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Sample records for monooxygenase induction means

  1. [A substrate-type induction of liver microsomal monooxygenases by phenobarbital].

    Science.gov (United States)

    Tsyrlov, I B; Gromova, O A; Rivkind, N B; Vakulin, G M; Liakhovich, V V

    1977-07-01

    A possibility of step-wise induction of microsomal monooxygenases after injection of phenobarbital in the presence of 3-methylcholanthrene-caused induction was studied. It was found that the ratio of the high- and low-spin types of cytochrome, rather than the position of the CO-peak of its reduced form is a criterion for functional specificity of hemoprotein. Induction by phenobarbital appears possible under conditions when the inductor binding to microsomal hemoprotein is lacking, since cytochrome P-488 has no binding sites for phenobarbital. It is assumed that under microsomal monooxygenases induction by phenobarbital activation of genome and subsequent protein synthesis are operated by the substrate rather than by products of its primary metabolism in microsomes.

  2. Molecular cloning and xenobiotic induction of seven novel cytochrome P450 monooxygenases in Aedes albopictus.

    Science.gov (United States)

    Chan, Hiang Hao; Wajidi, Mustafa Fadzil Farid; Zairi, Jaal

    2014-01-01

    Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450.

  3. Tolerance to Acetaminophen Hepatotoxicity in the Mouse Model of Autoprotection is Associated with Induction of Flavin-containing Monooxygenase-3 (FMO3) in Hepatocytes

    Science.gov (United States)

    Acetaminophen (APAP) pretreatment with a low hepatotoxic dose in mice results in resistance to a second, higher dose of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in...

  4. Induction of cytochrome P450-associated monooxygenases in northern leopard frogs, Rana pipiens, by 3,3',4,4',5-pentachlorobiphenyl

    Science.gov (United States)

    Huang, Y.-W.; Melancon, M.J.; Jung, R.E.; Karasov, W.H.

    1998-01-01

    Northern leopard frogs (Rana pipiens) were injected intraperitoneally either with a solution of polychlorinated biphenyl (PCB) 126 in corn oil at a concentration of 0.2, 0.7, 2.3 and 7.8 mg/kg body weight or with corn oil alone. Appropriate assay conditions with hepatic microsomes were determined for four cytochrome P450-associated monooxygenases: ethoxyresorufin-O-dealkylase (EROD), methoxy-ROD (MROD), benzyloxy-ROD (BROD) and pentoxy-ROD (PROD). One week after PCB administration, the specific activities of EROD, MROD, BROD and PROD were not elevated at doses ? 0.7 mg/kg (p > 0.05), but were significantly increased at doses ? 2.3 mg/kg compared to the control groups (p frogs.

  5. Multimodal Word Meaning Induction from Minimal Exposure to Natural Text

    Science.gov (United States)

    Lazaridou, Angeliki; Marelli, Marco; Baroni, Marco

    2017-01-01

    By the time they reach early adulthood, English speakers are familiar with the meaning of thousands of words. In the last decades, computational simulations known as distributional semantic models (DSMs) have demonstrated that it is possible to induce word meaning representations solely from word co-occurrence statistics extracted from a large…

  6. Learning Package by Means of the Inductive Teaching with Group Process

    Science.gov (United States)

    Sawangsri, Benchaporn

    2016-01-01

    This research focuses on the inductive teaching with group process and students' behavior as working in a group. There were four instruments under this study. The descriptive statistics were employed. The findings revealed that, firstly, the effectiveness of LPIDTGP [Learning Package by means of the Inductive Teaching with Group Process] is higher…

  7. Altering the substrate specificity and enantioselectivity of phenylacetone monooxygenase by structure-inspired enzyme redesign

    NARCIS (Netherlands)

    Torres Pazmino, Daniel E.; Snajdrova, Radka; Rial, Daniela V.; Mihovilovic, Marko D.; Fraaije, Marco W.

    Of all presently available Baeyer-Villiger monooxygenases, phenylacetone monooxygenase (PAMO) is the only representative for which a structure has been determined. While it is an attractive biocatalyst because of its thermostability, it is only active with a limited number of substrates. By means of

  8. Lyophilization conditions for the storage of monooxygenases

    NARCIS (Netherlands)

    van Beek, Hugo L.; Beyer, Nina; Janssen, Dick B.; Fraaije, Marco W.

    2015-01-01

    Cyclohexanone monooxygenase (CHMO) was used as a model enzyme to find suitable freeze-drying conditions for long-term storage of an isolated monooxygenase. CHMO is a Baeyer-Villiger monooxygenase (BVMO) known for its ability to catalyze a large number of oxidation reactions. With a focus on establis

  9. Induction

    DEFF Research Database (Denmark)

    Sprogøe, Jonas; Elkjaer, Bente

    2010-01-01

    The purpose of this paper is to explore how induction of newcomers can be understood as both organizational renewal and the maintenance of status quo, and to develop ways of describing this in terms of learning.......The purpose of this paper is to explore how induction of newcomers can be understood as both organizational renewal and the maintenance of status quo, and to develop ways of describing this in terms of learning....

  10. Misalignment detection in induction motors with flexible coupling by means of estimated torque analysis and MCSA

    Science.gov (United States)

    Verucchi, Carlos; Bossio, José; Bossio, Guillermo; Acosta, Gerardo

    2016-12-01

    In recent years, progress has been made in developing techniques to detect mechanical faults in actuators driven by induction motors. The latest developments show their capability to detect faults from the analysis of the motor electrical variables. The techniques are based on the analysis of the Motor Current Signature Analysis (MCSA) and the Load Torque Signature Analysis (LTSA), among others. Thus, failures such as misalignment between the motor and load, progressive gear teeth wear, and mass imbalances have been successfully detected. In case of misalignment between the motor and load, both angular and radial misalignment, the results presented in literature do not consider the characteristics of the coupling device. In this work, it is studied a mechanism in which the power transmission between the motor and load is performed by means of different types of couplings, mainly those most frequently used in industry. Results show that the conclusions drawn for a particular coupling are not necessarily applicable to others. Finally, this paper presents data of interest for the development of algorithms or expert systems for fault detection and diagnosis.

  11. Mean-field analysis of an inductive reasoning game: application to influenza vaccination.

    Science.gov (United States)

    Breban, Romulus; Vardavas, Raffaele; Blower, Sally

    2007-09-01

    Recently we have introduced an inductive reasoning game of voluntary yearly vaccination to establish whether or not a population of individuals acting in their own self-interest would be able to prevent influenza epidemics. Here, we analyze our model to describe the dynamics of the collective yearly vaccination uptake. We discuss the mean-field equations of our model and first order effects of fluctuations. We explain why our model predicts that severe epidemics are periodically expected even without the introduction of pandemic strains. We find that fluctuations in the collective yearly vaccination uptake induce severe epidemics with an expected periodicity that depends on the number of independent decision makers in the population. The mean-field dynamics also reveal that there are conditions for which the dynamics become robust to the fluctuations. However, the transition between fluctuation-sensitive and fluctuation-robust dynamics occurs for biologically implausible parameters. We also analyze our model when incentive-based vaccination programs are offered. When a family-based incentive is offered, the expected periodicity of severe epidemics is increased. This results from the fact that the number of independent decision makers is reduced, increasing the effect of the fluctuations. However, incentives based on the number of years of prepayment of vaccination may yield fluctuation-robust dynamics where severe epidemics are prevented. In this case, depending on prepayment, the transition between fluctuation-sensitive and fluctuation-robust dynamics may occur for biologically plausible parameters. Our analysis provides a practical method for identifying how many years of free vaccination should be provided in order to successfully ameliorate influenza epidemics.

  12. How effective is amniotomy as a means of induction of labour?

    LENUS (Irish Health Repository)

    Cooley, S M

    2012-02-01

    BACKGROUND: Amniotomy or artificial rupture of membranes is routinely used for induction of labour. AIMS: To assess the efficacy of amniotomy alone for induction. METHODS: A retrospective descriptive study of 3,586 cases of amniotomy for induction of labour between July 1996 and December 1999. RESULTS: In total, 26,670 women delivered in the National Maternity Hospital during the study period. Of these 4,928 women required induction of labour and 72.8% of these (n = 3,586) underwent amniotomy only for induction of labour. Spontaneous labour occurred in 90.1% of the women who underwent amniotomy within 24 h. Oxytocin as an induction agent was employed in 9.8% of cases. Overall, 80.5% of the women had a spontaneous delivery, 7.3% had a ventouse delivery, 4.3% had a forceps delivery, and 7.9% underwent a caesarean section. In total, 90.5% of multips and 63.4% of primips had a spontaneous vaginal delivery. CONCLUSIONS: Amniotomy is a simple, safe and effective method of induction of labour.

  13. Mutation of glutamic acid 103 of toluene o-xylene monooxygenase as a means to control the catabolic efficiency of a recombinant upper pathway for degradation of methylated aromatic compounds.

    Science.gov (United States)

    Cafaro, Valeria; Notomista, Eugenio; Capasso, Paola; Di Donato, Alberto

    2005-08-01

    Toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of Pseudomonas stutzeri OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and o-xylene. For example, the sequential monooxygenation of o-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the P. stutzeri meta pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from o-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from o-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.

  14. Structural and Catalytic Differences between Two FADH2-Dependent Monooxygenases: 2,4,5-TCP 4-Monooxygenase (TftD from Burkholderia cepacia AC1100 and 2,4,6-TCP 4-Monooxygenase (TcpA from Cupriavidus necator JMP134

    Directory of Open Access Journals (Sweden)

    ChulHee Kang

    2012-08-01

    Full Text Available 2,4,5-TCP 4-monooxygenase (TftD and 2,4,6-TCP 4-monooxygenase (TcpA have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP and 2,4,6-trichlorophenol (2,4,6-TCP. TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH2-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants.

  15. Structural and catalytic differences between two FADH(2)-dependent monooxygenases: 2,4,5-TCP 4-monooxygenase (TftD) from Burkholderia cepacia AC1100 and 2,4,6-TCP 4-monooxygenase (TcpA) from Cupriavidus necator JMP134.

    Science.gov (United States)

    Hayes, Robert P; Webb, Brian N; Subramanian, Arun Kumar; Nissen, Mark; Popchock, Andrew; Xun, Luying; Kang, Chulhee

    2012-01-01

    2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP). TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH(2))-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD) is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants.

  16. Inductive Sensor Performance in Partial Discharges and Noise Separation by Means of Spectral Power Ratios

    Directory of Open Access Journals (Sweden)

    Jorge Alfredo Ardila-Rey

    2014-02-01

    Full Text Available Partial discharge (PD detection is a standardized technique to qualify electrical insulation in machines and power cables. Several techniques that analyze the waveform of the pulses have been proposed to discriminate noise from PD activity. Among them, spectral power ratio representation shows great flexibility in the separation of the sources of PD. Mapping spectral power ratios in two-dimensional plots leads to clusters of points which group pulses with similar characteristics. The position in the map depends on the nature of the partial discharge, the setup and the frequency response of the sensors. If these clusters are clearly separated, the subsequent task of identifying the source of the discharge is straightforward so the distance between clusters can be a figure of merit to suggest the best option for PD recognition. In this paper, two inductive sensors with different frequency responses to pulsed signals, a high frequency current transformer and an inductive loop sensor, are analyzed to test their performance in detecting and separating the sources of partial discharges.

  17. Starch-degrading polysaccharide monooxygenases.

    Science.gov (United States)

    Vu, Van V; Marletta, Michael A

    2016-07-01

    Polysaccharide degradation by hydrolytic enzymes glycoside hydrolases (GHs) is well known. More recently, polysaccharide monooxygenases (PMOs, also known as lytic PMOs or LPMOs) were found to oxidatively degrade various polysaccharides via a copper-dependent hydroxylation. PMOs were previously thought to be either GHs or carbohydrate binding modules (CBMs), and have been re-classified in carbohydrate active enzymes (CAZY) database as auxiliary activity (AA) families. These enzymes include cellulose-active fungal PMOs (AA9, formerly GH61), chitin- and cellulose-active bacterial PMOs (AA10, formerly CBM33), and chitin-active fungal PMOs (AA11). These PMOs significantly boost the activity of GHs under industrially relevant conditions, and thus have great potential in the biomass-based biofuel industry. PMOs that act on starch are the latest PMOs discovered (AA13), which has expanded our perspectives in PMOs studies and starch degradation. Starch-active PMOs have many common structural features and biochemical properties of the PMO superfamily, yet differ from other PMO families in several important aspects. These differences likely correlate, at least in part, to the differences in primary and higher order structures of starch and cellulose, and chitin. In this review we will discuss the discovery, structural features, biochemical and biophysical properties, and possible biological functions of starch-active PMOs, as well as their potential application in the biofuel, food, and other starch-based industries. Important questions regarding various aspects of starch-active PMOs and possible economical driving force for their future studies will also be highlighted.

  18. Identification of Tumor Evolution Patterns by Means of Inductive Logic Programming

    Institute of Scientific and Technical Information of China (English)

    Vitoantonio Bevilacqua; Patrizia Chiarappa; Giuseppe Mastronardi; Filippo Menolascina; Angelo Paradiso; Stefania Tommasi

    2008-01-01

    In considering key events of genomic disorders in the development and progression of cancer, the correlation between genomic instability and carcinogenesis is currently under investigation. In this work, we propose an inductive logic programming approach to the problem of modeling evolution patterns for breast cancer. Using this approach, it is possible to extract fingerprints of stages of the disease that can be used in order to develop and deliver the most adequate therapies to patients. Furthermore, such a model can help physicians and biologists in the elucidation of molecular dynamics underlying the aberrations-waterfall model behind carcinogenesis. By showing results obtained on a real-world dataset, we try to give some hints about further approach to the knowledge-driven validations of such hypotheses.

  19. Shannon Entropy and K-Means Method for Automatic Diagnosis of Broken Rotor Bars in Induction Motors Using Vibration Signals

    Directory of Open Access Journals (Sweden)

    David Camarena-Martinez

    2016-01-01

    Full Text Available For industry, the induction motors are essential elements in production chains. Despite the robustness of induction motors, they are susceptible to failures. The broken rotor bar (BRB fault in induction motors has received special attention since one of its characteristics is that the motor can continue operating with apparent normality; however, at certain point the fault may cause severe damage to the motor. In this work, a methodology to detect BRBs using vibration signals is proposed. The methodology uses the Shannon entropy to quantify the amount of information provided by the vibration signals, which changes due to the presence of new frequency components associated with the fault. For automatic diagnosis, the K-means cluster algorithm and a decision-making unit that looks for the nearest cluster through the Euclidian distance are applied. Unlike other reported works, the proposal can diagnose the BRB condition during startup transient and steady state regimes of operation. Additionally, the proposal is also implemented into a field programmable gate array in order to offer a low-cost and low-complex online monitoring system. The obtained results demonstrate the proposal effectiveness to diagnose half, one, and two BRBs.

  20. The Origin and Evolution of Baeyer-Villiger Monooxygenases (BVMOs): An Ancestral Family of Flavin Monooxygenases.

    Science.gov (United States)

    Mascotti, Maria Laura; Lapadula, Walter Jesús; Juri Ayub, Maximiliano

    2015-01-01

    The Baeyer-Villiger Monooxygenases (BVMOs) are enzymes belonging to the "Class B" of flavin monooxygenases and are capable of performing exquisite selective oxidations. These enzymes have been studied from a biotechnological perspective, but their physiological substrates and functional roles are widely unknown. Here, we investigated the origin, taxonomic distribution and evolutionary history of the BVMO genes. By using in silico approaches, 98 BVMO encoding genes were detected in the three domains of life: Archaea, Bacteria and Eukarya. We found evidence for the presence of these genes in Metazoa (Hydra vulgaris, Oikopleura dioica and Adineta vaga) and Haptophyta (Emiliania huxleyi) for the first time. Furthermore, a search for other "Class B" monooxygenases (flavoprotein monooxygenases--FMOs--and N-hydroxylating monooxygenases--NMOs) was conducted. These sequences were also found in the three domains of life. Phylogenetic analyses of all "Class B" monooxygenases revealed that NMOs and BVMOs are monophyletic, whereas FMOs form a paraphyletic group. Based on these results, we propose that BVMO genes were already present in the last universal common ancestor (LUCA) and their current taxonomic distribution is the result of differential duplication and loss of paralogous genes.

  1. The Origin and Evolution of Baeyer-Villiger Monooxygenases (BVMOs: An Ancestral Family of Flavin Monooxygenases.

    Directory of Open Access Journals (Sweden)

    Maria Laura Mascotti

    Full Text Available The Baeyer-Villiger Monooxygenases (BVMOs are enzymes belonging to the "Class B" of flavin monooxygenases and are capable of performing exquisite selective oxidations. These enzymes have been studied from a biotechnological perspective, but their physiological substrates and functional roles are widely unknown. Here, we investigated the origin, taxonomic distribution and evolutionary history of the BVMO genes. By using in silico approaches, 98 BVMO encoding genes were detected in the three domains of life: Archaea, Bacteria and Eukarya. We found evidence for the presence of these genes in Metazoa (Hydra vulgaris, Oikopleura dioica and Adineta vaga and Haptophyta (Emiliania huxleyi for the first time. Furthermore, a search for other "Class B" monooxygenases (flavoprotein monooxygenases--FMOs--and N-hydroxylating monooxygenases--NMOs was conducted. These sequences were also found in the three domains of life. Phylogenetic analyses of all "Class B" monooxygenases revealed that NMOs and BVMOs are monophyletic, whereas FMOs form a paraphyletic group. Based on these results, we propose that BVMO genes were already present in the last universal common ancestor (LUCA and their current taxonomic distribution is the result of differential duplication and loss of paralogous genes.

  2. Conceptual Analysis of Inductive Meaning Constructing in Constructivist Interactions between Mentor and Learner

    DEFF Research Database (Denmark)

    Badie, Farshad

    model of knowing (and epistemology). I will focus on the fact that, a learner based on her/his pre-structured knowledge, on pre-conceptions and on personal knowings, could actively participate in an interaction with her/his mentor in order to construct her/his knowledge and to develop that construction...... Constructing (that is my own approach) as a significant product of constructivist interactions between learners and mentors, see [1, 2, 3]. I will—conceptually—describe the logical background of my approach in order to express and to analyse the formal and semantic terms within the progress. Additionally, I...... will employ some fundamentals from language of concepts in order to specify concepts, conceptions and conceptualisations within the model. This research has been structured over my designed semantic model of meaning construction within constructivist interactions [between mentors and learners]. The semantic...

  3. Power Quality Improvement and LVRT Capability Enhancement of Wind Farms by Means of an Inductive Filtering Method

    Directory of Open Access Journals (Sweden)

    Yanjian Peng

    2016-04-01

    Full Text Available Unlike the traditional method for power quality improvement and low-voltage ride through (LVRT capability enhancement of wind farms, this paper proposes a new wind power integrated system by means of an inductive filtering method, especially if it contains a grid-connected transformer, a static synchronous compensator (STATCOM and fully-tuned (FT branches. First, the main circuit topology of the new wind power integrated system is presented. Then, the mathematical model is established to reveal the mechanism of harmonic suppression and the reactive compensation of the proposed wind power integrated system, and then the realization conditions of the inductive filtering method is obtained. Further, the control strategy of STATCOM is introduced. Based on the measured data for a real wind farm, the simulation studies are carried out to illustrate the performance of the proposed new wind power integrated system. The results indicate that the new system can not only enhance the LVRT capability of wind farms, but also prevent harmonic components flowing into the primary (grid winding of the grid-connected transformer. Moreover, since the new method can compensate for reactive power in a wind farm, the power factor at the grid side can be improved effectively.

  4. [Advances in biomolecular machine: methane monooxygenases].

    Science.gov (United States)

    Lu, Jixue; Wang, Shizhen; Fang, Baishan

    2015-07-01

    Methane monooxygenases (MMO), regarded as "an amazing biomolecular machine", catalyze the oxidation of methane to methanol under aerobic conditions. MMO catalyze the oxidation of methane elaborately, which is a novel way to catalyze methane to methanol. Furthermore, MMO can inspire the biomolecular machine design. In this review, we introduced MMO including structure, gene and catalytic mechanism. The history and the taxonomy of MMO were also introduced.

  5. Polymerization by plasma of trichloroethylene by means of resistive and inductive coupling; Polimerizacion por plasmas de tricloroetileno por medio de acoplamiento resistivo e inductivo

    Energy Technology Data Exchange (ETDEWEB)

    Vasquez, M.; Cruz, G.; Olayo, M.G. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico); Timoshina, T. [IPN, ESIQIE, 07738 Mexico D.F. (Mexico); Morales, J.; Olayo, R. [UAM-I, 09340 Mexico D.F. (Mexico)

    2004-07-01

    It was carried out the polymerization for plasma of the trichloroethylene by means of two types of coupling, resistive and inductive with the objective of studying the structure, morphology and the electric properties of the polymers obtained under these conditions. The structure and morphology of the polymers were studied by means of EDS and FT-IR spectroscopies. (Author)

  6. StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System

    NARCIS (Netherlands)

    Tischler, D.; Kermer, R.; Groning, J.A.D.; Kaschabek, S.R.; Berkel, van W.J.H.; Schlomann, M.

