite oxidase catalyzes the physiologically vital oxidation of sulfite to sulfate. Recently, the crystal structure of chicken sulfite oxidase has been reported at 1.9 {angstrom} resolution. In contrast to the information available from previous X-ray absorption spectroscopic studies, the active site indicated by crystallography was a mono-oxo species. Because of this the possibility that the crystals did in fact contain a reduced molybdenum species was considered in the crystallographic work. The authors report herein an X-ray absorption spectroscopic study of polycrystalline sulfite oxidase prepared in the same manner as the previous single-crystal samples, and compare this with data for frozen solutions of oxidized and reduced enzyme.

  5. Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides.

    PubMed Central

    Matringe, M; Camadro, J M; Labbe, P; Scalla, R

    1989-01-01

    Diphenyl ether herbicides induce an accumulation of protoporphyrin IX in plant tissues. By analogy to human porphyria, the accumulation could be attributed to decreased (Mg or Fe)-chelatase or protoporphyrinogen oxidase activities. Possible effects of acifluorfen-methyl on these enzymes were investigated in isolated corn (maize, Zea mays) etioplasts, potato (Solanum tuberosum) and mouse mitochondria, and yeast mitochondrial membranes. Acifluorfen-methyl was strongly inhibitory to protoporphyrinogen oxidase activities whatever their origins [concn. causing 50% inhibition (IC50) = 4 nM for the corn etioplast enzyme]. By contrast, it was roughly 100,000 times less active on (Mg or Fe)-chelatase activities (IC50 = 80-100 microM). Our results lead us to propose protoporphyrinogen oxidase as a cellular target for diphenyl ether herbicides. PMID:2775186

  6. [Amperometric enzyme biosensor with a glucose oxidase-polyaniline membrane].

    PubMed

    Dziadevich, S V; Doldatkin, A P; Rossokhatyĭ, V K; Shram, N F; Shul'ga, A A; Strikha, V I

    1994-01-01

    An amperometric glucose biosensor was made by electrochemical polymerization of aniline onto the gold electrodes in presence of the enzyme glucose oxidase in the phosphate buffer solution with pH 7.0. Aniline is easily polymerized forming a thin film, which adheres tightly on the electrodes surface. During the electropolymerization process glucose oxidase was entrapped into polyaniline film which then became the catalyst of the enzyme reaction of glucose hydrolysis. Experiments were performed to determine optimal conditions of polyaniline-glucose oxidase film preparation. Glucose was amperometrically determined with the electrochemically fabricated biosensor in the concentration range 10(-4) M to 2 x 10(-2) M. The linearity of the enzyme electrode response ranged from 2 x 10(-4) M to 6 x 10(-3) M. The electrochemical synthesis of a polyaniline-enzyme thin film a high-technologic one and this permits fabricating various microbiosensors and multisensors in the continuous technological cycle. PMID:7754558

  7. The alternative oxidases: simple oxidoreductase proteins with complex functions.

    PubMed

    Young, Luke; Shiba, Tomoo; Harada, Shigeharu; Kita, Kiyoshi; Albury, Mary S; Moore, Anthony L

    2013-10-01

    The alternative oxidases are membrane-bound monotopic terminal electron transport proteins found in all plants and in some agrochemically important fungi and parasites including Trypansoma brucei, which is the causative agent of trypanosomiasis. They are integral membrane proteins and reduce oxygen to water in a four electron process. The recent elucidation of the crystal structure of the trypanosomal alternative oxidase at 2.85 Å (1 Å=0.1 nm) has revealed salient structural features necessary for its function. In the present review we compare the primary and secondary ligation spheres of the alternative oxidases with other di-iron carboxylate proteins and propose a mechanism for the reduction of oxygen to water. PMID:24059524

  8. 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

  9. 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.

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

    PubMed

    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

  11. 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

  12. Computer-controlled system for the study of oxidase reactions: application to the peroxidase-oxidase oscillator.

    PubMed

    McDonald, Andrew G; Tipton, Keith F

    2010-12-16

    An apparatus for the study of bisubstrate oxidase reactions at maintained steady-state substrate concentrations is described, and its specific application to the peroxidase-oxidase biochemical oscillator is reported. Instrument control and data acquisition are provided by custom software written in LabVIEW. The software allows measurement, recording, and control of dissolved oxygen through a Clark-type oxygen electrode, reaction monitoring by a UV/vis spectrophotometer, and controlled substrate delivery by a syringe infusion pump. For peroxidase from horseradish, the optimal pH for oscillatory behavior was found to be in the range 4.5-5.5. PMID:21049952

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

    PubMed Central

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

    2015-01-01

    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. PMID:26672986

  14. 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...

  15. 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...

  16. 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...

  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. 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

    2015-09-01

    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. PMID:26305170

  19. Structural Changes and Proton Transfer in Cytochrome c Oxidase

    PubMed Central

    Vilhjálmsdóttir, Jóhanna; Johansson, Ann-Louise; Brzezinski, Peter

    2015-01-01

    In cytochrome c oxidase electron transfer from cytochrome c to O2 is linked to transmembrane proton pumping, which contributes to maintaining a proton electrochemical gradient across the membrane. The mechanism by which cytochrome c oxidase couples the exergonic electron transfer to the endergonic proton translocation is not known, but it presumably involves local structural changes that control the alternating proton access to the two sides of the membrane. Such redox-induced structural changes have been observed in X-ray crystallographic studies at residues 423–425 (in the R. sphaeroides oxidase), located near heme a. The aim of the present study is to investigate the functional effects of these structural changes on reaction steps associated with proton pumping. Residue Ser425 was modified using site-directed mutagenesis and time-resolved spectroscopy was used to investigate coupled electron-proton transfer upon reaction of the oxidase with O2. The data indicate that the structural change at position 425 propagates to the D proton pathway, which suggests a link between redox changes at heme a and modulation of intramolecular proton-transfer rates. PMID:26310633

  20. Glucose oxidase-magnetite nanoparticle bioconjugate for glucose sensing.

    PubMed

    Rossi, Liane M; Quach, Ashley D; Rosenzweig, Zeev

    2004-10-01

    Immobilization of bioactive molecules on the surface of magnetic nanoparticles is of great interest, because the magnetic properties of these bioconjugates promise to greatly improve the delivery and recovery of biomolecules in biomedical applications. Here we present the preparation and functionalization of magnetite (Fe3O4) nanoparticles 20 nm in diameter and the successful covalent conjugation of the enzyme glucose oxidase to the amino-modified nanoparticle surface. Functionalization of the magnetic nanoparticle surface with amino groups greatly increased the amount and activity of the immobilized enzyme compared with immobilization procedures involving physical adsorption. The enzymatic activity of the glucose oxidase-coated magnetic nanoparticles was investigated by monitoring oxygen consumption during the enzymatic oxidation of glucose using a ruthenium phenanthroline fluorescent complex for oxygen sensing. The glucose oxidase-coated magnetite nanoparticles could function as nanometric glucose sensors in glucose solutions of concentrations up to 20 mmol L(-1). Immobilization of glucose oxidase on the nanoparticles also increased the stability of the enzyme. When stored at 4 degrees C the nanoparticle suspensions maintained their bioactivity for up to 3 months. PMID:15448967

  1. Molecular dynamics in cytochrome c oxidase Moessbauer spectra deconvolution

    SciTech Connect

    Bossis, Fabrizio; Palese, Luigi L.

    2011-01-07

    Research highlights: {yields} Cytochrome c oxidase molecular dynamics serve to predict Moessbauer lineshape widths. {yields} Half height widths are used in modeling of Lorentzian doublets. {yields} Such spectral deconvolutions are useful in detecting the enzyme intermediates. -- Abstract: In this work low temperature molecular dynamics simulations of cytochrome c oxidase are used to predict an experimentally observable, namely Moessbauer spectra width. Predicted lineshapes are used to model Lorentzian doublets, with which published cytochrome c oxidase Moessbauer spectra were simulated. Molecular dynamics imposed constraints to spectral lineshapes permit to obtain useful information, like the presence of multiple chemical species in the binuclear center of cytochrome c oxidase. Moreover, a benchmark of quality for molecular dynamic simulations can be obtained. Despite the overwhelming importance of dynamics in electron-proton transfer systems, limited work has been devoted to unravel how much realistic are molecular dynamics simulations results. In this work, molecular dynamics based predictions are found to be in good agreement with published experimental spectra, showing that we can confidently rely on actual simulations. Molecular dynamics based deconvolution of Moessbauer spectra will lead to a renewed interest for application of this approach in bioenergetics.

  2. 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...

  3. Phosphoproteins and the activation of the neutrophil respiratory burst oxidase

    SciTech Connect

    Okamura, N.; Curnutte, J.T.; Babior, B.M.

    1987-05-01

    The respiratory burst oxidase is a neutrophil enzyme that converts oxygen to O/sub 2//sup -/. It is dormant in resting cells but is activated when the cells are exposed to phorbol myristate acetate (PMA). PMA also induces the incorporation of /sup 32/P into certain neutrophil proteins. To determine whether phosphorylation of these proteins is related to oxidase activation, protein phosphorylation was studied in patients with chronic granulomatous disease (GCD), a group of inherited conditions in which oxidase activity is missing. In normals, neutrophil activation by PMA is associated with the phosphorylation inter alia of 48K proteins at pI 7.3 and 7.8. There is also inconstant phosphorylation of a 48K protein at pI 6.8. In 4 patients with X-linked chronic granulomatous disease (CGD), phosphorylation of pp48/6.8 and pp48/7.3 was absent, while in autosomal recessive CGD, phosphorylation of all 3 of these proteins was absent in 3 patients and significantly diminished in a fourth. These results suggest that the phosphorylation of these proteins is related to the activation of the respiratory burst oxidase. By peptide mapping, these 3 proteins appear to consist of a single peptide species whose pI variability may be due to post-translational modification. The only phosphoamino acid found in pp48/7.3 was phosphoserine.

  4. 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.

  5. Activity of glucose oxidase functionalized onto magnetic nanoparticles

    PubMed Central

    Kouassi, Gilles K; Irudayaraj, Joseph; McCarty, Gregory

    2005-01-01

    Background Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties. Methods Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy. Results The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles Conclusion Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications PMID:15762994

  6. 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...

  7. 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...

  8. 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...

  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, 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...

  11. CHARACTERISTICS OF POLYPHENOL OXIDASES FROM RED CLOVER (TRIFOLIUM PRATENSE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO, EC 1.14.18.1 or EC 1.10.3.1) catalyzes the oxidation of o-diphenols to o-quinones which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense). Production of o-quinones in red clover inhibits post-har...

  12. 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

  13. Structure of the trypanosome cyanide-insensitive alternative oxidase.

    PubMed

    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

    2013-03-19

    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

  14. Localization of the Dual Oxidase BLI-3 and Characterization of Its NADPH Oxidase Domain during Infection of Caenorhabditis elegans.

    PubMed

    van der Hoeven, Ransome; Cruz, Melissa R; Chávez, Violeta; Garsin, Danielle A

    2015-01-01

    Dual oxidases (DUOX) are enzymes that contain an NADPH oxidase domain that produces hydrogen peroxide (H2O2) and a peroxidase domain that can utilize H2O2 to carry out a variety of reactions. The model organism Caenorhabditis elegans produces the DUOX, BLI-3, which has roles in both cuticle development and in protection against infection. In previous work, we demonstrated that while certain peroxidases were protective against the human bacterial pathogen Enterococcus faecalis, the peroxidase domain of BLI-3 was not, leading to the postulate that the NADPH oxidase domain is the basis for BLI-3's protective effects. In this work, we show that a strain carrying a mutation in the NADPH oxidase domain of BLI-3, bli-3(im10), is more susceptible to E. faecalis and the human fungal pathogen Candida albicans. Additionally, less H2O2 is produced in response to pathogen using both an established Amplex Red assay and a strain of C. albicans, WT-OXYellow, which acts as a biosensor of reactive oxygen species (ROS). Finally, a C. elegans line containing a BLI-3::mCherry transgene was generated. Previous work suggested that BLI-3 is produced in the hypodermis and the intestine. Expression of the transgene was observed in both these tissues, and additionally in the pharynx. The amount and pattern of localization of BLI-3 did not change in response to pathogen exposure. PMID:25909649

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

    PubMed

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

    2014-10-01

    The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost. PMID:24862920

  16. 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...

  17. Role of reactive oxygen species produced by NADPH oxidase in gibberellin biosynthesis during barley seed germination.

    PubMed

    Kai, Kyohei; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Watabe, Gaku; Yuasa, Takashi; Iwaya-Inoue, Mari; Ishibashi, Yushi

    2016-05-01

    NADPH oxidase catalyzes the production of the superoxide anion (O2(-)), a reactive oxygen species (ROS), and regulates the germination of barley (Hordeum vulgare L.). Diphenyleneiodonium (DPI) chloride, an NADPH oxidase inhibitor, delayed barley germination, and exogenous H2O2 (an ROS) partially rescued it. Six enzymes, ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20-oxidase (GA20ox) and GA3-oxidase (GA3ox), catalyze the transformation of trans-geranylgeranyl diphosphate to active gibberellin, which promotes germination. Exogenous H2O2 promoted the expressions of HvKAO1 and HvGA3ox1 in barley embryos. These results suggest that ROS produced by NADPH oxidase are involved in gibberellin biosynthesis through the regulation of HvKAO1 and HvGA3ox1. PMID:27110861

  18. 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. PMID:26976571

  19. 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

  20. 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

  1. 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

  2. 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

  3. Oxygen reactivity of mammalian sulfite oxidase provides a concept for the treatment of sulfite oxidase deficiency.

    PubMed

    Belaidi, Abdel A; Röper, Juliane; Arjune, Sita; Krizowski, Sabina; Trifunovic, Aleksandra; Schwarz, Guenter

    2015-07-15

    Mammalian sulfite oxidase (SO) is a dimeric enzyme consisting of a molybdenum cofactor- (Moco) and haem-containing domain and catalyses the oxidation of toxic sulfite to sulfate. Following sulfite oxidation, electrons are passed from Moco via the haem cofactor to cytochrome c, the terminal electron acceptor. In contrast, plant SO (PSO) lacks the haem domain and electrons shuttle from Moco to molecular oxygen. Given the high similarity between plant and mammalian SO Moco domains, factors that determine the reactivity of PSO towards oxygen, remained unknown. In the present study, we generated mammalian haem-deficient and truncated SO variants and demonstrated their oxygen reactivity by hydrogen peroxide formation and oxygen-consumption studies. We found that intramolecular electron transfer between Moco and haem showed an inverse correlation to SO oxygen reactivity. Haem-deficient SO variants exhibited oxygen-dependent sulfite oxidation similar to PSO, which was confirmed further using haem-deficient human SO in a cell-based assay. This finding suggests the possibility to use oxygen-reactive SO variants in sulfite detoxification, as the loss of SO activity is causing severe neurodegeneration. Therefore we evaluated the potential use of PEG attachment (PEGylation) as a modification method for future enzyme substitution therapies using oxygen-reactive SO variants, which might use blood-dissolved oxygen as the electron acceptor. PEGylation has been shown to increase the half-life of other therapeutic proteins. PEGylation resulted in the modification of up to eight surface-exposed lysine residues of SO, an increased conformational stability and similar kinetic properties compared with wild-type SO. PMID:26171830

  4. Neolignans as xanthine oxidase inhibitors from Hyptis rhomboides.

    PubMed

    Tsai, Sheng-Fa; Lee, Shoei-Sheng

    2014-05-01

    The active fraction from the EtOH extract of Hyptis rhomboides against xanthine oxidase was identified by use of an HPLC microfractionation-centrifugal vacuum evaporation-bioassay hyphenated technique. Scale-up separation of the active subfractions using semi-preparative RP-HPLC provided 13 phenylpropanoid compounds, including O-styrenylneolignans, hyprhombins A-C, epihyprhombin B, and hyprhombin B methyl ester, and O-caffeoylnorneolignans, hyprhombins D and E. All of these compounds shared a common 1,4-benzodioxane skeleton, as established by spectroscopic analyses. Hyprhombin C and epihyprhombin B exhibited better anti-xanthine oxidase activity than allopurinol, with IC50 values of 0.6 and 2.0 μM, respectively. PMID:24559911

  5. Bilirubin oxidases in bioelectrochemistry: features and recent findings.

    PubMed

    Mano, Nicolas; Edembe, Lise

    2013-12-15

    Bilirubin oxidases, a sub class of the Multicopper oxidases family, were discovered in 1981 by Tanaka and Murao (Murao and Tanaka, 1981) and first used for the detection of bilirubin. Since 2001 and the pioneering work of Tsujimura, these BODs have attracted a lot of attention for the reduction of O2. Unlike laccases, these BODs are stable in physiological conditions (20mM phosphate buffer, pH 7.4, 0.14 M NaCl, 37 °C) and more than 120 papers have been published in the last 7 years. Here, we will first briefly describe some general features of BODs and then review the use of BODs for bilirubin biosensors and the recent achievements and progress toward the elaboration of efficient O2 reducing cathodes. PMID:23911663

  6. 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.

  7. 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.

  8. 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. PMID:26773413

  9. Glycosides and xanthine oxidase inhibitors from Conyza bonariensis.

    PubMed

    Kong, L D; Abliz, Z; Zhou, C X; Li, L J; Cheng, C H; Tan, R X

    2001-10-01

    Fractionation of the xanthine oxidase inhibitory methanol extract of Conyza bonariensis afforded three glycosides, in addition to nine known compounds including amyrin, beta-sitostero1 daucosterol, syringic acid 3-hydroxy-5-methoxybenzoic acid, eugenol 4-O-glucopyranoside, and luteolin, apigenin and takakin 8-O-glucuronide. The structures of the glycosides were established by a combination of spectroscopic methods (IR, MS, 1H and 13C NMR, DEPT, COSY, HMQC and HMBC) as 4-hydroxypyridin-3-carboxylic acid 4-O-glucopyranoside, 8-hydroxy-6,7-dihydrolinalool 8-O-glucopyranoside and bonaroside [viz. 1,3,4,12-tetrahydroxy-2-(9-hexadecenoylamino)octadecane 1-O-glucopyranoside]. The in vitro enzyme assay showed that syringic acid and takakin 8-O-glucuronide displayed weak inhibitory activity against xanthine oxidase with IC50 values of 500+/-41 microM and 170+/-12 microM, respectively. PMID:11576616

  10. A new crystal form of human diamine oxidase.

    PubMed

    McGrath, Aaron P; Hilmer, Kimberly M; Collyer, Charles A; Dooley, David M; Guss, J Mitchell

    2010-02-01

    Copper amine oxidases (CAOs) are ubiquitous in nature and catalyse the oxidative deamination of primary amines to the corresponding aldehydes. Humans have three viable CAO genes (AOC1-3). AOC1 encodes human diamine oxidase (hDAO), which is the frontline enzyme for histamine metabolism. hDAO is unique among CAOs in that it has a distinct substrate preference for diamines. The structure of hDAO in space group P2(1)2(1)2(1) with two molecules in the asymmetric unit has recently been reported. Here, the structure of hDAO refined to 2.1 A resolution in space group C222(1) with one molecule in the asymmetric unit is reported. PMID:20124708

  11. 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

  12. T lymphocyte killing by a xanthine-oxidase-containing immunotoxin.

    PubMed

    Battelli, M G; Abbondanza, A; Tazzari, P L; Bolognesi, A; Lemoli, R M; Stirpe, F

    1992-01-01

    We report on the preparation of an immunotoxin consisting of xanthine oxidase, a free-radical-producing enzyme, covalently linked to an anti-CD3 monoclonal antibody. The immunotoxin retained both enzymic and immunological properties and its toxicity to target cells (a) was greater than that of the free enzyme, (b) was proportional to the enzyme concentration, and (c) was reduced either in the absence of hypoxanthine or by an excess of free anti-CD3 monoclonal antibody. The cytotoxicity and selectivity of the hypoxanthine/conjugated xanthine oxidase system were potentiated by the addition of chelated iron and by washing away the unbound immunotoxin prior to the addition of substrate. The same system was not toxic to bone marrow progenitor cells. A possible use of this immunotoxin for the ex vivo purging of organs to be transplanted from T lymphocytes, to avoid the graft-versus-host reaction, is suggested. PMID:1394345

  13. 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

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

    PubMed Central

    2010-01-01

    Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III) to As(V) as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III). To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54) of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE) and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III) in this microorganism. PMID:20167112

  15. Crystal structures of intermediates in the nitroalkane oxidase reaction.

    PubMed

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

    2009-04-21

    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 A 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 [Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 125, 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

  16. 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.

  17. Characterization of two brassinosteroid C-6 oxidase genes in pea.

    PubMed

    Jager, Corinne E; Symons, Gregory M; Nomura, Takahito; Yamada, Yumiko; Smith, Jennifer J; Yamaguchi, Shinjiro; Kamiya, Yuji; Weller, James L; Yokota, Takao; Reid, James B

    2007-04-01

    C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea. PMID:17322341

  18. Use of a xanthine oxidase inhibitor in autoimmune hepatitis.

    PubMed

    Al-Shamma, Safa; Eross, Balint; Mclaughlin, Simon

    2013-03-01

    A 62-year-old woman with type 1 autoimmune hepatitis (AIH) failed to sustain remission when steroids were withdrawn from a regimen of steroids and azathioprine (AZA). Thiopurine metabolites revealed elevated 6-MMP (6-methyl mercaptopurine) and low 6-TGN (6-thioguanine nucleotide) consistent with AZA-induced hepatotoxicity. Introducing the xanthine oxidase inhibitor allopurinol led to rapid normalization of alanine aminotransferase (ALT) and discontinuation of steroids. PMID:23238820

  19. Structures and Mechanism of the Monoamine Oxidase Family

    PubMed Central

    Gaweska, Helena; Fitzpatrick, Paul F.

    2011-01-01

    Members of the monoamine oxidase family of flavoproteins catalyze the oxidation of primary and secondary amines, polyamines, amino acids, and methylated lysine side chains in proteins. The enzymes have similar overall structures, with conserved FAD-binding domains and varied substrate-binding sites. Multiple mechanisms have been proposed for the catalytic reactions of these enzymes. The present review compares the structures of different members of the family and the various mechanistic proposals. PMID:22022344

  20. 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.