    2010-01-01

    Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (S

  7. Effect of herbicide acetochlor on cytochrome P450 monooxygenases and GST of earthworms Eisenia fetida

    Institute of Scientific and Technical Information of China (English)

    XIAO Neng-wen; LIU Xiang-hui; LI Wei; GE Feng

    2006-01-01

    To assess the sublethal toxicity of the herbicide acetochlor to earthworms and to find out biomarkers possible inducted under acetochlor exposure, Eiseniafetida was exposed to artificial soils supplemented with different concentrations of acetochlor(5,10, 20, 40 and 80 mg/kg soil). Effects of the acetochlor on cytochrome P450 monooxygenases p-nitroanisole O-demethylase(ODM),aldrin epoxidase(AE) and glutathione-S-transferases (GSTs) activities were determined. The results revealed cytochrome P450 monooxygenases were elevated with increasing concentrations of acetochlor, and the AE activity increased significantly compared with control at the concentration of 80 mg/kg (P<0.05). However, ODM activity from E. fetida was not induced significantly by acetochlor at all treatments(P>0.05). Sodium dodecyl sulfate-polyacryamide gel electrophoresis(SDS-PAGE) showed that one protein band was visualized and no evident differences were found in protein profiles between treatments and control. The GST activity increased significantly with longer duration(P<0.05) and increasing concentrations of acetochlor exposure(P<0.05). This study showed that the monooxygenases and GSTs activities in E. fetida could be induced by acetochlor, and thus, the AE and GST could be used in sublethal assays for soil contamination surveys and GST could be used as biomarkers ofacetochlor exposure in E. fetida.

  8. Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

    NARCIS (Netherlands)

    Romero, Elvira; Castellanos, J Rubén Gómez; Mattevi, Andrea; Fraaije, Marco W

    2016-01-01

    Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity. However, the low stability of many Baeyer-Villiger monooxygenases is an obstacle for their exploitation in industry. Char

  9. Microbial flavoprotein monooxygenases as mimics of mammalian flavin-containing monooxygenases for the enantioselective preparation of drug metabolites

    NARCIS (Netherlands)

    Gul, Turan; Krzek, Marzena; Permentier, Hjalmar; Fraaije, Marco; Bischoff, Rainer

    2016-01-01

    Mammalian flavin-containing monooxygenases are difficult to obtain and study while they play a major role in detoxifying various xenobiotics. In order to provide alternative biocatalytic tools to generate FMO-derived drug metabolites, a collection of microbial flavoprotein monooxygenases, sequence-r

  10. Improving Awareness about the Meaning of the Principle of Mathematical Induction (Fomentar la Conciencia sobre el Significado del Principio de Inducción Matemática)

    OpenAIRE

    Annalisa Cusi; Nicolina A. Malara

    2009-01-01

    This work is based on our conviction that it is possible to minimize difficulties students face in learning the principle of mathematical induction by means of clarifying its logical aspects. Based on previous research and theory, we designed a method of fostering students’ understanding of the principle. We present results that support the effectiveness of our method with teachers in training who are not specializing in mathematics. Este trabajo está basado en nuestra convicción de que es po...

  11. Efficiency optimization of class-D biomedical inductive wireless power transfer systems by means of frequency adjustment.

    Science.gov (United States)

    Schormans, Matthew; Valente, Virgilio; Demosthenous, Andreas

    2015-01-01

    Inductive powering for implanted medical devices is a commonly employed technique, that allows for implants to avoid more dangerous methods such as the use of transcutaneous wires or implanted batteries. However, wireless powering in this way also comes with a number of difficulties and conflicting requirements, which are often met by using designs based on compromise. In particular, one aspect common to most inductive power links is that they are driven with a fixed frequency, which may not be optimal depending on factors such as coupling and load. In this paper, a method is proposed in which an inductive power link is driven by a frequency that is maintained at an optimum value f(opt), to ensure that the link is in resonance. In order to maintain this resonance, a phase tracking technique is employed at the primary side of the link; this allows for compensation of changes in coil separation and load. The technique is shown to provide significant improvements in maintained secondary voltage and efficiency for a range of loads when the link is overcoupled.

  12. A tale of two methane monooxygenases

    Science.gov (United States)

    Ross, Matthew O.

    2017-01-01

    Methane monooxygenase (MMO) enzymes activate O2 for oxidation of methane. Two distinct MMOs exist in nature, a soluble form that uses a diiron active site (sMMO) and a membrane-bound form with a catalytic copper center (pMMO). Understanding the reaction mechanisms of these enzymes is of fundamental importance to biologists and chemists, and is also relevant to the development of new biocatalysts. The sMMO catalytic cycle has been elucidated in detail, including O2 activation intermediates and the nature of the methane-oxidizing species. By contrast, many aspects of pMMO catalysis remain unclear, most notably the nuclearity and molecular details of the copper active site. Here, we review the current state of knowledge for both enzymes, and consider pMMO O2 activation intermediates suggested by computational and synthetic studies in the context of existing biochemical data. Further work is needed on all fronts, with the ultimate goal of understanding how these two remarkable enzymes catalyze a reaction not readily achieved by any other metalloenzyme or biomimetic compound. PMID:27878395

  13. Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology.

    Science.gov (United States)

    Gomiero, Chiara; Bertolutti, Giulia; Martinello, Tiziana; Van Bruaene, Nathalie; Broeckx, Sarah Y; Patruno, Marco; Spaas, Jan H

    2016-03-01

    Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFβ3 and bFGF2 + TGFβ3 + LLLT. Indeed, the supplement of bFGF2 and TGFβ3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFβ3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.

  14. Towards practical Baeyer-Villiger-monooxygenases: design of cyclohexanone monooxygenase mutants with enhanced oxidative stability.

    Science.gov (United States)

    Opperman, Diederik J; Reetz, Manfred T

    2010-12-10

    Baeyer-Villiger monooxygenases (BVMOs) catalyze the conversion of ketones and cyclic ketones into esters and lactones, respectively. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is known to show an impressive substrate scope as well as exquisite chemo-, regio-, and enantioselectivity in many cases. Large-scale synthetic applications of CHMO are hampered, however, by the instability of the enzyme. Oxidation of cysteine and methionine residues contributes to this instability. Designed mutations of all the methionine and cysteine residues in the CHMO wild type (WT) showed that the amino acids labile towards oxidation are mostly either surface-exposed or located within the active site, whereas the two methionine residues identified for thermostabilization are buried within the folded protein. Combinatorial mutations gave rise to two stabilized mutants with either oxidative or thermal stability, without compromising the activity or stereoselectivity of the enzyme. The most oxidatively stabilized mutant retained nearly 40 % of its activity after incubation with H(2)O(2) (0.2 M), whereas the wild-type enzyme's activity was completely abolished at concentrations as low as 5 mM H(2)O(2). We propose that oxidation-stable mutants might well be a "prerequisite" for thermostabilization, because laboratory-evolved thermostability in CHMO might be masked by a high degree of oxidation instability.

  15. Imaging electrical impedance from acoustic measurements by means of magnetoacoustic tomography with magnetic induction (MAT-MI).

    Science.gov (United States)

    Li, Xu; Xu, Yuan; He, Bin

    2007-02-01

    We have conducted computer simulation and experimental studies on magnetoacoustic-tomography with magnetic induction (MAT-MI) for electrical impedance imaging. In MAT-MI, the object to be imaged is placed in a static magnetic field, while pulsed magnetic stimulation is applied in order to induce eddy current in the object. In the static magnetic field, the Lorentz force acts upon the eddy current and causes acoustic vibrations in the object. The propagated acoustic wave is then measured around the object to reconstruct the electrical impedance distribution. In the present simulation study, a two-layer spherical model is used. Parameters of the model such as sample size, conductivity values, strength of the static and pulsed magnetic field, are set to simulate features of biological tissue samples and feasible experimental constraints. In the forward simulation, the electrical potential and current density are solved using Poisson's equation, and the acoustic pressure is calculated as the forward solution. The electrical impedance distribution is then reconstructed from the simulated pressure distribution surrounding the sample. The present computer simulation results suggest that MAT-MI can reconstruct conductivity images of biological tissue with high spatial resolution and high contrast. The feasibility of MAT-MI in providing high spatial resolution images containing impedance-related information has also been demonstrated in a phantom experiment.

  16. Extensive substrate profiling of cyclopentadecanone monooxygenase as Baeyer-Villiger biocatalyst reveals novel regiodivergent oxidations

    NARCIS (Netherlands)

    Fink, Michael J.; Fischer, Thomas C.; Rudroff, Florian; Dudek, Hanna; Fraaije, Marco W.; Mihovilovic, Marko D.

    2011-01-01

    Cyclopentadecanone monooxygenase (CPDMO) is one of the latest additions to the established library of Baeyer-Villiger monooxygenases. Desymmetrizations of substituted cyclobutanones and -hexanones as well as kinetic resolutions of racemic cycloketones are efficiently catalyzed by CPDMO. Moreover the

  17. Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

    NARCIS (Netherlands)

    Kamerbeek, NM; Olsthoorn, AJJ; Fraaije, MW; Janssen, DB; Kamerbeek, Nanne M.; Olsthoorn, Arjen J.J.

    2003-01-01

    The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of ca

  18. Hydroxylation of methane through component interactions in soluble methane monooxygenases.

    Science.gov (United States)

    Lee, Seung Jae

    2016-04-01

    Methane hydroxylation through methane monooxygenases (MMOs) is a key aspect due to their control of the carbon cycle in the ecology system and recent applications of methane gas in the field of bioenergy and bioremediation. Methanotropic bacteria perform a specific microbial conversion from methane, one of the most stable carbon compounds, to methanol through elaborate mechanisms. MMOs express particulate methane monooxygenase (pMMO) in most strains and soluble methane monooxygenase (sMMO) under copper-limited conditions. The mechanisms of MMO have been widely studied from sMMO belonging to the bacterial multicomponent monooxygenase (BMM) superfamily. This enzyme has diiron active sites where different types of hydrocarbons are oxidized through orchestrated hydroxylase, regulatory and reductase components for precise control of hydrocarbons, oxygen, protons, and electrons. Recent advances in biophysical studies, including structural and enzymatic achievements for sMMO, have explained component interactions, substrate pathways, and intermediates of sMMO. In this account, oxidation of methane in sMMO is discussed with recent progress that is critical for understanding the microbial applications of C-H activation in one-carbon substrates.

  19. Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase.

    Science.gov (United States)

    Romero, Elvira; Castellanos, J Rubén Gómez; Mattevi, Andrea; Fraaije, Marco W

    2016-12-19

    Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity. However, the low stability of many Baeyer-Villiger monooxygenases is an obstacle for their exploitation in industry. Characterization and crystal structure determination of a robust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts a variety of aliphatic, aromatic, and cyclic ketones, as well as prochiral sulfides. A compact substrate-binding cavity explains its preference for small rather than bulky substrates. Small-scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO. The exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.

  20. Improving Awareness about the Meaning of the Principle of Mathematical Induction (Fomentar la Conciencia sobre el Significado del Principio de Inducción Matemática

    Directory of Open Access Journals (Sweden)

    Annalisa Cusi

    2009-09-01

    Full Text Available This work is based on our conviction that it is possible to minimize difficulties students face in learning the principle of mathematical induction by means of clarifying its logical aspects. Based on previous research and theory, we designed a method of fostering students’ understanding of the principle. We present results that support the effectiveness of our method with teachers in training who are not specializing in mathematics. Este trabajo está basado en nuestra convicción de que es posible minimizar las dificultades de los alumnos cuando se enfrentan al aprendizaje del principio de inducción matemática mediante la clarificación de sus aspectos lógicos. Basándonos en la investigación y teoría previas, diseñamos un método para fomentar la comprensión del principio por los alumnos. Presentamos resultados que respaldan la efectividad de nuestro método con profesores en formación no especializados en matemáticas.

  1. Effects of pregnancy and protein-energy malnutrition on monooxygenase O-dealkylation activity in rat liver microsomes

    Directory of Open Access Journals (Sweden)

    S.N. Kuriyama

    2000-01-01

    Full Text Available Xenobiotic metabolism is influenced by a variety of physiological and environmental factors including pregnancy and nutritional status of the individual. Pregnancy has generally been reported to cause a depression of hepatic monooxygenase activities. Low-protein diets and protein-energy malnutrition have also been associated with a reduced activity of monooxygenases in nonpregnant animals. We investigated the combined effects of pregnancy and protein-energy malnutrition on liver monooxygenase O-dealkylation activity. On pregnancy day 0 rats were assigned at random to a group fed ad libitum (well-nourished, WN or to a malnourished group (MN which received half of the WN food intake (12 g/day. WN and MN rats were killed on days 0 (nonpregnant, 11 or 20 of pregnancy and ethoxy- (EROD, methoxy- (MROD and penthoxy- (PROD resorufin O-dealkylation activities were measured in liver microsomes. Only minor changes in enzyme activities were observed on pregnancy day 11, but a clear-cut reduction of monooxygenase activities (pmol resorufin min-1 mg protein-1 was noted near term (day 0 vs 20, means ± SD, Student t-test, P<0.05 in WN (EROD: 78.9 ± 15.1 vs 54.6 ± 10.2; MROD: 67.8 ± 10.0 vs 40.9 ± 7.2; PROD: 6.6 ± 0.9 vs 4.3 ± 0.8 and in MN (EROD: 89.2 ± 23.9 vs 46.9 ± 15.0; MROD: 66.8 ± 13.8 vs 27.9 ± 4.4; PROD: 6.3 ± 1.0 vs 4.1 ± 0.6 dams. On pregnancy day 20 MROD was lower in MN than in WN dams. Malnutrition did not increase the pregnancy-induced reduction of EROD and PROD activities. Thus, the present results suggest that the activities of liver monooxygenases are reduced in near-term pregnancy and that protein-energy malnutrition does not alter EROD or PROD in pregnant rats.

  2. Formation of carcinogenic and inactive chrysene metabolites by rat liver microsomes of various monooxygenase activities

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, J.; Grimmer, G.; Schmoldt, A.

    1982-12-01

    Microsomal oxidation of chrysene in rat liver occurs at various positions (1,2-; 3,4-; 5,6-). This has been verified by means of gas chromatography/mass spectrometry (GC/MS) and comparison with synthetic reference substances. After various rat pretreatments with inducers of the monooxygenase system the oxidation at the 3,4-position predominated in isolated microsomes. The formation of the ultimate carcinogen of chrysene - 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene - was not detectable in untreated rats. However, it was observed as 1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene-TMS-ether formed under workup and derivatisation conditions after pretreating the rats with phenobarbital, polychlorinated biphenyl, benzoflavone, or various polycyclic aromatic hydrocarbons. Polychlorinated biphenyls and benzoflavone were the most potent inducers for the formation of this metabolite.

  3. Exposing the Alkanesulfonate Monooxygenase Protein-Protein Interaction Sites.

    Science.gov (United States)

    Dayal, Paritosh V; Singh, Harsimran; Busenlehner, Laura S; Ellis, Holly R

    2015-12-29

    The alkanesulfonate monooxygenase enzymes (SsuE and SsuD) catalyze the desulfonation of diverse alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase enzyme. Previous studies have highlighted the presence of protein-protein interactions between SsuE and SsuD thought to be important in the flavin transfer event, but the putative interaction sites have not been identified. Protected sites on specific regions of SsuE and SsuD were identified by hydrogen-deuterium exchange mass spectrometry. An α-helix on SsuD containing conserved charged amino acids showed a decrease in percent deuteration in the presence of SsuE. The α-helical region of SsuD is part of an insertion sequence and is adjacent to the active site opening. A SsuD variant containing substitutions of the charged residues showed a 4-fold decrease in coupled assays that included SsuE to provide reduced FMN, but there was no activity observed with an SsuD variant containing a deletion of the α-helix under similar conditions. Desulfonation by the SsuD deletion variant was only observed with an increase in enzyme and substrate concentrations. Although activity was observed under certain conditions, there were no protein-protein interactions observed with the SsuD variants and SsuE in pull-down assays and fluorimetric titrations. The results from these studies suggest that optimal transfer of reduced flavin from SsuE to SsuD requires defined protein-protein interactions, but diffusion can occur under specified conditions. A basis is established for further studies to evaluate the structural features of the alkanesulfonate monooxygenase enzymes that promote desulfonation.

  4. Expression, purification and characterization of human Dopamine ß-monooxygenase

    DEFF Research Database (Denmark)

    Vendelboe, Trine Vammen

    This thesis deals with expression, purification and characterization of the copper containing enzyme dopamine ß-monooxygenase (DBM). DBM is an ascorbate dependent protein that requires Cu in the active site in order to be functional. DBM is made of four domains; An Nterminal DOMON domain, the two...... others, one of the reasons why these proteins are considered to follow the same mechanism. DBM converts dopamine (DA) into Norepinphrine (NE). Both substrate and product functions as neurotransmitters and the levels of these are involved in many different disorders such as depression and hypertension...

  5. Expression, purification and characterization of human Dopamine ß-monooxygenase

    DEFF Research Database (Denmark)

    Vendelboe, Trine Vammen

    This thesis deals with expression, purification and characterization of the copper containing enzyme dopamine ß-monooxygenase (DBM). DBM is an ascorbate dependent protein that requires Cu in the active site in order to be functional. DBM is made of four domains; An Nterminal DOMON domain, the two...... others, one of the reasons why these proteins are considered to follow the same mechanism. DBM converts dopamine (DA) into Norepinphrine (NE). Both substrate and product functions as neurotransmitters and the levels of these are involved in many different disorders such as depression and hypertension...

  6. Structural characterization of Lytic Polysaccharide MonoOxygenases

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of copper-containingmetalloenzymes that have been found to oxidatively degrade polysaccharides (and recently alsooligosaccharides). They dependent on redox partners to provide them with electrons and they utilizemolecular oxygen to cleave......) and their interaction with substratehave been structurally characterized. A number of structures of LsAA9A have been obtained in complexwith a range of cellulosic- and hemicellulosic substrates and with the active site Cu in different redox state.Two of the LsAA9A structures with the active site Cu in essentially a Cu...

  7. Selective Oxidations of Organoboron Compounds Catalyzed by Baeyer-Villiger Monooxygenases

    NARCIS (Netherlands)

    Brondani, Patricia B.; de Gonzalo, Gonzalo; Fraaije, Marco W.; Andrade, Leandro H.

    2011-01-01

    The applicability of Baeyer-Villiger monooxygenases (BVMOs) in organoboron chemistry has been explored through testing chemo-and enantioselective oxidations of a variety of boron-containing aromatic and vinylic compounds. Several BVMOs, namely: phenylacetone monooxygenase (PAMO), M446G PAMO mutant,

  8. Polycyclic Ketone Monooxygenase (PockeMO): A Robust Biocatalyst for the Synthesis of Optically Active Sulfoxides

    NARCIS (Netherlands)

    de Gonzalo, Gonzalo; Fürst, Maximilian; Fraaije, Marco

    2017-01-01

    A recently discovered, moderately thermostable Baeyer-Villiger monooxygenase, polycyclic ketone monooxygenase (PockeMO), from Thermothelomyces thermophila has been employed as a biocatalyst in a set of asymmetric sulfoxidations. The enzyme was able to catalyze the oxidation of various alkyl aryl

  9. Bioconversion of Mono- and Sesquiterpenoids by Recombinant Human Cytochrome P450 Monooxygenases

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Fichera, Mario A.; Malz, Frank; Ebbelaar, Monique; Bos, Rein; Woerdenbag, Herman J.; Quax, Wim J.; Kayser, Oliver

    2008-01-01

    Cytochrome P450 monooxygenases play an important role in the biosynthesis and metabolism of terpenoids. We explored the potential of recombinant human liver cytochrome P450 monooxygenases CYP1A2, CYP2C9, and CYP3A4, heterologously expressed in Escherichia coli, to convert mono- and sesquiterpenoids

  10. Regulated O2 activation in flavin-dependent monooxygenases.

    Science.gov (United States)

    Frederick, Rosanne E; Mayfield, Jeffery A; DuBois, Jennifer L

    2011-08-17

    Flavin-dependent monooxygenases (FMOs) are involved in important biosynthetic pathways in diverse organisms, including production of the siderophores used for the import and storage of essential iron in serious pathogens. We have shown that the FMO from Aspergillus fumigatus, an ornithine monooxygenase (Af-OMO), is mechanistically similar to its well-studied distant homologues from mammalian liver. The latter are highly promiscuous in their choice of substrates, while Af-OMO is unusually specific. This presents a puzzle: how do Af-OMO and other FMOs of the biosynthetic classes achieve such specificity? We have discovered substantial enhancement in the rate of O(2) activation in Af-OMO in the presence of L-arginine, which acts as a small molecule regulator. Such protein-level regulation could help explain how this and related biosynthetic FMOs manage to couple O(2) activation and substrate hydroxylation to each other and to the appropriate cellular conditions. Given the essentiality of Fe to Af and the avirulence of the Af-OMO gene knock out, inhibitors of Af-OMO are likely to be drug targets against this medically intractable pathogen.

  11. Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal ion and tetrahydrobiopterin dependence.

    Science.gov (United States)

    Watschinger, Katrin; Keller, Markus A; Hermetter, Albin; Golderer, Georg; Werner-Felmayer, Gabriele; Werner, Ernst R

    2009-01-01

    Glyceryl ether monooxygenase is a tetrahydrobiopterin-dependent membrane-bound enzyme which catalyses the cleavage of lipid ethers into glycerol and the corresponding aldehyde. Despite many different characterisation and purification attempts, so far no gene and primary sequence have been assigned to this enzyme. The seven other tetrahydrobiopterin-dependent enzymes can be divided in the family of aromatic amino acid hydroxylases - comprising phenylalanine hydroxylase, tyrosine hydroxylase and the two tryptophan hydroxylases - and into the three nitric oxide synthases. We tested the influences of different metal ions and metal ion chelators on glyceryl ether monooxygenase, phenylalanine hydroxylase and nitric oxide synthase activity to elucidate the relationship of glyceryl ether monooxygenase to these two families. 1,10-Phenanthroline, an inhibitor of non-heme iron-dependent enzymes, was able to potently block glyceryl ether monooxygenase as well as phenylalanine hydroxylase, but had no effect on inducible nitric oxide synthase. Two tetrahydrobiopterin analogues, N(5)-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin, had a similar impact on glyceryl ether monooxygenase activity, as has already been shown for phenylalanine hydroxylase. These observations point to a close analogy of the role of tetrahydrobiopterin in glyceryl ether monooxygenase and in aromatic amino acid hydroxylases and suggest that glyceryl ether monooxygenase may require a non-heme iron for catalysis.

  12. Aryl hydrocarbon mono-oxygenase activity in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Griffin, G.D.; Schuresko, D.D.

    1981-06-01

    Aryl hydrocarbon mono-oxygenase (AHM), an enzyme of key importance in metabolism of xenobiotic chemicals such as polynuclear aromatic hydrocarbons (PNA), is present in human lymphocytes. Studies investing the relation of activity of AHM in human lymphocytes to parameters such as disease state, PNA exposure, in vitro mitogen stimulation, etc. have been summarized in this report. Some studies have demonstrated increased AHM activity in lymphocytes from cigarette smokers (compared to nonsmokers), and in lung cancer patients when compared to appropriate control groups. These observations are confused by extreme variability in human lymphocyte AHM activities, such variability arising from factors such as genetic variation in AHM activity, variation in in vitro culture conditions which affect AHM activity, and the problematical relationship of common AHM assays to actual PNA metabolism taking place in lymphocytes. If some of the foregoing problems can be adequately addressed, lymphocyte AHM activity could hold the promise of being a useful biomarker system for human PNA exposure.

  13. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

    Science.gov (United States)

    Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé; Garajova, Sona; Foucat, Loïc; Falourd, Xavier; Saake, Bodo; Berrin, Jean-Guy; Cathala, Bernard

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers. PMID:28071716

  14. Structural characterization of Lytic Polysaccharide MonoOxygenases

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of copper-containingmetalloenzymes that have been found to oxidatively degrade polysaccharides (and recently alsooligosaccharides). They dependent on redox partners to provide them with electrons and they utilizemolecular oxygen to cleave......) and their interaction with substratehave been structurally characterized. A number of structures of LsAA9A have been obtained in complexwith a range of cellulosic- and hemicellulosic substrates and with the active site Cu in different redox state.Two of the LsAA9A structures with the active site Cu in essentially a Cu......(II) state show differences in thenature of the Cu-ligand with and without cellulosic substrate bound and provide structural insight into themechanistic action of LPMOs. Interestingly, for an LsAA9A complex structure with a hemicellulosicsubstrate (xylooligosaccharide) a corresponding difference...

  15. Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase.

    Science.gov (United States)

    Katopodis, A G; May, S W

    1990-05-15

    Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.

  16. Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: inducibility of cytochrome P450 dependent monooxygenase functions by beta-naphthoflavone, phenobarbital and dexamethasone.

    Science.gov (United States)

    Lupp, A; Lau, K; Trautmann, A K; Krausse, T; Klinger, W

    1999-01-01

    In the present study the effects of beta-naphthoflavone (BNF), phenobarbital (PB) and dexamethasone (DEX) on cytochrome P450 (P450) dependent monooxygenase functions were investigated in intrasplenic liver cell explants in comparison to adult liver. Fetal liver tissue suspensions were transplanted into the spleens of 60-90 days old adult male syngenic Fisher 344 inbred rats. 2, 4 or 6 months after surgery, transplant recipients and age matched controls were orally treated with BNF (1x50 mg/kg body weight (b.wt.)), PB (1x50 mg/kg b.wt.), DEX (for 3 days 4 mg/kg b.wt. per day), or the respective solvents (dimethylsulfoxide or 0.9% NaCl). The animals were sacrificed 24 (BNF, DEX) or 48 (PB) hours after the last treatment. P450 mediated monooxygenase functions were measured in spleen and liver 9000 g supernatants by three model reactions for different P450 subtypes: ethoxyresorufin O-deethylation (EROD; 1A), ethoxycoumarin O-deethylation (ECOD; 1A, 2A, 2B), and ethylmorphine N-demethylation (END; 3A). Spleen weights were significantly higher in transplanted rats, compared to controls, at all three time points after surgery. Induction with PB or DEX, and in some cases also with BNF, lead to a significant increase in liver weights of transplant recipients and control rats independent of the time after transplantation. In contrast, there was no influence on spleen weights due to BNF or PB. At all time points after surgery, with DEX a marked decrease in body weights, weights of adrenal glands and of lymphatic organs like thymus glands and spleens was observed, with the weights of the transplant containing spleens being still higher in comparison to control organs. Spleens of control animals displayed nearly no P450 mediated monooxygenase functions neither without nor with induction. After transplantation, however, significant EROD and ECOD, but hardly any END activities were seen in the host organs at all three time points after surgery. In transplant containing spleens

  17. Protein engineering of the cytochrome P450 monooxygenase from bacillus megaterium

    OpenAIRE

    Urlacher, Vlada B.; Schmid, Rolf D

    2004-01-01

    The role and importance of cytochrome P450 enzymes (CYP) in drug development, biodegradation processes and biocatalysis has been widely acknowledged. P450 monooxygenases exhibit an extremely wide substrate spectrum which is the basis of their ability to activate or detoxify a large variety of target molecules. P450 monooxygenases have been isolated from bacteria, yeasts, insects, as well as mammalian and plant tissues. Currently, the enzyme family is one of the best known gene subfamilies wit...

  18. Engineering Cyclohexanone Monooxygenase for the Production of Methyl Propanoate.

    Science.gov (United States)

    van Beek, Hugo L; Romero, Elvira; Fraaije, Marco W

    2017-01-20

    A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP(+) binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate. The resulting libraries were screened using whole cell biotransformations, and headspace gas chromatography-mass spectrometry was used to identify improved AcCHMO variants. This revealed that the I491A AcCHMO mutant exhibits a significant improvement over the wild type enzyme in the desired regioselectivity using 2-butanone as a substrate (40% vs 26% methyl propanoate, respectively). Another interesting mutant is the T56S AcCHMO mutant, which exhibits a higher conversion yield (92%) and kcat (0.5 s(-1)) than wild type AcCHMO (52% and 0.3 s(-1), respectively). Interestingly, the uncoupling rate for the T56S AcCHMO mutant is also significantly lower than that for the wild type enzyme. The T56S/I491A double mutant combined the beneficial effects of both mutations leading to higher conversion and improved regioselectivity. This study shows that even for a relatively small aliphatic substrate (2-butanone), catalytic efficiency and regioselectivity can be tuned by structure-inspired enzyme engineering.

  19. Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

    Energy Technology Data Exchange (ETDEWEB)

    Strushkevich, Natallia; MacKenzie, Farrell; Cherkesova, Tatyana; Grabovec, Irina; Usanov, Sergey; Park, Hee-Won (Toronto); (NAS-Belarus)

    2011-09-06

    In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C-C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1 - the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 {angstrom} away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.

  20. Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium.

    Science.gov (United States)

    Chéry, Cyrille C; Günther, Detlef; Cornelis, Rita; Vanhaecke, Frank; Moens, Luc

    2003-10-01

    The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation.

  1. Effects of Cu(2+) and Pb(2+) on different fish species: liver cytochrome P450-dependent monooxygenase activities and FTIR spectra.

    Science.gov (United States)

    Henczová, Mária; Deér, Aranka Kiss; Filla, Adrienn; Komlósi, Viktória; Mink, János

    2008-07-01

    The effects of Cu(2+)-sulfate and Pb(2+)-acetate on carp (Cyprinus carpio L.), silver carp (Hypopthalmichtys molitrix V.) and wels (Silurus glanis L.) were studied. The liver microsomal Cyt P450 content, the EROD, ECOD and APND monooxygenase activities were measured. In vivo treatment with 1 mg L(-1) Cu(2+) significantly elevated the activities of these enzymes and Cyt P450 content in silver carp livers. The high-dose Cu(2+) treatment (10 mg L(-1)) on silver carp caused two-fold higher induction in the P450 dependent monooxygenase isoensymes than in wels. Although the 2 mg kg(-1) treatment with Pb(2+) in carp elevated significantly the P450 content, the EROD isoenzyme activities were significantly decreased after 1 day, showing the destructive effect of metal ion on the enzyme system. In vitro, Cu(2+) and Pb(2+) decreased the Cyt P450 content in the carp liver microsomes and the absorption peak shifted to higher wavelength. Fourier Transform Infrared (FTIR) spectroscopy was used to detect the damaging effects of the heavy metals. According to the inhibitory potency to Cu(2+), the most sensitive isoenzyme was the EROD in wels, the least was the silver carp's isoenzyme. The investigated fish P450 isoenzymes showed, that the Cu(2+) was a stronger inhibitor than Pb(2+).

  2. Discovery and industrial applications of lytic polysaccharide mono-oxygenases.

    Science.gov (United States)

    Johansen, Katja S

    2016-02-01

    The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine. © 2016 Authors; published by Portland Press Limited.

  3. Targeted Deletion of Kynurenine 3-Monooxygenase in Mice

    Science.gov (United States)

    Giorgini, Flaviano; Huang, Shao-Yi; Sathyasaikumar, Korrapati V.; Notarangelo, Francesca M.; Thomas, Marian A. R.; Tararina, Margarita; Wu, Hui-Qiu; Schwarcz, Robert; Muchowski, Paul J.

    2013-01-01

    Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD+, did not differ between Kmo−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease. PMID:24189070

  4. Tetrachloroethylene, trichloroethylene, and chlorinated phenols induce toluene-o-xylene monooxygenase activity in Pseudomonas stutzeri OX1.

    Science.gov (United States)

    Ryoo, D; Shim, H; Arenghi, F L; Barbieri, P; Wood, T K

    2001-08-01

    Pseudomonas stutzeri OX1 naphthalene-oxidation activity is induced 3.0-fold by tetrachloroethylene (PCE) and 3.1-fold by trichloroethylene (TCE) at 100 microM. With the mutant P. stutzeri M1, which does not express toluene-o-xylene monooxygenase (ToMO, product of the tou operon), no naphthalene-oxidation activity induction by PCE and TCE was found; hence, PCE and TCE induce ToMO of P. stutzeri OX1. The chlorinated phenols 2-, 3-, and 4-chlorophenol induced ToMO expression 0.58-, 0.23- and 0.37-fold, respectively, compared to the direct inducer of the pathway, o-cresol. Using P. putida PaW340 (pPP4062, pFP3028), which has the tou promoter fused to the reporter catechol-2,3-dioxygenase, and the regulator gene touR, it was determined that the tou promoter was induced directly 5.7-, 7.1-, and 5.1-fold for 2-, 3-, and 4-chlorophenol, respectively (compared to an 8.8-fold induction with o-cresol). In addition, it was found that TCE and PCE do not directly induce the tou pathway and that components other than the tou structural and regulatory genes are necessary for induction. Gas chromatography results also showed that 100 microM TCE induced its own degradation (8-9%) in 16 h in P. stutzeri OX1, and all of the stoichiometric chloride from the degraded TCE was detected in solution.

  5. Monooxygenase activitity in Aedes aegypti population in Tembalang subdistrict, Semarang city

    Directory of Open Access Journals (Sweden)

    Dyah Widiastuti

    2015-06-01

    Full Text Available Dengue Haemorrhagic Fever (DHF is a major health problem in Tembalang sub district, Semarang City. Fogging with insecticide applications was done frequently as an effort to control Dengue vectors. The use of insecticides from the same class in a long time can lead to resistance in mosquitos’ population. The research aimed to observe the activity of monooxygenases in Aedes aegypti populations in Tembalang Subdistrict, Semarang. The study was conducted during February-November 2014 with a cross-sectional design in 10 villages in Tembalang Subdistirict, Semarang City. Field strains of Ae. aegypti eggs were collected using ovitraps. The collected eggs were grown under standard condition to adult mosquitoes. Mosquitos’ homogenate were stored at -85C and used for biochemical assays. The results showed there was increased monooxygenases activity in Ae. aegypti populations. Resistance to synthetic pyrethroid insecticide in Ae. aegypti mosquitoes population in Tembalang Subdistrict might be caused by the mechanism of detoxification enzymes in particular monooxygenases

  6. Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

    NARCIS (Netherlands)

    Schallmey, Anett; den Besten, Gijs; Teune, Ite G. P.; Kembaren, Roga F.; Janssen, Dick B.

    2011-01-01

    Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzym

  7. Catalytic and hydrodynamic properties of styrene monooxygenases from Rhocodoccus opacus 1CP are modulated by cofactor binding.

    NARCIS (Netherlands)

    Riedel, A.; Heine, T.; Westphal, A.H.; Conrad, C.; Rathsack, P.; Berkel, van W.J.H.; Tischler, D.

    2015-01-01

    Styrene monooxygenases (SMOs) are flavoenzymes catalyzing the epoxidation of styrene into styrene oxide. SMOs are composed of a monooxygenase (StyA) and a reductase (StyB). The latter delivers reduced FAD to StyA on the expense of NADH. We identified Rhodococcus opacus 1CP as the first microorganism

  8. Non-inductive components of electromagnetic signals associated with L'Aquila earthquake sequences estimated by means of inter-station impulse response functions

    Directory of Open Access Journals (Sweden)

    C. Di Lorenzo

    2011-04-01

    Full Text Available On 6 April 2009 at 01:32:39 UT a strong earthquake occurred west of L'Aquila at the very shallow depth of 9 km. The main shock local magnitude was Ml = 5.8 (Mw = 6.3. Several powerful aftershocks occurred the following days. The epicentre of the main shock occurred 6 km away from the Geomagnetic Observatory of L'Aquila, on a fault 15 km long having a NW-SE strike, about 140°, and a SW dip of about 42°. For this reason, L'Aquila seismic events offered very favourable conditions to detect possible electromagnetic emissions related to the earthquake. The data used in this work come from the permanent geomagnetic Observatories of L'Aquila and Duronia. Here the results concerning the analysis of the residual magnetic field estimated by means of the inter-station impulse response functions in the frequency band from 0.3 Hz to 3 Hz are shown.

  9. Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Paspaliari, Dafni Katerina; Loose, Jennifer S. M.; Larsen, Marianne Halberg

    2015-01-01

    B) and a multi-modular lytic polysaccharide monooxygenase (LmLPMO10). These enzymes have been related to virulence and their role in chitin metabolism is poorly understood. It is thus of interest to functionally characterize the individual enzymes in order to shed light on their roles in vivo. Our results......Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and Chi...

  10. Gravitational induction

    CERN Document Server

    Bini, Donato; Chicone, Carmen; Mashhoon, Bahram

    2008-01-01

    We study the linear post-Newtonian approximation to general relativity known as gravitoelectromagnetism (GEM); in particular, we examine the similarities and differences between GEM and electrodynamics. Notwithstanding some significant differences between them, we find that a special nonstationary metric in GEM can be employed to show {\\it explicitly} that it is possible to introduce gravitational induction within GEM in close analogy with Faraday's law of induction and Lenz's law in electrodynamics. Some of the physical implications of gravitational induction are briefly discussed.

  11. Inductive reasoning.

    Science.gov (United States)

    Hayes, Brett K; Heit, Evan; Swendsen, Haruka

    2010-03-01

    Inductive reasoning entails using existing knowledge or observations to make predictions about novel cases. We review recent findings in research on category-based induction as well as theoretical models of these results, including similarity-based models, connectionist networks, an account based on relevance theory, Bayesian models, and other mathematical models. A number of touchstone empirical phenomena that involve taxonomic similarity are described. We also examine phenomena involving more complex background knowledge about premises and conclusions of inductive arguments and the properties referenced. Earlier models are shown to give a good account of similarity-based phenomena but not knowledge-based phenomena. Recent models that aim to account for both similarity-based and knowledge-based phenomena are reviewed and evaluated. Among the most important new directions in induction research are a focus on induction with uncertain premise categories, the modeling of the relationship between inductive and deductive reasoning, and examination of the neural substrates of induction. A common theme in both the well-established and emerging lines of induction research is the need to develop well-articulated and empirically testable formal models of induction. Copyright © 2010 John Wiley & Sons, Ltd. For further resources related to this article, please visit the WIREs website.

  12. Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis

    DEFF Research Database (Denmark)

    Li, Jing; Kristiansen, Kim A.; Hansen, Bjarne Gram

    2011-01-01

    Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis th...

  13. Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Carlin, DA; Bertolani, SJ; Siegel, JB

    2015-01-01

    Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

  14. Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase.

    Science.gov (United States)

    Carlin, D A; Bertolani, S J; Siegel, J B

    2015-02-11

    Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

  15. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Lo Leggio, Leila; Simmons, Thomas J.; Poulsen, Jens-Christian Navarro

    2015-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here...... substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes....

  16. Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans.

    Science.gov (United States)

    Keller, N P; Watanabe, C M; Kelkar, H S; Adams, T H; Townsend, C A

    2000-01-01

    Sterigmatocystin (ST) and aflatoxin B(1) (AFB(1)) are two polyketide-derived Aspergillus mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by Aspergillus nidulans, is a late intermediate in the AFB(1) pathway of A. parasiticus and A. flavus. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in A. nidulans contains five genes, stcB, stcF, stcL, stcS, and stcW, encoding putative monooxygenase activities. Prior research showed that stcL and stcS mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at stcF, encoding a P-450 monooxygenase similar to A. parasiticus avnA, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies.

  17. Vector Potential, Electromagnetic Induction and "Physical Meaning"

    Science.gov (United States)

    Giuliani, G.

    2010-01-01

    A forgotten experiment by Andre Blondel (1914) proves, as held on the basis of theoretical arguments in a previous paper, that the time variation of the magnetic flux is not the cause of the induced emf; the physical agent is instead the vector potential through the term [equation omitted] (when the induced circuit is at rest). The "good…

  18. Alternative splicing governs sulfation of tyrosine or oligosaccharide on peptidylglycine alpha-amidating monooxygenase.

    Science.gov (United States)

    Yun, H Y; Keutmann, H T; Eipper, B A

    1994-04-08

    Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal alpha-amidation of neuro-endocrine peptides through the sequential action of monooxygenase and lyase domains contained within this bifunctional protein. Alternative splicing leads to the expression of soluble and integral membrane bifunctional PAM proteins as well as a soluble monofunctional monooxygenase. In order to determine how alternative splicing affects post-translational modification of PAM proteins, we investigated the sulfation of PAM proteins expressed in stably transfected hEK-293 cells. Metabolic labeling with [35S]SO4(2-) or [35S]methionine and immunoprecipitation demonstrated that [35S]SO4(2-) was efficiently incorporated into PAM proteins that have the noncatalytic exon A region following the monooxygenase domain (PAM-1 and PAM-4) and into a soluble bifunctional PAM protein (PAM-3). Alkaline hydrolysis, radiosequencing, and deglycosylation experiments demonstrated the presence of a sulfated tyrosine (Tyr965) in the COOH-terminal domain of PAM-3 and multiple sulfated O-glycans in the exon A region of PAM-1 and PAM-4. A mutant PAM-3 protein in which Tyr965 was changed to Ala965 (PAM-3/Y965A) was not sulfated and exhibited monooxygenase and lyase activities similar to those of wild type PAM-3. Pulse-chase and temperature block experiments showed that the PAM-3/Y965A protein exits the trans-Golgi network faster than wild type PAM-3. Thus inclusion of exon A results in the sulfation of O-glycans, while elimination of the transmembrane domain results in the sulfation of Tyr965.

  19. Inductive Learning

    Institute of Scientific and Technical Information of China (English)

    吴信东

    1993-01-01

    Machine learning(ML)is a major subfield of artificial intelligence(AI).It has been seen as a feasible way of avoiding the knowledge bottleneck problem in knowledge-based systems development.Research on ML has concentrated in the main on inductive learning,a paradigm for inducing rules from unordered sets of exmaples.AQ11 and ID3,the two most widespred algorithms in ML,are both inductive.This paper first summarizes AQ11,ID3 and the newly-developed extension matrix approach based HCV algorithm;and then reviews the recent development of inductive learing and automatic knowledge acquisition from data bases.

  20. Practicing induction:

    DEFF Research Database (Denmark)

    Sprogøe, Jonas; Rohde, Nicolas

    2009-01-01

    We claim that induction potentially triggers both individual and organizational learning and by drawing on practice-based theory we discuss how the interplay between individual and organization, what we call a generative dance, ignites both kinds of learning....

  1. Gravitational induction

    OpenAIRE

    Bini, Donato; Cherubini, Christian; Chicone, Carmen; Mashhoon, Bahram

    2008-01-01

    We study the linear post-Newtonian approximation to general relativity known as gravitoelectromagnetism (GEM); in particular, we examine the similarities and differences between GEM and electrodynamics. Notwithstanding some significant differences between them, we find that a special nonstationary metric in GEM can be employed to show {\\it explicitly} that it is possible to introduce gravitational induction within GEM in close analogy with Faraday's law of induction and Lenz's law in electrod...

  2. Effects of methotrexate on rat P-450 cytochrome mono-oxygenases; Action du methotrexate sur les monooxygenases a cytochromes P-450 chez le rat

    Energy Technology Data Exchange (ETDEWEB)

    Guitton, J.; Guilluy, R.; Brazier, J.L. [Faculte de Pharmacie, 69 - Lyon (France); Souillet, G. [Hopital Debrousse, 69 - Lyon (France); Riviere, J.L. [INRA, 69 - Marcy l`Etoile (France); Gerard, F. [Institut Pasteur, 69 - Lyon (France)

    1994-12-31

    Methotrexate, an anti-cancerous agent, acts as an anti-metabolite of the nucleic acids which synthesis is then inhibited. Using aminopyrine breath test after methotrexate processing, the effects of the molecule on activities of the hepatocyte P-450 cytochrome mono-oxygenases, are studied. Breath micro-tests with carbon 13-labelled aminopyrine have been carried out to observe the metabolism evolution. Micro-test results have been compared to microsomal enzymatic activities for various substrates, and also to P-450 cytochrome ratio. Results show that methotrexate induces a reduction in the P-450 cytochrome ratio, and thus reduce the hepatic biotransformation process. 1 fig., 30 refs.

  3. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Zhang, Zhenghong

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-...