  1. Functional characterization of alcohol oxidases from Aspergillus terreus MTCC 6324.

    PubMed

    Kumar, A Kiran; Goswami, Pranab

    2006-10-01

    Short chain alcohol oxidase (SCAO), long chain alcohol oxidase (LCAO), secondary alcohol oxidase (SAO), and aryl alcohol oxidase (AAO) activities were localized in the microsome of Aspergillus terreus during growth of the fungi on n-hexadecane. Zymogram analysis of the microsomes of n-hexadecane-grown cells in polyacrylamide gel electrophoresis showed distinct bands, H4, H3, H2, and H1, in a sequence of their molecular weight (Mr) from high to low. The Mr of the isozymes corresponding to the bands H4, H3, and H2 were close to each other and were higher than 272 kDa. While, the Mr of the isozyme H1 was found to be approximately 48 kDa. H1 gave activity only as SCAO. Although the substrates for other bands were varied, strong (S), medium (M), and weak (W) activity for the bands were as follows: H2: SAO (S), AAO (S), LCAO (M), SCAO (S); H3: LCAO (S), SCAO (S); H4: SCAO (S), LCAO (W), SAO (W). The pH and temperature optima of these isozymes were found to be 8.5+/-0.5 and 30+/-1 degrees C, respectively. The stability of the isozymes was drastically decreased beyond 30 degrees C. The SAO showed 33% enantiomeric excess for the R(-)2-octanol over S(+)2-octanol, which may be correlated with the lower Michaelis-Menten constant (K (M)) values of the enzyme for the R(-)2-octanol than the S(+)2-octanol. The fluorescence emission spectra of the chromatographically purified SCAO at 443 nm excitation were similar to that obtained with authentic flavin adenine dinucleotide. PMID:16547701

  2. Four novel mutations of the coproporphyrinogen III oxidase gene.

    PubMed

    Aurizi, C; Lupia Palmieri, G; Barbieri, L; Macrì, A; Sorge, F; Usai, G; Biolcati, G

    2009-01-01

    Here we report the characterization of four novel mutations and a previously described one of the coproporphyrinogen III oxidase (CPO) gene in five Italian patients affected by Hereditary Coproporphyria (HCP). Three of the novel genetic variants are missense mutations (p.Gly242Cys; p.Leu398Pro; p.Ser245Phe) and one is a frameshift mutation (p.Gly188TrpfsX45). PMID:19267996

  3. Alkylamino derivatives of 4-aminomethylpyridine as inhibitors of copper-containing amine oxidases.

    PubMed

    Bertini, Vincenzo; Buffoni, Franca; Ignesti, Giovanni; Picci, Nevio; Trombino, Sonia; Iemma, Francesca; Alfei, Silvana; Pocci, Marco; Lucchesini, Francesco; De Munno, Angela

    2005-02-10

    The first substratelike, reversible inhibitors of different copper amine oxidases (CAOs) with IC50 (M) as low as 2.0 x 10(-8) corresponding to derivatives of 4-aminomethylpyridine with alkoxy (1a-d), alkylthio (2a,b), and alkylamino (3a-e, 4a-j) groups in the positions 3 and 5 have been prepared and studied. The inhibitors 1a-d are active on benzylamine oxidase and semicarbazide-sensitive amine oxidase and are very selective with respect to diamine oxidase, lysyl oxidase, and monoamine oxidases. The inhibitors 2a,b are selective for benzylamine oxidase whereas 2a is also a new type of good substrate of diamine oxidase. The inhibitors 3a-e and 4a-j are substratelike, reversible, nonselective inhibitors of various CAOs including pea seedling amine oxidase and Hansenula polymorpha amine oxidase, whose enzymatic sites are known from X-ray structure determinations. The inhibitors 3b,c and 4b,c are excellent substratelike tools for studies correlating CAOs that afford crystals suitable for X-ray structure determinations with CAOs from mammals. PMID:15689151

  4. [Respiratory oxidases: the enzymes which use most of the oxygen which living things breathe].

    PubMed

    Toledo-Cuevas, E M

    1997-01-01

    The respiratory oxidases are the last enzymes of the aerobic respiratory chain. They catalize the reduction of molecular oxygen to water, with generation of an electrochemical gradient useful for the energy demanding cellular processes. Most of the oxidases belong to the heme-copper superfamily. They possess a heme-copper center, constituted of a high spin heme and a CuB center, where the reduction of oxygen takes place and probably where the link to proton pumping is located. The superfamily is divided in two classes: the quinol- and the cytochrome c-oxidases. The latter are divided in the aa3 and the cbb3-type cytochrome c oxidases. The main difference between quinol- and the aa3-type cytochrome c-oxidases is the CuA center, which is absent in the quinol oxidases. The cbb3-type cytochrome oxidases have the binuclear center, but lack the CuA center. They also does not have the classical subunits II and III. These differences seem not to affect the oxygen reduction or the proton pumping. Probably the oxidases have evolved from some denitrification enzymes and prior the photosynthetic process. Also is possible that the cbb3-type cytochrome oxidases or others very similar have been the first oxidases to appear. PMID:10932727

  5. Characterization of Euphorbia characias Latex Amine Oxidase1

    PubMed Central

    Padiglia, Alessandra; Medda, Rosaria; Lorrai, Anita; Murgia, Barbara; Pedersen, Jens Z.; Finazzi Agró, Alessandro; Floris, Giovanni

    1998-01-01

    A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 × 105 m−1 cm−1 and 6000 m−1 cm−1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone. PMID:9701592

  6. Potential xanthine oxidase inhibitory activity of endophytic Lasiodiplodia pseudotheobromae.

    PubMed

    Kapoor, Neha; Saxena, Sanjai

    2014-07-01

    Xanthine oxidase is considered as a potential target for treatment of hyperuricemia. Hyperuricemia is predisposing factor for gout, chronic heart failure, atherosclerosis, tissue injury, and ischemia. To date, only two inhibitors of xanthine oxidase viz. allopurinol and febuxostat have been clinically approved for used as drugs. In the process of searching for new xanthine oxidase inhibitors, we screened culture filtrates of 42 endophytic fungi using in vitro qualitative and quantitative XO inhibitory assays. The qualitative assay exhibited potential XO inhibition by culture filtrates of four isolates viz. #1048 AMSTITYEL, #2CCSTITD, #6AMLWLS, and #96 CMSTITNEY. The XO inhibitory activity was present only in the chloroform extract of the culture filtrates. Chloroform extract of culture filtrate #1048 AMSTITYEL exhibited the highest inhibition of XO with an IC50 value of 0.61 μg ml(-1) which was better than allopurinol exhibiting an IC50 of 0.937 μg ml(-1) while febuxostat exhibited a much lower IC50 of 0.076 μg ml(-1). Further, molecular phylogenetic tools and morphological studies were used to identify #1048 AMSTITYEL as Lasiodiplodia pseudotheobromae. This is the first report of an endophytic Lasiodiplodia pseudotheobromae from Aegle marmelos exhibiting potential XO Inhibitory activity. PMID:24801403

  7. Phagocyte NADPH oxidase: a multicomponent enzyme essential for host defenses.

    PubMed

    El-Benna, Jamel; Dang, Pham My-Chan; Gougerot-Pocidalo, Marie-Anne; Elbim, Carole

    2005-01-01

    Phagocytes such as neutrophils and monocytes play an essential role in host defenses against microbial pathogens. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, the hydroxyl radical, and hypochlorous acid, together with microbicidal peptides and proteases, constitute their antimicrobial arsenal. The enzyme responsible for superoxide anion production and, consequently, ROS generation, is called NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of cytosolic proteins (p47phox, p67phox, p40phox, and rac1/2) and membrane proteins (p22phox and gp91phox, which form cytochrome b558) which assemble at membrane sites upon cell activation. The importance of this enzyme in host defenses is illustrated by a life-threatening genetic disorder called chronic granulomatous disease in which the phagocyte enzyme is dysfunctional, leading to life-threatening bacterial and fungal infections. Also, because ROS can damage surrounding tissues, their production, and thus NADPH oxidase activation, must be tightly regulated. This review describes the structure and activation of the neutrophil NADPH enzyme complex. PMID:15995580

  8. Androgen receptor and monoamine oxidase polymorphism in wild bonobos.

    PubMed

    Garai, Cintia; Furuichi, Takeshi; Kawamoto, Yoshi; Ryu, Heungjin; Inoue-Murayama, Miho

    2014-12-01

    Androgen receptor gene (AR), monoamine oxidase A gene (MAOA) and monoamine oxidase B gene (MAOB) have been found to have associations with behavioral traits, such as aggressiveness, and disorders in humans. However, the extent to which similar genetic effects might influence the behavior of wild apes is unclear. We examined the loci AR glutamine repeat (ARQ), AR glycine repeat (ARG), MAOA intron 2 dinucleotide repeat (MAin2) and MAOB intron 2 dinucleotide repeat (MBin2) in 32 wild bonobos, Pan paniscus, and compared them with those of chimpanzees, Pan troglodytes, and humans. We found that bonobos were polymorphic on the four loci examined. Both loci MAin2 and MBin2 in bonobos showed a higher diversity than in chimpanzees. Because monoamine oxidase influences aggressiveness, the differences between the polymorphisms of MAin2 and MBin2 in bonobos and chimpanzees may be associated with the differences in aggression between the two species. In order to understand the evolution of these loci and AR, MAOA and MAOB in humans and non-human primates, it would be useful to conduct future studies focusing on the potential association between aggressiveness, and other personality traits, and polymorphisms documented in bonobos. PMID:25606465

  9. Reaction mechanism of mammalian mitochondrial cytochrome c oxidase.

    PubMed

    Yoshikawa, Shinya; Muramoto, Kazumasa; Shinzawa-Itoh, Kyoko

    2012-01-01

    Cytochrome c oxidase (COX) is the terminal oxidase of the mitochondrial respiratory system. This enzyme reduces molecular oxygen (O(2)) to water in a reaction coupled with the pumping of protons across the mitochondrial inner membrane. Progress in investigating the reaction mechanism of this enzyme has been limited by the resolution of its X-ray structure. Bovine heart COX has provided the highest resolution (1.8 Å) X-ray structure presently available among the terminal oxidases. The reaction mechanism of the bovine heart enzyme has been the most extensively studied, particularly with respect to (1) the reduction of O(2) to water without release of reactive oxygen species, (2) the mechanism of coupling between the O(2) reduction process and proton pumping, (3) the structural basis for unidirectional proton transfer (proton pumping), and (4) the effective prevention of proton leakage from the proton-pumping pathway to the proton pathway used for generation of water molecules. In this chapter, we will review recent structural studies of bovine heart COX and discuss the mechanisms described earlier in context of the structural data. PMID:22729860

  10. Effects of a 500 Mc s -1 additional cavity on spontaneous coherent synchrotron oscillations in the Super-ACO storage ring

    NASA Astrophysics Data System (ADS)

    Bergher, M.

    An additional 500 Mc s -1 cavity, fifth harmonics of the main cavity, was installed on the Super-ACO storage ring in order to shorten the bunch length. This cavity was introduced to obtain shorter wavelengths in the UV for the FEL and shorter flashes of light for the experiments using the two-bunches-mode functioning for the time-resolved measurements. Spontaneous coherent synchrotron oscillations (SCSO) were profoundly modified by the presence of this cavity. These instabilities are probably the consequence of the formation of micro-bunches. The vanishing of these micro-bunches is associated to the emission of coherent synchrotron radiation, which gives the SCSO a cyclic character. The identification of the resonant elements responsible for these cyclic instabilities can help us to suppress or substantial ameliorate the SCSO by acting selectively on these resonant elements. This method can be applied to other storage or damping rings that show the same type of instabilities.

  11. Absolute calibration of soft x-ray detectors (photocathodes, XUV photodiodes, thinned CCD, ...) with the synchrotron radiation of SUPER ACO at the LURE, Orsay

    NASA Astrophysics Data System (ADS)

    Reverdin, C.; Troussel, P.; Bourgade, J. L.; Le Guern, F.; Mens, A.; Schirmann, D.; Dalmasso, J. M.; Gontier, D.; Mazataud, D.

    1994-10-01

    To interpret the experimental results in laser matter interaction experiments, the absolute spectral response of soft x-ray detectors is often needed. For this purpose CEL-V uses calibration lines of synchrotron radiation of SUPER-ACO at the LURE. The energy of output photons can be selected from 50 eV to 1000 eV. The output photon flux is absolutely calibrated with a bolometer or a soft x-ray photodiode. Then we measure the response of the studied detector installed at the same location. Measurements of quantum efficiencies of photocathodes (Al and CsI on Al) and of the response of a thinned CCD are presented versus photon energy.

  12. 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

  13. 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.

  14. Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

    PubMed

    Dudakova, Lubica; Liskova, Petra; Trojek, Tomas; Palos, Michalis; Kalasova, Sarka; Jirsova, Katerina

    2012-11-01

    Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per μg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic

  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. Photochemical reactions of cytochrome oxidase at low temperatures.

    PubMed

    Orii, Y

    1978-01-01

    The unique of CO-cytochrome oxidase as first noted by Yonetani et al. (22) is that after its photodissociation at low temperatures recombination occurs as the sample temperature is raised, but at temperatures considerably higher than those for other CO-heme and CO-hemoprotein complexes; that is, the half recombination temperature is 180 K contrary to 25-30 K for other CO complexes. The photodissociability, however, disappeared when monomeric cytochrome oxidase was treated with pCMB to remove an intrinsic copper, the significance of which in CO complex formation was thus demonstrated. It is proposed that the copper is situated close to heme a and traps the photodissociated CO. The access of the trapped CO to the heme a to resume the original binding is effected only when sufficient energy for thermal agitation is provided by elevating the sample temperature. During the course of this study, new photo- and thermochromic properties were observed with the reduced enzyme by cooling it in liquid nitrogen after preincubation at pH 8.6 to 10.5. The characteristic bands appeared at 575 and 428 nm and diminished when this ample was illuminated at 26 K. As the sample temperature was raised these bands were restored with a half transition temperature of 80 K. When the reduced oxidase had been complexed with CO, cyanide or azide, or treated with pCMB, such a unique species did not appear. The enthalpy change of 1.16 kcal/mol for the formation of this species as well as the above-described properties suggests that the hydrogen bond between the formyl side group of heme a and one of seven sulfhydryl groups in cytochrome oxidase is responsible for the appearance and disappearance of this new species. Based on these results a schematic model has been proposed for the photo- and thermochromism of cytochrome oxidase at cryogenic temperatures and for the microenvironment of the prosthetic heme a and copper in this enzyme. On the other hand, contrary to the central dogma of Warburg

  17. Catalase-peroxidases (KatG) exhibit NADH oxidase activity.

    PubMed

    Singh, Rahul; Wiseman, Ben; Deemagarn, Taweewat; Donald, Lynda J; Duckworth, Harry W; Carpena, Xavi; Fita, Ignacio; Loewen, Peter C

    2004-10-01

    Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG. PMID:15280362

  18. 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

  19. Cell-free NADPH oxidase activation assays: "in vitro veritas".

    PubMed

    Pick, Edgar

    2014-01-01

    The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

  20. 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

  1. "Ping-pong gaze" secondary to monoamine oxidase inhibitor overdose.

    PubMed

    Attaway, Amy; Sroujieh, Laila; Mersfelder, Tracey L; Butler, Christopher; Ouellette, Daniel

    2016-01-01

    An infrequent manifestation of monoamine oxidase inhibitor (MAOI) toxicity is "ping-pong gaze" (PPG). We describe the case of a 26-year-old female who was found unresponsive after taking 40 tablets of phenelzine. On presentation to the hospital, her eyes were moving in characteristic "ping pong" fashion. After 6 hours her gaze terminated. The following day her neurologic exam was benign and she had no long-term sequelae. While the etiology of PPG is unknown, it is most often seen with irreversible structural brain damage. However, a detailed literature review revealed that previous cases of MAOI toxicity where the patient survived have all had complete neurologic recovery. PMID:27127395

  2. Xanthine oxidase inhibitors from Vietnamese Blumea balsamifera L.

    PubMed

    Nguyen, Mai Thanh Thi; Nguyen, Nhan Trung

    2012-08-01

    From the MeOH extract of the aerial part of Blumea balsamifera L., a new dihydroflavonol, (2R,3S)-(-)-4'-O-methyldihydroquercetin (1), together with seven known compounds has been isolated. Their structures were elucidated on the basis of spectroscopic data. Compounds 1-4 and 6-8 displayed significant xanthine oxidase inhibitory activity in a concentration-dependent manner, and compounds 1, 6 and 8 showed more potent inhibitory activity, with IC₅₀ values ranging from 0.23 to 1.91 µM, than that of a positive control allopurinol (IC₅₀ 2.50 µM). PMID:22821854

  3. The Role of Monoamine Oxidase Inhibitors in Current Psychiatric Practice

    PubMed Central

    Fiedorowicz, Jess G.; Swartz, Karen L.

    2007-01-01

    The use of monoamine oxidase inhibitors (MAOIs) by psychiatrists has declined over the past several decades with the expansion of psychiatrists’ pharmacologic armamentarium. This trend has also been driven by concern about food and drug interactions and side effects, as well as waning physician experience with these medications. Many psychiatrists, in fact, never prescribe MAOIs. Recent research has liberalized the MAOI diet and identified symptom presentations more likely to respond to these medications. Thus, clinicians must continue to familiarize themselves with the properties of and indications for prescribing MAOIs. PMID:15552546

  4. Purification and properties of human amniotic fluid diamine oxidase.

    PubMed

    Tufvesson, G

    1978-09-01

    Diamine oxidase (DAO) was purified from amniotic fluid. The activity was separated in two DAO fractions with pI values of 5.8 and 4.0. Molecular weight were found to be 245,000 and 485,000, respectively, with subunit molecular weight of 110,000. This indicated that they probably are dimer and tetramer of the same DAO subunit. The enzyme was active against putrescine and histamine and was strongly inhibited by carbonyl group reagents. A Ping Pong Bi Ter enzyme reaction mechanism is probable. The diamine, with one amino group protonized, is suggested to be responsible for interaction with the enzyme. PMID:30164

  5. Electronic Structure Contributions to Reactivity in Xanthine Oxidase Family Enzymes

    PubMed Central

    Stein, Benjamin W.; Kirk, Martin L.

    2016-01-01

    We review the xanthine oxidase (XO) family of pyranopterin molybdenum enzymes with a specific emphasis on electronic structure contributions to reactivity. In addition to xanthine and aldehyde oxidoreductases, which catalyze the 2-electron oxidation of aromatic heterocycles and aldehyde substrates, this mini-review highlights recent work on the closely related carbon monoxide dehydrogenase (CODH) that catalyzes the oxidation of CO using a unique Mo-Cu heterobimetallic active site. A primary focus of this mini-review relates to how spectroscopy and computational methods have been used to develop an understanding of critical relationships between geometric structure, electronic structure, and catalytic function. PMID:25425163

  6. Reductive trapping of substrate to bovine plasma amine oxidase

    SciTech Connect

    Hartmann, C.; Klinman, J.P.

    1987-01-25

    Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O/sub 2/ to H/sub 2/O/sub 2/. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation between a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of (/sup 14/C)benzylamine and (/sup 3/H)NaCNBH/sub 3/. The use of the relatively weak reductant, NaCNBH/sub 3/, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH/sub 3/ in the presence of substrate, leading to the incorporation of 1 mol of (/sup 14/C)benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with (/sup 3/H)NaCNBH/sub 3/, analogous to an earlier study with (/sup 3/H)NaCNBH/sub 4/ (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.

  7. Photosensitivity in sponge due to cytochrome c oxidase?

    PubMed

    Björn, Lars Olof; Rasmusson, Allan G

    2009-06-01

    An action spectrum for photosensitivity in sponge larvae published by Leys et al. [J. Comp. Physiol., A, 2002, 188, 199-202] was interpreted by the authors as being due to a combination of light absorption by flavin or carotenoid in the blue region, and another pigment such as pterin in the long-wavelength region. Here we show here that their action spectrum closely matches the absorption spectrum of reduced cytochrome c oxidase that is present in sponges, and compare with other photoreactions which are thought to be due to this chromoprotein. PMID:19492101

  8. Partial conversion of Hansenula polymorpha amine oxidase into a "plant" amine oxidase: implications for copper chemistry and mechanism.

    PubMed

    Welford, Richard W D; Lam, Angel; Mirica, Liviu M; Klinman, Judith P

    2007-09-25

    The mechanism of the first electron transfer from reduced cofactor to O2 in the catalytic cycle of copper amine oxidases (CAOs) remains controversial. Two possibilities have been proposed. In the first mechanism, the reduced aminoquinol form of the TPQ cofactor transfers an electron to the copper, giving radical semiquinone and Cu(I), the latter of which reduces O2 (pathway 1). The second mechanism invokes direct transfer of the first electron from the reduced aminoquinol form of the TPQ cofactor to O2 (pathway 2). The debate over these mechanisms has arisen, in part, due to variable experimental observations with copper amine oxidases from plant versus other eukaryotic sources. One important difference is the position of the aminoquinol/Cu(II) to semiquinone/Cu(I) equilibrium on anaerobic reduction with amine substrate, which varies from almost 0% to 40% semiquinone/Cu(I). In this study we have shown how protein structure controls this equilibrium by making a single-point mutation at a second-sphere ligand to the copper, D630N in Hansenula polymorpha amine oxidase, which greatly increases the concentration of the cofactor semiquinone/Cu(I) following anaerobic reduction by substrate. The catalytic properties of this mutant, including 18O kinetic isotope effects, point to a conservation of pathway 2, despite the elevated production of the cofactor semiqunone/Cu(I). Changes in kcat/Km[O2] are attributed to an impact of D630N on an increased affinity of O2 for its hydrophobic pocket. The data in this study indicate that changes in cofactor semiquinone/Cu(I) levels are not sufficient to alter the mechanism of O2 reduction and illuminate how subtle features are able to control the reduction potential of active site metals in proteins. PMID:17760423

  9. 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. PMID:26367330

  10. 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...

  11. The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mitochondrial alternative oxidase (AOX) is a non-energy conserving ubiquinol oxidase found in most fungal genomes studied to date. With the development of fungicides containing cytochrome-dependent respiratory chain (CRC) inhibitors, a strong interest in studying AOX functions in phytopathogenic...

  12. Generation of protonic potential by the bd-type quinol oxidase of Azotobacter vinelandii.

    PubMed

    Bertsova, Y V; Bogachev, A V; Skulachev, V P

    1997-09-01

    Inside-out subcellular vesicles of Azotobacter vinelandii are found to produce delta pH and delta psi (interior acidic and positive) when oxidising malate or menadiol. These effects are inherent in both Cyd+ Cyo- (lacking the o-type oxidase) and Cyd- Cyo+ (lacking the bd-type oxidase) strains. They appear to be myxothiazol-sensitive in the Cyd- Cyo+ strain but not in the Cyd+ Cyo- strain. The H+/e- ratio for the terminal part of respiratory chain of a bd-type oxidase overproducing strain is established as being close to 1. It is also shown that NADH oxidation by the vesicles from the Cyd- Cyo+ strain is sensitive to low concentrations of myxothiazol and antimycin A whereas that of the Cyd+ Cyo- strain is resistant to these Q-cycle inhibitors. It is concluded that (i) the bd-type oxidase of A. vinelandii is competent in generating a protonic potential but its efficiency is lower than that of the o-type oxidase and (ii) Q-cycle does operate in the o-type cytochrome oxidase terminated branch of the A. vinelandii respiratory chain and does not in the bd-type quinol oxidase terminated branch. These relationships are discussed in the context of the respiratory protection function of the bd-type oxidase in A. vinelandii. PMID:9315721

  13. Cold-adapted arsenite oxidase from a psychrotolerant Polaromonas species.

    PubMed

    Osborne, Thomas H; Heath, Matthew D; Martin, Andrew C R; Pankowski, Jaroslaw A; Hudson-Edwards, Karen A; Santini, Joanne M

    2013-04-01

    Polaromonas sp. str. GM1 is an aerobic, psychrotolerant, heterotrophic member of the Betaproteobacteria and is the only isolate capable of oxidising arsenite at temperatures below 10 °C. Sequencing of the aio gene cluster in GM1 revealed the presence of the aioB and aioA genes, which encode the arsenite oxidase but the regulatory genes typically found upstream of aioB in other members of the Proteobacteria were absent. The GM1 Aio was purified to homogeneity and was found to be a heterodimer. The enzyme contained Mo and Fe as cofactors and had, using the artificial electron acceptor 2,6-dichlorophenolindophenol, a Km for arsenite of 111.70 ± 0.88 μM and a Vmax of 12.16 ± 0.30 U mg(-1), which is the highest reported specific activity for any known Aio. The temperature-activity profiles of the arsenite oxidases from GM1 and the mesophilic betaproteobacterium Alcaligenes faecalis were compared and showed that the GM1 Aio was more active at low temperatures than that of A. faecalis. A homology model of the GM1 Aio was made using the X-ray crystal structure of the Aio from A. faecalis as the template. Structural changes that account for cold adaptation were identified and it was found that these resulted in increased enzyme flexibility and a reduction in the hydrophobicity of the core. PMID:23150098

  14. 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. PMID:19648408

  15. 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.