  4. Speciation of nickel in airborne particulate matter by means of sequential extraction in a micro flow system and determination by graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Fuichtjohann, L; Jakubowski, N; Gladtke, D; Klocko, D; Broekaert, J A

    2001-12-01

    A four-stage sequential extraction procedure for the speciation of nickel has been applied to ambient aerosol samples. The determination of the soluble, sulfidic, metallic and oxidic Ni fractions in particulate matter was carried out by graphite furnace (electrothermal) atomic absorption spectrometry (ETAAS) and inductively coupled plasma mass spectrometry (ICP-MS). An EDTA solution, a mixture of diammonium citrate and hydrogen peroxide, and a KCuCl3 solution were used as leaching agents for the determination of the soluble, sulfidic and metallic species, respectively, and nitric acid was used for the determination of oxidic compounds after microwave digestion of particulate matter sampled on filters. A new micro scale filter holder placed in a closed flow injection analysis (FIA) system for use in nickel speciation by means of sequential extraction, and the results of the optimisation of the extraction conditions are described. The temperature program for ETAAS was optimised for all extraction solutions with the aid of temperature curves. Pyrolysis temperatures of 900. 600 and 1,000 degrees C were found to be optimum for EDTA, hydrogen peroxide plus ammonium citrate and KCuCl3-containing solutions, respectively. Airborne dust was sampled on lilters at two locations near to a metallurgical plant in Dortmund, Germany. Concentrations in the low ng m(-3) range down to the detections limits (0.1-0.3 ng m(-3)) and various nickel species were found to be present in the collected dust. The mean fractions of total nickel (sampling period of one month) were found to contain 36+20% of soluble, 6 +/- 4% of sulfidic, 11 +/- 15% of metallic and 48 +/- 18% of oxidic nickel.

  5. Rapid Amplification of 5′ cDNA End of S. Liaotungensis Choline Monooxygenase Using Inverse PCR RACE

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Based on part of a known cDNA sequence of Suaeda Liaotungensis choline monooxygenase, the authors successfully cloned the 5′ cDNA end of Suaeda Lianotungensis choline monooxygenase using Inverse PCR RACE with a specially designed 5′-phosphated RT primer and two pairs of specific inverse PCR primers. Compared with the anchored PCR RACE, inverse PCR RACE has better specificity and higher amplification.

  6. Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.

    Science.gov (United States)

    O'Dell, William B; Agarwal, Pratul K; Meilleur, Flora

    2017-01-16

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed "pre-bound" molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme-substrate complex. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Improved homology model of cyclohexanone monooxygenase from Acinetobacter calcoaceticus based on multiple templates.

    Science.gov (United States)

    Bermúdez, Eduardo; Ventura, Oscar N; Eriksson, Leif A; Saenz-Méndez, Patricia

    2014-04-01

    A new homology model of cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus is derived based on multiple templates, and in particular the crystal structure of CHMO from Rhodococcus sp. The derived model was fully evaluated, showing that the quality of the new structure was improved over previous models. Critically, the nicotinamide cofactor is included in the model for the first time. Analysis of several molecular dynamics snapshots of intermediates in the enzymatic mechanism led to a description of key residues for cofactor binding and intermediate stabilization during the reaction, in particular Arg327 and the well known conserved motif (FxGxxxHxxxW) in Baeyer-Villiger monooxygenases, in excellent agreement with known experimental and computational data.

  8. The mechanism of methane and dioxygen activation in the catalytic cycle of methane monooxygenase.

    Science.gov (United States)

    Shteinman, A A

    1995-03-27

    The binuclear structure of the active center of methane monooxygenase plays a determining role in dioxygen activation and in selectivity and specificity of alkane oxidation with this enzyme. A new mechanism is suggested for binding and activation of O2, which involves side-on binding of O2-(2) to iron atoms followed by its conversion to the bis-mu-oxo complex considered as an alternative of ferryl in CH4 activation. This mechanism results in the sequence of the cleavage of the O-O bond of peroxide O/O2-instead of the opposite sequence O2-/O, which takes place in the case of heme monooxygenase cytochrome P-450. Therefore, in this case there is no necessity of the charge relay system [N.B. Gerber and S.G. Sligar, J. Am. Chem. Soc. 114 (1992) 8742] for the transformation of O2 to an active intermediate. The experiment for checking this hypothesis is suggested.

  9. Discovery, application and protein engineering of Baeyer-Villiger monooxygenases for organic synthesis.

    Science.gov (United States)

    Balke, Kathleen; Kadow, Maria; Mallin, Hendrik; Sass, Stefan; Bornscheuer, Uwe T

    2012-08-21

    Baeyer-Villiger monooxygenases (BVMOs) are useful enzymes for organic synthesis as they enable the direct and highly regio- and stereoselective oxidation of ketones to esters or lactones simply with molecular oxygen. This contribution covers novel concepts such as searching in protein sequence databases using distinct motifs to discover new Baeyer-Villiger monooxygenases as well as high-throughput assays to facilitate protein engineering in order to improve BVMOs with respect to substrate range, enantioselectivity, thermostability and other properties. Recent examples for the application of BVMOs in synthetic organic synthesis illustrate the broad potential of these biocatalysts. Furthermore, methods to facilitate the more efficient use of BVMOs in organic synthesis by applying e.g. improved cofactor regeneration, substrate feed and in situ product removal or immobilization are covered in this perspective.

  10. Manipulation of neuropeptide biosynthesis through the expression of antisense RNA for peptidylglycine alpha-amidating monooxygenase.

    Science.gov (United States)

    Mains, R E; Bloomquist, B T; Eipper, B A

    1991-02-01

    Stable cell lines with significantly elevated or diminished levels of a key neuropeptide processing enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), were generated by transfection of a mouse pituitary cell line with expression vectors containing PAM cDNA in the sense or antisense orientation. By evaluating the ability of these cell lines to alpha-amidate endogenous neuropeptides, a rate-limiting role for PAM in neuropeptide alpha-amidation was demonstrated. Overexpression of either the full-length PAM precursor with its trans-membrane domain or a soluble protein containing only the monooxygenase domain of PAM led to increased alpha-amidation of endogenous neuropeptides. Overexpression of the full-length PAM led to an unexpected decrease in the endoproteolytic processing of endogenous prohormone; conversely, underexpression of PAM led to significantly enhanced endoproteolytic processing of endogenous prohormone. These data suggest that PAM may have additional functions in peptide processing.

  11. Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs

    OpenAIRE

    Gilbert, Bettina; McDonald, Ian R.; Finch, Ruth; Stafford, Graham P.; Nielsen, Allan K.; Murrell, J. Colin

    2000-01-01

    The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the α-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, co...

  12. The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.; Beecher, Jean; Donadio, Giuliana; Saneei, Vahid; Dcunha, Stephlina A.; McGhie, Emma J.; Sayer, Christopher; Davenport, Colin F. [University of Exeter, Stocker Road, Exeter EX4 4QD (United Kingdom); Lau, Peter C. [National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC H4P 2R2 (Canada); Hasegawa, Yoshie; Iwaki, Hiroaki [Kansai University (Japan); Kadow, Maria; Balke, Kathleen; Bornscheuer, Uwe T. [Greifswald University, Felix-Hausdorff-Strasse 4, 17487 Greifswald (Germany); Bourenkov, Gleb [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Notkestrasse 85, 22607 Hamburg (Germany); Littlechild, Jennifer A., E-mail: j.a.littlechild@exeter.ac.uk [University of Exeter, Stocker Road, Exeter EX4 4QD (United Kingdom)

    2015-10-31

    The first crystal structure of a type II Baeyer–Villiger monooxygenase reveals a different ring orientation of its FMN cofactor compared with other related bacterial luciferase-family enzymes. The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

  13. Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhi; Rupasinghe, Sanjeewa G.; Schuler, Mary A.; Nair, Satish K. (UIUC)

    2012-02-08

    The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-{angstrom} resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.

  14. Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

    Energy Technology Data Exchange (ETDEWEB)

    Musumeci, Matías; Lozada, Mariana; Rial, Daniela; Mac Cormack, Walter; Jansson, Janet; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe

    2017-04-01

    The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

  15. Induction Brazing

    DEFF Research Database (Denmark)

    Henningsen, Poul

    . The method has proven to give successful results in brazing tube-plate joints of copper-brass, copper-stainless steel, stainless steel-brass, and stainless steel-stainless steel. A new design of an adjustable flux concentrator for induction heating tube-to-plate joints is proposed and tested on a variety......Induction brazing is a fast and appropriate method for industrial joining of complex geometries and metal combinations. In all types of brazing processes it is important to heat the joint interface of the two materials to the same, high temperature. If one of the specimens is warmer than the other...... materials has large influence on the heating time and temperature distribution in induction heating. In order to ensure high and uniform temperature distribution near the interface of a joint between dissimilar materials the precise coil geometry and position is of great importance. The present report...

  16. Sequent calculus proof systems for inductive definitions

    OpenAIRE

    Brotherston, James

    2006-01-01

    Inductive definitions are the most natural means by which to represent many families of structures occurring in mathematics and computer science, and their corresponding induction / recursion principles provide the fundamental proof techniques by which to reason about such families. This thesis studies formal proof systems for inductive definitions, as needed, e.g., for inductive proof support in automated theorem proving tools. The systems are formulated as sequent calculi for...

  17. Induction practice -

    DEFF Research Database (Denmark)

    Rohde, Nicolas; Sprogøe, Jonas

    2007-01-01

    that induction potentially triggers both individual and organizational learning and by drawing on practice-based theory we discuss how the interplay between individual and the organization, what we call agenerative dance, ignites both kinds of learning. We focus on and describe the interplay , ignites both kinds...... of learning. We focus on and describe the interplay that takes place in particular induction events and analyze the "dance" through the lens of learning. The paper concludes with a brief discussion about the implications for practitioners and the challenges and future research prospects we have encountered....... We draw on an empirical enquiry in two organizational settings, a Danish management consulting company and a German retail bank....

  18. mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species.

    Science.gov (United States)

    Brzostowicz, Patricia C; Walters, Dana M; Thomas, Stuart M; Nagarajan, Vasantha; Rouvière, Pierre E

    2003-01-01

    mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

  19. Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes.

    Science.gov (United States)

    Rodriguez, R J; Proteau, P J; Marquez, B L; Hetherington, C L; Buckholz, C J; O'Connell, K L

    1999-08-01

    Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation.

  20. Metabolism of ketoconazole and deacetylated ketoconazole by rat hepatic microsomes and flavin-containing monooxygenases.

    Science.gov (United States)

    Rodriguez, R J; Acosta, D

    1997-06-01

    Ketoconazole (KT) has been reported to cause hepatotoxicity, which is probably not mediated through an immunoallergic mechanism. Although KT is extensively metabolized by hepatic microsomal enzymes, the nature, route of formation, and toxicity of suspected metabolites are largely unknown. Recent reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice, which, like lipophilic 4-alkylpiperazines, is susceptible to successive oxidative attacks on the N-1 position producing ring-opened dialdehydes. The rate of formation of DAK from hepatic rat microsomal incubations of KT was determined by HPLC. The rate of disappearance for KT was almost equal to the rate of DAK formation: 5.96 and 5.88 microM/hr, respectively. Also, the potential bioactivation of DAK was evaluated by measuring substrate activity of DAK with purified pig liver flavin-containing monooxygenase (FMO) and rat liver microsomes. Activity was measured by following DAK-dependent oxygen uptake polarographically at 37 degrees C in pyrophosphate buffer (pH 8.8) containing the glucose-6-phosphate NADPH-generating system. The K(M)'s of DAK were 34.6 and 77.4 microM for the purified FMO and rat microsomal FMO, respectively. Lastly, DAK was found to be metabolized by an NADPH-dependent rat liver microsomal monooxygenases at pH 8.8 to two metabolites as determined by HPLC. Heat inactivation of rat liver microsomal FMO abolished the formation of these metabolites from DAK. SKF-525A and anti-rat NADPH cytochrome P450 reductase did not inhibit this reaction. These results suggest that deacetylation of KT yields a major product, DAK, for further metabolism by microsomal monooxygenases that seem to be FMO-related.

  1. Activation of р-450-depended monooxygenases changing immunotoxicity of phosphoroorganic compounds due to their metabolism character

    Directory of Open Access Journals (Sweden)

    P.F. Zabrodsky

    2010-03-01

    Full Text Available It was established that the application of the monooxygenase system inductors (MSI of phenobarbital and benzonal up to acute poisoning of animals by trichlorfom in a dose of 1,0 LD50, metabolized in the organism till production of compounds with higher toxicity caused its immunotoxic properties increase. The experiment was carried out on outbred white rats. the acute dimethyldichlorvinylphosphate (1,0 LD50 poisoning, biotransformation of which proceeded with formation of less-toxic and non-toxic compounds after MSI introduction, caused its decrease of suppression influence on immunity system indices

  2. Gene cloning and functional analysis of triple alkane monooxygenases from Geobacillus thermoleovorans B23

    OpenAIRE

    2014-01-01

    An extremely thermophilic bacterium, Geobacillus thermoleovorans B23 which was isolated from a deep subterranean oil reservoir at Niigata, Japan, is capable of degrading broad range alkanes (C11-C32) at 70℃ by terminal oxidation pathway, followed by β-oxidation pathway. Whole genome sequence analysis revealed that B23 did not have alkB-type alkane monooxygenases genes like most alkane degrading bacteria but it carried three gene homologs namely ladAαB23, ladAβB23 and ladBB23 on its chromosome...

  3. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

    Science.gov (United States)

    Lo Leggio, Leila; Simmons, Thomas J; Poulsen, Jens-Christian N; Frandsen, Kristian E H; Hemsworth, Glyn R; Stringer, Mary A; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S; De Maria, Leonardo; Harris, Paul V; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

    2015-01-22

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.

  4. Genomic organization and splicing variants of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

    DEFF Research Database (Denmark)

    Williamson, M; Hauser, F; Grimmelikhuijzen, C J

    2000-01-01

    of primitive nervous systems. In mammals, peptide amidation is catalyzed by two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) that act sequentially. These two activities are contained within one bifunctional enzyme...... of exon-8, which was present in the previously characterized PHM cDNA (CP1-A). CP1-A and -B have 97% amino acid sequence identity, whereas both splicing variants have around 42% sequence identity with the PHM part of rat PAM. Essential amino acid residues for the catalytic activity and the 3D structure...

  5. Biosynthesis of psoralens. Psoralen 5-monooxygenase activity from elicitor-treated Ammi majus cells.

    Science.gov (United States)

    Hamerski, D; Matern, U

    1988-11-01

    Microsomes prepared from cultured Ammi majus cells that had been challenged for 14 h with an elicitor derived from the cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) converted psoralen to bergaptol (5-hydroxypsoralen) in the presence of NADPH and oxygen. The enzymatic activity was characterized as an inducible cytochrome-P-450-dependent monooxygenase associated with the endoplasmic reticulum. All of the steps involved in bergapten (5-methoxypsoralen) biosynthesis in Ammi majus have now been demonstrated in vitro. The results suggest that bergaptol and not hydroxymarmesin in the precursor of bergapten.

  6. Interaction of the mechanism-based inactivator acetylene with ammonia monooxygenase of Nitrosomonas europaea.

    Science.gov (United States)

    Gilch, Stefan; Vogel, Manja; Lorenz, Matthias W; Meyer, Ortwin; Schmidt, Ingo

    2009-01-01

    The ammonia monooxygenase (AMO) of Nitrosomonas europaea is a metalloenzyme that catalyses the oxidation of ammonia to hydroxylamine. We have identified histidine 191 of AmoA as the binding site for the oxidized mechanism-based inactivator acetylene. Binding of acetylene changed the molecular mass of His-191 from 155.15 to 197.2 Da (+42.05), providing evidence that acetylene was oxidized to ketene (CH2CO; 42.04 Da) which binds specifically to His-191. It must be assumed that His-191 is part of the acetylene-activating site in AMO or at least directly neighbours this site.

  7. Oxidation of chlorinated olefins by Escherichia coli transformed with dimethyl sulfide monooxygenase genes or cumene dioxygenase genes.

    Science.gov (United States)

    Takami, Wako; Yoshida, Takako; Nojiri, Hideaki; Yamane, Hisakazu; Omori, Toshio

    1999-04-01

    In the present work, it was shown that the dimethyl sulfide (DMS) monooxygenase and the cumene dioxygenase catalyzed oxidation of various chlorinated ethenes, propenes, and butenes. The specific activities of these oxygenases were determined for C(2) to C(4) chlorinated olefins, and the oxidation rates ranged from 0.19 to 4.18 nmol.min(-1).mg(-1) of dry cells by the DMS monooxygenase and from 0.19 to 1.29 nmol.min(-1).mg(-1) of dry cells by the cumene dioxygenase. The oxidation products were identified by gas chromatography-mass spectrometry. Most chlorinated olefins were monooxygenated by the DMS monooxygenase to yield chlorinated epoxides. In the case of the cumene dioxygenase, the substrates lacking any chlorine atom on double-bond carbon atoms were dioxygenated, and those with chlorine atoms attaching to double-bond carbon atoms were monooxygenated to yield allyl alcohols.

  8. Induction Brazing

    DEFF Research Database (Denmark)

    Henningsen, Poul

    , or if the hottest area is located outside the joint interface, a number of defects may appear: the braze metal may flow away from the joint, the flux may burn off, poor binding of the braze metal may appear or the braze metal may be overheated. Joint geometry as well as electro-magnetic properties of the work piece......Induction brazing is a fast and appropriate method for industrial joining of complex geometries and metal combinations. In all types of brazing processes it is important to heat the joint interface of the two materials to the same, high temperature. If one of the specimens is warmer than the other...

  9. Purification and characterization of a Baeyer-Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monoterpene degradation pathways

    NARCIS (Netherlands)

    Werf, van der M.J.

    2000-01-01

    A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMK

  10. Purification and characterization of a Baeyer-Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways

    NARCIS (Netherlands)

    Werf, M.J. van der

    2000-01-01

    A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMK

  11. CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes

    Science.gov (United States)

    Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was fo...

  12. A comparison between vital capacity induction and tidal breathing induction techniques for the induction of anesthesia and compound A production

    Institute of Scientific and Technical Information of China (English)

    LIU Shu-jie; LI Yue; SUN Bo; WANG Chang-song; GONG Yu-lei; ZHOU Yan-mei; LI En-you

    2010-01-01

    Background Vital capacity induction and tidal breathing induction are currently administered for inhalation induction of anesthesia with sevoflurane. The aim of this study was to compare them using sevoflurane with respect to induction time,complications of inhalation induction, and compound A production in adult patients.Methods Fifty-one women with American Society of Anesthesiologists physical status Ⅰ-Ⅱ undergoing mammary gland tumorectomy were randomly assigned to receive either vital capacity induction or tidal breathing induction with 8% sevoflurane at 6 L/min followed by laryngeal mask airway insertion. Induction times, complications of inhalation induction,and vital signs were recorded. Inspired concentrations of compound A were assayed and sofnolime temperatures were monitored at one-minute intervals after sevoflurane administration.Results The time to loss of eyelash reflex was significantly shorter with the vital capacity induction technique than with the tidal breathing induction technique ((43.8±13.4) seconds vs. (70.8±16.4) seconds, respectively; P <0.01).Cardiovascular stability was similar in both groups. The incidence of complications was significantly less with the vital capacity induction technique than with the tidal breathing induction technique (7.7% vs. 32%, respectively; P <0.01).However, the mean and maximum concentrations of compound A during induction were significantly higher in the vital capacity group than those in the tidal breathing group (P <0.05); compound A concentration at the beginning of anesthesia maintenance was (40.73±10.83) ppm in the vital capacity group and (29.45±7.51) ppm in tidal breathing group (P=0.019).Conclusion For inhalation induction of anesthesia, the vital capacity induction was faster and produced fewer complications than that for tidal breathing induction, but increased compound A production in the circuit system.

  13. Investigation of the enzymology and pharmacology of novel substrates and inhibitors of dopamine beta-monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, S.F.

    1987-01-01

    Dopamine beta-monooxygenase (DBM) was shown to catalyze the selenoxidation of 2-(phenylseleno)ethylamines, selenium-containing analogues of dopamine, by the normal monooxygenase pathway. The compounds 2-(phenylseleno)-ethylamine (PAESe), 2-(4'-hydroxyphenylseleno)ethylamine (pOH PAESe), and 1-(phenylseleno)-2-propylamine (Me PAESe) were synthesized and fully characterized as DBM substrates. Two other classes of compounds were investigated as potential alternate substrates for DBM. The possibility of stereoselective sulfonylation of 2-(phenylsulfenyl)- ethylamine (PAESO) was considered. A unique class of compounds, 2-(phenylthio)ethanols were designed and synthesized as DBM substrates but were found to be a novel class of potent competitive inhibitors of DBM with respect to tyramine. Preliminary experiments were also performed in an effort to demonstrate that the potent antihypertensive and indirect-acting sympathomimetic activity of 2-(phenylthio)ethylamine (PAES) was a result of DBM-oxygenation of this compound in vivo. The specific reserpine-sensitive uptake of (/sup 3/H)-norepinephrine into rat brain synaptosomes was demonstrated as was the synaptosomal conversion of (/sup 3/H)-dopamine to (/sup 3/H)-norepinephrine.

  14. Increased monooxygenase activity associated with resistance to permethrin in Pediculus humanus capitis (Anoplura: Pediculidae) from Argentina.

    Science.gov (United States)

    González Audino, P; Barrios, S; Vassena, C; Mougabure Cueto, G; Zerba, E; Picollo, M I

    2005-05-01

    We studied the profile of permethrin resistance in populations of head lice infesting children 6-12 yr old in schools and their homes in and around Buenos Aires, Argentina. Five permethrin-resistant populations with different levels of resistance were collected: Hogar Loyola (HL), Republica de Turquia (RT), Hogar Mitre (HM), Guardia de Honor (GH), and Ricardo Guiraldes (RG). One susceptible population, Bandera Argentina (BA), also was collected. Their level of resistance was evaluated, and results showed resistance ratios of 13 for HL, 16 for RT, 22 for HM, 61 for GH, and 69 for RG. To elucidate the possible involvement of the cytochrome P450 monooxygenase system in conferring permethrin resistance, ethoxycoumarin-O-deethylase (ECOD) activity was measured in abdomens of individual third instars and adults by using a fluorometric assay. The ECOD activity was lower in the susceptible BA population (4.7 ng per louse) than in the resistant ones (13.7 ng per louse for RG, 12.3 ng per louse for GH, 8.6 ng per louse for RT, and 8.2 ng per louse for HL). ECOD activity was significantly correlated with the level of resistance in the field populations (r = 0.97, P = 0.0009), suggesting a role for cytochrome monooxygenase P450 system in permethrin resistance by head louse, Pediculus humanus capitis De Geer.