  16. 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.

  17. 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.

  18. Oxidative Half-reaction of Arabidopsis thaliana Sulfite Oxidase

    PubMed Central

    Byrne, Robert S.; Hänsch, Robert; Mendel, Ralf R.; Hille, Russ

    2009-01-01

    Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. The plant form of the enzyme, on the other hand, lacks a prosthetic group other than its molybdenum center and utilizes molecular oxygen as the physiological oxidant. Hydrogen peroxide is the ultimate product of the reaction. Here, we present data demonstrating that superoxide is produced essentially quantitatively both in the course of the reaction of reduced enzyme with O2 and during steady-state turnover and only subsequently decays (presumably noncatalytically) to form hydrogen peroxide. Rapid-reaction kinetic studies directly following the reoxidation of reduced enzyme demonstrate a linear dependence of the rate constant for the reaction on [O2] with a second-order rate constant of kox = 8.7 × 104 ± 0.5 × 104 m−1s−1. When the reaction is carried out in the presence of cytochrome c to follow superoxide generation, biphasic time courses are observed, indicating that a first equivalent of superoxide is generated in the oxidation of the fully reduced Mo(IV) state of the enzyme to Mo(V), followed by a slower oxidation of the Mo(V) state to Mo(VI). The physiological implications of plant sulfite oxidase as a copious generator of superoxide are discussed. PMID:19875441

  19. Evolution of the primate cytochrome c oxidase subunit II gene.

    PubMed

    Adkins, R M; Honeycutt, R L

    1994-03-01

    We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. PMID:8006990

  20. 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

  1. Substrate specificity of copper-containing plant amine oxidases.

    PubMed

    Pietrangeli, P; Federico, R; Mondovì, B; Morpurgo, L

    2007-07-01

    The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions. PMID:17521737

  2. Renalase Prevents AKI Independent of Amine Oxidase Activity

    PubMed Central

    Wang, Ling; Velazquez, Heino; Moeckel, Gilbert; Chang, John; Ham, Ahrom; Lee, H. Thomas; Safirstein, Robert

    2014-01-01

    AKI is characterized by increased catecholamine levels and hypertension. Renalase, a secretory flavoprotein that oxidizes catecholamines, attenuates ischemic injury and the associated increase in catecholamine levels in mice. However, whether the amine oxidase activity of renalase is involved in preventing ischemic injury is debated. In this study, recombinant renalase protected human proximal tubular (HK-2) cells against cisplatin- and hydrogen peroxide–induced necrosis. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals subjected to cisplatin-induced AKI. Interestingly, compared with the intact renalase protein, a 20–amino acid peptide (RP-220), which is conserved in all known renalase isoforms, but lacks detectable oxidase activity, was equally effective at protecting HK-2 cells against toxic injury and preventing ischemic injury in wild-type mice. Furthermore, in vitro treatment with RP-220 or recombinant renalase rapidly activated Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinases and downregulated c-Jun N-terminal kinase. In summary, renalase promotes cell survival and protects against renal injury in mice through the activation of intracellular signaling cascades, independent of its ability to metabolize catecholamines, and we have identified the region of renalase required for these effects. Renalase and related peptides show potential as therapeutic agents for the prevention and treatment of AKI. PMID:24511138

  3. Function and structure of rat hepatic coproporphyrinogen oxidase.

    PubMed

    Sorianello, E M; Mazzetti, M B

    2000-10-01

    Rat hepatic coproporphyrinogen oxidase, the sixth enzyme in the heme biosynthetic pathway, was purified 1340-fold with a yield of 39.7%. To obtain the soluble enzyme, different methods were applied to disrupt mitochondria, with sonication giving the highest yield (85%). The minimum catalytic form of enzyme was a dimer with a molecular mass of 77 +/- 4 kDa. The existence of aggregated forms was possible since in fractions of gel filtration elution activity was observed with higher molecular mass. We determined a Stokes radius of 36.3 A, a sedimentation coefficient (S20,w) of 5.06 S, and frictional ratio of 1.29, suggesting a nearly globular shape of the protein. Regardless of the type of salt, high ionic strength inhibits the enzyme, probably modifying its native structure. Experiments with amino acid modifiers showed that histidine, arginine, and tryptophan are involved in the catalytic process. Non-ionic detergents and phospholipids activated the enzyme, probably because they reproduce its natural hydrophobic environment. The present study describes a simple method for the purification of rat liver coproporphyrinogen oxidase, introducing for the first time data on the structure and function of the protein in a tissue often used as a laboratory model in biological studies, and contributing to the study of human hereditary coproporphyria. PMID:11079369

  4. 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. PMID:21511361

  5. 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

  6. [Molecular docking analysis of xanthine oxidase inhibition by constituents of cichory].

    PubMed

    Wang, Xue-jie; Lin, Zhi-jian; Zhang, Bing; Zhu, Chun-sheng; Niu, Hong-juan; Zhou, Yue; Nie, An-zheng; Wang, Yu

    2015-10-01

    Human xanthine oxidase is considered to be a target for therapy of hyperuricemia. Cichorium intybus is a Chinese plant medicine which widely used in Xinjiang against various diseases. In order to screen the inhibitors of xanthine oxidase from C. intybus and to explore main pharmacological actions of cichory a compound collection of C. intybus was built via consulting related references about chemical research on cichory. The three-dimensional crystal structure of xanthine oxidase (PDB code: 1N5X) from Protein Data Bank was downloaded.. Autodock 4.2 was employed to screen the inhibitors of xanthine oxidase from cichory 70 compounds were found to possess quite low binding free energy comparing with TEI (febuxostat). C. intybus contains constituents possessing potential inhibitive activity against xanthine oxidase. It can explain the main pharmacological actions of cichory which can significantly lower the level of serum uric acid. PMID:26975108

  7. 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. PMID:27083394

  8. NADPH Oxidase 1 and Its Derived Reactive Oxygen Species Mediated Tissue Injury and Repair

    PubMed Central

    Fu, Xiu-Jun; Peng, Ying-Bo; Hu, Yi-Ping; Shi, You-Zhen; Yao, Min; Zhang, Xiong

    2014-01-01

    Reactive oxygen species are mostly viewed to cause oxidative damage to various cells and induce organ dysfunction after ischemia-reperfusion injury. However, they are also considered as crucial molecules for cellular signal transduction in biology. NADPH oxidase, whose only function is reactive oxygen species production, has been extensively investigated in many cell types especially phagocytes. The deficiency of NADPH oxidase extends the process of inflammation and delays tissue repair, which causes chronic granulomatous disease in patients. NADPH oxidase 1, one member of the NADPH oxidase family, is not only constitutively expressed in a variety of tissues, but also induced to increase expression in both mRNA and protein levels under many circumstances. NADPH oxidase 1 and its derived reactive oxygen species are suggested to be able to regulate inflammation reaction, cell proliferation and migration, and extracellular matrix synthesis, which contribute to the processes of tissue injury and repair. PMID:24669283

  9. Infantile isolated sulphite oxidase deficiency: report of a case with negative sulphite test and normal sulphate excretion.

    PubMed

    van der Klei-van Moorsel, J M; Smit, L M; Brockstedt, M; Jakobs, C; Dorche, C; Duran, M

    1991-01-01

    We present the clinical and biochemical data of a patient with infantile isolated sulphite oxidase deficiency with late onset of symptoms. A comparison of the biochemical parameters is made with the neonatal type of this disease and with the data of described patients with the combined defect of sulphite oxidase and xanthine oxidase, due to molybdenum cofactor deficiency. False-negative sulphite dip stick test as a pitfall in the diagnosis of sulphite oxidase deficiency is discussed. PMID:2044591

  10. High concentrations of antibodies to xanthine oxidase in human and animal sera. Molecular characterization.

    PubMed Central

    Bruder, G; Jarasch, E D; Heid, H W

    1984-01-01

    The widespread occurrence of antibodies (IgG) specific to xanthine oxidase in both normal (nonimmune) human and animal sera, and in antisera raised against a diversity of unrelated antigens is described. A study of sera from 81 humans revealed that xanthine oxidase-specific IgG represents a high proportion (1-8%) of total IgG. No obvious correlation to pathological events or symptoms of disease could be found. These xanthine oxidase-specific antibodies could be isolated by immunoaffinity chromatography on purified human or bovine xanthine oxidase and showed specific binding to the enzyme polypeptide of Mr 155,000 in immunoblotting experiments. By immunofluorescence microscopy they displayed the same cell type-specific reaction as experimentally induced antibodies, i.e., the staining of lactating mammary gland epithelium and capillary endothelium. The naturally occurring xanthine oxidase-specific antibodies consisted of polyclonal IgG of various subclasses. F(ab')2 preparations gave immune-reactions identical to those of IgG. The human xanthine oxidase-specific IgG cross-reacted with the bovine enzyme and both human and animal antibodies partially inhibited its activity. The xanthine oxidase activity of human milk lipid globules and supernatant fractions from various human tissues was extremely low when compared with that of the bovine antigen. The enzyme protein, however, was effectively precipitated from these sources by both the human and bovine antibodies. We suggest that the exceptionally high concentrations of antibodies against one protein, xanthine oxidase, are due to self-immunization to the xanthine oxidase antigen present in endothelial cells of capillaries. We do not exclude, however, nutritional contributions of bovine milk antigen to the appearance of xanthine oxidase antibodies in human sera. The possible biological functions of this immunological reaction are discussed. Images PMID:6381540

  11. Xanthine oxidase in human skeletal muscle following eccentric exercise: a role in inflammation.

    PubMed Central

    Hellsten, Y; Frandsen, U; Orthenblad, N; Sjødin, B; Richter, E A

    1997-01-01

    1. The present study tested the hypothesis that the level of xanthine oxidase is elevated in injured human skeletal muscle in association with inflammatory events. Seven male subjects performed five bouts of strenuous one-legged eccentric exercise. Muscle biopsies from both the exercised and the control leg, together with venous blood samples, were obtained prior to exercise and at 45 min, 24, 48 and 96 h after exercise. The time courses of xanthine oxidase immunoreactivity and indicators of muscle damage and inflammation were examined. 2. The number of xanthine oxidase structures observed by immunohistological methods in the exercised muscle was up to eightfold higher than control from day 1 to day 4 after exercise (P < 0.05). The increase was attributed to an enhanced expression of xanthine oxidase in microvascular endothelial cells and an invasion of leucocytes containing xanthine oxidase. 3. The concentration of plasma interleukin-6 was significantly higher 90 min after exercise than before exercise (P < 0.05) and remained higher than pre-exercise levels throughout the 4 days. On day 4 the plasma creatine kinase activity was approximately 150-fold higher (P < 0.05) than resting levels. 4. Despite the increase in xanthine oxidase in the muscle there were no detectable changes in the levels of muscle malondialdehyde or in plasma antioxidant capacity up to 4 days post-exercise. 5. It is concluded that eccentric exercise leads to an increased level of xanthine oxidase in human muscle and that the increase is associated with secondary inflammatory processes. The increase in xanthine oxidase in the muscle occurs mainly in microvascular endothelial cells, but occurs also via infiltrating leucocytes containing xanthine oxidase. A role for leucocytes in xanthine oxidase induction in endothelium is proposed. Images Figure 2 PMID:9023782

  12. Spatiotemporal Localization of d-Amino Acid Oxidase and d-Aspartate Oxidases during Development in Caenorhabditis elegans

    PubMed Central

    Saitoh, Yasuaki; Katane, Masumi; Kawata, Tomonori; Maeda, Kazuhiro; Sekine, Masae; Furuchi, Takemitsu; Kobuna, Hiroyuki; Sakamoto, Taro; Inoue, Takao; Arai, Hiroyuki; Nakagawa, Yasuhito

    2012-01-01

    Recent investigations have shown that a variety of d-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. d-Amino acid oxidase (DAO) and d-aspartate oxidase (DDO) are degradative enzymes stereospecific for d-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of d-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several d- and l-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and d-amino acids in multicellular organisms. PMID:22393259

  13. Spatiotemporal localization of D-amino acid oxidase and D-aspartate oxidases during development in Caenorhabditis elegans.

    PubMed

    Saitoh, Yasuaki; Katane, Masumi; Kawata, Tomonori; Maeda, Kazuhiro; Sekine, Masae; Furuchi, Takemitsu; Kobuna, Hiroyuki; Sakamoto, Taro; Inoue, Takao; Arai, Hiroyuki; Nakagawa, Yasuhito; Homma, Hiroshi

    2012-05-01

    Recent investigations have shown that a variety of D-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. D-Amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are degradative enzymes stereospecific for D-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of D-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several D- and L-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and D-amino acids in multicellular organisms. PMID:22393259

  14. Cyanobacterial lactate oxidases serve as essential partners in N2 fixation and evolved into photorespiratory glycolate oxidases in plants.

    PubMed

    Hackenberg, Claudia; Kern, Ramona; Hüge, Jan; Stal, Lucas J; Tsuji, Yoshinori; Kopka, Joachim; Shiraiwa, Yoshihiro; Bauwe, Hermann; Hagemann, Martin

    2011-08-01

    Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N(2)-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N(2)-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N(2) fixation was more sensitive to O(2) in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O(2)-scavenging enzyme to protect nitrogenase in extant N(2)-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration. PMID:21828292

  15. 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. PMID:21734717

  16. 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

  17. Two-color experiments combining the UV storage ring free-electron laser and the SA5 IR beamline at Super-ACO

    NASA Astrophysics Data System (ADS)

    Nahon, Laurent; Renault, Eric; Couprie, Marie-Emmanuelle; Nutarelli, Daniele; Garzella, David; Billardon, Michel; Carr, G. Lawrence; Williams, Gwyn P.; Dumas, Paul

    1999-10-01

    The UV-storage ring Free Electron Laser (FEL) operating at Super-ACO is a tunable, coherent and intense (up to 300 mW) photon source in the near-UV range (300 - 350 nm). Besides, it has the unique feature to be synchronized in a one-to-one shot ratio with the Synchrotron Radiation (SR) at the high repetition rate of 8.32 MHz. This FEL + SR combination appears to be very powerful for the performance of pump- probe time-resolved and/or frequency-resolved experiments on the sub-ns and ns time-scales. In particular, there is a strong scientific case for the combination of the recently- commissioned SA5 Infra-Red Synchrotron Radiation beamline with the UV-FEL, for the performance of transient IR- absorption spectroscopy on FEL-excited samples with a Fourier-transform spectrometer coupled with a microscope allowing high spectral and spatial resolution. The principle and interest of the two-color combination altogether with the description of both the FEL and the SA5 IR beamline are presented. The first synchronization signal between the IR and the UV beams is shown. The correct spatial overlap between the UV (FEL) and the IR (SR) photon beams is demonstrated by monitoring via IR-spectro-microscopy the time evolution of a single mineral particulate (kaolinite) under UV-FEL irradiation.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26637599

  19. 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

    2016-03-01

    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. PMID:26840063

  20. 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

    2016-02-01

    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. PMID:26637599

  1. 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

  2. Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice.

    PubMed

    Nomura, Johji; Busso, Nathalie; Ives, Annette; Matsui, Chieko; Tsujimoto, Syunsuke; Shirakura, Takashi; Tamura, Mizuho; Kobayashi, Tsunefumi; So, Alexander; Yamanaka, Yoshihiro

    2014-01-01

    Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE(-/-) mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE(-/-) mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis. PMID:24686534

  3. 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.

  4. Biogenesis and Assembly of Eukaryotic Cytochrome c Oxidase Catalytic Core

    PubMed Central

    Soto, Ileana C.; Fontanesi, Flavia; Liu, Jingjing; Barrientos, Antoni

    2011-01-01

    Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin which assembly is intricate and highly regulated. The COX catalytic core is formed by three mitochondrial DNA encoded subunits, Cox1, Cox2 and Cox3, conserved in the bacterial enzyme. Their biogenesis requires the action of messenger-specific and subunit-specific factors which facilitate the synthesis, membrane insertion, maturation or assembly of the core subunits. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to identify these ancillary factors. Here we review the current state of knowledge of the biogenesis and assembly of the eukaryotic COX catalytic core and discuss the degree of conservation of the players and mechanisms operating from yeast to human. PMID:21958598

  5. Quantitative structure-activity studies on monoamine oxidase inhibitors.

    PubMed

    Johnson, C L

    1976-05-01

    Quantitative structure-activity studies were carried out on a series of N-isopropylaryl hydrazides which inhibits monoamine oxidase (MAO). The inhibitory potencies of these compounds of MAO were found to correlate with the electron-withdrawing capacity of the aryl ring substituents as estimated by both empirical Hammet sigma constants and electronic indices from molecular orbital calculations. Based on these correlations and previously published data on other classes of MAO inhibitors, a general model for the inhibitor pharmacophore is proposed: potent MAO of an aromatic ring; electron-withdrawing groups on the aromatic ring or replacing the phenyl ring with certain types of heterocyclic rings will tend to increase the potency. PMID:1271400

  6. Traumatic Brain Injury and NADPH Oxidase: A Deep Relationship

    PubMed Central

    Prata, Cecilia; Vieceli Dalla Sega, Francesco; Piperno, Roberto; Hrelia, Silvana

    2015-01-01

    Traumatic brain injury (TBI) represents one of the major causes of mortality and disability in the world. TBI is characterized by primary damage resulting from the mechanical forces applied to the head as a direct result of the trauma and by the subsequent secondary injury due to a complex cascade of biochemical events that eventually lead to neuronal cell death. Oxidative stress plays a pivotal role in the genesis of the delayed harmful effects contributing to permanent damage. NADPH oxidases (Nox), ubiquitary membrane multisubunit enzymes whose unique function is the production of reactive oxygen species (ROS), have been shown to be a major source of ROS in the brain and to be involved in several neurological diseases. Emerging evidence demonstrates that Nox is upregulated after TBI, suggesting Nox critical role in the onset and development of this pathology. In this review, we summarize the current evidence about the role of Nox enzymes in the pathophysiology of TBI. PMID:25918580

  7. 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.

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

    PubMed Central

    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.; Çoku, 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

    2009-01-01

    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-tRNAGlu 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 tRNAGlu may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis. PMID:19720722

  9. NADPH Oxidase: A Potential Target for Treatment of Stroke

    PubMed Central

    Zhang, Li; Wu, Jie; Duan, Xiaochun; Tian, Xiaodi; Shen, Haitao; Sun, Qing; Chen, Gang

    2016-01-01

    Stroke is the third leading cause of death in industrialized nations. Oxidative stress is involved in the pathogenesis of stroke, and excessive generation of reactive oxygen species (ROS) by mitochondria is thought to be the main cause of oxidative stress. NADPH oxidase (NOX) enzymes have recently been identified and studied as important producers of ROS in brain tissues after stroke. Several reports have shown that knockout or deletion of NOX exerts a neuroprotective effect in three major experimental stroke models. Recent studies also confirmed that NOX inhibitors ameliorate brain injury and improve neurological outcome after stroke. However, the physiological and pathophysiological roles of NOX enzymes in the central nervous system (CNS) are not known well. In this review, we provide a comprehensive summary of our current understanding about expression and physiological function of NOX enzymes in the CNS and its pathophysiological roles in the three major types of stroke: ischemic stroke, intracerebral hemorrhage, and subarachnoid hemorrhage. PMID:26941888

  10. 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.

  11. Peroxidase and peroxidase-oxidase activities of isolated human myeloperoxidases.

    PubMed Central

    Svensson, B E; Domeij, K; Lindvall, S; Rydell, G

    1987-01-01

    Isolated neutrophils from healthy donors were used for the isolation of four highly purified forms of myeloperoxidase as determined by spectral (A430/A280 ratio 0.80-0.87) and enzyme-activity measurements. Although the myeloperoxidases exhibited different elution profiles on cation-exchange chromatography, gel filtration indicated similar relative molecular masses. When these forms were assayed for peroxidase and peroxidase-oxidase activities with several substrates, they all exhibited virtually the same specific activities. These results suggest that possible functional differences between the enzymes may be related to differences in their sites of action rather than to differences in enzyme activity. Myeloperoxidase from a patient with chronic myeloid leukaemia also revealed a similar heterogeneity on cation-exchange chromatography. However, this myeloperoxidase contained in addition one form with a lower and one form with a higher relative molecular mass, as indicated by gel-filtration chromatography. PMID:3036098

  12. Rational surface silane modification for immobilizing glucose oxidase.

    PubMed

    Tian, Feibao; Guo, Yi; Lin, Feifei; Zhang, Yumei; Yuan, Qipeng; Liang, Hao

    2016-06-01

    Glucose oxidase (GOx) has many significant applications in biosensor and biocatalysis. In this study, we firstly quantitatively analyzed the binding efficiency of (3-aminopropyl) trimethoxysilane (APTES) modified onto the surface of GOx. It was found that the contents of the grafted silane did not significantly influence the relative activities and tertiary structures of all surface modified GOxs. Immobilization ratio and relative activity of all instances of APTES modified GOx increased, compared with those of native enzyme. However, good stability of immobilized GOx at extreme pH and high temperature could only be obtained when modified protein with low binding silane content. At pH 2.0, the immobilized GOx with low binding content showed a more than 600% activity, compared to the free enzyme. Therefore, rational surface modification would be beneficial to improving the activity and stability of immobilized enzyme as well as increasing loading amount. PMID:26921503

  13. Physiological role of alternative oxidase (from yeasts to plants).

    PubMed

    Rogov, A G; Zvyagilskaya, R A

    2015-04-01

    Mitochondria of all so far studied organisms, with the exception of Archaea, mammals, some yeasts, and protists, contain, along with the classical phosphorylating cytochrome pathway, a so-called cyanide-insensitive alternative oxidase (AOX) localized on the matrix side of the mitochondrial inner membrane, and electron transport through which is not coupled with ATP synthesis and energy accumulation. Mechanisms underlying plentiful functions of AOX in organisms at various levels of organization ranging from yeasts to plants are considered. First and foremost, AOX provides a chance of cell survival after inhibiting the terminal components of the main respiratory chain or losing the ability to synthesize these components. The vitally important role of AOX is obvious in thermogenesis of thermogenic plant organs where it becomes the only terminal oxidase with a very high activity, and the energy of substrate oxidation by this respiratory pathway is converted into heat, thus promoting evaporation of volatile substances attracting pollinating insects. AOX plays a fundamentally significant role in alleviating or preventing oxidative stress, thus ensuring the defense against a wide range of stresses and adverse environmental conditions, such as changes in temperature and light intensities, osmotic stress, drought, and attack by incompatible strains of bacterial pathogens, phytopathogens, or their elicitors. Participation of AOX in pathogen survival during its existence inside the host, in antivirus defense, as well as in metabolic rearrangements in plants during embryogenesis and cell differentiation is described. Examples are given to demonstrate that AOX might be an important tool to overcome the adverse aftereffects of restricted activity of the main respiratory chain in cells and whole animals. PMID:25869356

  14. Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.

    PubMed

    Arango Gutierrez, Erik; Mundhada, Hemanshu; Meier, Thomas; Duefel, Hartmut; Bocola, Marco; Schwaneberg, Ulrich

    2013-12-15

    Glucose oxidase is an oxidoreductase exhibiting a high β-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed. A diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. For directed GOx evolution a microtiter plate detection system based on the quinone diimine mediator was developed and the well-known ABTS-assay was applied in microtiter plate format to validate oxygen independency of improved GOx variants. Two iterative rounds of random diversity generation and screening yielded to two subsets of amino acid positions which mainly improved activity (A173, A332) and oxygen independency (F414, V560). Simultaneous site saturation of all four positions with a reduced subset of amino acids using the OmniChange method yielded finally variant V7 with a 37-fold decreased oxygen dependency (mediator activity: 7.4 U/mg WT, 47.5 U/mg V7; oxygen activity: 172.3 U/mg WT, 30.1 U/mg V7). V7 is still highly β-D-glucose specific, highly active with the quinone diimine mediator and thermal resistance is retained (prerequisite for GOx coating of diabetes test stripes). The latter properties and V7's oxygen insensitivity make V7 a very promising candidate to replace standard GOx in diabetes care applications. PMID:23835222

  15. 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. PMID:22975122

  16. Cytochrome c oxidase: Evolution of control via nuclear subunit addition☆

    PubMed Central

    Pierron, Denis; Wildman, Derek E.; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I.

    2014-01-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the “higher” organism possessing large brains and muscles. The main function of these subunits appears to be “only” to control the activity of the mitochondrial subunits. We propose that this control function is an as yet underappreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a “domestication scenario” such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. This article is part of a Special Issue entitled

  17. D-amino acid oxidase from the yeast Trigonopsis variabilis.

    PubMed

    Kubicek-Pranz, E M; Röhr, M

    1985-04-01

    D-Amino acid oxidase (EC 1.4.3.3) has been purified from the yeast Trigonopsis variabilis by the application of ion-exchange chromatography on DEAE-cellulose, salt precipitation, gel filtration, and hydroxyapatite adsorption. Alternatively the last two steps can be substituted by a single fast protein liquid chromatographic ion-exchange step (Mono Q). The enzyme appeared homogeneous in PAGE, but small amounts of impurities (not exceeding 5% of total protein) appeared in sodium dodecyl sulfate (SDS)-PAGE. Its Mr in SDS-PAGE is 39,000; it exhibits an isoelectric point of 4.8 and contains 7% (w/v) covalently bound carbohydrate. Its absorption spectrum is similar to hog kidney D-amino acid oxidase, indicating the presence of bound FAD, which, however, could not be separated from the enzyme under non-denaturing conditions. The enzyme is inhibited by SH-oxidizing agents, but not by metal-chelate formers and not by benzoate or toluene. It uses O2 exclusively as the only H acceptor. Km and Vmax values were determined for 15 D-amino acids, which, among 23 tested, were substrates of the enzyme. The enzyme has highest affinity for D-phenylalanine and D-leucine, but maximal activity is obtained with D-citrulline and D-isoleucine. The specific activity of the purified preparation is even higher than that of the commercially available hog kidney enzyme (21.7 vs 16 U/mg). The yeast enzyme may be a useful analytical and preparative tool in view of the difference between its substrate specificity and that of the hog enzyme. PMID:2865242

  18. NADPH oxidase 2 plays a role in experimental corneal neovascularization.

    PubMed

    Chan, Elsa C; van Wijngaarden, Peter; Chan, Elsie; Ngo, Darleen; Wang, Jiang-Hui; Peshavariya, Hitesh M; Dusting, Gregory J; Liu, Guei-Sheung

    2016-05-01

    Corneal neovascularization, the growth of new blood vessels in the cornea, is a leading cause of vision impairment after corneal injury. Neovascularization typically occurs in response to corneal injury such as that caused by infection, physical trauma, chemical burns or in the setting of corneal transplant rejection. The NADPH oxidase enzyme complex is involved in cell signalling for wound-healing angiogenesis, but its role in corneal neovascularization has not been studied. We have now analysed the role of the Nox2 isoform of NADPH oxidase in corneal neovascularization in mice following chemical injury. C57BL/6 mice aged 8-14 weeks were cauterized with an applicator coated with 75% silver nitrate and 25% potassium nitrate for 8 s. Neovascularization extending radially from limbal vessels was observed in corneal whole-mounts from cauterized wild type mice and CD31+ vessels were identified in cauterized corneal sections at day 7. In contrast, in Nox2 knockout (Nox2 KO) mice vascular endothelial growth factor-A (Vegf-A), Flt1 mRNA expression, and the extent of corneal neovascularization were all markedly reduced compared with their wild type controls. The accumulation of Iba-1+ microglia and macrophages in the cornea was significantly less in Nox2 KO than in wild type mice. In conclusion, we have demonstrated that Nox2 is implicated in the inflammatory and neovascular response to corneal chemical injury in mice and clearly VEGF is a mediator of this effect. This work raises the possibility that therapies targeting Nox2 may have potential for suppressing corneal neovascularization and inflammation in humans. PMID:26814205

  19. Apocynin: chemical and biophysical properties of a NADPH oxidase inhibitor.

    PubMed

    Petrônio, Maicon S; Zeraik, Maria Luiza; Fonseca, Luiz Marcos da; Ximenes, Valdecir F

    2013-01-01

    Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (e275nm = 1.1 × 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M-1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin. PMID:23455672

  20. Protonation State-Dependent Communication in Cytochrome c Oxidase.

    PubMed

    Helabad, Mahdi Bagherpoor; Ghane, Tahereh; Reidelbach, Marco; Woelke, Anna Lena; Knapp, Ernst Walter; Imhof, Petra

    2016-08-01

    Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both redox enzyme and proton pump, proton transfer out of either of the channels toward the BNC or into the protein toward a proton loading site, and ultimately through the membrane, must be highly regulated. The O→E intermediate of cytochrome c oxidase is the first redox state in its catalytic cycle, where proton transfer through the K-channel, from K362 to Y288 at the BNC, is important. Molecular dynamics simulations of this intermediate with 16 different combinations of protonation states of key residues in the D- and K-channel show the mutual impact of the two proton-conducting channels to be protonation state-dependent. Strength as well as means of communication, correlations in positions, or connections along the hydrogen-bonded network, change with the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286, which would in principle allow proton transfer to the BNC, but no proton pumping until a proton has reached E286. PMID:27508434

  1. Engineering Pyranose 2-Oxidase for Modified Oxygen Reactivity

    PubMed Central

    Brugger, Dagmar; Krondorfer, Iris; Shelswell, Christopher; Huber-Dittes, Benjamin; Haltrich, Dietmar; Peterbauer, Clemens K.

    2014-01-01

    Pyranose 2-oxidase (POx), a member of the GMC family of flavoproteins, catalyzes the regioselective oxidation of aldopyranoses at position C2 to the corresponding 2-ketoaldoses. During the first half-reaction, FAD is reduced to FADH2 and reoxidized in the second half-reaction by reducing molecular oxygen to H2O2. Alternative electron acceptors including quinones, radicals or chelated metal ions show significant and in some cases even higher activity. While oxygen as cheap and abundantly available electron acceptor is favored for many processes, reduced oxygen reactivity is desirable for some applications such as in biosensors/biofuel cells because of reduced oxidative damages to the biocatalyst from concomitant H2O2 production as well as reduced electron “leakage” to oxygen. The reactivity of flavoproteins with oxygen is of considerable scientific interest, and the determinants of oxygen activation and reactivity are the subject of numerous studies. We applied site-saturation mutagenesis on a set of eleven amino acids around the active site based on the crystal structure of the enzyme. Using microtiter plate screening assays with peroxidase/2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 2,6-dichlorophenolindophenol, variants of POx with decreased oxidase activity and maintained dehydrogenase activity were identified. Variants T166R, Q448H, L545C, L547R and N593C were characterized with respect to their apparent steady-state constants with oxygen and the alternative electron acceptors DCPIP, 1,4-benzoquinone and ferricenium ion, and the effect of the mutations was rationalized based on structural properties. PMID:25296188

  2. Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum.

    PubMed

    Sutay Kocabas, Didem; Bakir, Ufuk; Phillips, Simon E V; McPherson, Michael J; Ogel, Zumrut B

    2008-06-01

    A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases. PMID:18369615

  3. Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach.

    PubMed

    Agostinelli, E; Tempera, G; Viceconte, N; Saccoccio, S; Battaglia, V; Grancara, S; Toninello, A; Stevanato, R

    2010-02-01

    The polyamines spermine, spermidine and putrescine are ubiquitous cell components. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper-containing amine oxidases. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. In tumors, polyamines and amine oxidases are increased as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H(2)O(2) and aldehydes produced by the reaction. This study demonstrated that multidrug-resistant (MDR) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding wild-type (WT) ones to H(2)O(2) and aldehydes, the products of BSAO-catalyzed oxidation of spermine. Transmission electron microscopy (TEM) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. Increasing the incubation temperature from 37 to 42 degrees Celsius enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumor growth, particularly well if the enzyme has been conjugated to a biocompatible hydrogel polymers. Since both wild-type and MDR cancer cells after pre-treatment with MDL 72527, a lysosomotropic compound, are sensitized to subsequent exposure to BSAO/spermine, it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumor cells. It is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting. PMID:20012114

  4. 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.

  5. Bienzyme biosensors for glucose, ethanol and putrescine built on oxidase and sweet potato peroxidase.

    PubMed

    Castillo, Jaime; Gáspár, Szilveszter; Sakharov, Ivan; Csöregi, Elisabeth

    2003-05-01

    Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H(2)O(2) produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors. PMID:12706582

  6. Engineering Human Urate Oxidase: Towards Reactivating It as an Important Therapeutic Enzyme.

    PubMed

    Dabbagh, Fatemeh; Ghoshoon, Mohammad B; Hemmati, Shiva; Zamani, Mozhdeh; Mohkam, Milad; Ghasemi, Younes

    2015-01-01

    Urate oxidase is considered as an important therapeutic enzyme used to control hyperuricemia. In spite of widespread distribution in numerous (micro)organisms, active urate oxidase is absent in higher primates (humans and apes) due to gene mutations. Considering the therapeutic significance of urate oxidase, further understanding on the inactivation process of the enzyme during primate evolution is critical. This study, therefore, aims to express genetically modified human urate oxidase in the methylotrophic yeast Pichia pastoris. Accordingly, the genetically modified human urate oxidase was successfully expressed intracellularly and extracellularly under the control of an alcohol oxidase promoter and was subjected to the enzyme activity assay. The results demonstrated that reactivating the non-functional human urate oxidase gene fully or even moderately by simply replacing the premature stop codons is impossible. This finding confirms the idea that a number of successive loss-of-function missense mutations occurred during evolution, making higher primates functional uricase-deficit and vulnerable to hyperuricemic disorders. PMID:26343133

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

    PubMed

    Lin, L S; Varner, J E

    1991-05-01

    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." PMID:16668145

  8. Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

    PubMed Central

    Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

    2011-01-01

    Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the

  9. The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance

    PubMed Central

    Leclerc, Julia; Rosenfeld, Eric; Trainini, Mathieu; Martin, Bénédicte; Meuric, Vincent; Bonnaure-Mallet, Martine; Baysse, Christine

    2015-01-01

    Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance. PMID:26629705

  10. Lack of GLYCOLATE OXIDASE1, but Not GLYCOLATE OXIDASE2, Attenuates the Photorespiratory Phenotype of CATALASE2-Deficient Arabidopsis.

    PubMed

    Kerchev, Pavel; Waszczak, Cezary; Lewandowska, Aleksandra; Willems, Patrick; Shapiguzov, Alexey; Li, Zhen; Alseekh, Saleh; Mühlenbock, Per; Hoeberichts, Frank A; Huang, Jingjing; Van Der Kelen, Katrien; Kangasjärvi, Jaakko; Fernie, Alisdair R; De Smet, Riet; Van de Peer, Yves; Messens, Joris; Van Breusegem, Frank

    2016-07-01

    The genes coding for the core metabolic enzymes of the photorespiratory pathway that allows plants with C3-type photosynthesis to survive in an oxygen-rich atmosphere, have been largely discovered in genetic screens aimed to isolate mutants that are unviable under ambient air. As an exception, glycolate oxidase (GOX) mutants with a photorespiratory phenotype have not been described yet in C3 species. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the peroxisomal CATALASE2 (cat2-2) that display stunted growth and cell death lesions under ambient air, we isolated a second-site loss-of-function mutation in GLYCOLATE OXIDASE1 (GOX1) that attenuated the photorespiratory phenotype of cat2-2 Interestingly, knocking out the nearly identical GOX2 in the cat2-2 background did not affect the photorespiratory phenotype, indicating that GOX1 and GOX2 play distinct metabolic roles. We further investigated their individual functions in single gox1-1 and gox2-1 mutants and revealed that their phenotypes can be modulated by environmental conditions that increase the metabolic flux through the photorespiratory pathway. High light negatively affected the photosynthetic performance and growth of both gox1-1 and gox2-1 mutants, but the negative consequences of severe photorespiration were more pronounced in the absence of GOX1, which was accompanied with lesser ability to process glycolate. Taken together, our results point toward divergent functions of the two photorespiratory GOX isoforms in Arabidopsis and contribute to a better understanding of the photorespiratory pathway. PMID:27225899

  11. Comparative characterization of three D-aspartate oxidases and one D-amino acid oxidase from Caenorhabditis elegans.

    PubMed

    Katane, Masumi; Saitoh, Yasuaki; Seida, Yousuke; Sekine, Masae; Furuchi, Takemitsu; Homma, Hiroshi

    2010-06-01

    Previously, we cloned cDNAs for four Caenorhabditis elegans genes (F20 Hp, C47Ap, F18Ep, and Y69Ap genes) that were annotated in the database as encoding D-amino acid oxidase (DAO) or D-aspartate oxidase (DDO) proteins. These genes were expressed in Escherichia coli, and the recombinant C47Ap and F18Ep were shown to have functional DDO activities, while Y69Ap had functional DAO activity. In this study, we improved the E. coli culture conditions for the production of recombinant F20 Hp and, following purification of the protein, revealed that it has functional DDO activity. The kinetic properties of recombinant C47Ap (DDO-1), F18Ep (DDO-2), F20 Hp (DDO-3), and Y69Ap (DAO) were also determined and compared with recombinant human DDO and DAO. In contrast to the low catalytic efficiency of human DDO for D-Glu, all three C. elegans DDOs showed higher catalytic efficiencies for D-Glu than D-Asp or N-methyl-D-Asp. The catalytic efficiency of C. elegans DAO for D-Ser was substantially lower than that of human DAO, while the C. elegans DAO was more efficient at deamination of basic D-amino acids (D-Arg and D-His) than human DAO. Collectively, our results indicate that C. elegans contains at least three genes that encode functional DDOs, and one gene encoding a functional DAO, and that these enzymes have different and distinctive properties. PMID:20564561

  12. 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. PMID:25301889

  13. Disruption of the CYTOCHROME C OXIDASE DEFICIENT1 Gene Leads to Cytochrome c Oxidase Depletion and Reorchestrated Respiratory Metabolism in Arabidopsis1[C][W

    PubMed Central

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

    2014-01-01

    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. PMID:25301889

  14. 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.

  15. Management of Neonatal Respiratory Distress Syndrome Employing ACoRN Respiratory Sequence Protocol versus Early Nasal Continuous Positive Airway Pressure Protocol

    PubMed Central

    Niknafs, Pedram; Faghani, Asadallah; Afjeh, Seyed-Abolfazl; Moradinazer, Mehdi; Bahman-Bijari, Bahareh

    2014-01-01

    Objective: Respiratory distress syndrome (RDS) is a common cause of respiratory distress in premature infants. This study was designed to evaluate two different RDS treatment protocols by comparing the outcomes. Methods: This study was a double center cross sectional study performed from June to December 2012. During that period, 386 neonates with RDS were hospitalized and treated according to two different therapeutic protocols so-called Acute Care of at-Risk Newborns (ACoRN) respiratory sequence protocol (group I) and Early Nasal Continuous Positive Airway Pressure (E-NCPAP) protocol (group II). The variables and main outcomes of this study were gestational age, birth weight, bronchopulmonary dysplasia (BPD), pulmonary hemorrhage (PH), intraventricular hemorrhage (IVH), air leak and mortality rate (MR). Findings : Out of 386 infants, 202 infants were in group I (male 60.4%, female 39.6%, mean gestational age 316/7 weeks, mean birth weight=1688 grams) and group II included 184 infants (male 61.4%, female 38.6%, mean gestational age 32 weeks, mean birth weight 1787 grams), P= 0.07. The ratios of BPD of group I to group II and PH of group I to group two were not significant (P=0.63 and P=0.84, respectively). Air leak ratio in group I was higher than in group II (P=0.001). Although IVH ratio in group II was higher than in group I (P=0.01), grade III and IV IVH was higher in group I (30% vs. 4.6%). In case of MR, it was higher in group I than in group II (P=0.001). Conclusion: According to the findings the incidence of air leak, grade III and IV IVH and MR was less common in E-NCPAP protocol, so it may show the effectiveness of this protocol. The authors suggest that more researches are needed for more accurate results. PMID:25793046

  16. 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.

  17. 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...

  18. Identification of a superoxide-generating NADPH oxidase system in human fibroblasts.

    PubMed Central

    Meier, B; Cross, A R; Hancock, J T; Kaup, F J; Jones, O T

    1991-01-01

    Human fibroblasts have the capacity to release superoxide radicals upon stimulation of an electron transport system similar to the NADPH oxidase of leukocytes. Two components of the NADPH oxidase system, (1) a flavoprotein of 45 kDa which binds diphenylene iodonium (a compound described as a specific inhibitor of the leukocyte NADPH oxidase), and (2) a low-potential cytochrome b, are present in fibroblast membranes. Fibroblasts exhibit these compounds at lower concentrations than do polymorphonuclear leukocytes or B-lymphocytes. The superoxide-generating system is rather uniformly associated with the outer cell membrane, as shown by light and electron microscopy. Superoxide release upon stimulation with various agents was prevented by the addition of micromolar concentrations of diphenylene iodonium, making an NADPH oxidase a likely source. Images Fig. 4. PMID:1850240

  19. Optimization of glucose oxidase production by Aspergillus niger using genetic- and process-engineering techniques.

    PubMed

    Hellmuth, K; Pluschkell, S; Jung, J K; Ruttkowski, E; Rinas, U

    1995-11-01

    Wild-type Aspergillus niger NRRL-3 was transformed with multiple copies of the glucose oxidase structural gene (god). The gene was placed under the control of the gpdA promoter of A. nidulans. For more efficient secretion the alpha-amylase signal peptide from A. oryzae was inserted in front of god. Compared to the wild type, the recombinant strain NRRL-3 (GOD3-18) produced up to four times more extracellular glucose oxidase under identical culture conditions. Addition of yeast extract (2 gl-1) to a mineral salts medium containing only glucose as carbon source increased volumetric and specific extracellular glucose oxidase activities by 130% and 50% respectively. With the same medium composition and inoculum size, volumetric and specific extracellular glucose oxidase activities increased more than ten times in bioreactor cultivations compared to shake-flask cultures. PMID:8590664

  20. 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. PMID:2410565

  1. Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases.

    PubMed

    Safarian, Schara; Rajendran, Chitra; Müller, Hannelore; Preu, Julia; Langer, Julian D; Ovchinnikov, Sergey; Hirose, Taichiro; Kusumoto, Tomoichirou; Sakamoto, Junshi; Michel, Hartmut

    2016-04-29

    The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper-containing oxidases, even though the structures are completely different. PMID:27126043

  2. 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.

  3. [Thermostabilities of plant phenol oxidase and peroxidase, determining the technology of their use in food industry].

    PubMed

    Mchedlishvili, N I; Omiadze, N T; Gulua, L K; Sadunishvili, T A; Zamtaradze, R K; Abutidze, M O; Bendeliani, E G; Kvesitadze, G I

    2005-01-01

    Stabilities of phenol oxidase and peroxidase from tea plant (Camellia sinensis L.) clone Kolkhida leaves, apple (Malus domestica L.) cultivar Kekhura fruits, walnut (Juglans regia L.) green pericarp, and horseradish (Armoracia lapathifolia Gilib) roots were studied using different storage temperature modes and storage duration. It was demonstrated that both enzymes retained residual activities (approximately 10%) upon 20-min incubation at 80 degrees C. Phenol oxidases from tea, walnut, and, especially, apple, as well as tea peroxidase were stable during storage. A technology for treatment of plant oxidases was proposed, based on the use of a natural inhibitor phenol oxidase and peroxidase, isolated from tea leaves, which solving the problem of residual activities of these enzymes, arising during pasteurization and storage of beverages and juices. It was demonstrated that browning of apple juice during pasteurization and beer turbidity during storage could be efficiently prevented using the natural inhibitor of these enzymes. PMID:15859458

  4. Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography.

    PubMed

    Yamamoto, T; Moriwaki, Y; Takahashi, S; Tsutsumi, Z; Yamakita, J; Nasako, Y; Hiroishi, K; Higashino, K

    1996-06-01

    An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 microliters of plasma and 240 microliters of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 microM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects. PMID:8811453

  5. Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Battelli, M G; Abbondanza, A; Musiani, S; Buonamici, L; Strocchi, P; Tazzari, P L; Gramantieri, L; Stirpe, F

    1999-03-01

    Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range. PMID:10217635

  6. Enzymatic Characterization and In Vivo Function of Five Terminal Oxidases in Pseudomonas aeruginosa

    PubMed Central

    Kawakami, Takuro; Osamura, Tatsuya; Hirai, Takehiro; Sakai, Yoshiaki; Ishii, Masaharu

    2014-01-01

    The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo3-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa3-type cytochrome c oxidase (aa3), and two cbb3-type cytochrome c oxidases (cbb3-1 and cbb3-2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant 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 cbb3-1 and cbb3-2, indicating that Cyo, CIO, and aa3 are low-affinity enzymes and that cbb3-1 and cbb3-2 are high-affinity enzymes. Although cbb3-1 and cbb3-2 exhibited different expression patterns in response to oxygen concentration, they had similar Km values for oxygen. Both cbb3-1 and cbb3-2 utilized cytochrome c4 as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb3-1 and cbb3-2 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. PMID:25182500

  7. Herbivore-plant interactions: mixed-function oxidases and secondary plant substances.

    PubMed

    Brattsten, L B; Wilkinson, C F; Eisner, T

    1977-06-17

    The mixed-function oxidases of a polyphagous insect larva (the southern armyworm, Spodoptera eridania) were found to be induced by a diversity of secondary plant substances. The induction proceeds rapidly and in response to a small quantity of secondary substance. Following induction, the larva is less susceptible to dietary poisoning. It is argued that mixed-function oxidases play a major role in protecting herbivores against chemical stress from secondary plant substances. PMID:17831753

  8. Neuronal NAD(P)H Oxidases Contribute to ROS Production and Mediate RGC Death after Ischemia

    PubMed Central

    Dvoriantchikova, Galina; Grant, Jeff; Santos, Andrea Rachelle C.; Hernandez, Eleut; Ivanov, Dmitry

    2012-01-01

    Purpose. To study the role of neuronal nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase–dependent reactive oxygen species (ROS) production in retinal ganglion cell (RGC) death after ischemia. Methods. Ischemic injury was induced by unilateral elevation of intraocular pressure via direct corneal cannulation. For in vitro experiments, RGCs isolated by immunopanning from retinas were exposed to oxygen and glucose deprivation (OGD). The expression levels of NAD(P)H oxidase subunits were evaluated by quantitative PCR, immunocytochemistry, and immunohistochemistry. The level of ROS generated was assayed by dihydroethidium. The NAD(P)H oxidase inhibitors were then tested to determine if inhibition of NAD(P)H oxidase altered the production of ROS within the RGCs and promoted cell survival. Results. It was reported that RGCs express catalytic Nox1, Nox2, Nox4, Duox1, as well as regulatory Ncf1/p47phox, Ncf2/p67phox, Cyba/p22phox, Noxo1, and Noxa1 subunits of NAD(P)H oxidases under normal conditions and after ischemia. However, whereas RGCs express only low levels of catalytic Nox2, Nox4, and Duox1, and regulatory Ncf1/p47, Ncf2/p67 subunits, they exhibit significantly higher levels of catalytic subunit Nox1 and the subunits required for optimal activity of Nox1. It was observed that the nonselective NAD(P)H oxidase inhibitors VAS-2870, AEBSF, and the Nox1 NAD(P)H oxidase–specific inhibitor ML-090 decreased the ROS burst stimulated by OGD, which was associated with a decreased level of RGC death. Conclusions. The findings suggest that NAD(P)H oxidase activity in RGCs renders them vulnerable to ischemic death. Importantly, high levels of Nox1 NAD(P)H oxidase subunits in RGCs suggest that this enzyme could be a major source of ROS in RGCs produced by NAD(P)H oxidases. PMID:22467573

  9. Herpecaudin from Herpetospermum caudigerum, a Xanthine Oxidase Inhibitor with a Novel Isoprenoid Scaffold.

    PubMed

    Jiang, He Zhong; Tan, Rui; Jiao, Rui Hua; Deng, Xin Zhao; Tan, Ren Xiang

    2016-07-01

    Herpecaudin (3), a xanthine oxidase inhibitor with an unprecedented scaffold, was discovered from Herpetospermum caudigerum seeds. The structure was determined by spectroscopic and X-ray single crystallographic methods. A possible biogenetic pathway leading to herpecaudin is proposed, starting from congeners 23,24-dihydrocucurbitacin E (1) and endecaphyllacin B (2), and involving retro-aldol cleavage as a key step. All three compounds proved to be active and represent new scaffolds of non-purine analogue xanthine oxidase inhibitors. PMID:27272398

  10. Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617.