  15. Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue

    Directory of Open Access Journals (Sweden)

    Hugo L. van Beek

    2014-01-01

    Full Text Available Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO, a Baeyer–Villiger monooxygenase (BVMO from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C–A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.

  16. Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue.

    Science.gov (United States)

    van Beek, Hugo L; Wijma, Hein J; Fromont, Lucie; Janssen, Dick B; Fraaije, Marco W

    2014-01-01

    Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer-Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C-A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.

  17. Lactone-bound structures of cyclohexanone monooxygenase provide insight into the stereochemistry of catalysis.

    Science.gov (United States)

    Yachnin, Brahm J; McEvoy, Michelle B; MacCuish, Roderick J D; Morley, Krista L; Lau, Peter C K; Berghuis, Albert M

    2014-12-19

    The Baeyer-Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO(Tight) and CHMO(Loose) structures. The CHMO(Tight) structure represents the enzyme state in which substrate acceptance and stereospecificity is determined, providing a foundation for engineering BVMOs with altered substrate spectra and/or stereospecificity. The CHMO(Loose) structure is the first structure where the product is solvent accessible. This structure represents the enzyme state upon binding and release of the substrate and product. In addition, the role of the invariant Arg329 in chaperoning the substrate/product during the catalytic cycle is highlighted. Overall, these data provide a structural framework for the engineering of BVMOs with altered substrate spectra and/or stereospecificity.

  18. Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader.

    Science.gov (United States)

    Coleman, Nicholas V; Yau, Sheree; Wilson, Neil L; Nolan, Laura M; Migocki, Margaret D; Ly, Mai-Anh; Crossett, Ben; Holmes, Andrew J

    2011-06-01

    Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.

  19. Biooxidation of n-butane to 1-butanol by engineered P450 monooxygenase under increased pressure.

    Science.gov (United States)

    Nebel, Bernd A; Scheps, Daniel; Honda Malca, Sumire; Nestl, Bettina M; Breuer, Michael; Wagner, Hans-Günter; Breitscheidel, Boris; Kratz, Detlef; Hauer, Bernhard

    2014-12-10

    In addition to the traditional 1-butanol production by hydroformylation of gaseous propene and by fermentation of biomass, the cytochrome P450-catalyzed direct terminal oxidation of n-butane into the primary alcohol 1-butanol constitutes an alternative route to provide the high demand of this basic chemical. Moreover the use of n-butane offers an unexploited ubiquitous feed stock available in large quantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvement of the native redox system, a highly ω-regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12g/L butanol, of which 2.99g/L comprise for 1-butanol, has been obtained after 20h reaction time. Due to the poor solubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase the conversion. After optimization a maximum product content of 4.35g/L 1-butanol from a total amount of 4.53g/L butanol catalyzed by the self-sufficient fusion monooxygenase has been obtained at 15bar pressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced fivefold using the engineered monooxygenase whole cell system by using the high-pressure reaction assembly.

  20. Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro.

    Science.gov (United States)

    Brummund, Jan; Müller, Monika; Schmitges, Thomas; Kaluzna, Iwona; Mink, Daniel; Hilterhaus, Lutz; Liese, Andreas

    2016-09-10

    Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations. In order to regenerate the natural cofactor NADPH, a glucose dehydrogenase was applied. The low water soluble terpene α-ionone was used as substrate for the model reaction system. The studies reveal that an addition of surfactants in combination with low volumetric amounts of co-solvent can significantly increase substrate availability and reaction rates. Furthermore, these additives facilitated a reliable sampling procedure during the process. Another key factor for the process design was the oxygen supply. Based on various investigations, a bubble-aerated stirred tank reactor in batch mode represents a promising reactor concept for P450 oxidations. Main restriction of the investigated reaction system was the low process stability of the P450 monooxygenase, characterized by maximum total turnover numbers of ∼4100molα-ionone/molP450.

  1. Optimization of a Cytochrome-P450-Monooxygenase-1A-Mediated EROD Assay in the Cape Hake Species Merluccius capensis and Merluccius paradoxus (Pisces

    Directory of Open Access Journals (Sweden)

    Louise De Almeida

    2011-01-01

    Full Text Available Cytochrome P450 monooxygenase 1A (CYP1A is induced by several planar toxic compounds, for example, polychlorinated biphenyls (PCBs and the induction of this protein is often measured in terms of CYP1A-mediated 7-ethoxyresorufin-O-deethylase (EROD activity. This study was aimed at developing this assay in the Cape hake species Merluccius capensis and Merluccius paradoxus (considered one stock. Microsomal fractions were obtained from frozen fish liver samples by differential centrifugation. Fluorimetric and spectrophotometric analysis of the EROD assay resulted in the spectrophotometric (at 572 nm detection method being selected, as this method resulted in a lower degree of variability and demonstrated higher reproducibility. The activity in the EROD assay was enhanced in the presence of NADPH, and the addition of dicumarol (phase II enzyme inhibitor to the reaction mixtures prevented the underestimation of this assay by the inhibition of DT-diaphorase. In summary, an EROD assay was established for use in Cape hake species.

  2. PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites

    NARCIS (Netherlands)

    Hendrickx, B; Dejonghe, W; Faber, F; Boenne, W; Bastiaens, L; Verstraete, W; Top, EM; Springael, D

    2006-01-01

    tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel elec

  3. A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

    NARCIS (Netherlands)

    Cankar, K.; van Houwelingen, A.; Bosch, H.J.; Sonke, T.; Bouwmeester, H.; Beekwilder, J.P.

    2011-01-01

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene syntha

  4. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase

    DEFF Research Database (Denmark)

    Pierce, Brian; Wittrup Agger, Jane; Wichmann, Jesper

    2017-01-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety...

  5. 3-Ketosteroid 9 alpha-hydroxylase enzymes : Rieske non-heme monooxygenases essential for bacterial steroid degradation

    NARCIS (Netherlands)

    Petrusma, Mirjan; van der Geize, Robert; Dijkhuizen, Lubbert

    2014-01-01

    Various micro-organisms are able to use sterols/steroids as carbon- and energy sources for growth. 3-Ketosteroid 9 alpha-hydroxylase (KSH), a two component Rieske non-heme monooxygenase comprised of the oxygenase KshA and the reductase KshB, is a key-enzyme in bacterial steroid degradation. It initi

  6. Mammalian peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen

    DEFF Research Database (Denmark)

    Brenet, Fabienne; Dussault, Nadège; Borch, Jonas

    2005-01-01

    Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslat...

  7. Joint Functions of Protein Residues and NADP(H) in Oxygen Activation by Flavin-containing Monooxygenase

    NARCIS (Netherlands)

    Orru, Roberto; Torres Pazmino, Daniel; Fraaije, Marco W.; Mattevi, Andrea

    2010-01-01

    The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H)

  8. 4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB as an oxidative biocatalyst in the synthesis of optically active sulfoxides

    NARCIS (Netherlands)

    Gonzalo, Gonzalo de; Torres Pazmiño, Daniel E.; Ottolina, Gianluca; Fraaije, Marco W.; Carrea, Giacomo

    2006-01-01

    Recombinant 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB has been tested as a catalyst in sulfoxidation reactions on a set of aromatic sulfides. With a few exceptions, excellent enantioselectivities in the synthesis of chiral phenyl and benzyl sulfoxides were achieved

  9. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Science.gov (United States)

    Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of soybean genome sequence allows us to ident...

  10. Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4.

    Science.gov (United States)

    Newman, L M; Wackett, L P

    1995-10-31

    Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2

  11. Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes.

    Science.gov (United States)

    Cafaro, Valeria; Izzo, Viviana; Scognamiglio, Roberta; Notomista, Eugenio; Capasso, Paola; Casbarra, Annarita; Pucci, Piero; Di Donato, Alberto

    2004-04-01

    Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.

  12. Kynurenine 3-monooxygenase polymorphisms: relevance for kynurenic acid synthesis in patients with schizophrenia and healthy controls

    DEFF Research Database (Denmark)

    Holtze, Maria; Saetre, Peter; Engberg, Göran;

    2012-01-01

    on the activity of kynurenine 3-monooxygenase (KMO), the enzyme converting kynurenine to 3-hydroxykynurenine. Methods: We analyzed the association between KMO gene polymorphisms and CSF concentrations of KYNA in patients with schizophrenia and healthy controls. Fifteen single nucleotide polymorphisms (SNPs) were......Background: Patients with schizophrenia show increased brain and cerebrospinal fluid (CSF) concentrations of the endogenous N-methyl-D-aspartate receptor antagonist kynurenic acid (KYNA). This compound is an end-metabolite of the kynurenine pathway, and its formation indirectly depends...... selected covering KMO and were analyzed in UNPHASED. Results: We included 17 patients with schizophrenia and 33 controls in our study. We found an association between a KMO SNP (rs1053230), encoding an amino acid change of potential importance for substrate interaction, and CSF concentrations of KYNA...

  13. Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

    Science.gov (United States)

    Roccatano, Danilo

    2015-07-01

    The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure-dynamics-function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions.

  14. Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase.

    Science.gov (United States)

    Whittington, D A; Rosenzweig, A C; Frederick, C A; Lippard, S J

    2001-03-27

    To investigate the role of protein cavities in facilitating movement of the substrates, methane and dioxygen, in the soluble methane monooxygenase hydroxylase (MMOH), we determined the X-ray structures of MMOH from Methylococcus capsulatus (Bath) cocrystallized with dibromomethane or iodoethane, or by using crystals pressurized with xenon gas. The halogenated alkanes bind in two cavities within the alpha-subunit that extend from one surface of the protein to the buried dinuclear iron active site. Two additional binding sites were located in the beta-subunit. Pressurization of two crystal forms of MMOH with xenon resulted in the identification of six binding sites located exclusively in the alpha-subunit. These results indicate that hydrophobic species bind preferentially in preexisting cavities in MMOH and support the hypothesis that such cavities may play a functional role in sequestering and enhancing the availability of the physiological substrates for reaction at the active site.

  15. Expression and purification of the metal-containing monooxygenases tryptophan hydroxylase and dopamine β-hydroxylase

    DEFF Research Database (Denmark)

    Karlsen, Pernille Efferbach

    to abnormal levels of the neurotransmitters serotonin, dopamine and norepinephrine and the regulation of tryptophan hydroxylase and dopamine β-hydroxylase. These include depression, anxiety disorders, obsessive compulsive disorder (OCD), schizophrenia, Parkinson's disease and attention deficit......-containing enzyme which belongs to the aromatic amino acid hydroxylase (AAAH) family. It exist in two isoforms, TPH1 and TPH2, which are expressed in different tissues and have different properties. TPH is known as a very diffcult protein to work with especially due to instability and only truncated forms of TPH1...... to the family of ascorbate dependent type II Cu monooxygenases. Very little knowledge exist on DβH and most of it comes from investigations of related proteins. Attempts to express human DβH in bacterial systems have been done in the Metalloprotein Chemistry and Engineering Group, but at present no system...

  16. Cytochrome P450 monooxygenases involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium.

    Science.gov (United States)

    Chigu, Nomathemba Loice; Hirosue, Sinji; Nakamura, Chie; Teramoto, Hiroshi; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2010-08-01

    Cytochrome P450 monooxygenases (P450s) involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium were identified by comprehensive screening of both catalytic potentials and transcriptomic profiling. Functional screening of P. chrysosporium P450s (PcCYPs) revealed that 14 PcCYP species catalyze stepwise conversion of anthracene to anthraquinone via intermediate formation of anthrone. Moreover, transcriptomic profiling explored using a complementary DNA microarray system demonstrated that 12 PcCYPs are up-regulated in response to exogenous addition of anthracene. Among the up-regulated PcCYPs, five species showed catalytic activity against anthracene. Based upon both catalytic and transcriptional properties, these five species are most likely to play major roles in anthracene metabolic processes in vivo. Thus, the combination of functional screening and a microarray system may provide a novel strategy for obtaining a thorough understanding of the catalytic functions and biological impacts of PcCYPs.

  17. Lytic polysaccharide monooxygenases: a crystallographer's view on a new class of biomass-degrading enzymes

    Directory of Open Access Journals (Sweden)

    Kristian E. H. Frandsen

    2016-11-01

    Full Text Available Lytic polysaccharide monooxygenases (LPMOs are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future.

  18. FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase.

    Science.gov (United States)

    Tischler, Dirk; Schlömann, Michael; van Berkel, Willem J H; Gassner, George T

    2013-11-29

    StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst.

  19. Flavin-dependent monooxygenases as a detoxification mechanism in insects: new insights from the arctiids (lepidoptera.

    Directory of Open Access Journals (Sweden)

    Sven Sehlmeyer

    Full Text Available Insects experience a wide array of chemical pressures from plant allelochemicals and pesticides and have developed several effective counterstrategies to cope with such toxins. Among these, cytochrome P450 monooxygenases are crucial in plant-insect interactions. Flavin-dependent monooxygenases (FMOs seem not to play a central role in xenobiotic detoxification in insects, in contrast to mammals. However, the previously identified senecionine N-oxygenase of the arctiid moth Tyria jacobaeae (Lepidoptera indicates that FMOs have been recruited during the adaptation of this insect to plants that accumulate toxic pyrrolizidine alkaloids. Identification of related FMO-like sequences of various arctiids and other Lepidoptera and their combination with expressed sequence tag (EST data and sequences emerging from the Bombyx mori genome project show that FMOs in Lepidoptera form a gene family with three members (FMO1 to FMO3. Phylogenetic analyses suggest that FMO3 is only distantly related to lepidopteran FMO1 and FMO2 that originated from a more recent gene duplication event. Within the FMO1 gene cluster, an additional gene duplication early in the arctiid lineage provided the basis for the evolution of the highly specific biochemical, physiological, and behavioral adaptations of these butterflies to pyrrolizidine-alkaloid-producing plants. The genes encoding pyrrolizidine-alkaloid-N-oxygenizing enzymes (PNOs are transcribed in the fat body and the head of the larvae. An N-terminal signal peptide mediates the transport of the soluble proteins into the hemolymph where PNOs efficiently convert pro-toxic pyrrolizidine alkaloids into their non-toxic N-oxide derivatives. Heterologous expression of a PNO of the generalist arctiid Grammia geneura produced an N-oxygenizing enzyme that shows noticeably expanded substrate specificity compared with the related enzyme of the specialist Tyria jacobaeae. The data about the evolution of FMOs within lepidopteran insects

  20. Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes.

    Science.gov (United States)

    Hrycay, Eugene G; Bandiera, Stelvio M

    2015-01-01

    This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex.

  1. Homology modeling and protein engineering of alkane monooxygenase in Burkholderia thailandensis MSMB121: in silico insights.

    Science.gov (United States)

    Jain, Chakresh Kumar; Gupta, Money; Prasad, Yamuna; Wadhwa, Gulshan; Sharma, Sanjeev Kumar

    2014-07-01

    The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans, Burkhulderia, Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)-a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans. Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305-370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism.

  2. Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase.

    Science.gov (United States)

    Bailey, Lucas J; Elsen, Nathaniel L; Pierce, Brad S; Fox, Brian G

    2008-01-01

    Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded approximately 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed approximately 1 mol of the S=1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K(M-) and apparent k(cat)-values of 0.15 microM and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.

  3. Role for threonine 201 in the catalytic cycle of the soluble diiron hydroxylase toluene 4-monooxygenase.

    Science.gov (United States)

    Elsen, Nathaniel L; Bailey, Lucas J; Hauser, Andrew D; Fox, Brian G

    2009-05-12

    The active site residue Thr-201 in toluene 4-monooxygenase hydroxylase (T4moH) has a structural counterpart in the active sites of all diiron monooxygenases. Thus, our previous finding that mutation of this residue to Ala, Gly, or Ser had no impact on steady-state catalysis or coupling was surprising. In this work, we provide kinetic, biochemical, and structural evidence that one role of Thr-201 may be to stabilize a peroxo-level intermediate during enzyme catalysis. During reactions in the absence of substrate, T201 T4moH slowly consumed O(2) but only a negligible amount of H(2)O(2) was released. In contrast, T201A T4moH gave stoichometric release of H(2)O(2) during reaction in the absence of substrate. Both enzyme isoforms were tightly coupled during steady-state catalysis with saturating toluene and other optimal substrates and exhibited near-identical kinetic parameters. However, rapid mix single-turnover studies showed that T201A T4moH had a faster first-order rate constant for product formation than T201 T4moH did. Comparison of X-ray crystal structures of resting and reduced T201A T4moH in complex with T4moD with comparable structures of T201 T4moHD revealed changes in the positions of several key active site residues relative to the comparable structures of T201 T4moH with T4moD. This combination of catalytic and structural studies offers important new insight into the role of the role of conserved Thr-201, and its contributions to the catalytic reaction cycle.

  4. Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem.

    Science.gov (United States)

    Minerdi, Daniela; Zgrablic, Ivan; Castrignanò, Silvia; Catucci, Gianluca; Medana, Claudio; Terlizzi, Maria Elena; Gribaudo, Giorgio; Gilardi, Gianfranco; Sadeghi, Sheila J

    2015-10-12

    Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance.

  5. The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants.

    Directory of Open Access Journals (Sweden)

    Giuliana Donadio

    Full Text Available Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.

  6. The biochemical mechanism of auxin biosynthesis by an arabidopsis YUCCA flavin-containing monooxygenase.

    Science.gov (United States)

    Dai, Xinhua; Mashiguchi, Kiyoshi; Chen, Qingguo; Kasahara, Hiroyuki; Kamiya, Yuji; Ojha, Sunil; DuBois, Jennifer; Ballou, David; Zhao, Yunde

    2013-01-18

    Auxin regulates every aspect of plant growth and development. Previous genetic studies demonstrated that YUCCA (YUC) flavin-containing monooxygenases (FMOs) catalyze a rate-limiting step in auxin biosynthesis and that YUCs are essential for many developmental processes. We proposed that YUCs convert indole-3-pyruvate (IPA) to indole-3-acetate (IAA). However, the exact biochemical mechanism of YUCs has remained elusive. Here we present the biochemical characterization of recombinant Arabidopsis YUC6. Expressed in and purified from Escherichia coli, YUC6 contains FAD as a cofactor, which has peaks at 448 nm and 376 nm in the UV-visible spectrum. We show that YUC6 uses NADPH and oxygen to convert IPA to IAA. The first step of the YUC6-catalyzed reaction is the reduction of the FAD cofactor to FADH(-) by NADPH. Subsequently, FADH(-) reacts with oxygen to form a flavin-C4a-(hydro)peroxy intermediate, which we show has a maximum absorbance at 381 nm in its UV-visible spectrum. The final chemical step is the reaction of the C4a-intermediate with IPA to produce IAA. Although the sequences of the YUC enzymes are related to those of the mammalian FMOs, which oxygenate nucleophilic substrates, YUC6 oxygenates an electrophilic substrate (IPA). Nevertheless, both classes of enzymes form quasi-stable C4a-(hydro)peroxyl FAD intermediates. The YUC6 intermediate has a half-life of ∼20 s whereas that of some FMOs is >30 min. This work reveals the catalytic mechanism of the first known plant flavin monooxygenase and provides a foundation for further investigating how YUC activities are regulated in plants.

  7. Laser flash induced electron transfer in P450cam monooxygenase: putidaredoxin reductase-putidaredoxin interaction.

    Science.gov (United States)

    Sevrioukova, I F; Hazzard, J T; Tollin, G; Poulos, T L

    2001-09-04

    The P450cam monooxygenase from Pseudomonas putida consists of three redox proteins: NADH-putidaredoxin reductase (Pdr), putidaredoxin (Pdx), and cytochrome P450cam. The redox properties of the FAD-containing Pdr and the mechanism of Pdr-Pdx complex formation are the least studied aspects of this system. We have utilized laser flash photolysis techniques to produce the one-electron-reduced species of Pdr, to characterize its spectral and electron-transferring properties, and to investigate the mechanism of its interaction with Pdx. Upon flash-induced reduction by 5-deazariboflavin semiquinone, the flavoprotein forms a blue neutral FAD semiquinone (FADH(*)). The FAD semiquinone was unstable and partially disproportionated into fully oxidized and fully reduced flavin. The rate of FADH(*) decay was dependent on ionic strength and NAD(+). In the mixture of Pdr and Pdx, where the flavoprotein was present in excess, electron transfer (ET) from FADH(*) to the iron-sulfur cluster was observed. The Pdr-to-Pdx ET rates were maximal at an ionic strength of 0.35 where a kinetic dissociation constant (K(d)) for the transient Pdr-Pdx complex and a limiting k(obs) value were equal to 5 microM and 226 s(-1), respectively. This indicates that FADH(*) is a kinetically significant intermediate in the turnover of P450cam monooxygenase. Transient kinetics as a function of ionic strength suggest that, in contrast to the Pdx-P450cam redox couple where complex formation is predominantly electrostatic, the Pdx-Pdr association is driven by nonelectrostatic interactions.