    PubMed

    Arnaud, S; Malatesta, F; Guigliarelli, B; Gayda, J P; Bertrand, P; Miraglio, R; Denis, M

    1991-06-01

    The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 617 ends with a single terminal oxidase. It is a heme-containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475-479]. The purification of this oxidase was achieved in a single step through by DEAE-Trisacryl chromatography. SDS/PAGE showed the presence of four subunits. The pI was found to be 4.45 and a Mr of 130,000 was determined by gel filtration. The amino acid composition of the purified terminal oxidase has been determined. About 52% of the residues are hydrophobic, strengthening the membranous nature of this bacterial oxidase. Room temperature optical spectra are typical of heme b with a 560-nm band for the reduced form in the alpha range. The prosthetic group is made of two hemes b, one high-spin (S = 5/2, gl = 5.9, g parallel approximately 2.0), the other low-spin (S = 1/2, gz = 2.94, gy = 2.27). No other metal centre was detected by EPR. The two hemes remained unresolved in optical spectra, even at low temperature, and throughout redox titration. They behaved potentiometrically like a one-electron, single redox couple, with Em = 87 +/- 10 mV at pH 7.2 and 293 K. The purified oxidase did not oxidize ferrocytochrome c, but displayed quinol oxidase activity both with the native quinone (2419 nmol O2.min-1.mg protein-1 and commercially available coenzyme (101.74 nmol O2.min-1.mg protein-1). Exposure of the reduced enzyme to CO induced the collapse of alpha and beta bands as occurred during reoxidation. In contrast, NaCN and NaN3 fully inhibited the oxidase activity. Results are discussed with respect to other purified quinol oxidases. PMID:1645655

  11. Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

    PubMed Central

    Grimm, Melissa J.; Lewandowski, David C.; Pham, Christine T. N.; Blackwell, Timothy S.; Petraitiene, Ruta; Petraitis, Vidmantas; Walsh, Thomas J.; Urban, Constantin F.; Segal, Brahm H.

    2011-01-01

    NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox−/−) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)−/−×cathepsin G (CG)−/− mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox−/− mice, whereas NE−/−×CG−/− mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens. PMID:22163282

  12. Dual oxidase regulates neutrophil recruitment in allergic airways.

    PubMed

    Chang, Sandra; Linderholm, Angela; Franzi, Lisa; Kenyon, Nicholas; Grasberger, Helmut; Harper, Richart

    2013-12-01

    Enhanced reactive oxygen species production in allergic airways is well described and correlates with increased airway contractions, inflammatory cell infiltration, goblet cell metaplasia, and mucus hypersecretion. There is also an abundance of interleukin-4/interleukin-13 (IL-4/IL-13)- or interleukin-5-secreting cells that are thought to be central to the pathogenesis of allergic asthma. We postulated that the dual oxidases (DUOX1 and DUOX2), members of the nicotinamide adenine dinucleotide phosphate oxidase family that release hydrogen peroxide (H2O2) in the respiratory tract, are critical proteins in the pathogenesis of allergic airways. DUOX activity is regulated by cytokines, including IL-4 and IL-13, and DUOX-mediated H2O2 influences several important features of allergic asthma: mucin production, IL-8 secretion, and wound healing. The objective of this study was to establish the contribution of DUOXs to the development of allergic asthma in a murine model. To accomplish this goal, we utilized a DUOXA-deficient mouse model (Duoxa(-/-)) that lacked maturation factors for both DUOX1 and DUOX2. Our results are the first to demonstrate evidence of DUOX protein and DUOX functional activity in murine airway epithelium. We also demonstrate that DUOXA maturation factors are required for airway-specific H2O2 production and localization of DUOX to cilia of fully differentiated airway epithelial cells. We compared wild-type and Duoxa(-/-) mice in an ovalbumin exposure model to determine the role of DUOX in allergic asthma. In comparison to DUOX-intact mice, Duoxa(-/-) mice had reduced mucous cell metaplasia and lower levels of TH2 cytokine levels in bronchoalveolar fluid. In addition, increased airway resistance in response to methacholine was observed in Duoxa(+/+) mice, as expected, but was absent in Duoxa(-/-) mice. Surprisingly, Duoxa(-/-) mice had decreased influx of neutrophils in bronchoalveolar fluid and lung tissue sections associated with a lower level of the

  13. Dual Oxidase Regulates Neutrophil Recruitment in Allergic Airways

    PubMed Central

    Chang, Sandra; Linderholm, Angela; Franzi, Lisa; Kenyon, Nicholas; Grasberger, Helmut; Harper, Richart

    2013-01-01

    Enhanced reactive oxygen species production in allergic airways is well described, and correlates with increased airway contractions, inflammatory cell infiltration, goblet cell metaplasia, and mucus hypersecretion. There is also an abundance of interleukin-4/interleukin-13 (IL-4/IL-13) or interleukin-5-secreting cells that are thought to be central to the pathogenesis of allergic asthma. We postulated that dual oxidases (DUOX1 and DUOX2), members of the nicotinamide adenine dinucleotide phosphate oxidase family that release hydrogen peroxide (H2O2) in the respiratory tract, are critical proteins in the pathogenesis of allergic airways. DUOX activity is regulated by cytokines including IL-4 and IL-13, and DUOX-mediated H2O2 influences several important features of allergic asthma: mucin production, IL-8 secretion, and wound healing. The objective of this study was to establish the contribution of DUOX to the development of allergic asthma in a murine model. To accomplish this goal, we utilized a DUOXA-deficient mouse model (Duoxa−/−) that lacked maturation factors for both DUOX1 and DUOX2. Our results are the first to demonstrate evidence of DUOX protein and DUOX functional activity in murine airway epithelium. We also demonstrate that DUOXA maturation factors are required for airway-specific H2O2 production and localization of DUOX to cilia of fully differentiated airway epithelial cells. We compared wild-type and Duoxa−/− mice in an ovalbumin exposure model to determine the role of DUOX in allergic asthma. In comparison to DUOX-intact mice, Duoxa−/− mice had reduced mucous cell metaplasia, and lower levels of TH2 cytokine levels in bronchoalveolar fluid. In addition, increased airway resistance in response to methacholine was observed in Duoxa+/+ mice as expected, but was absent in Duoxa−/− mice. Surprisingly, Duoxa−/− mice had decreased influx of neutrophils in bronchoalveolar fluid and lung tissue sections associated with a lower level of

  14. 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. PMID:26340429

  15. NADPH oxidase of human dendritic cells: role in Candida albicans killing and regulation by interferons, dectin-1 and CD206.

    PubMed

    Donini, Marta; Zenaro, Elena; Tamassia, Nicola; Dusi, Stefano

    2007-05-01

    Human monocyte-derived DC express the enzyme NADPH oxidase, responsible for ROS production. We show that Candida albicans did not activate NADPH oxidase in DC, and was poorly killed by these cells. However, Candida-killing activity increased upon DC stimulation with the NADPH oxidase activator PMA and was further enhanced by DC treatment with IFN-alpha or IFN-gamma. This fungicidal activity took place at high DC-to-Candida ratio, but decreased at low DC-to-yeast ratio, when Candida inhibited the NADPH oxidase by contrasting the assembly of the enzyme on DC plasma membrane. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated the PMA-dependent DC candidacidal capacity. Engagement of beta-glucan receptor dectin-1 induced NADPH oxidase activation in DC that was depressed by mannose-binding receptor CD206 co-stimulation. Candida was internalized by DC through mannose-binding receptors, but not through dectin-1, thus explaining why Candida did not elicit NADPH oxidase activity. Our results indicate that NADPH oxidase is involved in DC Candida-killing activity, which is increased by IFN. However, Candida escapes the oxidative damage by inhibiting NADPH oxidase and by entering DC through receptors not involved in NADPH oxidase activation. PMID:17407098

  16. 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

  17. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    PubMed Central

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  18. Oxidase-functionalized Fe(3)O(4) nanoparticles for fluorescence sensing of specific substrate.

    PubMed

    Liu, Cheng-Hao; Tseng, Wei-Lung

    2011-10-01

    This study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe(3)O(4) nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H(2)O(2), the reaction pH between Fe(3)O(4) NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe(3)O(4) NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe(3)O(4) NPs and oxidase for the quantification of specific substrates through the H(2)O(2)-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 μM using glucose oxidase-Fe(3)O(4), galactose oxidase-Fe(3)O(4), and choline oxidase-Fe(3)O(4) composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method. PMID:21843679

  19. Cloning, expression and biochemical characterization of the cholesterol oxidase CgChoA from Chryseobacterium gleum

    PubMed Central

    2014-01-01

    Background Cholesterol oxidases are important enzymes for applications such as the analysis of cholesterol in clinical samples, the synthesis of steroid derived drugs, and are considered as potential antibacterial drug targets. Results The gene choA encoding a cholesterol oxidase from Chryseobacterium gleum DSM 16776 was cloned into the pQE-30 expression vector and heterologously expressed in Escherichia coli JM109 co-transformed with pRARE2. The N-terminally His-tagged cholesterol oxidase (CgChoA) was assigned to be a monomer in solution by size exclusion chromatography, showed a temperature optimum of 35°C, and a pH optimum at 6.75 using 0.011 M MOPS buffer under the tested conditions. The purified protein showed a maximum activity of 15.5 U/mg. CgChoA showed a Michaelis-Menten like kinetic behavior only when the substrate was dissolved in water and taurocholate (apparent Km = 0.5 mM). In addition, the conversion of cholesterol by CgChoA was studied via biocatalytic batches at analytical scale, and cholest-4-en-3-one was confirmed as product by HPLC-MS. Conclusion CgChoA is a true cholesterol oxidase which activity ranges among the high performing described cholesterol oxidases from other organisms. Thus, the enzyme broadens the available toolbox of cholesterol oxidases for e.g. synthetic and biosensing applications. PMID:24885249

  20. Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity

    PubMed Central

    Campillo-Brocal, Jonatan Cristian; Lucas-Elio, Patricia; Sanchez-Amat, Antonio

    2013-01-01

    Abstract A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases. PMID:23873697

  1. A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

    PubMed Central

    Buschmann, Sabine; Richers, Sebastian; Ermler, Ulrich; Michel, Hartmut

    2014-01-01

    The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-d-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. PMID:24488923

  2. Ectopic Expression of Pumpkin Gibberellin Oxidases Alters Gibberellin Biosynthesis and Development of Transgenic Arabidopsis Plants1

    PubMed Central

    Radi, Abeer; Lange, Theo; Niki, Tomoya; Koshioka, Masaji; Lange, Maria João Pimenta

    2006-01-01

    Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development. PMID:16384902

  3. 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. PMID:25573142

  4. Regulation of NADPH Oxidase in Vascular Endothelium: The Role of Phospholipases, Protein Kinases, and Cytoskeletal Proteins

    PubMed Central

    Pendyala, Srikanth; Usatyuk, Peter V.; Gorshkova, Irina A.; Garcia, Joe G.N.

    2009-01-01

    The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47phox, p67phox, and Rac1) and membrane-associated components (Noxes and p22phox). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase–derived ROS in the pathobiology of lung diseases. Antioxid. Redox Signal. 11, 841–860. PMID:18828698

  5. 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

  6. 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. PMID:27066025

  7. Coherent reaction dynamics in a bacterial cytochrome c oxidase.

    PubMed

    Liebl, U; Lipowski, G; Négrerie, M; Lambry, J C; Martin, J L; Vos, M H

    1999-09-01

    Biological reactions in protein complexes involve structural dynamics spanning many orders of magnitude in time. In standard descriptions of catalysis by enzymes, the transition state between reactant and product is reached by thermal, stochastic motion. In the ultrashort time domain, however, the protein moiety and cofactor motions leading to altered conformations can be coherent rather than stochastic in nature. Such coherent motions may play a key role in controlling the accessibility of the transition state and explain the high efficiency of the reaction. Here we present evidence for coherent population transfer to the product state during an ultrafast reaction catalysed by a key enzyme in aerobic organisms. Using the enzyme cytochrome c oxidase aa3 from the bacterium Paracoccus denitrificans, we have studied haem dynamics during the photo-initiated ultrafast transfer of carbon monoxide from haem a3 to CuB by femtosecond spectroscopy. The ground state of the unliganded a3 species is populated in a stepwise manner in time, indicating that the reaction is mainly governed by coherent vibrations (47cm(-1)). The reaction coordinate involves conformational relaxation of the haem group and we suggest that ligand transfer also contributes. PMID:10490029

  8. 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

  9. 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.

  10. The Function of Ascorbate Oxidase in Tobacco1

    PubMed Central

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

    2003-01-01

    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-γ-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. PMID:12857842

  11. 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

  12. 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. PMID:24412427

  13. 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.

  14. 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.

  15. Phagocyte NADPH oxidase, chronic granulomatous disease and mycobacterial infections.

    PubMed

    Deffert, Christine; Cachat, Julien; Krause, Karl-Heinz

    2014-08-01

    Infection of humans with Mycobacterium tuberculosis remains frequent and may still lead to death. After primary infection, the immune system is often able to control M. tuberculosis infection over a prolonged latency period, but a decrease in immune function (from HIV to immunosenescence) leads to active disease. Available vaccines against tuberculosis are restricted to BCG, a live vaccine with an attenuated strain of M. bovis. Immunodeficiency may not only be associated with an increased risk of tuberculosis, but also with local or disseminated BCG infection. Genetic deficiency in the reactive oxygen species (ROS)-producing phagocyte NADPH oxidase NOX2 is called chronic granulomatous disease (CGD). CGD is among the most common primary immune deficiencies. Here we review our knowledge on the importance of NOX2-derived ROS in mycobacterial infection. A literature review suggests that human CGD patient frequently have an increased susceptibility to BCG and to M. tuberculosis. In vitro studies and experiments with CGD mice are incomplete and yielded - at least in part - contradictory results. Thus, although observations in human CGD patients leave little doubt about the role of NOX2 in the control of mycobacteria, further studies will be necessary to unequivocally define and understand the role of ROS. PMID:24916152

  16. cDNA cloning and expression of potato polyphenol oxidase.

    PubMed

    Hunt, M D; Eannetta, N T; Yu, H; Newman, S M; Steffens, J C

    1993-01-01

    Polyphenol oxidases (PPOs) of plants are copper metalloproteins which catalyze the oxidation of mono- and o-diphenols to o-diquinones. Although PPOs are believed to be primarily responsible for the deleterious browning of many fruit and vegetable crops and are thought to be involved in plant-pest interactions, direct evidence for these roles is lacking. We report the cloning of two PPO cDNAs from Solanum tuberosum leaves. These cDNAs exhibit 97% and 98% sequence similarity at the DNA and deduced amino acid levels, respectively. Putative copper-binding regions of both cDNAs are very similar to those of mammalian, bacterial and Neurospora tyrosinases. Both leaf PPO cDNAs appear to encode polypeptides which are processed to a mature molecular weight of 57,000. In potato leaves, petioles, roots, and flowers, PPO is encoded by ca. 2 kb transcripts. Leaf PPO mRNA is developmentally regulated and only detectable in young foliage. In contrast, the protein profile of immunologically detectable PPO remains constant from the apical node through the eleventh leaf node. PMID:7678763

  17. Brain monoamine oxidase A activity predicts trait aggression.

    PubMed

    Alia-Klein, Nelly; Goldstein, Rita Z; Kriplani, Aarti; Logan, Jean; Tomasi, Dardo; Williams, Benjamin; Telang, Frank; Shumay, Elena; Biegon, Anat; Craig, Ian W; Henn, Fritz; Wang, Gene-Jack; Volkow, Nora D; Fowler, Joanna S

    2008-05-01

    The genetic deletion of monoamine oxidase A (MAO A), an enzyme that breaks down the monoamine neurotransmitters norepinephrine, serotonin, and dopamine, produces aggressive phenotypes across species. Therefore, a common polymorphism in the MAO A gene (MAOA, Mendelian Inheritance in Men database number 309850, referred to as high or low based on transcription in non-neuronal cells) has been investigated in a number of externalizing behavioral and clinical phenotypes. These studies provide evidence linking the low MAOA genotype and violent behavior but only through interaction with severe environmental stressors during childhood. Here, we hypothesized that in healthy adult males the gene product of MAO A in the brain, rather than the gene per se, would be associated with regulating the concentration of brain amines involved in trait aggression. Brain MAO A activity was measured in vivo in healthy nonsmoking men with positron emission tomography using a radioligand specific for MAO A (clorgyline labeled with carbon 11). Trait aggression was measured with the multidimensional personality questionnaire (MPQ). Here we report for the first time that brain MAO A correlates inversely with the MPQ trait measure of aggression (but not with other personality traits) such that the lower the MAO A activity in cortical and subcortical brain regions, the higher the self-reported aggression (in both MAOA genotype groups) contributing to more than one-third of the variability. Because trait aggression is a measure used to predict antisocial behavior, these results underscore the relevance of MAO A as a neurochemical substrate of aberrant aggression. PMID:18463263

  18. Lysyl oxidase: from basic science to future cancer treatment.

    PubMed

    Nishioka, Takeshi; Eustace, Amanda; West, Catharine

    2012-01-01

    In this mini-review, we discuss the physiological and pathological roles of lysyl oxidase (LOX) and its family, LOX-like proteins (LOXL), in relation to prognosis of major cancers. The number of reports on LOX family is numerous. We have decided to review the articles that were recently published (i.e. past 5 years). Experimental techniques in molecular biology have advanced surprisingly in the past decade. Accordingly, the results of the studies are more reliable. Most studies reached the same conclusion; a higher LOX- or LOXL- expression is associated with a poor prognosis. Molecular experiments have already started aiming for clinical application, and the results are encouraging. Suppressing LOX or LOXL activities resulted in lower cell motility in collagen gel and, moreover, succeeded in reducing metastases in mice. LOX family members were originally recognized as molecules that cross-link collagen fibers in the extracellular matrix. Recent studies demonstrated that they are also involved in a phenomenon called Epithelial Mesenchymal Transition (EMT). This may affect cell movement and cancer cell invasiveness. LOX and LOXL2 are regulated by hypoxia, a major factor in the failure of cancer treatment. Here we discuss the molecular biology of the LOX family in relation to its role in tumor biology. PMID:22453058

  19. Brain Monoamine Oxidase-A Activity Predicts Trait Aggression

    PubMed Central

    Alia-Klein, Nelly; Goldstein, Rita Z.; Kriplani, Aarti; Logan, Jean; Tomasi, Dardo; Williams, Benjamin; Telang, Frank; Shumay, Elena; Biegon, Anat; Craig, Ian W.; Henn, Fritz; Wang, Gene-Jack; Volkow, Nora D.; Fowler, Joanna S.

    2008-01-01

    The genetic deletion of monoamine oxidase A (MAO A, an enzyme which breaks down the monoamine neurotransmitters norepinephrine, serotonin and dopamine) produces aggressive phenotypes across species. Therefore, a common polymorphism in the MAO A gene (MAOA, MIM 309850, referred to as high or low based on transcription in non-neuronal cells) has been investigated in a number of externalizing behavioral and clinical phenotypes. These studies provide evidence linking the low MAOA genotype and violent behavior but only through interaction with severe environmental stressors during childhood. Here, we hypothesized that in healthy adult males the gene product of MAO A in the brain, rather than the gene per se, would be associated with regulating the concentration of brain amines involved in trait aggression. Brain MAO A activity was measured in-vivo in healthy non-smoking men with positron emission tomography using a radioligand specific for MAO A (clorgyline labeled with carbon 11). Trait aggression was measured with the Multidimensional Personality Questionnaire (MPQ). Here we report for the first time that brain MAO A correlates inversely with the MPQ trait measure of aggression (but not with other personality traits) such that the lower the MAO A activity in cortical and subcortical brain regions the higher the self-reported aggression (in both MAOA genotype groups) contributing to more than a third of the variability. Since trait aggression is a measure used to predict antisocial behavior, these results underscore the relevance of MAO A as a neurochemical substrate of aberrant aggression. PMID:18463263

  20. 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

  1. 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. PMID:23471247

  2. Polyphenol oxidase as a biochemical seed defense mechanism.

    PubMed

    Fuerst, E Patrick; Okubara, Patricia A; Anderson, James V; Morris, Craig F

    2014-01-01

    Seed dormancy and resistance to decay are fundamental survival strategies, which allow a population of seeds to germinate over long periods of time. Seeds have physical, chemical, and biological defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. Here, we hypothesize that seeds also possess enzyme-based biochemical defenses, based on induction of the plant defense enzyme, polyphenol oxidase (PPO), when wild oat (Avena fatua L.) caryopses and seeds were challenged with seed-decaying Fusarium fungi. These studies suggest that dormant seeds are capable of mounting a defense response to pathogens. The pathogen-induced PPO activity from wild oat was attributed to a soluble isoform of the enzyme that appeared to result, at least in part, from proteolytic activation of a latent PPO isoform. PPO activity was also induced in wild oat hulls (lemma and palea), non-living tissues that cover and protect the caryopsis. These results are consistent with the hypothesis that seeds possess inducible enzyme-based biochemical defenses arrayed on the exterior of seeds and these defenses represent a fundamental mechanism of seed survival and longevity in the soil. Enzyme-based biochemical defenses may have broader implications since they may apply to other defense enzymes as well as to a diversity of plant species and ecosystems. PMID:25540647

  3. Characterization of active site residues of nitroalkane oxidase.

    PubMed

    Valley, Michael P; Fenny, Nana S; Ali, Shah R; Fitzpatrick, Paul F

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  4. Reexamining Michaelis-Menten enzyme kinetics for xanthine oxidase

    PubMed Central

    Chinn, Tamara M.