  8. Callus induction and plant regeneration of Oncidium‘Gower Ramsey’ by means of protocorm-like body culture%文心兰类原球茎的愈伤组织诱导及其植株再生

    Institute of Scientific and Technical Information of China (English)

    黄霞; 卢禹

    2016-01-01

    该研究首次以文心兰的类原球茎( protocorm-like bodies, PLBs)为外植体进行愈伤组织诱导及其植株再生培养,并分析了不同浓度的TDZ和2,4-D配比对愈伤组织增殖的影响。结果表明:以1/2MS为基本培养基,添加1 mg.L-1 TDZ与3 mg.L-12,4-D,从接种的 PLBs上可以诱导出乳白色的、较疏松的愈伤组织,诱导频率达到100%。愈伤组织继代培养时,在2,4-D浓度为0.5~2.0 mg.L-1的范围内,其增殖主要受TDZ浓度的影响,TDZ浓度从1.0 mg.L-1降低到0.5 mg.L-1,愈伤组织鲜重增殖倍数显著增加,由最低的4.50倍增加到最高的6.04倍。愈伤组织增殖的最适培养基为1/2MS +0.5 mg.L-1 TDZ +1.0 mg.L-12,4-D。将在最适愈伤组织增殖培养基上继代培养约1个月的愈伤组织转移到T2培养基(3.5 g.L-1花宝1号+20 g.L-1红薯+25 g.L-1香蕉+1 g.L-1 tryptone +20 g.L-1蔗糖+3.5 g.L-1 phytagel)上,黑暗培养1个月后,每克鲜重的愈伤组织约诱导出1328.67个PLBs。将诱导出的PLBs转移到新鲜的T2培养基上光照培养1个月,萌发率为90.12%。而将小植株转移到添加1 g.L-1活性炭的1/2MS培养基上,成苗率达到100%。该研究结果成功建立了文心兰的高频愈伤组织诱导及其植株再生体系,为文心兰基因工程育种提供了一个高效、稳定的转化受体系统。%Oncidium ‘Gower Ramsey ’ is a species of orchid with high ornamental value and strong market competitiveness. Gene engineering breeding technology provides an effective way for the genetic improvement of ornamental plants. And callus is an ideal transformation receptor. Up to now, callus induction and plant regeneration of Onciudium have been successfully achieved using stem segment, root tip and shoot tip. But there are some shortcomings for these in vitro re-generation systems, such as low frequency and long period of callus induction, difficulty in maintaining embryogenic state of callus, etc. In this research, for

  9. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

    NARCIS (Netherlands)

    Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2014-01-01

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

  10. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase

    NARCIS (Netherlands)

    Loose, Jennifer S. M.; Forsberg, Zarah; Fraaije, Marco W.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2014-01-01

    The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass

  11. Investigation of etching and deposition processes of Cl{sub 2}/O{sub 2}/Ar inductively coupled plasmas on silicon by means of plasma-surface simulations and experiments

    Energy Technology Data Exchange (ETDEWEB)

    Tinck, S; Bogaerts, A [Research Group PLASMANT, Department of Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp (Belgium); Boullart, W, E-mail: stefan.tinck@ua.ac.b [IMEC, Kapeldreef 75, B-3001 Leuven (Belgium)

    2009-05-07

    In this paper, a simulation method is described to predict the etching behaviour of Cl{sub 2}/O{sub 2}/Ar inductively coupled plasmas on a Si substrate, as used in shallow trench isolation for the production of electronic devices. The hybrid plasma equipment model (HPEM) developed by Kushner et al is applied to calculate the plasma characteristics in the reactor chamber and two additional Monte Carlo simulations are performed to predict the fluxes, angles and energy of the plasma species bombarding the Si substrate, as well as the resulting surface processes such as etching and deposition. The simulations are performed for a wide variety of operating conditions such as gas composition, chamber pressure, power deposition and substrate bias. It is predicted by the simulations that when the fraction of oxygen in the gas mixture is too high, the oxidation of the Si substrate is superior to the etching of Si by chlorine species, resulting in an etch rate close to zero as is also observed in the experiments.

  12. A putative flavin-containing mono-oxygenase as a marker for certain defense and cell death pathways

    DEFF Research Database (Denmark)

    Olszak, Brian; Malinovsky, Frederikke Gro; Brodersen, Peter

    2006-01-01

    mRNA of the putative Arabidopsis flavin mono-oxygenase encoded by At1g19250 (here designated AtFMO GenBank accession no. NM_101783 ) accumulated to high levels in the acd11 and lsd1 mutants that exhibit run-away cell death, but not in mutants with constitutive defense responses. AtFMO mRNA accumu......mRNA of the putative Arabidopsis flavin mono-oxygenase encoded by At1g19250 (here designated AtFMO GenBank accession no. NM_101783 ) accumulated to high levels in the acd11 and lsd1 mutants that exhibit run-away cell death, but not in mutants with constitutive defense responses. AtFMO m...

  13. Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E. coli.

    Science.gov (United States)

    Kadow, Maria; Balke, Kathleen; Willetts, Andrew; Bornscheuer, Uwe T; Bäckvall, J-E

    2014-05-01

    The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.

  14. The p450 monooxygenase BcABA1 is essential for abscisic acid biosynthesis in Botrytis cinerea

    DEFF Research Database (Denmark)

    Siewers, V.; Smedsgaard, Jørn; Tudzynski, P.

    2004-01-01

    The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids...... but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this "direct" pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced...... the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential...

  15. Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1

    OpenAIRE

    Koh, Sung-Cheol; Bowman, John P.; Sayler, Gary S.

    1993-01-01

    A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specif...

  16. Influence of recipient gender on intrasplenic fetal liver tissue transplants in rats: cytochrome P450-mediated monooxygenase functions.

    Science.gov (United States)

    Lupp, Amelie; Hugenschmidt, Sabine; Rost, Michael; Müller, Dieter

    2004-05-01

    Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern with consecutive differences in P450-mediated monooxygenase activities, which have been shown to be due to a differential profile of growth hormone (GH) secretion. Parallel to previous investigations on P450 isoforms expression, the aim of the present study was to elucidate the influence of recipient gender on P450-mediated monooxygenase activities in intrasplenic liver tissue transplants in comparison to orthotopic liver. Fetal liver tissue suspensions of mixed gender were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting transplant-recipients and age-matched controls were treated with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the vehicles and sacrificed 24 or 48 h thereafter. P450-dependent monooxygenase activities were assessed by a series of model reactions for different P450 subtypes in liver and spleen 9000 g supernatants. In spleens of male and female control rats only very low monooxygenase activities were detectable, whereas with most model reactions distinct activities were observed in transplant-containing organs. Livers and transplant-containing spleens from male rats displayed higher basal ethoxycoumarin O-deethylase and testosterone 2alpha-, 2beta-, 6beta-, 14alpha-, 15alpha-, 15beta-, 16alpha-, 16beta- and 17-hydroxylase activities than those from females. On the other hand, like the respective livers, spleens from female transplant-recipients demonstrated more pronounced p-nitrophenol- and testosterone 6alpha- and 7alpha-hydroxylase activities than those from male hosts. With nearly all model reactions gender-specific differences in inducibility by BNF, PB or DEX could be demonstrated in livers as well as in transplant-containing spleens. These results further confirm that the P450 system of intrasplenic liver tissue transplants and the respective orthotopic livers is similarly influenced

  17. Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi

    OpenAIRE

    Khajamohiddin Syed; Karabo Shale; Nataraj Sekhar Pagadala; Jack Tuszynski

    2014-01-01

    Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin's theory of natural selection to id...

  18. Copper-Dioxygen Complex Mediated C-H Bond Oxygenation: Relevance for Particulate Methane Monooxygenase (pMMO)

    OpenAIRE

    Himes, Richard A.; Karlin, Kenneth D.

    2009-01-01

    Particulate methane monooxygenase (pMMO), an integral membrane protein found in methanotrophic bacteria, catalyzes the oxidation of methane to methanol. Expression and greater activity of the enzyme in the presence of copper ion suggest that pMMO is a cuprous metalloenzyme. Recent advances – especially the first crystal structures of pMMO – have energized the field, but the nature of the active site(s) and the mechanism of methane oxidation remain poorly understood – yet hotly contested. Here...

  19. Role of hepatic monooxygenases in generating estrogenic metabolites from methoxychlor and from its identified contaminants.

    Science.gov (United States)

    Bulger, W H; Feil, V J; Kupfer, D

    1985-01-01

    Previous investigations demonstrated that methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] contains estrogenic contaminants and that methoxychlor per se is not an estrogen but is a proestrogen being metabolized in vivo into estrogenic products. The present study examined structurally identified methoxychlor contaminants as to their estrogenic or proestrogenic properties. Also, the estrogenic activity of demethylated metabolites of methoxychlor and of one contaminant was determined. To examine these properties, we utilized an assay developed by us that monitors whether a given compound, incubated with isolated rat uteri, can diminish the uterine cytosolic estrogen receptor and elevate the nuclear estrogen receptor and whether metabolic intervention by hepatic microsomal monooxygenase(s) is required by the respective compound for this cellular redistribution of the receptor. Of the 15 compounds examined which constitute with methoxychlor 99.5% of total technical grade methoxychlor, two compounds, 1,1-dichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethene (mono-OH-MDDE) and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-methoxychlor), were active per se and two compounds, 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethene (MDDE) and methoxychlor, required metabolic transformation for estrogenic activity to be manifested. Subsequently, it was shown that the mono- and bis-OH metabolites of MDDE and of methoxychlor were active estrogens and that the order of activity, either by the above procedure or in terms of relative binding affinity to rat uterine cytosolic receptor, was as follows: bis-OH-MDDE much greater than bis-OH-methoxychlor greater than mono-OH-MDDE greater than mono-OH-methoxychlor. Following the in vitro observations, the activity of MDDE and bis-OH-MDDE was determined in vivo in immature rats. It appears that both compounds are estrogenic, yielding marked elevation in ornithine decarboxylase (EC 4.1.1.17) levels and moderate

  20. Molecular determinants of the regioselectivity of toluene/o-xylene monooxygenase from Pseudomonas sp. strain OX1.

    Science.gov (United States)

    Notomista, Eugenio; Cafaro, Valeria; Bozza, Giuseppe; Di Donato, Alberto

    2009-02-01

    Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori.

  1. Lexicographic Path Induction

    DEFF Research Database (Denmark)

    Schürmann, Carsten; Sarnat, Jeffrey

    2009-01-01

    Programming languages theory is full of problems that reduce to proving the consistency of a logic, such as the normalization of typed lambda-calculi, the decidability of equality in type theory, equivalence testing of traces in security, etc. Although the principle of transfinite induction...... an induction principle that combines the comfort of structural induction with the expressive strength of transfinite induction. Using lexicographic path induction, we give a consistency proof of Martin-Löf’s intuitionistic theory of inductive definitions. The consistency of Heyting arithmetic follows directly...

  2. Molecular phylogeny, long-term evolution, and functional divergence of flavin-containing monooxygenases.

    Science.gov (United States)

    Hao, Da Cheng; Chen, Shi Lin; Mu, Jun; Xiao, Pei Gen

    2009-11-01

    Flavin-containing monooxygenases (FMOs) metabolize xenobiotic compounds, many of which are clinically important, as well as endogenous substrates as part of a discrete physiological process. The FMO gene family is conserved and ancient with representatives present in all phyla so far examined. However, there is a lack of information regarding the long-term evolution and functional divergence of these proteins. This study represents the first attempt to characterize the long-term evolution followed by the members of this family. Our analysis shows that there is extensive silent divergence at the nucleotide level suggesting that this family has been subject to strong purifying selection at the protein level. Invertebrate FMOs have a polyphyletic origin. The functional divergence of FMOs 1-5 started before the split between amphibians and mammals. The vertebrate FMO5 is more ancestral than other four FMOs. Moreover, the existence of higher levels of codon bias was detected at the N-terminal ends, which can be ascribed to the critical role played by the FAD binding motif in this region. Finally, critical amino acid residues for FMO functional divergence (type I & II) after gene duplication were detected and characterized.

  3. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

    Science.gov (United States)

    Kittl, Roman; Kracher, Daniel; Burgstaller, Daniel; Haltrich, Dietmar; Ludwig, Roland

    2012-10-26

    Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.

  4. Evolutionary recruitment of a flavin-dependent monooxygenase for stabilization of sequestered pyrrolizidine alkaloids in arctiids.

    Science.gov (United States)

    Langel, Dorothee; Ober, Dietrich

    2011-09-01

    Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants.

  5. The TMAO-Generating Enzyme Flavin Monooxygenase 3 Is a Central Regulator of Cholesterol Balance

    Directory of Open Access Journals (Sweden)

    Manya Warrier

    2015-01-01

    Full Text Available Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO have recently been linked to cardiovascular disease (CVD risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3 as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

  6. Kynurenine–3–monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis

    Science.gov (United States)

    Mole, Damian J; Webster, Scott P; Uings, Iain; Zheng, Xiaozhong; Binnie, Margaret; Wilson, Kris; Hutchinson, Jonathan P; Mirguet, Olivier; Walker, Ann; Beaufils, Benjamin; Ancellin, Nicolas; Trottet, Lionel; Bénéton, Véronique; Mowat, Christopher G; Wilkinson, Martin; Rowland, Paul; Haslam, Carl; McBride, Andrew; Homer, Natalie ZM; Baily, James E; Sharp, Matthew GF; Garden, O James; Hughes, Jeremy; Howie, Sarah EM; Holmes, Duncan S; Liddle, John; Iredale, John P

    2015-01-01

    Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death1,2 Acute mortality from AP-MODS exceeds 20%3 and for those who survive the initial episode, their lifespan is typically shorter than the general population4. There are no specific therapies available that protect individuals against AP-MODS. Here, we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism5, is central to the pathogenesis of AP-MODS. We created a mouse strain deficient for Kmo with a robust biochemical phenotype that protected against extrapancreatic tissue injury to lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in levels of kynurenine pathway metabolites in vivo and afforded therapeutic protection against AP-MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS and open up a new area for drug discovery in critical illness. PMID:26752518

  7. Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases.

    Science.gov (United States)

    Johnson, Reed M; Wen, Zhimou; Schuler, Mary A; Berenbaum, May R

    2006-08-01

    Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.

  8. Switching the Regioselectivity of a Cyclohexanone Monooxygenase toward (+)-trans-Dihydrocarvone by Rational Protein Design.

    Science.gov (United States)

    Balke, Kathleen; Schmidt, Sandy; Genz, Maika; Bornscheuer, Uwe T

    2016-01-15

    The regioselectivity of the Baeyer-Villiger monooxygenase-catalyzed oxidation is governed mostly by electronic effects leading to the migration of the higher substituted residue. However, in some cases, substrate binding occurs in a way that the less substituted residue lies in an antiperiplanar orientation to the peroxy bond in the Criegee intermediate yielding in the formation of the "abnormal" lactone product. We are the first to demonstrate a complete switch in the regioselectivity of the BVMO from Arthrobacter sp. (CHMOArthro) as exemplified for (+)-trans-dihydrocarvone by redesigning the active site of the enzyme. In the designed triple mutant, the substrate binds in an inverted orientation leading to a ratio of 99:1 in favor of the normal lactone instead of exclusive formation of the abnormal lactone in case of the wild type enzyme. In order to validate our computational study, the beneficial mutations were successfully transferred to the CHMO from Acinetobacter sp. (CHMOAcineto), again yielding in a complete switch of regioselectivity.

  9. Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor.

    Science.gov (United States)

    Mirza, I Ahmad; Yachnin, Brahm J; Wang, Shaozhao; Grosse, Stephan; Bergeron, Hélène; Imura, Akihiro; Iwaki, Hiroaki; Hasegawa, Yoshie; Lau, Peter C K; Berghuis, Albert M

    2009-07-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  10. Aerobic degradation of tetrachloroethylene by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1.

    Science.gov (United States)

    Ryoo, D; Shim, H; Canada, K; Barbieri, P; Wood, T K

    2000-07-01

    Tetrachloroethylene (PCE) is thought to have no natural source, so it is one of the most difficult contaminants to degrade biologically. This common groundwater pollutant was thought completely nonbiodegradable in the presence of oxygen. Here we report that the wastewater bacterium Pseudomonas stutzeri OX1 degrades aerobically 0. 56 micromol of 2.0 micromol PCE in 21 h (Vmax approximately 2.5 nmol min(-1) mg(-1) protein and KM approximately 34 microM). These results were corroborated by the generation of 0.48 micromol of the degradation product, chloride ions. This degradation was confirmed to be a result of expression of toluene-o-xylene monooxygenase (ToMO) by P. stutzeri OX1, since cloning and expressing this enzyme in Escherichia coli led to the aerobic degradation of 0.19 micromol of 2.0 micromol PCE and the generation of stoichiometric amounts of chloride. In addition, PCE induces formation of ToMO, which leads to its own degradation in P. stutzeri OX1. Degradation intermediates reduce the growth rate of this strain by 27%.

  11. Phenol and 2-naphthol production by toluene 4-monooxygenases using an aqueous/dioctyl phthalate system.

    Science.gov (United States)

    Tao, Ying; Bentley, William E; Wood, Thomas K

    2005-09-01

    A two-phase system is developed here for converting: (1) benzene to phenol and (2) naphthalene to 2-naphthol, using whole cells expressing wild-type toluene 4-monooxygenase (T4MO) and the alpha subunit variant TmoA I100A from Pseudomonas mendocina KR1. Using the T4MO TmoA I100A variant, the solubility of naphthalene was enhanced and the toxicity of the naphthols was prevented by the use of a water/dioctyl phthalate (80:20, vol%) system which yielded 21-fold more 2-naphthol. More than 99% 2-naphthol was extracted to the dioctyl phthalate phase, dihydroxynaphthalene formation was prevented, 92% 2-naphthol was formed, and 12% naphthalene was converted. Similarly, using 50 vol% dioctyl phthalate, an initial concentration of 3.0 g l(-1) (39 mM), and wild-type T4MO, a 51+/-9% conversion of benzene was obtained and phenol was produced at a purity of 97%. Relative to the one-phase system, there was a 12-fold reduction in the formation of the byproduct catechol.

  12. Suppressed expression of choline monooxygenase in sugar beet on the accumulation of glycine betaine.

    Science.gov (United States)

    Yamada, Nana; Takahashi, Hiroyuki; Kitou, Kunihide; Sahashi, Kosuke; Tamagake, Hideto; Tanaka, Yoshito; Takabe, Teruhiro

    2015-11-01

    Glycine betaine (GB) is an important osmoprotectant and synthesized by two-step oxidation of choline. Choline monooxygenase (CMO) catalyzes the first step of the pathway and is believed to be a rate limiting step for GB synthesis. Recent studies have shown the importance of choline-precursor supply for GB synthesis. In order to investigate the role of CMO for GB accumulation in sugar beet (Beta vulgaris), transgenic plants carrying the antisense BvCMO gene were developed. The antisense BvCMO plants showed the decreased activity of GB synthesis from choline compared to wild-type (WT) plants which is well related to the suppressed level of BvCMO protein. However, GB contents were similar between transgenic and WT plants with the exception of young leaves and storage roots. Transgenic plants showed enhanced susceptibility to salt stress than WT plants. These results suggest the importance of choline-precursor-supply for GB accumulation, and young leaves and storage root are sensitive sites for GB accumulation.

  13. Identification of selectivity determinants in CYP monooxygenases by modelling and systematic analysis of sequence and structure.

    Science.gov (United States)

    Seifert, Alexander; Pleiss, Jurgen

    2012-02-01

    Cytochrome P450 monooxygenases (CYPs) form a large, ubiquitous enzyme family and are of great interest in red and white biotechnology. To investigate the effect of protein structure on selectivity, the binding of substrate molecules near to the active site was modelled by molecular dynamics simulations. From a comprehensive and systematic comparison of more than 6300 CYP sequences and 31 structures using the Cytochrome P450 Engineering Database (CYPED), residues were identified which are predicted to point close to the heme centre and thus restrict accessibility for substrates. As a result, sequence-structure-function relationships are described that can be used to predict selectivity-determining positions from CYP sequences and structures. Based on this analysis, a minimal library consisting of bacterial CYP102A1 (P450(BM3)) and 24 variants was constructed. All variants were functionally expressed in E. coli, and the library was screened with four terpene substrates. Only 3 variants showed no activity towards all 4 terpenes, while 11 variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type. The minimal library also contains variants that show interesting side products which are not generated by the wild type enzyme. By two additional rounds of molecular modelling, diversification, and screening, the selectivity of one of these variants for a new product was optimised with a minimal screening effort. We propose this as a generic approach for other CYP substrates.

  14. Squalene mono-oxygenase, a key enzyme in cholesterol synthesis, is stabilized by unsaturated fatty acids.

    Science.gov (United States)

    Stevenson, Julian; Luu, Winnie; Kristiana, Ika; Brown, Andrew J

    2014-08-01

    SM (squalene mono-oxygenase) catalyses the first oxygenation step in cholesterol synthesis, immediately before the formation of the steroid backbone at lanosterol. SM is an important control point in the pathway, and is regulated at the post-translational level by accelerated cholesterol-dependent ubiquitination and proteasomal degradation, which is associated with the accumulation of squalene. Using model cell systems, we report that SM is stabilized by unsaturated fatty acids. Treatment with unsaturated fatty acids such as oleate, but not saturated fatty acids, increased protein levels of SM or SM-N100-GFP (the first 100 amino acids of SM fused to GFP) at the post-translational level and partially overcame cholesterol-dependent degradation, as well as reversing cholesterol-dependent squalene accumulation. Maximum stabilization required activation of fatty acids, but not triacylglycerol or phosphatidylcholine synthesis. The mechanism of oleate-mediated stabilization appeared to occur through reduced ubiquitination by the E3 ubiquitin ligase MARCH6. Stabilization of a cholesterol biosynthetic enzyme by unsaturated fatty acids may help maintain a constant cholesterol/phospholipid ratio.

  15. Oxygenation of Organoboronic Acids by a Nonheme Iron(II) Complex: Mimicking Boronic Acid Monooxygenase Activity.

    Science.gov (United States)

    Chatterjee, Sayanti; Paine, Tapan Kanti

    2015-10-19

    Phenolic compounds are important intermediates in the bacterial biodegradation of aromatic compounds in the soil. An Arthrobacter sp. strain has been shown to exhibit boronic acid monooxygenase activity through the conversion of different substituted phenylboronic acids to the corresponding phenols using dioxygen. While a number of methods have been reported to cleave the C-B bonds of organoboronic acids, there is no report on biomimetic iron complex exhibiting this activity using dioxygen as the oxidant. In that direction, we have investigated the reactivity of a nucleophilic iron-oxygen oxidant, generated upon oxidative decarboxylation of an iron(II)-benzilate complex [(Tp(Ph2))Fe(II)(benzilate)] (Tp(Ph2) = hydrotris(3,5-diphenyl-pyrazol-1-yl)borate), toward organoboronic acids. The oxidant converts different aryl/alkylboronic acids to the corresponding oxygenated products with the incorporation of one oxygen atom from dioxygen. This method represents an efficient protocol for the oxygenation of boronic acids with dioxygen as the terminal oxidant.

  16. C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress

    Directory of Open Access Journals (Sweden)

    Nisha Hirani

    2016-05-01

    Full Text Available Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation.

  17. Localization of integral membrane peptidylglycine alpha-amidating monooxygenase in neuroendocrine cells.

    Science.gov (United States)

    Milgram, S L; Kho, S T; Martin, G V; Mains, R E; Eipper, B A

    1997-03-01

    Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.

  18. Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase.

    Science.gov (United States)

    Alam, M R; Steveson, T C; Johnson, R C; Bäck, N; Abraham, B; Mains, R E; Eipper, B A

    2001-03-01

    The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.

  19. Signaling Mediated by the Cytosolic Domain of Peptidylglycine α-Amidating Monooxygenase

    Science.gov (United States)

    Alam, M. Rashidul; Steveson, Tami C.; Johnson, Richard C.; Bäck, Nils; Abraham, Benjamin; Mains, Richard E.; Eipper, Betty A.

    2001-01-01

    The luminal domains of membrane peptidylglycine α-amidating monooxygenase (PAM) are essential for peptide α-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion. PMID:11251076

  20. Menkes protein contributes to the function of peptidylglycine alpha-amidating monooxygenase.

    Science.gov (United States)

    Steveson, Tami C; Ciccotosto, Giuseppe D; Ma, Xin-Ming; Mueller, Gregory P; Mains, Richard E; Eipper, Betty A

    2003-01-01

    Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine alpha-amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.

  1. What Does the Mean Mean?

    Science.gov (United States)

    Watier, Nicholas N.; Lamontagne, Claude; Chartier, Sylvain

    2011-01-01

    The arithmetic mean is a fundamental statistical concept. Unfortunately, social science students rarely develop an intuitive understanding of the mean and rely on the formula to describe or define it. According to constructivist pedagogy, educators that have access to a variety of conceptualizations of a particular concept are better equipped to…

  2. Reproduction of duckbill catfish Sorubim lima in captivity (Siluriformes, Pimelodidae by means of hormonal induction/ Reprodução em cativeiro do jurupecê Sorubim lima (Siluriformes, Pimelodidae por meio de indução hormonal

    Directory of Open Access Journals (Sweden)

    Sandro Geraldo de Castro Britto

    Full Text Available Sorubim lima is a migratory catfish that do not reproduce in lenthic environments such as those formed after construction of hydroelectric power plants. An alternative for conservation of the species in these environments is the stocking with fingerlings produced in captivity. The technique used to reproduce it in captivity was the induction by carp pituitary hormone, with dosage of 5 mg.kg-1 for females (1 mg.kg-1 in the first dose and 4 mg.kg-1 in the second and 3 mg.kg-1 for males (1 mg.kg-1 in the first dose and 2 mg.kg-1 in the second, with an interval of 14 hours. The determination of extrusion moment of oocytes was made by monitoring the migration of nucleus from central to peripheral position. After UTA 264.5 (accumulated thermal units the oocytes were obtained by compression of the ventral region of the female, but males were sacrificed for extraction of sperm. The eggs were kept in an incubator and the time of hatching of larvae was 370 UTA. The larvae were initially fed with plankton and showed no cannibalism. Moreover, they were not demanding about the food, accepting prepared rations after fifteenth day of life. The weight gain of larvae was very low, even close to 20 mm in length (14 days, not exceeding 0.05 mg per day. After that both weight and length increased rapidly.Sorubim lima é um bagre migratório que não se reproduz em ambientes lênticos como os que são formados após a construção de usinas hidrelétricas. Uma das alternativas para conservação da espécie nesses ambientes é a estocagem com alevinos produzidos em cativeiro. A técnica utilizada para a sua reprodução em cativeiro foi a da indução por hormônio hipofisário de carpa, com dosagem de 5 mg.kg-1 para fêmeas (1 mg.kg-1 na primeira dose e 4 mg.kg-1 na segunda e 3 mg.kg-1 para machos (1 mg.kg-1 na primeira dose e 2 mg.kg-1 na segunda, com intervalo de 14 horas. A determinação do momento da extrusão dos óvulos foi feita pelo acompanhamento da migra

  3. Default Meanings

    Institute of Scientific and Technical Information of China (English)

    K. M. Jaszczolt

    2006-01-01

    In this paper I present some aspects of the controversy surrounding the semantics/pragmatics boundary and offer a possible solution in the form of Default Semantics. First, I present a brief introduction to Paul Grice's approach to meaning as intentional communication. Next, I address the problem of the unit of which truth conditions should be predicated, assuming after Grice that the most successful approach to meaning has to make use of truth conditions.Further, I extend the discussion to some aspects of neo-Gricean pragmatics, most notably the issue of pragmatic processing that contributes to the truth-conditional representation and the proposed shortcuts through this processing in the form of default meanings. In the main part, I present the theory of Default Semantics ( Jaszczolt 2005a) , assess some arguments in favour of such an interactive theory of meaning that combines information from various sources, and address the issue of compositionality of meaning so conceived. In the final part, I apply the theory of Default Semantics to expressions with future-time reference in English in order to demonstrate the advantages of a framework that combines various types of information about utterance meaning in one, merged semantic representation. The main advantage turns out to be, in this domain, the unified treatment of all expressions with future-time reference.

  4. Intonational meaning.

    Science.gov (United States)

    Prieto, Pilar

    2015-01-01

    Traditionally, prosodic studies have focused on the study of intonational form and the study of intonational meaning has been relatively neglected. Similarly, the fields of semantics and pragmatics have paid little attention to the pragmatic uses of intonation. As a result, there is no firm agreement within the linguistic community on how to integrate the analysis of intonational meaning across languages into a unified prosodic, semantic, and pragmatic approach. This article provides an overview of the literature on intonational meaning, describing the recent advances made in the fields of prosody, semantics/pragmatics, and syntax. Several theoretical approaches to explaining the semantics and pragmatics of intonation are presented. A common feature to most frameworks is that intonation (1) should be regarded as an integral part of linguistic grammar; and (2) typically encodes meanings related to the modal aspect of propositions. However, features such as compositionality, duality of structure, and context-dependency are still hotly debated issues. These features will be discussed from different theoretical perspectives, and we will identify potential advances related to the full integration of intonational meaning into dynamic and multidimensional models of meaning.

  5. Inductive Monitoring System (IMS)

    Data.gov (United States)

    National Aeronautics and Space Administration — IMS: Inductive Monitoring System The Inductive Monitoring System (IMS) is a tool that uses a data mining technique called clustering to extract models of normal...

  6. Inductive situation calculus

    OpenAIRE

    Denecker, Marc; Ternovska, Eugenia

    2004-01-01

    Temporal reasoning has always been a major test case for knowledge representation formalisms. In this paper, we develop an inductive variant of the situation calculus in ED-logic, classical logic extended with inductive definitions. This logic has been proposed recently and is an extension of classical logic. It allows for a uniform representation of various forms of definitions, including monotone inductive definitions and non-monotone forms of inductive definitions such as iterated inductio...

  7. Hypotheses and Inductive Predictions

    NARCIS (Netherlands)

    ROMEYN, J.-W.

    2008-01-01

    ABSTRACT. This paper studies the use of hypotheses schemes in generating inductive predictions. After discussing Carnap–Hintikka inductive logic, hypotheses schemes are defined and illustrated with two partitions. One partition results in the Carnapian continuum of inductive methods, the other resul

  8. Hypotheses and Inductive Predictions

    NARCIS (Netherlands)

    ROMEYN, J.-W.

    2008-01-01

    ABSTRACT. This paper studies the use of hypotheses schemes in generating inductive predictions. After discussing Carnap–Hintikka inductive logic, hypotheses schemes are defined and illustrated with two partitions. One partition results in the Carnapian continuum of inductive methods, the other resul

  9. Inductive Reasoning and Writing

    Science.gov (United States)

    Rooks, Clay; Boyd, Robert

    2003-01-01

    Induction, properly understood, is not merely a game, nor is it a gimmick, nor is it an artificial way of explaining an element of reasoning. Proper understanding of inductive reasoning--and the various types of reasoning that the authors term inductive--enables the student to evaluate critically other people's writing and enhances the composition…

  10. Contextual Meaning and Word Meaning

    Institute of Scientific and Technical Information of China (English)

    Istvan Kecskes

    2006-01-01

    The paper has three goals: 1 ) Explain the role of context and word in meaning construction and comprehension. 2) Present a model that can explain meaning generated by both monolingual and multilingual meaning construction systems. 3) Discuss how the model can be applied to explain major issues in pragmatics.Pragmatics is understood here in a narrow sense as defined by Sperber & Noveck: "...pragmatics is the study of how linguistic properties and contextual factors interact in the interpretation of utterances" ( Sperber & Noveck 2004:1 ). It is argued that world knowledge is available to interlocutors in two forms: as encapsulated in lexical items based on prior encounters and experience (conventionalized cognitive context), and as provided by the actual situational context framed by the situation in which the interaction takes place. Meaning formally expressed in the linguistic interactional context is created on-the-spot, and is the result of the interaction of the two sides of world knowledge and the actual situational context.The paper makes three claims: First, supremacy of context is not unconditional in language processing. Second,salient meaning rather than literal meaning of lexical units plays a central role in comprehension. Third, intercultural communication differs from intracultural communication in the conceptual content of conventionalized cognitive contexts rather than in communicative means.

  11. Induction machine handbook

    CERN Document Server

    Boldea, Ion

    2002-01-01

    Often called the workhorse of industry, the advent of power electronics and advances in digital control are transforming the induction motor into the racehorse of industrial motion control. Now, the classic texts on induction machines are nearly three decades old, while more recent books on electric motors lack the necessary depth and detail on induction machines.The Induction Machine Handbook fills industry's long-standing need for a comprehensive treatise embracing the many intricate facets of induction machine analysis and design. Moving gradually from simple to complex and from standard to

  12. 死亡视角下的生命意义——村上春树作品有感%Meaning of life under the perspective of death——Inductive Haruki Murakami's works

    Institute of Scientific and Technical Information of China (English)

    刘亦奋

    2015-01-01

    Haruki Murakami is Japanese writer research philosophy of death, the creation of works, has a strong scent of death infiltration in it. Haruki Murakami is affected by the Japanese culture, focus on the study of death imagery, derived from the Japanese traditional culture to the cult of death. These studies about death, Murakami inspired by western literary thoughts in the depth of consciousness, in the field of its research implements the thought the collision of death, to dig out the meaning of life from it. In Haruki Murakami's works to analyze, this paper extracted from death, for death under the perspective of life significance is analyzed.%村上春树是研究死亡哲学的日本作家,其所创作的作品中,都有浓厚的死亡气息渗透于其中.村上春树受到日本文化的影响,着重于研究死亡意象,源自于日本传统文化中对死亡的崇拜.关于死亡这些的研究,村上春树在意识深处受到西方文学思想的启发,在其研究领域中实现了死亡思想的碰撞,从中挖掘出生命的意义.本论文针对村上春树的作品以分析,从中提炼死亡意向,对于死亡视角下的生命意义进行分析.

  13. Theoretical exploration of the oxidative properties of a [(tren Me1)CuO2]+ adduct relevant to copper monooxygenase enzymes: insights into competitive dehydrogenation versus hydroxylation reaction pathways.

    Science.gov (United States)

    de la Lande, Aurélien; Parisel, Olivier; Gérard, Hélène; Moliner, Vicente; Reinaud, Olivia

    2008-01-01

    Singlet and triplet H-transfer reaction paths from C-H and N-H bonds were examined by means of DFT and spin-flip TD-DFT computations on the [(tren Me1)CuO2]+ adduct. The singlet energy surfaces allow its evolution towards H2O2 and an imine species. Whereas N-H cleavage appears to be a radical process, C-H rupture results in a carbocation intermediate stabilized by an adjacent N atom and an electrostatic interaction with the [CuIOOH] metal core. Upon injection of an additional electron, the latter species straightforwardly forms a hydroxylated product. Based on these computational results, a new mechanistic description of the reactivity of copper monooxygenases is proposed.

  14. Anthranoyl-CoA monooxygenase/reductase from Azoarcus evansii possesses both FMN and FAD in two distinct and independent active sites.

    Science.gov (United States)

    Bergner, Thomas; Pavkov-Keller, Tea; Kreuzer, Katharina; Kowaliuk, Jakob; Plank, Markus; Runggatscher, Kathrin; Turrini, Nikolaus G; Zucol, Benjamin; Wallner, Silvia; Faber, Kurt; Gruber, Karl; Macheroux, Peter

    2015-08-01

    Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to 5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.

  15. Pharmacological kynurenine 3-monooxygenase enzyme inhibition significantly reduces neuropathic pain in a rat model.

    Science.gov (United States)

    Rojewska, Ewelina; Piotrowska, Anna; Makuch, Wioletta; Przewlocka, Barbara; Mika, Joanna

    2016-03-01

    Recent studies have highlighted the involvement of the kynurenine pathway in the pathology of neurodegenerative diseases, but the role of this system in neuropathic pain requires further extensive research. Therefore, the aim of our study was to examine the role of kynurenine 3-monooxygenase (Kmo), an enzyme that is important in this pathway, in a rat model of neuropathy after chronic constriction injury (CCI) to the sciatic nerve. For the first time, we demonstrated that the injury-induced increase in the Kmo mRNA levels in the spinal cord and the dorsal root ganglia (DRG) was reduced by chronic administration of the microglial inhibitor minocycline and that this effect paralleled a decrease in the intensity of neuropathy. Further, minocycline administration alleviated the lipopolysaccharide (LPS)-induced upregulation of Kmo mRNA expression in microglial cell cultures. Moreover, we demonstrated that not only indirect inhibition of Kmo using minocycline but also direct inhibition using Kmo inhibitors (Ro61-6048 and JM6) decreased neuropathic pain intensity on the third and the seventh days after CCI. Chronic Ro61-6048 administration diminished the protein levels of IBA-1, IL-6, IL-1beta and NOS2 in the spinal cord and/or the DRG. Both Kmo inhibitors potentiated the analgesic properties of morphine. In summary, our data suggest that in neuropathic pain model, inhibiting Kmo function significantly reduces pain symptoms and enhances the effectiveness of morphine. The results of our studies show that the kynurenine pathway is an important mediator of neuropathic pain pathology and indicate that Kmo represents a novel pharmacological target for the treatment of neuropathy.

  16. Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs.

    Science.gov (United States)

    Gilbert, B; McDonald, I R; Finch, R; Stafford, G P; Nielsen, A K; Murrell, J C

    2000-03-01

    The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for sigma(70) promoters were identified, suggesting that these pmo genes are recognized by sigma(70) and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.

  17. Flavin-containing monooxygenase S-oxygenation of a series of thioureas and thiones

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, Marilyn C.; Siddens, Lisbeth K. [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331-7301 (United States); Krueger, Sharon K. [The Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-7301 (United States); Stevens, J. Fred [The Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-7301 (United States); College of Pharmacy, Oregon State University, Corvallis, OR 97331-7301 (United States); Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331-7301 (United States); Kedzie, Karen [Department of Biological Sciences, Allergan, Inc., Irvine, CA 92623-9534 (United States); Fang, Wenkui K.; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael [Department of Chemical Sciences, Allergan, Inc., Irvine, CA 92623-9534 (United States); Gil, Daniel [Department of Biological Sciences, Allergan, Inc., Irvine, CA 92623-9534 (United States); Williams, David E., E-mail: david.williams@oregonstate.edu [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331-7301 (United States); The Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-7301 (United States); Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331-7301 (United States)

    2014-07-15

    Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC–MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2–7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with K{sub m}s ranging from 7 to 160 μM and turnover numbers of 30–40 min{sup −1}. The product formed was identified by LC–MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1.

  18. Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry.

    Science.gov (United States)

    Westereng, Bjørge; Arntzen, Magnus Ø; Agger, Jane Wittrup; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H

    2017-01-01

    Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the number of LPMOs that are active on other polysaccharides is increasing. The products generated by LPMOs from cellulose are either oxidized in the downstream end (at C1) or upstream end (at C4), or at both ends. These modifications only result in small structural changes, which makes both chromatographic separation and product identification by mass spectrometry challenging. The changes in physicochemical properties that are associated with oxidation need to be considered when choosing analytical approaches. C1 oxidation leads to a sugar that is no longer reducing but instead has an acidic functionality, whereas C4 oxidation leads to products that are inherently labile at high and low pH and that exist in a keto-gemdiol equilibrium that is strongly shifted toward the gemdiol in aqueous solutions. Partial degradation of C4-oxidized products leads to the formation of native products, which could explain why some authors claim to have observed glycoside hydrolase activity for LPMOs. Notably, apparent glycoside hydrolase activity may also be due to small amounts of contaminating glycoside hydrolases since these normally have much higher catalytic rates than LPMOs. The low catalytic turnover rates of LPMOs necessitate the use of sensitive product detection methods, which limits the analytical possibilities considerably. Modern liquid chromatography and mass spectrometry have become essential tools for evaluating LPMO activity, and this chapter provides an overview of available methods together with a few novel tools. The methods described constitute a suite of techniques for analyzing oxidized carbohydrate products, which can be applied to LPMOs as well as other carbohydrate

  19. Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases.

    Science.gov (United States)

    Rodriguez, R J; Miranda, C L

    2000-09-01

    N-Deacetyl ketoconazole (DAK) is the major metabolite of orally administered ketoconazole. This major metabolite has been demonstrated to be further metabolized predominately by the flavin-containing monooxygenases (FMOs) to the secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole (N-hydroxy-DAK) by adult and postnatal rat hepatic microsomes. Our current investigation evaluated the FMO isoform specificity of DAK in a pyrophosphate buffer (pH 8.8) containing the glucose 6-phosphate NADPH-generating system. cDNA-expressed human FMOs (FMO1, FMO3, and FMO5) and cDNA-expressed rabbit FMOs (FMO1, FMO2, FMO3, and FMO5) were used to assess the metabolism of DAK to its subsequent FMO-mediated metabolites by HPLC analysis. Human and rabbit cDNA-expressed FMO3 resulted in extensive metabolism of DAK in 1 h (71.2 and 64.5%, respectively) to N-hydroxy-DAK (48.2 and 47.7%, respectively) and two other metabolites, metabolite 1 (11.7 and 7.8%, respectively) and metabolite 3 (10.5 and 10.0%, respectively). Previous studies suggest that metabolite 1 is the nitrone formed after successive FMO-mediated metabolism of N-hydroxy-DAK. Moreover, these studies display similar metabolic profiles seen with adult and postnatal rat hepatic microsomes. The human and rabbit FMO1 metabolized DAK predominately to the N-hydroxy-DAK in 1 h (36.2 and 25.3%, respectively) with minimal metabolism to the other metabolites (DAK to N-hydroxy-DAK (15.9%) and metabolite 1 (6.6%). Last, DAK did not appear to be a substrate for human or rabbit FMO5. Heat inactivation of cDNA-expressed FMOs abolished DAK metabolite formation. These results suggest that DAK is a substrate for human and rabbit FMO1 and FMO3, rabbit FMO2, but not human or rabbit FMO5.

  20. Component interactions in the soluble methane monooxygenase system from Methylococcus capsulatus (Bath).

    Science.gov (United States)

    Gassner, G T; Lippard, S J

    1999-09-28

    The soluble methane monooxygenase system of Methylococcus capsulatus (Bath) includes three protein components: a 251-kDa non-heme dinuclear iron hydroxylase (MMOH), a 39-kDa iron-sulfur- and FAD-containing reductase (MMOR), and a 16-kDa regulatory protein (MMOB). The thermodynamic stability and kinetics of formation of complexes between oxidized MMOH and MMOB or MMOR were measured by isothermal titration calorimetry and stopped-flow fluorescence spectroscopy at temperatures ranging from 3.3 to 45 degrees C. The results, in conjunction with data from equilibrium analytical ultracentrifugation studies of MMOR and MMOB, indicate that free MMOR and MMOB exist as monomers in solution and bind MMOH with 2:1 stoichiometry. The role of component interactions in the catalytic mechanism of sMMO was investigated through simultaneous measurement of oxidase and hydroxylase activities as a function of varied protein component concentrations during steady-state turnover. The partitioning of oxidase and hydroxylase activities of sMMO is highly dependent on both the MMOR concentration and the nature of the organic substrate. In particular, NADH oxidation is significantly uncoupled from methane hydroxylation at MMOR concentrations exceeding 20% of the hydroxylase concentration but remains tightly coupled to propylene epoxidation at MMOR concentrations ranging up to the MMOH concentration. The steady-state kinetic data were fit to numerical simulations of models that include both the oxidase activities of free MMOR and of MMOH/MMOR complexes and the hydroxylase activity of MMOH/MMOB complexes. The data were well described by a model in which MMOR and MMOB bind noncompetitively at distinct interacting sites on the hydroxylase. MMOB manifests its regulatory effects by differentially accelerating intermolecular electron transfer from MMOR to MMOH containing bound substrate and product in a manner consistent with its activating and inhibitory effects on the hydroxylase.