    2013-01-01

    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. PMID:23471247

  5. Unfolding and refolding of active apple polyphenol oxidase.

    PubMed

    Mari, S; Marquès, L; Breton, F; Karamanos, Y; Macheix, J J

    1998-11-01

    For the first time, unfolding (6 M guanidine) and refolding of partially proteolysed purified polyphenol oxidase (PPOr) was achieved, with 88% of activity recovered. Optimal refolding conditions consisted in stepwise dialysis of guanidine treated extracts, the dialysis buffers containing 1 M (NH4)2SO4 and 100 microM CuSO4. However, CuSO4 had limited effect on the recovering of PPOr activity, whereas (NH4)2SO4 was essential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have shown (i) a compact tertiary structure and (ii) the presence of disulfide bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 41 and 42 kDa, respectively. Resistance to proteolytic cleavage was used to study the conformational changes induced by the denaturing treatments. Folded PPOr was resistant to further proteolysis whereas unfolded PPO was totally digested, indicating the role of tertiary structure of PPOr in the resistance to proteases. PMID:9842726

  6. Monoamine oxidase A (MAO A) inhibitors decrease glioma progression.

    PubMed

    Kushal, Swati; Wang, Weijun; 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

    2016-03-22

    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

  7. 2-Benzylidene-1-indanone derivatives as inhibitors of monoamine oxidase.

    PubMed

    Nel, Magdalena S; Petzer, Anél; Petzer, Jacobus P; Legoabe, Lesetja J

    2016-10-01

    In the present study, a series of twenty-two 2-benzylidene-1-indanone derivatives were synthesised and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The 2-benzylidene-1-indanone derivatives are structurally related to a series of benzylideneindanone derivatives which has previously been found to be MAO-B inhibitors. This study finds that the 2-benzylidene-1-indanones are MAO-B specific inhibitors with IC50 values <2.74μM. Among the compounds evaluated, twelve compounds exhibited IC50<0.1μM and may be considered as high potency inhibitors. The 2-benzylidene-1-indanone derivatives also inhibited MAO-A with the most potent inhibition exhibited by 5g (IC50=0.131μM). An analysis of the structure-activity relationships for MAO-B inhibition show that substitution on the A-ring with a 5-hydroxy group and on the B-ring with halogens and the methyl group yield high potency inhibition. It may therefore be concluded that 2-benzylidene-1-indanone analogues are promising leads for design of therapies for disorders such as Parkinson's disease. PMID:27578245

  8. Defensive Mutualism Rescues NADPH Oxidase Inactivation in Gut Infection.

    PubMed

    Pircalabioru, Gratiela; Aviello, Gabriella; Kubica, Malgorzata; Zhdanov, Alexander; Paclet, Marie-Helene; Brennan, Lorraine; Hertzberger, Rosanne; Papkovsky, Dmitri; Bourke, Billy; Knaus, Ulla G

    2016-05-11

    NOX/DUOX family of NADPH oxidases are expressed in diverse tissues and are the primary enzymes for the generation of reactive oxygen species (ROS). The intestinal epithelium expresses NOX1, NOX4, and DUOX2, whose functions are not well understood. To address this, we generated mice with complete or epithelium-restricted deficiency in the obligatory NOX dimerization partner Cyba (p22(phox)). We discovered that NOX1 regulates DUOX2 expression in the intestinal epithelium, which magnified the epithelial ROS-deficiency. Unexpectedly, epithelial deficiency of Cyba resulted in protection from C. rodentium and L. monocytogenes infection. Microbiota analysis linked epithelial Cyba deficiency to an enrichment of H2O2-producing bacterial strains in the gut. In particular, elevated levels of lactobacilli physically displaced and attenuated C. rodentium virulence by H2O2-mediated suppression of the virulence-associated LEE pathogenicity island. This transmissible compensatory adaptation relied on environmental factors, an important consideration for prevention and therapy of enteric disease. PMID:27173933

  9. Evolution of histamine oxidase activity for biotechnological applications.

    PubMed

    Rosini, Elena; Tonin, Fabio; Vasylieva, Natalia; Marinesco, Stephane; Pollegioni, Loredano

    2014-01-01

    Histamine is present to various degrees in many foods, and concentrations in fish samples are considered a good indicator of freshness and hygienic food quality. Seeking for innovative methods to quantify histamine in foods, we used a synthetic gene designed on the sequence of histamine oxidase from Arthrobacter crystallopoietes (HOD) as the starting point in this study to develop a biosensor. HOD was expressed in Escherichia coli cells with a yield of ∼7 mg protein/L of fermentation broth. Recombinant wild-type HOD oxidized histamine and tyramine whereas it was inactive toward putrescine and cadaverine (two amines present in fish samples). The putative residues involved in substrate binding were identified by an in silico docking procedure based on a model of the structure of HOD: site-saturation mutagenesis was performed on 8 positions. The most significant changes in kinetic properties were observed for the P143M HOD: this variant showed higher histamine affinity and lower substrate inhibition by tyramine than wild-type enzyme. Biosensor prototypes were produced using both the wild-type and the P143M variant HOD. These biosensors showed a good sensitivity and selectivity with respect to biogenic amines present in food specimens. Accordingly, the HOD-based biosensor was successfully used to assess histamine in fish samples, yielding values in good agreement with those obtained by HPLC analyses but in a few seconds and at a significantly lower cost per analysis. PMID:23995223

  10. 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%. PMID:26856529

  11. Xanthine oxidase inhibitory activity of Vietnamese medicinal plants.

    PubMed

    Nguyen, Mai Thanh Thi; Awale, Suresh; Tezuka, Yasuhiro; Tran, Quan Le; Watanabe, Hiroshi; Kadota, Shigetoshi

    2004-09-01

    Among 288 extracts, prepared from 96 medicinal plants used in Vietnamese traditional medicine to treat gout and related symptoms, 188 demonstrated xanthine oxidase (XO) inhibitory activity at 100 microg/ml, with 46 having greater than 50% inhibition. At 50 microg/ml, 168 of the extracts were active, with 21 possessing more than 50% inhibition. At 25 microg/ml, 146 extracts exhibited inhibitory activity, with 8 showing over 50% inhibition, while 126 extracts presented activity at 10 microg/ml, with 2 having greater than 50% inhibition. The MeOH extracts of Artemisia vulgaris, Caesalpinia sappan (collected at the Seven-Mountain area), Blumea balsamifera (collected in Lam Dong province), Chrysanthemum sinense and MeOH-H(2)O extract of Tetracera scandens (Khanh Hoa province) exhibited strong XO inhibitory activity with IC(50) values less than 20 microg/ml. The most active extract was the MeOH extract of the flower of C. sinense with an IC(50) value of 5.1 microg/ml. Activity-guided fractionation of the MeOH extract led to the isolation of caffeic acid (1), luteolin (2), eriodictyol (3), and 1,5-di-O-caffeoylquinic acid (4). All these compounds showed significant XO inhibitory activity in a concentration-dependent manner, and the activity of 2 was more potent (IC(50) 1.3 microM) than the clinically used drug, allopurinol (IC(50) 2.5 microM). PMID:15340229

  12. Cataplexy and monoamine oxidase deficiency in Norrie disease.

    PubMed

    Vossler, D G; Wyler, A R; Wilkus, R J; Gardner-Walker, G; Vlcek, B W

    1996-05-01

    Norrie disease (ND) is an X-linked recessive disorder causing ocular atrophy, mental retardation, deafness, and dysmorphic features. Virtually absent monoamine oxidase (MAO) type-A and -B activity has been found in some boys with chromosome deletions. We report the coexistence of cataplexy and abnormal REM sleep organization with ND. Three related boys, referred for treatment of medically refractory atonic spells and apneas, underwent extended EEG-video-polysomnographic monitoring. They demonstrated attacks of cataplexy and inappropriate periods of REM sleep during which they were unarousable. One boy also had generalized tonic-clonic seizures. Previous testing revealed that all three have complete ND gene deletions. In all subjects, platelet MAO-B activity was absent, serum serotonin levels were markedly increased, and plasma catecholamine levels were normal. Data from the canine narcolepsy syndrome model implicate abnormal catecholaminergic and cholinergic activities in the pathogenesis of cataplexy. Our findings suggest that abnormal MAO activity or an imbalance between serotonin and other neurotransmitter levels may be involved in the pathogenesis of human cataplexy. PMID:8628463

  13. 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

  14. 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

  15. NADPH Oxidase-Dependent Superoxide Production in Plant Reproductive Tissues

    PubMed Central

    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. PMID:27066025

  16. Purification and properties of dihydrogeodin oxidase from Aspergillus terreus.

    PubMed

    Fujii, I; Iijima, H; Tsukita, S; Ebizuka, Y; Sankawa, U

    1987-01-01

    The last step of (+)-geodin biosynthesis is a phenol oxidative coupling, which is one of the most important reactions in biosynthesis of natural products. The enzyme named dihydrogeodin oxidase catalyzes the regio- and stereospecific phenol oxidative coupling reaction to form (+)-geodin from dihydrogeodin. The enzyme was purified from the cell-free extract of Aspergillus terreus, a (+)-geodin producer, by ammonium sulfate fractionation, acid treatment, and column chromatographies on DEAE-cellulose, Hydroxyapatite, chromatofocusing, and Toyopearl HW-55S. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 153,000 by gel filtration on a Toyopearl HW-55S column and 76,000 by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer. The purified enzyme showed an intense blue color and had absorption maxima at 280 and 600 nm, which suggested it to be a blue copper protein. The copper content was found to be 8 atoms per subunit by atomic absorption analysis and no significant amount of other metals was detected by ICP emission spectrometry. The electron paramagnetic resonance spectrum showed the presence of type 1 and type 2 copper atoms in the enzyme molecule. Sodium azide and ethylxanthate inhibited the enzyme activity, but potassium cyanide and diethyldithiocarbamate, both known as potent copper enzyme inhibitors, were not inhibitory. PMID:3032923

  17. 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.

  18. Low monoamine oxidase B in peripheral organs in smokers

    PubMed Central

    Fowler, Joanna S.; Logan, Jean; Wang, Gene-Jack; Volkow, Nora D.; Telang, Frank; Zhu, Wei; Franceschi, Dinko; Pappas, Naomi; Ferrieri, Richard; Shea, Colleen; Garza, Victor; Xu, Youwen; Schlyer, David; Gatley, S. John; Ding, Yu-Shin; Alexoff, David; Warner, Donald; Netusil, Noelwah; Carter, Pauline; Jayne, Millard; King, Payton; Vaska, Paul

    2003-01-01

    One of the major mechanisms for terminating the actions of catecholamines and vasoactive dietary amines is oxidation by monoamine oxidase (MAO). Smokers have been shown to have reduced levels of brain MAO, leading to speculation that MAO inhibition by tobacco smoke may underlie some of the behavioral and epidemiological features of smoking. Because smoking exposes peripheral organs as well as the brain to MAO-inhibitory compounds, we questioned whether smokers would also have reduced MAO levels in peripheral organs. Here we compared MAO B in peripheral organs in nonsmokers and smokers by using positron emission tomography and serial scans with the MAO B-specific radiotracers,l-[11C]deprenyl and deuterium-substituted l-[11C]deprenyl (l-[11C]deprenyl-D2). Binding specificity was assessed by using the deuterium isotope effect. We found that smokers have significantly reduced MAO B in peripheral organs, particularly in the heart, lungs, and kidneys, when compared with nonsmokers. Reductions ranged from 33% to 46%. Because MAO B breaks down catecholamines and other physiologically active amines, including those released by nicotine, its inhibition may alter sympathetic tone as well as central neurotransmitter activity, which could contribute to the medical consequences of smoking. In addition, although most of the emphases on the carcinogenic properties of smoke have been placed on the lungs and the upper airways, this finding highlights the fact that multiple organs in the body are also exposed to pharmacologically significant quantities of chemical compounds in tobacco smoke. PMID:12972641

  19. Monoamine oxidase A gene (MAOA) predicts behavioral aggression following provocation

    PubMed Central

    McDermott, Rose; Tingley, Dustin; Cowden, Jonathan; Frazzetto, Giovanni; Johnson, Dominic D. P.

    2009-01-01

    Monoamine oxidase A gene (MAOA) has earned the nickname “warrior gene” because it has been linked to aggression in observational and survey-based studies. However, no controlled experimental studies have tested whether the warrior gene actually drives behavioral manifestations of these tendencies. We report an experiment, synthesizing work in psychology and behavioral economics, which demonstrates that aggression occurs with greater intensity and frequency as provocation is experimentally manipulated upwards, especially among low activity MAOA (MAOA-L) subjects. In this study, subjects paid to punish those they believed had taken money from them by administering varying amounts of unpleasantly hot (spicy) sauce to their opponent. There is some evidence of a main effect for genotype and some evidence for a gene by environment interaction, such that MAOA is less associated with the occurrence of aggression in a low provocation condition, but significantly predicts such behavior in a high provocation situation. This new evidence for genetic influences on aggression and punishment behavior complicates characterizations of humans as “altruistic” punishers and supports theories of cooperation that propose mixed strategies in the population. It also suggests important implications for the role of individual variance in genetic factors contributing to everyday behaviors and decisions. PMID:19168625

  20. Cytochrome c Oxidase Biogenesis: New levels of Regulation

    PubMed Central

    Fontanesi, Flavia; Soto, Ileana C.; Barrientos, Antoni

    2008-01-01

    Summary Eukaryotic cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a multimeric enzyme of dual genetic origin, whose assembly is a complicated and highly regulated process. COX displays a concerted accumulation of its constitutive subunits. Data obtained from studies performed with yeast mutants indicate that most catalytic core unassembled subunits are post-translationally degraded. Recent data obtained in the yeast Saccharomyces cerevisiae have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis targeting subunit 1 or Cox1p. Cox1p is a mitochondrially encoded catalytic subunit of COX which acts as a seed around which the full complex is assembled. A regulatory mechanism exists by which Cox1p synthesis is controlled by the availability of its assembly partners. The unique properties of this regulatory mechanism offer a means to catalyze multiple-subunit assembly. New levels of COX biogenesis regulation have been recently proposed. For example, COX assembly and stability of the fully assembled enzyme depend on the presence in the mitochondrial compartments of two partners of the oxidative phosphorylation system, the mobile electron carrier cytochrome c and the mitochondrial ATPase. The different mechanisms of regulation of COX assembly are reviewed and discussed. PMID:18465791

  1. Polyphenol oxidase as a biochemical seed defense mechanism

    PubMed Central

    Fuerst, E. Patrick; Okubara, Patricia A.; Anderson, James V.; Morris, Craig F.

    2014-01-01

    Seed dormancy and resistance to decay are fundamental survival strategies, which allow a population of seeds to germinate over long periods of time. Seeds have physical, chemical, and biological defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. Here, we hypothesize that seeds also possess enzyme-based biochemical defenses, based on induction of the plant defense enzyme, polyphenol oxidase (PPO), when wild oat (Avena fatua L.) caryopses and seeds were challenged with seed-decaying Fusarium fungi. These studies suggest that dormant seeds are capable of mounting a defense response to pathogens. The pathogen-induced PPO activity from wild oat was attributed to a soluble isoform of the enzyme that appeared to result, at least in part, from proteolytic activation of a latent PPO isoform. PPO activity was also induced in wild oat hulls (lemma and palea), non-living tissues that cover and protect the caryopsis. These results are consistent with the hypothesis that seeds possess inducible enzyme-based biochemical defenses arrayed on the exterior of seeds and these defenses represent a fundamental mechanism of seed survival and longevity in the soil. Enzyme-based biochemical defenses may have broader implications since they may apply to other defense enzymes as well as to a diversity of plant species and ecosystems. PMID:25540647

  2. Inhibition of apple polyphenol oxidase activity by sodium chlorite.

    PubMed

    Lu, Shengmin; Luo, Yaguang; Feng, Hao

    2006-05-17

    Sodium chlorite (SC) was shown to have strong efficacy both as a sanitizer to reduce microbial growth on produce and as a browning inhibitor on fresh-cut apples in previous experiments. This study was undertaken to investigate the inhibitory effect of SC on polyphenol oxidase (PPO) and the associated mechanisms. The experiment showed that SC had a strong inhibition of apple PPO. The extent of inhibition was influenced by SC concentration and pH. Inhibition was most prominent at pH 4.5, at which approximately 30% of enzyme activity was lost in the presence of 10 mM SC, followed closely by that at pH 4.0 with a 26% reduction in PPO activity. The inhibition mode was determined using Dixon and Lineweaver-Burk plots, which established SC to be a mixed inhibitor of apple PPO for the oxidation of catechol. Preincubation of PPO with 8 mM SC for 8 min caused a maximum of 46% activity reduction compared to noninhibited control. However, preincubation of SC with catechol for 8 min resulted in no additional loss of PPO activity. These findings provide further evidence that the inhibition of PPO activity by SC is due to the inhibition of the enzyme itself rather than removal of the substrate. PMID:19127746

  3. Examination of Cholesterol oxidase attachment to magnetic nanoparticles

    PubMed Central

    Kouassi, Gilles K; Irudayaraj, Joseph; McCarty, Gregory

    2005-01-01

    Magnetic nanoparticles (Fe3O4) were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe3O4 nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications. PMID:15661076

  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 effects of ingested aluminium on brain cytochrome oxidase activity.

    PubMed

    Mohan, N; Alleyne, T; Adogwa, A

    2009-11-01

    Aluminium has a unique combination of physical and chemical properties which has enabled man to put this metal to very wide and varied use. However prolonged exposure to aluminium ions may lead to adverse health effects. In this study, we evaluated the effects of dietary aluminium on the protein composition and the intrinsic activity of cytochrome oxidase (COX) for brain mitochondria. New Zealand white rabbits were maintained on a diet of commercial rabbit pellets and distilled water for a period of 12 weeks. For the experimental group, AlCl3, 330 mg/kg/L was added to the drinking water. When compared to the control, mitochondria isolated from the brains of the AICl3 fed rabbits showed no change in Km but an approximate 35% decrease in both the low and high affinity Vmax values. Also, whereas the protein composition of the mitochondria from both sources appeared to be normal, isolation of highly purified COX proved to be difficult and for the AlCl3 fed rabbits, a number of the enzyme's low molecular weight subunits were absent. These results appear to confirm a relationship between long term aluminium consumption and low brain COX activity; they further suggest that an altered COX structure may be the cause of the low enzymic activity. PMID:20441059

  6. Ataxia telangiectasia mutated influences cytochrome c oxidase activity.

    PubMed

    Patel, Akshar Y; McDonald, Todd M; Spears, Larry D; Ching, James Kain; Fisher, Jonathan S

    2011-02-25

    Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1h with the ATM inhibitor KU55933 caused a ∼50% reduction (P<0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P<0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity. PMID:21266166

  7. 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. PMID:20429683

  8. Purification and characterization of polyphenol oxidase from fresh ginseng

    PubMed Central

    Kim, Jae-Joon; Kim, Woo-Yeon

    2013-01-01

    Polyphenol oxidase (PPO) was purified from fresh ginseng roots using acetone precipitation, carboxymethyl (CM)-Sepharose chromatography, and phenyl-Sepharose chromatography. Two isoenzymes (PPO 1 and PPO 2) were separated using an ion-exchange column with CM-Sepharose. PPO 1 was purified up to 13.2-fold with a 22.6% yield. PPO 2 bound to CM-Sepharose, eluted with NaCl, and was purified up to 22.5-fold with a 17.4% yield. PPO 2 was further chromatographed on phenyl-Sepharose. The molecular weight of the purified PPO 2 from fresh ginseng was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was about 40 kDa. The optimum temperature and pH were 20℃ and 7.0, respectively, using catechol as a substrate. Pyrogallol showed the highest substrate specificity. The effect of a PPO inhibitor showed that its activity increased slightly in the presence of a low concentration of citric acid. High concentrations of acidic compounds and sulfite agents significantly inhibited purified ginseng PPO 2. PMID:23717165

  9. 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. PMID:25308684

  10. Redox transitions between oxygen intermediates in cytochrome-c oxidase.

    PubMed Central

    Verkhovsky, M I; Morgan, J E; Wikström, M

    1996-01-01

    Some intermediates in the reduction of O2 to water by cytochrome-c oxidase have been characterized by optical, Raman, and magnetic circular dichroism spectroscopy. The so-called "peroxy" (P) and "ferryl" (F) forms of the enzyme, which have been considered to be intermediates of the oxygen reaction, can be generated when the oxidized enzyme reacts with H2O2, or when the two-electron reduced ("CO mixed-valence") enzyme reacts with O2. The structures as well as the overall redox states of P and F have recently been controversial. We show here, using tris(2,2'-bipyridyl)ruthenium(II) as a photoinducible reductant, that one-electron reduction of P yields F, and that one-electron reduction of F yields the oxidized enzyme. This confirms that the overall redox states of P and F differ from the oxidized enzyme by two and one electron equivalents, respectively. The structures of the P and F states are discussed. PMID:8901563

  11. Nanoconfinement effects: glucose oxidase reaction kinetics in nanofluidics.

    PubMed

    Wang, Chen; Sheng, Zhen-Huan; Ouyang, Jun; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

    2012-02-01

    Size-tunable nanofluidic devices coupled to an electrochemical detector have been designed and then used to study glucose oxidase (GOx) reaction kinetics confined in nanospaces. The devices are fabricated via a photochemical decomposition reaction, which forms nanochannels covered with carboxyl groups. The generated carboxyl groups enable us to chemically pattern biological molecules on the polymer surfaces via covalent bonding. With this approach, the activity of the immobilized biological molecules confined in nanospaces with different sizes has been investigated. GOx species are chemically immobilized on the surface of the nanochannels, catalyzing the oxidation of substrate glucose as it flows through the channels. The enzyme reaction product, hydrogen peroxide, passing through the nanochannels, reaches an electrochemical detector, giving rise to an increase in anodic current. This steady-state electrochemical current, which responds to various glucose concentrations, can be used to evaluate the GOx activity under confinement conditions. The results show significant nanoconfinement effects that are dependent on the channel size where the reaction occurs, demonstrating the importance of spatial confinement on the GOx reaction kinetics. The present approach provides an effective method for the study of enzyme activity and other bioassay systems, such as cell assays, drug discovery, and clinical diagnosis. PMID:22311832

  12. Inhibition of NADPH oxidase by glucosylceramide confers chemoresistance

    PubMed Central

    Barth, Brian M; Gustafson, Sally J; Young, Megan M; Fox, Todd E; Shanmugavelandy, Sriram S; Kaiser, James M; Cabot, Myles C; Kuhn, Thomas B

    2010-01-01

    The bioactive sphingolipid ceramide induces oxidative stress by disrupting mitochondrial function and stimulating NADPH oxidase (NOX) activity, both implicated in cell death mechanisms. Many anticancer chemotherapeutics (anthracyclines, Vinca alkaloids, paclitaxel and fenretinide), as well as physiological stimuli such as tumor necrosis factor α (TNFα), stimulate ceramide accumulation and increase oxidative stress in malignant cells. Consequently, ceramide metabolism in malignant cells and, in particular the upregulation of glucosylceramide synthase (GCS), has gained considerable interest in contributing to chemoresistance. We hypothesized that increases in GCS activity and thus glucosylceramide, the product of GCS activity, represents an important resistance mechanism in glioblastoma. In our study, we determined that increased GCS activity effectively blocked reactive oxygen species formation by NOX. We further showed, in both glioblastoma and neuroblastoma cells that glucosylceramide directly interfered with NOX assembly, hence delineating a direct resistance mechanism. Collectively, our findings indicated that pharmacological or molecular targeting of GCS, using non-toxic nanoliposome delivery systems, successfully augmented NOX activity, and improved the efficacy of known chemotherapeutic agents. PMID:20935456

  13. Heterodimer Formation of BnPKSA or BnPKSB with BnACOS5 Constitutes a Multienzyme Complex in Tapetal Cells and is Involved in Male Reproductive Development in Brassica napus.

    PubMed

    Qin, Maomao; Tian, Tiantian; Xia, Shengqian; Wang, Zhixin; Song, Liping; Yi, Bin; Wen, Jing; Shen, Jinxiong; Ma, Chaozhi; Fu, Tingdong; Tu, Jinxing

    2016-08-01

    Multienzyme associations localized to specific subcellular sites are involved in several critical functions in cellular metabolism, such as plant survival and reproduction. To date, few multienzyme complexes involved in male fertility have been examined in Brassica napus Here, we reported that in B. napus, the members of a multienzyme complex work in an interaction pattern different from that in Arabidopsis thaliana for sporopollenin biosynthesis. 7365A, a male-sterile mutant with a relatively smooth anther cuticle, was found to have a dramatic reduction in both cutin monomers and wax composition. Proteomic comparison between the mutant 7365A and wild-type 7365B showed down-regulation of three sporopollenin biosynthetic enzymes, namely BnPKSA, BnPKSB and BnTKPR; these enzymes were tightly co-expressed with BnACOS5. BnPKSA and BnPKSB showed similar expression patterns but distinct accumulation levels, suggesting that they had partially distinct functions during sporopollenin biosynthesis. In vitro and in vivo analyses demonstrated that BnPKSB directly interacted with BnPKSA and BnACOS5, but no such interactions were found in the present investigation for BnTKPR1. Interestingly, the interaction between PKSA and PKSB has not been discovered in Arabidopsis, which may indicate a new interaction representing an additional efficient regulation method in B. napus Taken together, we propose that BnPKSA and BnPKSB may comprise a heterodimer combined with BnACOS5, constituting a sporopollenin metabolon in tapetal cells that is related to male reproductive development in B. napus. PMID:27335346

  14. 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

  15. Direct Identification of a Bacterial Manganese(II) Oxidase, the Multicopper Oxidase MnxG, from Spores of Several Different Marine Bacillus Species▿ †

    PubMed Central

    Dick, Gregory J.; Torpey, Justin W.; Beveridge, Terry J.; Tebo, Bradley M.

    2008-01-01

    Microorganisms catalyze the formation of naturally occurring Mn oxides, but little is known about the biochemical mechanisms of this important biogeochemical process. We used tandem mass spectrometry to directly analyze the Mn(II)-oxidizing enzyme from marine Bacillus spores, identified as an Mn oxide band with an in-gel activity assay. Nine distinct peptides recovered from the Mn oxide band of two Bacillus species were unique to the multicopper oxidase MnxG, and one peptide was from the small hydrophobic protein MnxF. No other proteins were detected in the Mn oxide band, indicating that MnxG (or a MnxF/G complex) directly catalyzes biogenic Mn oxide formation. The Mn(II) oxidase was partially purified and found to be resistant to many proteases and active even at high concentrations of sodium dodecyl sulfate. Comparative analysis of the genes involved in Mn(II) oxidation from three diverse Bacillus species revealed a complement of conserved Cu-binding regions not present in well-characterized multicopper oxidases. Our results provide the first direct identification of a bacterial enzyme that catalyzes Mn(II) oxidation and suggest that MnxG catalyzes two sequential one-electron oxidations from Mn(II) to Mn(III) and from Mn(III) to Mn(IV), a novel type of reaction for a multicopper oxidase. PMID:18165363

  16. hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria*

    PubMed Central

    Clemente, Paula; Peralta, Susana; Cruz-Bermudez, Alberto; Echevarría, Lucía; Fontanesi, Flavia; Barrientos, Antoni; Fernandez-Moreno, Miguel A.; Garesse, Rafael

    2013-01-01

    Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency. PMID:23362268

  17. 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

  18. A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

    PubMed

    Jeong, Eunjoo; Houn, Thavrak; Kuk, Yongin; Kim, Eun-Seon; Chandru, Hema Kumar; Baik, Myunggi; Back, Kyoungwhan; Guh, Ja-Ock; Han, Oksoo

    2003-10-01

    In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides. PMID:12941291

  19. Production of the carbonate radical anion during xanthine oxidase turnover in the presence of bicarbonate.

    PubMed

    Bonini, Marcelo G; Miyamoto, Sayuri; Di Mascio, Paolo; Augusto, Ohara

    2004-12-10

    Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants. PMID:15448145

  20. The intimate and controversial relationship between voltage-gated proton channels and the phagocyte NADPH oxidase.

    PubMed

    DeCoursey, Thomas E

    2016-09-01

    One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase complex and voltage-gated proton channels (HV 1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV 1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987-1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV 1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV 1, and HV 1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV 1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase - an industrial strength producer of reactive oxygen species (ROS) - to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come. PMID:27558336

  1. 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.

  2. 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.

  3. Analysis of TETRAKETIDE α-PYRONE REDUCTASE function in Arabidopsis thaliana reveals a previously unknown, but conserved, biochemical pathway in sporopollenin monomer biosynthesis.

    PubMed

    Grienenberger, Etienne; Kim, Sung Soo; Lallemand, Benjamin; Geoffroy, Pierrette; Heintz, Dimitri; Souza, Clarice de Azevedo; Heitz, Thierry; Douglas, Carl J; Legrand, Michel

    2010-12-01

    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

  4. Respiratory conservation of energy with dioxygen: cytochrome C oxidase.

    PubMed

    Yoshikawa, Shinya; Shimada, Atsuhiro; Shinzawa-Itoh, Kyoko

    2015-01-01

    Cytochrome c oxidase (CcO) is the terminal oxidase of cell respiration which reduces molecular oxygen (O₂) to H2O coupled with the proton pump. For elucidation of the mechanism of CcO, the three-dimensional location and chemical reactivity of each atom composing the functional sites have been extensively studied by various techniques, such as crystallography, vibrational and time-resolved electronic spectroscopy, since the X-ray structures (2.8 Å resolution) of bovine and bacterial CcO have been published in 1995.X-ray structures of bovine CcO in different oxidation and ligand binding states showed that the O₂reduction site, which is composed of Fe (heme a 3) and Cu (CuB), drives a non-sequential four-electron transfer for reduction of O₂to water without releasing any reactive oxygen species. These data provide the crucial structural basis to solve a long-standing problem, the mechanism of the O₂reduction.Time-resolved resonance Raman and charge translocation analyses revealed the mechanism for coupling between O₂reduction and the proton pump: O₂is received by the O₂reduction site where both metals are in the reduced state (R-intermediate), giving the O₂-bound form (A-intermediate). This is spontaneously converted to the P-intermediate, with the bound O₂fully reduced to 2 O²⁻. Hereafter the P-intermediate receives four electron equivalents from the second Fe site (heme a), one at a time, to form the three intermediates, F, O, and E to regenerate the R-intermediate. Each electron transfer step from heme a to the O₂reduction site is coupled with the proton pump.X-ray structural and mutational analyses of bovine CcO show three possible proton transfer pathways which can transfer pump protons (H) and chemical (water-forming) protons (K and D). The structure of the H-pathway of bovine CcO indicates that the driving force of the proton pump is the electrostatic repulsion between the protons on the H-pathway and positive charges of heme a, created

  5. 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.

  6. 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.

  7. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination

    PubMed Central

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells. PMID:26579718

  8. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination.

    PubMed

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells. PMID:26579718

  9. Greater Monoamine Oxidase A Binding in Alcohol Dependence

    PubMed Central

    Matthews, Brittany A.; Kish, Stephen J.; Xu, Xin; Boileau, Isabelle; Rusjan, Pablo M.; Wilson, Alan A.; DiGiacomo, Dan; Houle, Sylvain; Meyer, Jeffrey H.

    2016-01-01

    Background Alcohol dependence (AD) is a multiorgan disease in which excessive oxidative stress and apoptosis are implicated. Monoamine oxidase A (MAO-A) is an important enzyme on the outer mitochondrial membrane that participates in the cellular response to oxidative stress and mitochondrial toxicity. It is unknown whether MAO-A levels are abnormal in AD. We hypothesized that MAO-A VT, an index of MAO-A level, is elevated in the prefrontal cortex (PFC) during AD, because markers of greater oxidative stress and apoptosis are reported in the brain in AD and a microarray analysis reported greater MAO-A messenger RNA in the PFC of rodents exposed to alcohol vapor. Methods Sixteen participants with alcohol dependence and 16 healthy control subjects underwent [11C]-harmine positron emission tomography. All were nonsmoking, medication- and drug-free, and had no other past or present psychiatric or medical illnesses. Results MAO-A VT was significantly greater in the PFC (37%, independent samples t test, t30 = 3.93, p < .001), and all brain regions analyzed (mean 32%, multivariate analysis of variance, F7,24 = 3.67, p = .008). Greater duration of heavy drinking correlated positively with greater MAO-A VT in the PFC (r = .67, p = .005) and all brain regions analyzed (r = .73 to .57, p = .001–.02). Conclusions This finding represents a new pathological marker present in AD that is therapeutically targetable through direct inhibition or by novel treatments toward oxidative/pro-apoptotic processes implicated by MAO-A overexpression. PMID:24269057

  10. A subset of N-substituted phenothiazines inhibits NADPH oxidases.

    PubMed

    Seredenina, Tamara; Chiriano, Gianpaolo; Filippova, Aleksandra; Nayernia, Zeynab; Mahiout, Zahia; Fioraso-Cartier, Laetitia; Plastre, Olivier; Scapozza, Leonardo; Krause, Karl-Heinz; Jaquet, Vincent

    2015-09-01

    NADPH oxidases (NOXs) constitute a family of enzymes generating reactive oxygen species (ROS) and are increasingly recognized as interesting drug targets. Here we investigated the effects of 10 phenothiazine compounds on NOX activity using an extensive panel of assays to measure production of ROS (Amplex red, WST-1, MCLA) and oxygen consumption. Striking differences between highly similar phenothiazines were observed. Two phenothiazines without N-substitution, including ML171, did not inhibit NOX enzymes, but showed assay interference. Introduction of an aliphatic amine chain on the N atom of the phenothiazine B ring (promazine) conferred inhibitory activity toward NOX2, NOX4, and NOX5 but not NOX1 and NOX3. Addition of an electron-attracting substituent in position 2 of the C ring extended the inhibitory activity to NOX1 and NOX3, with thioridazine being the most potent inhibitor. In contrast, the presence of a methylsulfoxide group at the same position (mesoridazine) entirely abolished NOX-inhibitory activity. A cell-free NOX2 assay suggested that inhibition by N-substituted phenothiazines was not due to competition with NADPH. A functional implication of NOX-inhibitory activity of thioridazine was demonstrated by its ability to block redox-dependent myofibroblast differentiation. Our results demonstrate that NOX-inhibitory activity is not a common feature of all antipsychotic phenothiazines and that substitution on the B-ring nitrogen is crucial for the activity, whereas that on the second position of the C ring modulates it. Our findings contribute to a better understanding of NOX pharmacology and might pave the path to discovery of more potent and selective NOX inhibitors. PMID:26013584

  11. 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.

  12. 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%. PMID:15137808

  13. 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

  14. Computer simulation of water in cytochrome c oxidase.

    PubMed

    Zheng, Xuehe; Medvedev, Dmitry M; Swanson, Jessica; Stuchebrukhov, Alexei A

    2003-03-01

    Statistical mechanics and molecular dynamics simulations have been carried out to study the distribution and dynamics of internal water molecules in bovine heart cytochrome c oxidase (CcO). CcO is found to be capable of holding plenty of water, which in subunit I alone amounts to about 165 molecules. The dynamic characterization of these water molecules is carried out. The nascent water molecules produced in the redox reaction at the heme a(3)-CuB binuclear site form an intriguing chain structure. The chain begins at the position of Glu242 at the end of the D channel, and has a fork structure, one branch of which leads to the binuclear center, and the other to the propionate d of heme a(3). The branch that leads to the binuclear center has dynamic access both to the site where the formation of water occurs, and to delta-nitrogen of His291. From the binuclear center, the chain continues to run into the K channel. The stability of this hydrogen bond network is examined dynamically. The catalytic site is located at the hydrophobic region, and the nascent water molecules are produced at the top of the energy hill. The energy gradient is utilized as the mechanism of water removal from the protein. The water exit channels are explored using high-temperature dynamics simulations. Two putative channels for water exit from the catalytic site have been identified. One is leading directly toward Mg(2+) site. However, this channel is only open when His291 is dissociated from CuB. If His291 is bound to CuB, the only channel for water exit is the one that originates at E242 and leads toward the middle of the membrane. This is the same channel that is presumably used for oxygen supply. PMID:12615353

  15. Isolation of regulatory-competent, phosphorylated cytochrome C oxidase.

    PubMed

    Lee, Icksoo; Salomon, Arthur R; Yu, Kebing; Samavati, Lobelia; Pecina, Petr; Pecinova, Alena; Hüttemann, Maik

    2009-01-01

    The role of posttranslational modifications, specifically reversible phosphorylation as a regulatory mechanism operating in the mitochondria, is a novel research direction. The mitochondrial oxidative phosphorylation system is a particularly interesting unit because it is responsible for the production of the vast majority of cellular energy in addition to free radicals, two factors that are aberrant in numerous human diseases and that may be influenced by reversible phosphorylation of the oxidative phosphorylation complexes. We here describe a detailed protocol for the isolation of mammalian liver and heart mitochondria and subsequently cytochrome c oxidase (CcO) under conditions maintaining the physiological phosphorylation state. The protocol employs the use of activated vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent the activation of calcium-dependent protein phosphatases. CcO purified without manipulation of signaling pathways shows strong tyrosine phosphorylation on subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced by the cAMP- and TNFalpha-dependent pathways in liver. Using our protocol on cow liver tissue we further show the identification of a new phosphorylation site on CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of the precursor peptide) via immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This phosphorylation site is located close to the ATP and ADP binding site, which adjusts CcO activity to cellular energy demand, and we propose that phosphorylation of tyrosine 11 enables allosteric regulation. PMID:19426869

  16. NADPH Oxidase and the Cardiovascular Toxicity Associated with Smoking

    PubMed Central

    Kim, Mikyung; Han, Chang-ho

    2014-01-01

    Smoking is one of the most serious but preventable causes of cardiovascular disease (CVD). Key aspects of pathological process associated with smoking include endothelial dysfunction, a prothrombotic state, inflammation, altered lipid metabolism, and hypoxia. Multiple molecular events are involved in smokinginduced CVD. However, the dysregulations of reactive oxygen species (ROS) generation and metabolism mainly contribute to the development of diverse CVDs, and NADPH oxidase (NOX) has been established as a source of ROS responsible for the pathogenesis of CVD. NOX activation and resultant ROS production by cigarette smoke (CS) treatment have been widely observed in isolated blood vessels and cultured vascular cells, including endothelial and smooth muscle cells. NOX-mediated oxidative stress has also been demonstrated in animal studies. Of the various NOX isoforms, NOX2 has been reported to mediate ROS generation by CS, but other isoforms were not tested thoroughly. Of the many CS constituents, nicotine, methyl vinyl ketone, and α,β-unsaturated aldehydes, such as, acrolein and crotonaldehyde, appear to be primarily responsible for NOX-mediated cytotoxicity, but additional validation will be needed. Human epidemiological studies have reported relationships between polymorphisms in the CYBA gene encoding p22phox, a catalytic subunit of NOX and susceptibility to smoking-related CVDs. In particular, G allele carriers of A640G and -930A/G polymorphisms were found to be vulnerable to smoking-induced cardiovascular toxicity, but results for C242T studies are conflicting. On the whole, evidence implicates the etiological role of NOX in smoking-induced CVD, but the clinical relevance of NOX activation by smoking and its contribution to CVD require further validation in human studies. A detailed understanding of the role of NOX would be helpful to assess the risk of smoking to human health, to define high-risk subgroups, and to develop strategies to prevent or treat

  17. Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

    PubMed Central

    2010-01-01

    Background In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties. Results The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e.g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity. Conclusions Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins. PMID:20092622

  18. 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. PMID:19863756

  19. NADPH Oxidases in Heart Failure: Poachers or Gamekeepers?

    PubMed Central

    Zhang, Min; Perino, Alessia; Ghigo, Alessandra; Hirsch, Emilio

    2013-01-01

    Abstract Significance: Oxidative stress is involved in the pathogenesis of heart failure but clinical antioxidant trials have been unsuccessful. This may be because effects of reactive oxygen species (ROS) depend upon their source, location, and concentration. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) proteins generate ROS in a highly regulated fashion and modulate several components of the heart failure phenotype. Recent Advances: Two Nox isoforms, Nox2 and Nox4, are expressed in the heart. Studies using gene-modified mice deficient in Nox2 activity indicate that Nox2 activation contributes to angiotensin II–induced cardiomyocyte hypertrophy, atrial fibrillation, and the development of interstitial fibrosis but may also positively modulate physiological excitation-contraction coupling. Nox2 contributes to myocyte death under stress situations and plays important roles in postmyocardial infarction remodeling, in part by modulating matrix metalloprotease activity. In contrast to Nox2, Nox4 is constitutively active at a low level and induces protective effects in the heart under chronic stress, for example, by maintaining myocardial capillary density. However, high levels of Nox4 could have detrimental effects. Critical Issues: The effects of Nox proteins during the development of heart failure likely depend upon the isoform, activation level, and cellular distribution, and may include beneficial as well as detrimental effects. More needs to be learnt about the precise regulation of abundance and biochemical activity of these proteins in the heart as well as the downstream signaling pathways that they regulate. Future Directions: The development of specific approaches to target individual Nox isoforms and/or specific cell types may be important for the achievement of therapeutic efficacy in heart failure. Antioxid. Redox Signal. 18, 1024–1041. PMID:22747566

  20. Mitochondrial Copper Metabolism and Delivery to Cytochrome c Oxidase

    PubMed Central

    Horn, Darryl; Barrientos, Antoni

    2010-01-01

    Summary Metals are essential elements of all living organisms. Among them, copper is required for a multiplicity of functions including mitochondrial oxidative phosphorylation and protection against oxidative stress. Here we will focus on describing the pathways involved in the delivery of copper to cytochrome c oxidase (COX), a mitochondrial metalloenzyme acting as the terminal enzyme of the mitochondrial respiratory chain. The catalytic core of COX is formed by three mitochondrially-encoded subunits and contains three copper atoms. Two copper atoms bound to subunit 2 constitute the CuA site, the primary acceptor of electrons from ferrocytochrome c. The third copper, CuB, is associated with the high-spin heme a3 group of subunit 1. Recent studies, mostly performed in the yeast Saccharomyces cerevisiae, have provided new clues about 1- the source of the copper used for COX metallation; 2- the roles of Sco1p and Cox11p, the proteins involved in the direct delivery of copper to the CuA and CuB sites, respectively; 3- the action mechanism of Cox17p, a copper chaperone that provides copper to Sco1p and Cox11p; 4- the existence of at least four Cox17p homologues carrying a similar twin CX9C domain suggestive of metal binding, Cox19p, Cox23p, Pet191p and Cmc1p, that could be part of the same pathway; and 5- the presence of a disulfide relay system in the intermembrane space of mitochondria that mediates import of proteins with conserved cysteines motifs such as the CX9C characteristic of Cox17p and its homologues. The different pathways are reviewed and discussed in the context of both mitochondrial COX assembly and copper homeostasis. PMID:18459161

  1. Cytochrome c oxidase: evolution of control via nuclear subunit addition.

    PubMed

    Pierron, Denis; Wildman, Derek E; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I

    2012-04-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the "higher" organism possessing large brains and muscles. The main function of these subunits appears to be "only" to control the activity of the mitochondrial subunits. We propose that this control function is an as yet under appreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a "domestication scenario" such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. PMID:21802404

  2. 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

  3. Expression dynamics of NADPH oxidases during early zebrafish development.

    PubMed

    Weaver, Cory J; Leung, Yuk Fai; Suter, Daniel M

    2016-07-01

    Nicotinamide dinucleotide phosphate oxidases (NOX) control various cellular signaling cascades. In the nervous system, there is recent evidence that NOX-derived reactive oxygen species (ROS) regulate neurite outgrowth, regeneration, and stem cell proliferation; however, a comprehensive NOX gene expression analysis is missing for all major model systems. Zebrafish embryos provide an excellent model system to study neurodevelopment and regeneration because they develop quickly and are well suited for in vivo imaging and molecular approaches. Although the sequences of five NOX genes (nox1, nox2/cybb, nox4, nox5, and duox) have been identified in the zebrafish genome, nothing is known about their expression pattern. Here, we used quantitative polymerase chain reaction combined with in situ hybridization to develop a catalog of nox1, nox2/cybb, nox5, and duox expression in zebrafish during early nervous system development from 12 to 48 hours post fertilization. We found that expression levels of nox1, nox5, and duox are dynamic during the first 2 days of development, whereas nox2/cybb levels remain remarkably stable. By sectioning in situ hybridized embryos, we found a pattern of broad and overlapping NOX isoform expression at 1 and 1.5 days post fertilization. After 2 days of development, a few brain regions displayed increased NOX expression levels. Collectively, these results represent the first comprehensive analysis of NOX gene expression in the zebrafish and will provide a basis for future studies aimed at determining the functions of NOX enzymes in neurodevelopment and regeneration. J. Comp. Neurol. 524:2130-2141, 2016. © 2015 Wiley Periodicals, Inc. PMID:26662995

  4. Xanthine Oxidase and Cardiovascular Risk in Obese Children

    PubMed Central

    Tam, Harrison K.; Kelly, Aaron S.; Metzig, Andrea M.; Steinberger, Julia

    2014-01-01

    Abstract Background: Pathological mechanisms of how childhood obesity leads to increased risk of cardiovascular disease (CVD) are not fully characterized. Oxidative-stress–related enzymes, such as xanthine oxidase (XO), have been linked to obesity, endothelial dysfunction, and CVD in adults, but little is known about this pathway in children. The aim of this study was to determine whether differential XO activity is associated with endothelial dysfunction, CVD risk factors, or cytokine levels. Methods: Fasting plasma samples were obtained from obese (BMI ≥95th percentile; n=20) and age- and gender-matched healthy weight (BMI >5th and <85th percentile; n=22) children and adolescents (mean age, 12±3 years) to quantify XO activity. In addition, fasting cholesterol, insulin, glucose, blood pressure, endothelial function, and cytokine levels were assessed. Results: We observed a 3.8-fold increase in plasma XO activity in obese, compared to healthy weight, children (118±21 vs. 31±9 nU/mg of protein; p<0.001). Plasma XO activity was correlated with BMI z-score (r=0.41), waist circumference (r=0.41), high-density lipoprotein cholesterol (r=−0.32), oxidized low-density lipoprotein (r=0.57), adiponectin (r=−0.53), and monocyte chemotactic protein-1 (r=−0.59). Conclusion: XO activity is highly elevated in obese children and correlates with CVD risk factors, suggesting that XO may play a role in increasing cardiovascular risk early in life in the context of obesity. PMID:24568669

  5. Lathyrus cicera copper amine oxidase reactions with tryptamine.

    PubMed

    Pietrangeli, Paola; Bellelli, Andrea; Fattibene, Paola; Mondovì, Bruno; Morpurgo, Laura

    2012-04-01

    Lathyrus cicera copper amine oxidase (LCAO) rapidly formed the typical Cu(I)-TPQ semiquinone UV-visible spectrum, identical to that formed by other substrates, upon O(2) exhaustion by turnover with excess tryptamine. A new band at 630 nm formed more slowly, with intensity dependent on aldehyde and H(2)O(2) concentrations. On prolonged incubation, all bands decayed in parallel, together with loss of enzymatic activity. The blue color disappeared on addition of KCN, a Cu(I) stabilizing agent, while the intensity of the radical visible bands increased. This shows that the 630 nm absorbing species is a Cu(II) derivative, as confirmed by the unchanged intensity of the EPR spectrum of the frozen blue solution from that of the native protein. Rapid kinetics experiments showed that this species derives from a reduced form of the protein, plus aldehyde and H(2)O(2) and that it is not in dynamic equilibrium with the radical. Given the similar population of the semiquinone radical with all substrates, it is possible that the reaction with aldehyde and H(2)O(2) occurs in all cases although substrates lacking the indole group only produce the Cu(I)-semiquinone band. The radical participation to the catalytic activity is demonstrated by the observation that its relative population (controlled by the pH) parallels changes in the reoxidation rate constant, while the 630 nm absorbing species is implied in the inactivation process, which depends on H(2)O(2) and aldehyde concentration. The results of the paper are consistent with half-of-the-site reactivity, i.e. the two subunits of LCAO are kinetically and spectroscopically distinct from each other. PMID:22369770

  6. 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

  7. 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.

  8. The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide.

    PubMed

    Korshunov, Sergey; Imlay, Karin R C; Imlay, James A

    2016-07-01

    When sulfur compounds are scarce or difficult to process, Escherichia coli adapts by inducing the high-level expression of sulfur-compound importers. If cystine then becomes available, the cystine is rapidly overimported and reduced, leading to a burgeoning pool of intracellular cysteine. Most of the excess cysteine is exported, but some is adventitiously degraded, with the consequent release of sulfide. Sulfide is a potent ligand of copper and heme moieties, raising the prospect that it interferes with enzymes. We observed that when cystine was provided and sulfide levels rose, E. coli became strictly dependent upon cytochrome bd oxidase for continued respiration. Inspection revealed that low-micromolar levels of sulfide inhibited the proton-pumping cytochrome bo oxidase that is regarded as the primary respiratory oxidase. In the absence of the back-up cytochrome bd oxidase, growth failed. Exogenous sulfide elicited the same effect. The potency of sulfide was enhanced when oxygen concentrations were low. Natural oxic-anoxic interfaces are often sulfidic, including the intestinal environment where E. coli dwells. We propose that the sulfide resistance of the cytochrome bd oxidase is a key trait that permits respiration in such habitats. PMID:26991114

  9. Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study

    PubMed Central

    Masoud, Rawand; Bizouarn, Tania; Houée-Levin, Chantal

    2014-01-01

    The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2•−, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O2•−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase. PMID:25462061

  10. 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.

  11. NADPH oxidase activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

    PubMed Central

    MacFarlane, P M; Satriotomo, I; Windelborn, J A; Mitchell, G S

    2009-01-01

    Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O2, 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (∼C4) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C4 spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91phox, whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47phox and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C4 ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury. PMID:19237427

  12. 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

  13. Comparative modeling of the latent form of a plant catechol oxidase using a molluskan hemocyanin structure.

    PubMed

    Gerdemann, Carsten; Eicken, Christoph; Galla, Hans Joachim; Krebs, Bernt

    2002-04-10

    The structure of the precursor form of catechol oxidase from sweet potatoes (Ipomoea batatas) has been modeled on the basis of the 3D structural data of mature catechol oxidase [Nat. Struct. Biol. 5 (1998) 1084] and of hemocyanin from giant octopus (Octopus dofleini) [J. Mol. Biol. 278 (1998) 855]. A C-terminal extension peptide is found in the cDNA sequence but not in the purified, mature form of catechol oxidase. Superimposition of the 3D structures of the native hemocyanin and catechol oxidase reveals a close relationship except for an additional C-terminal domain only found in the hemocyanin structure. As sequence alignment shows good homology this domain of the hemocyanin structure was used as a template to model the 3D structure of the C-terminal extension peptide of catechol oxidase. As hemocyanins show no or only weak catecholase activity due to this domain this indicates an inhibitory function of this extension peptide. Beside this possible shielding function for the precursor form, evidence for a function in copper-uptake also increases due to the location of three histidine residues in the model. PMID:11931976

  14. NADPH oxidase of guinea-pig macrophages catalyses the reduction of ubiquinone-1 under anaerobic conditions.

    PubMed Central

    Murakami, M; Nakamura, M; Minakami, S

    1986-01-01

    The stimulation-specific NADPH-dependent reduction of ubiquinone-1 (Q-1) in guinea-pig macrophages was studied. The activity was due neither to any modified product of the phagocytosis-specific NADPH oxidase nor to non-specific diaphorases of the cells, since the activity was measured in sonicated or detergent-disrupted cells by subtracting the activity in the resting cells from that in cells activated by phorbol 12-myristate 13-acetate. The activity was not mediated by superoxide anions, since strict anaerobic conditions were employed. The anaerobic reduction of Q-1 was NADPH-specific, like superoxide formation under aerobic conditions, and its maximal velocity was also essentially the same as that of superoxide formation. The oxidase does not directly reduce Q-1 under aerobic conditions [Nakamura, Murakami, Umei & Minakami (1985) FEBS Lett. 186, 215-218], and the electron transfer from NADPH to cytochrome c by the oxidase under aerobic conditions was not enhanced by the addition of Q-1. The observations indicate that the phagocytosis-specific NADPH oxidase reduces Q-1 and that oxygen competes with the reduction of Q-1. Q-1 seems to accept electrons not from the intermediary electron carriers of the oxidase but from the terminal oxygen-reducing site of the enzyme. PMID:3026322

  15. 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.

  16. 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. PMID:26257292

  17. Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides.

    PubMed

    Camadro, J M; Matringe, M; Thome, F; Brouillet, N; Mornet, R; Labbe, P

    1995-05-01

    Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein. PMID:7758461

  18. Redox-Dependent Conformational Changes in Cytochrome c Oxidase Suggest a Gating Mechanism for Proton Uptake

    SciTech Connect

    Qin, Ling; Liu, Jian; Mills, Denise A.; Proshlyakov, Denis A.; Hiser, Carrie; Ferguson-Miller, Shelagh

    2009-08-05

    A role for conformational change in the coupling mechanism of cytochrome c oxidase is the subject of controversy. Relatively small conformational changes have been reported in comparisons of reduced and oxidized crystal structures of bovine oxidase but none in bacterial oxidases. Comparing the X-ray crystal structures of the reduced (at 2.15 {angstrom} resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, we observe a displacement of heme a3 involving both the porphyrin ring and the hydroxyl farnesyl tail, accompanied by protein movements in nearby regions, including the mid part of helix VIII of subunit I which harbors key residues of the K proton uptake path, K362 and T359. The conformational changes in the reduced form are reversible upon reoxidation. They result in an opening of the top of the K pathway and more ordered waters being resolved in that region, suggesting an access path for protons into the active site. In all high-resolution structures of oxidized R. sphaeroides cytochrome c oxidase, a water molecule is observed in the hydrophobic region above the top of the D path, strategically positioned to facilitate the connection of residue E286 of subunit I to the active site or to the proton pumping exit path. In the reduced and reduced plus cyanide structures, this water molecule disappears, implying disruption of proton conduction from the D path under conditions when the K path is open, thus providing a mechanism for alternating access to the active site.

  19. NADPH Oxidase Biology and the Regulation of Tyrosine Kinase Receptor Signaling and Cancer Drug Cytotoxicity

    PubMed Central

    Paletta-Silva, Rafael; Rocco-Machado, Nathália; Meyer-Fernandes, José Roberto

    2013-01-01

    The outdated idea that reactive oxygen species (ROS) are only dangerous products of cellular metabolism, causing toxic and mutagenic effects on cellular components, is being replaced by the view that ROS have several important functions in cell signaling. In aerobic organisms, ROS can be generated from different sources, including the mitochondrial electron transport chain, xanthine oxidase, myeloperoxidase, and lipoxygenase, but the only enzyme family that produces ROS as its main product is the NADPH oxidase family (NOX enzymes). These transfer electrons from NADPH (converting it to NADP−) to oxygen to make O2•−. Due to their stability, the products of NADPH oxidase, hydrogen peroxide, and superoxide are considered the most favorable ROS to act as signaling molecules. Transcription factors that regulate gene expression involved in carcinogenesis are modulated by NADPH oxidase, and it has emerged as a promising target for cancer therapies. The present review discusses the mechanisms by which NADPH oxidase regulates signal transduction pathways in view of tyrosine kinase receptors, which are pivotal to regulating the hallmarks of cancer, and how ROS mediate the cytotoxicity of several cancer drugs employed in clinical practice. PMID:23434665

  20. The Occurrence of Glycolate Dehydrogenase and Glycolate Oxidase in Green Plants

    PubMed Central

    Frederick, Sue Ellen; Gruber, Peter J.; Tolbert, N. E.

    1973-01-01

    Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O2 uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance. Activities of catalase, a marker enzyme for peroxisomes, were also determined and were generally lower in the algae than in the land plants or aquatic angiosperms. Among the algae, however, there were no consistent correlations between levels of catalase and the type of enzyme which oxidized glycolate. PMID:16658555

  1. 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. PMID:27364008

  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. Single mutations that redirect internal proton transfer in the ba3 oxidase from Thermus thermophilus

    PubMed Central

    Smirnova, Irina; Chang, Hsin-Yang; von Ballmoos, Christoph; Ädelroth, Pia; Gennis, Robert B.; Brzezinski, Peter

    2014-01-01

    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” occur simultaneously. However, with the ba3 oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than 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 the 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, respectively. 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. PMID:24004023

  4. Localization of NADPH Oxidase in Sympathetic and Sensory Ganglion Neurons and Perivascular Nerve Fibers

    PubMed Central

    Cao, Xian; Demel, Stacie L.; Quinn, Mark T.; Galligan, James J.; Kreulen, David L.

    2009-01-01

    Superoxide anion (O2−•) production was previously reported to be increased in celiac ganglia (CG) during DOCA-salt hypertension, possibly via activation of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. This suggested a role for neuronal NADPH oxidase in autonomic neurovascular control. However, the expression and localization of NADPH oxidase in the peripheral neurons is not fully known. The purpose of this study was to examine the subcellular localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. In rat CG, p22phox and neuropeptide Y (NPY) were colocalized in all neurons. P22phox was also localized to dorsal root ganglia (DRG) neurons that contain calcitonin gene related peptide (CGRP). In mesenteric arteries, p22phox and p47phox were colocalized with NPY or CGRP in perivascular nerve terminals. A similar pattern of nerve terminal staining of p22phox and p47phox was also found in cultured CG neurons and nerve growth factor (NGF)-differentiated PC12 cells. These data demonstrate a previously uncharacterized localization of NADPH oxidase in perivascular nerve fibers. The presence of a O2−• – generating enzyme in close vicinity to the sites of neurotransmitter handling in the nerve fibers suggests the possibility of novel redox-mediated mechanisms in peripheral neurovascular control. PMID:19716351

  5. Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: kinetics and operational stability.

    PubMed

    Nordkvist, Mikkel; Nielsen, Per Munk; Villadsen, John

    2007-07-01

    Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase was studied. The K(m)-value for lactose, obtained by a traditional enzymatic assay, was 0.066 mM at pH 6.4 and 38 degrees C. The effect of oxygen on the enzymatic rate of reaction as well as the operational stability of the enzyme was studied by performing reactions at constant pH and temperature in a stirred tank reactor. Catalase was included in all reactions to avoid inhibition and deactivation of the oxidase by hydrogen peroxide. At pH 6.4 and 38 degrees C, K(m) for oxygen was 0.97 mM, while the catalytical rate constant, k(cat), was 94 s(-1). Furthermore, we found that the operational stability of the oxidase was dependent on the type of base used for neutralization of the acid produced. Thus, when 2 M NaOH was used for neutralization of a reaction medium containing 50 mM phosphate buffer, significant deactivation of the oxidase was observed. Also, we found that the oxidase was protected against deactivation by base at high lactose concentrations. A simple model is proposed to explain the obtained results. PMID:17154316

  6. Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

    PubMed Central

    Gohlke, U; Warne, A; Saraste, M

    1997-01-01

    The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals. PMID:9135135

  7. 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.

  8. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

    PubMed Central

    Liko, Idlir; Degiacomi, Matteo T.; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V.

    2016-01-01

    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. PMID:27364008

  9. 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

  10. Involvement of NADPH oxidases in suppression of cyclooxygenase-2 promoter-dependent transcriptional activities by sesamol

    PubMed Central

    Shimizu, Satomi; Ishigamori, Rikako; Fujii, Gen; Takahashi, Mami; Onuma, Wakana; Terasaki, Masaru; Yano, Tomohiro; Mutoh, Michihiro

    2015-01-01

    Cyclooxygenase-2 (COX-2) has been shown to play an important role in colon carcinogenesis. Moreover, one of the components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NADPH oxidase 1 (NOX1), dominantly expressed in the colon, is implicated in the pathogenesis of colon cancer. We have reported that sesamol, one of the lignans in sesame seeds, suppressed COX-2 gene transcriptional activity in human colon cancer cells, and also suppressed intestinal polyp formation in Apc-mutant mice. In the present study, we investigated the involvement of NADPH oxidase in the inhibition of COX-2 transcriptional activity by sesamol. We found that several NADPH oxidase inhibitors, such as apocynin, showed suppressive effects on COX-2 transcriptional activity. Moreover, sesamol significantly suppressed NOX1 mRNA levels in a dose-dependent manner. In addition, we demonstrated that knockdown of NOX1 successfully suppressed COX-2 transcriptional activity. These results suggest that inhibition of NADPH oxidase, especially NOX1, may be involved in the mechanism of the suppression of COX-2 transcriptional activity by sesamol. PMID:25759517

  11. Alternative Oxidase Expression in the Mouse Enables Bypassing Cytochrome c Oxidase Blockade and Limits Mitochondrial ROS Overproduction

    PubMed Central

    El-Khoury, Riyad; Dufour, Eric; Rak, Malgorzata; Ramanantsoa, Nelina; Grandchamp, Nicolas; Csaba, Zsolt; Duvillié, Bertrand; Bénit, Paule; Gallego, Jorge; Gressens, Pierre; Sarkis, Chamsy; Jacobs, Howard T.; Rustin, Pierre

    2013-01-01

    Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects. PMID:23300486

  12. Oligomerization of heme o synthase in cytochrome oxidase biogenesis is mediated by cytochrome oxidase assembly factor Coa2.

    PubMed

    Khalimonchuk, Oleh; Kim, Hyung; Watts, Talina; Perez-Martinez, Xochitl; Winge, Dennis R

    2012-08-01

    The synthesis of the heme a cofactor used in cytochrome c oxidase (CcO) is dependent on the sequential action of heme o synthase (Cox10) and heme a synthase (Cox15). The active state of Cox10 appears to be a homo-oligomeric complex, and formation of this complex is dependent on the newly synthesized CcO subunit Cox1 and the presence of an early Cox1 assembly intermediate. Cox10 multimerization is triggered by progression of Cox1 from the early assembly intermediate to downstream intermediates. The CcO assembly factor Coa2 appears important in coupling the presence of newly synthesized Cox1 to Cox10 oligomerization. Cells lacking Coa2 are impaired in Cox10 complex formation as well as the formation of a high mass Cox15 complex. Increasing Cox1 synthesis in coa2Δ cells restores respiratory function if Cox10 protein levels are elevated. The C-terminal segment of Cox1 is important in triggering Cox10 oligomerization. Expression of the C-terminal 54 residues of Cox1 appended to a heterologous matrix protein leads to efficient Cox10 complex formation in coa2Δ cells, but it fails to induce Cox15 complex formation. The state of Cox10 was evaluated in mutants, which predispose human patients to CcO deficiency and the neurological disorder Leigh syndrome. The presence of the D336V mutation in the yeast Cox10 backbone results in a catalytically inactive enzyme that is fully competent to oligomerize. Thus, Cox10 oligomerization and catalytic activation are separate processes and can be uncoupled. PMID:22669974

  13. Inductive effects of rifapentine on mice hepatic mixed function oxidase system.

    PubMed

    Liu, D Y; Wang, Y S

    1990-03-01

    Rifapentine (R773, DL473) is a long-acting antituberculous drug used in China. In our experiments we have found some manifestations of induction of hepatic mixed function oxidase system in mice following pretreatment with rifapentine or phenobarbital. Both rifapentine and phenobarbital significantly increased the rate of antipyrine and pentobarbital metabolism in vivo. They also increased liver weight, the content of liver microsomal protein and cytochrome P-450, the activity of NADPH-cytochrome C reductase and NADPH oxidase. SDS-polyacylamide gel electrophoresis showed that the relative proportions of some polypeptide bands in mice microsomal fraction were significantly changed following rifapentine or phenobarbital pretreatment. The results indicate that rifapentine, like phenobarbital, is a potent inducer of hepatic mixed function oxidase system in mice and that it should be used carefully in clinical therapy, when combined with other drugs. PMID:2319833

  14. Purification and characterization of pea seedling amine oxidase for crystallization studies.

    PubMed Central

    McGuirl, M A; McCahon, C D; McKeown, K A; Dooley, D M

    1994-01-01

    Pea (Pisum sativum L.) seedling amine oxidase (EC 1.4.3.6) is the first amine oxidase to be crystallized that diffracts to atomic resolution (2.5 A). Extensive modifications of a published purification procedure were necessary to obtain protein that would give diffraction-quality crystals. Here we report the improved purification and also use this high-purity protein to reexamine some fundamental characteristics of pea seedling amine oxidase. The extinction coefficient at 280 nm (epsilon 1%(280)) and the molecular mass of the protein are investigated by a variety of techniques, yielding epsilon 1%(280) = 20 cm-1 and a mass 150 +/- 6 kD. In addition, the stoichiometry of the metal and organic cofactors, Cu(II) and 6-hydroxy dopa (Topa) quinone, respectively, is examined. The ratio of Cu(II):Topa:protein monomer is found to be 1:1:1. PMID:7824646

  15. Purification and spectroscopic studies on catechol oxidase from lemon balm (Melissa officinalis).

    PubMed

    Rompel, Annette; Büldt-Karentzopoulos, Klaudia; Molitor, Christian; Krebs, Bernt

    2012-09-01

    A catechol oxidase from lemon balm (Melissa officinalis) moCO which only catalyzes the oxidation of catechols to quinones without hydroxylating tyrosine was purified. The molecular mass of the M. officinalis enzyme of 39,370 Da was obtained by MALDI mass spectrometry and the isoelectric point was determined to be 3.4. Addition of 2 eq. H(2)O(2) to the enzyme leads to oxy catechol oxidase. In the UV/Vis spectrum two new absorption bands occur at 343 nm (ε=8510 M(-1)cm(-1)) and 580 nm (ε=580 M(-1)cm(-1)) due to O(2)(2-)Cu (II) charge transfer transitions in accordance with the oxy forms of other type 3 copper proteins. The N-terminal sequence has been determined by Edman degradation to NPVQAPELDKCGTAT, exhibiting a proline at the second and sixth position conserved in other polyphenol oxidases. PMID:22727580

  16. Cytochrome C oxidase activity in germinating Phaseolus vulgaris l. seeds: Effects of carbon monoxide

    SciTech Connect

    Caughey, W.S. ); Sowa, S.; Roos, E.E.

    1989-04-01

    Cytochrome c oxidase is a key bioenergetic enzyme required for seed germination. The enzyme was isolated from 2-day germinating beans and biochemically compared to its bovine heart counterpart. Carbon monoxide, which binds to the heme a{sub 3} site of cytochrome c oxidase, we used to probe O{sub 2} utilization activity in isolated enzyme, mitochondrial particles, and whole seeds. Bean seeds under 80% CO/20% O{sub 2} exhibited 46% growth inhibition as determined by root length. Reversible, dose-dependent partial inhibition of bean seed mitochondrial respiration was observed in the presence of CO; heart mitochondria had a more sensitive, less reversible response. Effects of CO on bean and bovine heart enzyme were similar. The close correlation of CO effects observed on seedling growth, mitochondrial respiration and cytochrome oxidase activity indicate an important role for this enzyme during the early stages of seed germination.

  17. Identification of candidate aldehyde oxidases from the silkworm Bombyx mori potentially involved in antennal pheromone degradation.

    PubMed

    Pelletier, Julien; Bozzolan, Françoise; Solvar, Marthe; François, Marie-Christine; Jacquin-Joly, Emmanuelle; Maïbèche-Coisne, Martine

    2007-12-01

    Signal inactivation is a crucial step in the dynamic of olfactory process and involves various Odorant-Degrading Enzymes. In the silkworm Bombyx mori, one of the best models for studying olfaction in insects, the involvement of an antennal-specific aldehyde oxidase in the degradation of the sex pheromone component bombykal has been demonstrated over the three past decades by biochemical studies. However, the corresponding enzyme has never been characterized at the molecular level. Bioinformatic screening of B. mori genome and molecular approaches have been used to isolate several candidate sequences of aldehyde oxidases. Two interesting antennal-expressed genes have been further characterized and their putative functions are discussed in regard to their respective expression pattern and to our knowledge on aldehyde oxidase properties. Interestingly, one gene appeared as specifically expressed in the antennae of B. mori and associated in males with the bombykal-sensitive sensilla, strongly suggesting that it could encode for the previously biochemically characterized enzyme. PMID:17904312

  18. Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

    SciTech Connect

    Robinson, K.; Pilot, T.F.; Meany, J.E. )

    1990-01-01

    The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.

  19. Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes.

    PubMed

    Li, Yanhua; Gao, Zengqiang; Hou, Haifeng; Li, Lei; Zhang, Jihui; Yang, Haihua; Dong, Yuhui; Tan, Huarong

    2011-02-18

    Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His179, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure. PMID:21147069

  20. Copper radical oxidases and related extracellular oxidoreductases of wood-decay Agaricomycetes.

    PubMed

    Kersten, Phil; Cullen, Dan

    2014-11-01

    Extracellular peroxide generation, a key component of oxidative lignocellulose degradation, has been attributed to various enzymes including the copper radical oxidases. Encoded by a family of structurally related sequences, the genes are widely distributed among wood decay fungi including three recently completed polypore genomes. In all cases, core catalytic residues are conserved, but five subfamilies are recognized. Glyoxal oxidase, the most intensively studied representative, has been shown physiologically connected to lignin peroxidase. Relatively little is known about structure-function relationships among more recently discovered copper radical oxidases. Nevertheless, differences in substrate preferences have been observed in one case and the proteins have been detected in filtrates of various wood-grown cultures. Such diversity may reflect adaptations to host cell wall composition and changing environmental conditions. PMID:24915038