  1. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates

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    Wenwen Kong

    2016-08-01

    Full Text Available Glucosinolates (GSLs, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived GSLs—S-oxygenation of methylthioalkyl GSLs to methylsulfinylalkyl GSLs—was found to be catalyzed by five flavin-containing monooxygenases (FMOs, FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6 and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of MT GSL to the sum of MT and MS GSL, suggesting that the introduction of the two genes converted MT GSL into MS GSL. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid (ABA, 1-aminocyclopropane-1-carboxylic acid (ACC, jasmonic acid (JA, salicylic acid (SA, indole-3-acetic acid (IAA, and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of GSL modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of GSL modifications and the importance of evolution of these duplicated genes.

  2. Effect of Human Flavin-Containing Monooxygenase 3 Polymorphism on the Metabolism of Aurora Kinase Inhibitors

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    Sheila J. Sadeghi

    2013-01-01

    Full Text Available Aurora kinases were recently identified as a potential target in anticancer therapy and, amongst their available inhibitors, Tozasertib (VX-680 and Danusertib (PHA-739358 have been indicated as possible substrates of human flavin-containing monooxygenase 3 (hFMO3. Here we report the in vitro rate of oxidation of these drugs by wild-type hFMO3 and its polymorphic variant V257M. The conversion of Tozasertib and Danusertib to their corresponding metabolites, identified by LC-MS, by the purified wild-type and V257M hFMO3 show significant differences. In the case of Tozasertib, the V257M variant shows a catalytic efficiency, expressed as kcat/Km, similar to the wild-type: 0.39 ± 0.06 min−1µM−1 for V257M compared to 0.33 ± 0.04 min−1µM−1 for the wild type. On the other hand, in the case of Danusertib, V257M shows a 3.4× decrease in catalytic efficiency with kcat/Km values of 0.05 ± 0.01 min−1µM−1 for V257M and 0.17 ± 0.03 min−1µM−1 for the wild type. These data reveal how a simple V257M substitution ascribed to a single nucleotide polymorphism affects the N-oxidation of relevant anticancer drugs, with important outcome in their therapeutic effects. These findings demonstrate that codon 257 is important for activity of the hFMO3 gene and the codon change V to M has an effect on the catalytic efficiency of this enzyme.

  3. Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

    Science.gov (United States)

    Pham, Minh D; Lin, Ya-Ping; Van Vuong, Quan; Nagababu, Penumaka; Chang, Brian T-A; Ng, Kok Yaoh; Chen, Chein-Hung; Han, Chau-Chung; Chen, Chung-Hsuan; Li, Mai Suan; Yu, Steve S-F; Chan, Sunney I

    2015-12-01

    Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.

  4. Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths.

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    Linzhu Wang

    Full Text Available Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.

  5. Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths.

    Science.gov (United States)

    Wang, Linzhu; Beuerle, Till; Timbilla, James; Ober, Dietrich

    2012-01-01

    Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.

  6. The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase.

    Science.gov (United States)

    Schmidt, Sandy; Genz, Maika; Balke, Kathleen; Bornscheuer, Uwe T

    2015-11-20

    Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.

  7. Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

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    Xiong Jie

    2009-05-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses to xenobiotics, making it an ideal model system to study the evolutionary and functional diversity of the P450 monooxygenase gene family. Results A total of 44 putative functional cytochrome P450 genes were identified and could be classified into 13 families and 21 sub-families according to standard nomenclature. The characteristics of both the conserved intron-exon organization and scaffold localization of tandem repeats within each P450 family clade suggested that the enlargement of T. thermophila P450 families probably resulted from recent separate small duplication events. Gene expression patterns of all T. thermophila P450s during three important cell physiological stages (vegetative growth, starvation and conjugation were analyzed based on EST and microarray data, and three main categories of expression patterns were postulated. Evolutionary analysis including codon usage preference, site-specific selection and gene-expression evolution patterns were investigated and the results indicated remarkable divergences among the T. thermophila P450 genes. Conclusion The characterization, expression and evolutionary analysis of T. thermophila P450 monooxygenase genes in the current study provides useful information for understanding the characteristics and diversities of the P450 genes in the Ciliophora, and provides the baseline for functional analyses of individual P450 isoforms in this model ciliate species.

  8. Comparative Analysis of the Conventional and Novel pmo (Particulate Methane Monooxygenase) Operons from Methylocystis Strain SC2

    OpenAIRE

    Ricke, Peter; Erkel, Christoph; Kube, Michael; Reinhardt, Richard; Liesack, Werner

    2004-01-01

    In addition to the conventional pmoA gene (pmoA1) encoding the active site polypeptide of particulate methane monooxygenase, a novel pmoA gene copy (pmoA2) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the pmoA1 and pmoA2 gene copies in the type II MOB Methylocystis strain SC2 are each part of a complete pmoCAB gene clu...

  9. Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

    OpenAIRE

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M.; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnab...

  10. Development of a Series of Kynurenine 3-Monooxygenase Inhibitors Leading to a Clinical Candidate for the Treatment of Acute Pancreatitis.

    Science.gov (United States)

    Walker, Ann L; Ancellin, Nicolas; Beaufils, Benjamin; Bergeal, Marylise; Binnie, Margaret; Bouillot, Anne; Clapham, David; Denis, Alexis; Haslam, Carl P; Holmes, Duncan S; Hutchinson, Jonathan P; Liddle, John; McBride, Andrew; Mirguet, Olivier; Mowat, Christopher G; Rowland, Paul; Tiberghien, Nathalie; Trottet, Lionel; Uings, Iain; Webster, Scott P; Zheng, Xiaozhong; Mole, Damian J

    2017-04-27

    Recently, we reported a novel role for KMO in the pathogenesis of acute pancreatitis (AP). A number of inhibitors of kynurenine 3-monooxygenase (KMO) have previously been described as potential treatments for neurodegenerative conditions and particularly for Huntington's disease. However, the inhibitors reported to date have insufficient aqueous solubility relative to their cellular potency to be compatible with the intravenous (iv) dosing route required in AP. We have identified and optimized a novel series of high affinity KMO inhibitors with favorable physicochemical properties. The leading example is exquisitely selective, has low clearance in two species, prevents lung and kidney damage in a rat model of acute pancreatitis, and is progressing into preclinical development.

  11. Development of a series of aryl pyrimidine kynurenine monooxygenase inhibitors as potential therapeutic agents for the treatment of Huntington's disease.

    Science.gov (United States)

    Toledo-Sherman, Leticia M; Prime, Michael E; Mrzljak, Ladislav; Beconi, Maria G; Beresford, Alan; Brookfield, Frederick A; Brown, Christopher J; Cardaun, Isabell; Courtney, Stephen M; Dijkman, Ulrike; Hamelin-Flegg, Estelle; Johnson, Peter D; Kempf, Valerie; Lyons, Kathy; Matthews, Kimberly; Mitchell, William L; O'Connell, Catherine; Pena, Paula; Powell, Kendall; Rassoulpour, Arash; Reed, Laura; Reindl, Wolfgang; Selvaratnam, Suganathan; Friley, Weslyn Ward; Weddell, Derek A; Went, Naomi E; Wheelan, Patricia; Winkler, Christin; Winkler, Dirk; Wityak, John; Yarnold, Christopher J; Yates, Dawn; Munoz-Sanjuan, Ignacio; Dominguez, Celia

    2015-02-12

    We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system.

  12. Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

    OpenAIRE

    2000-01-01

    4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH2)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH2 in ...

  13. Differential Reactivity between Two Copper Sites in Peptidylglycine r-Hydroxylating Monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    E Chufan; S Prigge; X Siebert; B Eipper; R Mains; L Amzel

    2011-12-31

    Peptidylglycine {alpha}-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the C{alpha} of C-terminal glycine-extended peptides and proteins, the first step in the activation of many peptide hormones, growth factors, and neurotransmitters. The crystal structure of the enzyme revealed two nonequivalent Cu sites (Cu{sub M} and Cu{sub H}) separated by {approx}11 {angstrom}. In the resting state of the enzyme, Cu{sub M} is coordinated in a distorted tetrahedral geometry by one methionine, two histidines, and a water molecule. The coordination site of the water molecule is the position where external ligands bind. The Cu{sub H} has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu{sub M} is identified as the metal involved in catalysis, while Cu{sub H} is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO{sub 2}{sup -}), azide (N{sub 3}{sup -}), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO{sub 2}, shows that nitrite anion coordinates Cu{sub M} in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind Cu{sub H}, despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate Cu{sub M} but not Cu{sub H} in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN{sub 3} and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the Cu{sub H} is also observed in the reduced form of the enzyme: CO binds Cu{sub M} but not Cu{sub H} in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5

  14. Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates involved in catalysis.

    Science.gov (United States)

    Sheng, D; Ballou, D P; Massey, V

    2001-09-18

    Cyclohexanone monooxygenase (CHMO), a bacterial flavoenzyme, carries out an oxygen insertion reaction on cyclohexanone to form a seven-membered cyclic product, epsilon-caprolactone. The reaction catalyzed involves the four-electron reduction of O2 at the expense of a two-electron oxidation of NADPH and a two-electron oxidation of cyclohexanone to form epsilon-caprolactone. Previous studies suggested the participation of either a flavin C4a-hydroperoxide or a flavin C4a-peroxide intermediate during the enzymatic catalysis [Ryerson, C. C., Ballou, D. P., and Walsh, C. (1982) Biochemistry 21, 2644-2655]. However, there was no kinetic or spectral evidence to distinguish between these two possibilities. In the present work we used double-mixing stopped-flow techniques to show that the C4a-flavin-oxygen adduct, which is formed rapidly from the reaction of oxygen with reduced enzyme in the presence of NADP, can exist in two states. When the reaction is carried out at pH 7.2, the first intermediate is a flavin C4a-peroxide with maximum absorbance at 366 nm; this intermediate becomes protonated at about 3 s(-1) to form what is believed to be the flavin C4a-hydroperoxide with maximum absorbance at 383 nm. These two intermediates can be interconverted by altering the pH, with a pK(a) of 8.4. Thus, at pH 9.0 the flavin C4a-peroxide persists mainly in the deprotonated form. Further kinetic studies also demonstrated that only the flavin C4a-peroxide intermediate could oxygenate the substrate, cyclohexanone. The requirement in catalysis of the deprotonated flavin C4a-peroxide, a nucleophile, is consistent with a Baeyer-Villiger rearrangement mechanism for the enzymatic oxygenation of cyclohexanone. In the course of these studies, the Kd for cyclohexanone to the C4a-peroxyflavin form of CHMO was determined to be approximately 1 microM. The rate-determining step in catalysis was shown to be the release of NADP from the oxidized enzyme.

  15. Kinetics and activation thermodynamics of methane monooxygenase compound Q formation and reaction with substrates.

    Science.gov (United States)

    Brazeau, B J; Lipscomb, J D

    2000-11-07

    The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of

  16. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris; Hyman, Michael R.; Löffler, F. E.

    2016-01-29

    Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and

  17. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.

    Science.gov (United States)

    Bennett, Kristen; Sadler, Natalie C; Wright, Aaron T; Yeager, Chris; Hyman, Michael R

    2016-04-01

    Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2 (-)) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4 (+)-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. Copyright © 2016, American Society for Microbiology. All

  18. Cloning, characterization and expression of $OsFMO_{(t)}$ in rice encoding a flavin monooxygenase

    Indian Academy of Sciences (India)

    Jicai Yi; Lanna Liu; Youpei Cao; Jiazuo Li; Mantong Mei

    2013-12-01

    Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of $OsFMO_{(t)}$, a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. $OsFMO_{(t)}$ was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a ‘G×××G’ characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO$_{\\text{(t)}}$ and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of $OsFMO_{(t)}$ in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO$_{\\text{(t)}}$ protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of $OsFMO_{(t)}$ was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by $OsFMO_{(t)}$ is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.

  19. Structure and mechanism of styrene monooxygenase reductase: new insight into the FAD-transfer reaction.

    Science.gov (United States)

    Morrison, Eliot; Kantz, Auric; Gassner, George T; Sazinsky, Matthew H

    2013-09-03

    The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH → FADox charge-transfer intermediate and catalyzes a hydride-transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s(-1), in a step that is coupled to the rapid dissociation of NAD(+). Electrochemical and equilibrium-binding studies indicate that NSMOA binds FADhq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)-hydroperoxide intermediate. The half-life of apoSMOB generated in the FAD-transfer reaction is increased ∼21-fold, supporting a protein-protein interaction between apoSMOB and the peroxide intermediate of NSMOA. The mechanisms of FAD dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. On the basis of these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S state) and more reactive, transfer state (T state). The dissociation of NAD(+) after the hydride-transfer reaction transiently populates the T state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB-FADhq shifts the FADhq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB-FADox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions.

  20. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes.

    Science.gov (United States)

    Pierce, Brian C; Agger, Jane Wittrup; Zhang, Zhenghong; Wichmann, Jesper; Meyer, Anne S

    2017-09-08

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes was evaluated in combination with endoglucanase and beta-glucosidase. Two enzymes (TrCel61A and Aspte6) showed the ability to release more than 36% of the pretreated soy spent flake glucose - a greater than 75% increase over the same treatment without LPMO addition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Directed evolution of phenylacetone monooxygenase as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone.

    Science.gov (United States)

    Parra, Loreto P; Acevedo, Juan P; Reetz, Manfred T

    2015-07-01

    Phenylacetone monooxygenase (PAMO) is an exceptionally robust Baeyer-Villiger monooxygenase, which makes it ideal for potential industrial applications. However, its substrate scope is limited, unreactive cyclohexanone being a prominent example. Such a limitation is unfortunate, because this particular transformation in an ecologically viable manner would be highly desirable, the lactone and the respective lactam being of considerable interest as monomers in polymer science. We have applied directed evolution in search of an active mutant for this valuable C-C activating reaction. Using iterative saturation mutagenesis (ISM), several active mutants were evolved, with only a minimal trade-off in terms of stability. The best mutants allow for quantitative conversion of 2 mM cyclohexanone within 1 h reaction time. In order to circumvent the NADP(+) regeneration problem, whole E. coli resting cells were successfully applied. Molecular dynamics simulations and induced fit docking throw light on the origin of enhanced PAMO activity. The PAMO mutants constitute ideal starting points for future directed evolution optimization necessary for an industrial process.

  2. Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules.

    Science.gov (United States)

    Bučko, Marek; Schenkmayerová, Andrea; Gemeiner, Peter; Vikartovská, Alica; Mihovilovič, Marko D; Lacík, Igor

    2011-08-10

    An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.

  3. Isolation and initial characterization of a novel type of Baeyer-Villiger monooxygenase activity from a marine microorganism.

    Science.gov (United States)

    Willetts, Andrew; Joint, Ian; Gilbert, Jack A; Trimble, William; Mühling, Martin

    2012-07-01

    A novel type of Baeyer-Villiger monooxygenase (BVMO) has been found in a marine strain of Stenotrophomonas maltophila strain PML168 that was isolated from a temperate intertidal zone. The enzyme is able to use NADH as the source of reducing power necessary to accept the atom of diatomic oxygen not incorporated into the oxyfunctionalized substrate. Growth studies have establish that the enzyme is inducible, appears to serve a catabolic role, and is specifically induced by one or more unidentified components of seawater as well as various anthropogenic xenobiotic compounds. A blast search of the primary sequence of the enzyme, recovered from the genomic sequence of the isolate, has placed this atypical BVMO in the context of the several hundred known members of the flavoprotein monooxygenase superfamily. A particular feature of this BVMO lies in its truncated C-terminal domain, which results in a relatively small protein (357 amino acids; 38.4 kDa). In addition, metagenomic screening has been conducted on DNA recovered from an extensive range of marine environmental samples to gauge the relative abundance and distribution of similar enzymes within the global marine microbial community. Although low, abundance was detected in samples from many marine provinces, confirming the potential for biodiscovery in marine microorganisms.

  4. Controlled oxidation of aliphatic CH bonds in metallo-monooxygenases: mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons.

    Science.gov (United States)

    Chen, Yao-Sheng; Luo, Wen-I; Yang, Chung-Ling; Tu, Yi-Jung; Chang, Chun-Wei; Chiang, Chih-Hsiang; Chang, Chi-Yao; Chan, Sunney I; Yu, Steve S-F

    2014-05-01

    The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions.

  5. Inductive Logic and Statistics

    NARCIS (Netherlands)

    Romeijn, J. -W.

    2009-01-01

    This chapter concerns inductive logic in relation to mathematical statistics. I start by introducing a general notion of probabilistic induc- tive inference. Then I introduce Carnapian inductive logic, and I show that it can be related to Bayesian statistical inference via de Finetti's representatio

  6. DNA adducts, benzo(a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy).

    Science.gov (United States)

    Pisoni, M; Cogotzi, L; Frigeri, A; Corsi, I; Bonacci, S; Iacocca, A; Lancini, L; Mastrototaro, F; Focardi, S; Svelto, M

    2004-10-01

    The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a "cleaner" reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity (B(a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B(a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of beta-N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B(a)PMO activity in digestive gland in both years (r = 0.60 in 2001; r = 0.73 in 2002). Increases were observed in B(a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B(a)PMO activity over the two monitoring campaigns. The membrane labilization

  7. Progress in Induction Linacs

    Energy Technology Data Exchange (ETDEWEB)

    Caporaso, G J

    2000-09-27

    This presentation will be a broad survey of progress in induction technology over the past four years. Much work has been done on accelerators for hydrodynamic test radiography and other applications. Solid-state pulsers have been developed which can provide unprecedented flexibility and precision in pulse format and accelerating voltage for both ion and electron induction machines. Induction linacs can now be built which can operate with MHz repetition rates. Solid-state technology has also made possible the development of fast kickers for precision control of high current beams. New insulator technology has been developed which will improve conventional induction linacs in addition to enabling a new class of high gradient induction linacs.

  8. Inductance loop and partial

    CERN Document Server

    Paul, Clayton R

    2010-01-01

    "Inductance is an unprecedented text, thoroughly discussing "loop" inductance as well as the increasingly important "partial" inductance. These concepts and their proper calculation are crucial in designing modern high-speed digital systems. World-renowned leader in electromagnetics Clayton Paul provides the knowledge and tools necessary to understand and calculate inductance." "With the present and increasing emphasis on high-speed digital systems and high-frequency analog systems, it is imperative that system designers develop an intimate understanding of the concepts and methods in this book. Inductance is a much-needed textbook designed for senior and graduate-level engineering students, as well as a hands-on guide for working engineers and professionals engaged in the design of high-speed digital and high-frequency analog systems."--Jacket.

  9. Enzymatic Synthesis of Enantiomerically Pure beta-Amino Ketones, beta-Amino Esters, and beta-Amino Alcohols with Baeyer-Villiger Monooxygenases

    NARCIS (Netherlands)

    Rehdorf, Jessica; Mihovilovic, Marko D.; Fraaije, Marco W.; Bornscheuer, Uwe T.

    2010-01-01

    The enzymatic kinetic resolution of a broad set of beta-amino ketones was investigated by using a collection of 16 Baeyer-Villiger monooxygenases from different bacterial origins, which display various substrate specificities. Within this platform of enzymes excellent enantioselectivities (E>200) we

  10. Pseudomonas aeruginosa LysR PA4203 regulator NmoR acts as a repressor of the PA4202 nmoA> gene, encoding a nitronate monooxygenase

    DEFF Research Database (Denmark)

    Vercammen, Ken; Wei, Qing; Charlier, Daniel;

    2015-01-01

    The PA4203 gene encodes a LysR regulator and lies between the ppgL gene (PA4204), which encodes a periplasmic gluconolactonase, and, in the opposite orientation, the PA4202 (nmoA) gene, coding for a nitronate monooxygenase, and ddlA (PA4201), encoding a d-alanine alanine ligase. The intergenic re...

  11. Direct reading inductance meter

    Science.gov (United States)

    Kolby, R. B. (Inventor)

    1977-01-01

    A direct reading inductance meter comprised of a crystal oscillator and an LC tuned oscillator is presented. The oscillators function respectively to generate a reference frequency, f(r), and to generate an initial frequency, f(0), which when mixed produce a difference equal to zero. Upon connecting an inductor of small unknown value in the LC circuit to change its resonant frequency to f(x), a difference frequency (f(r)-f(x)) is produced that is very nearly a linear function of the inductance of the inductor. The difference frequency is measured and displayed on a linear scale in units of inductance.

  12. Induction motor control design

    CERN Document Server

    Marino, Riccardo; Verrelli, Cristiano M

    2010-01-01

    ""Nonlinear and Adaptive Control Design for Induction Motors"" is a unified exposition of the most important steps and concerns in the design of estimation and control algorithms for induction motors. A single notation and modern nonlinear control terminology is used to make the book accessible to readers who are not experts in electric motors at the same time as giving a more theoretical control viewpoint to those who are. In order to increase readability, the book concentrates on the induction motor, eschewing the much more complex and less-well-understood control of asynchronous motors. The

  13. Properties of inductive reasoning.

    Science.gov (United States)

    Heit, E

    2000-12-01

    This paper reviews the main psychological phenomena of inductive reasoning, covering 25 years of experimental and model-based research, in particular addressing four questions. First, what makes a case or event generalizable to other cases? Second, what makes a set of cases generalizable? Third, what makes a property or predicate projectable? Fourth, how do psychological models of induction address these results? The key results in inductive reasoning are outlined, and several recent models, including a new Bayesian account, are evaluated with respect to these results. In addition, future directions for experimental and model-based work are proposed.

  14. Electromagnetic induction in Australia

    Science.gov (United States)

    Lilley, F. E. M.

    Electromagnetic induction at the terrestrial surface is a general and ubiquitous process. This note, which covers research on the subject in Australia, reflects the writer's own interest and refers particularly to induction by natural source fields in the period range of 1 minute to 1 day.Such source fields arise external to Earth, in the ionosphere and beyond, in the magnetosphere. The process of electromagnetic induction by these fields involves the flow through Earth of tens of thousands of amperes, over scale lengths of thousands of kilometers.

  15. Tectonics: The meaning of form

    DEFF Research Database (Denmark)

    Christiansen, Karl; Brandt, Per Aage

    Tectonics – The meaning of form deals with one of the core topics of architecture: the relationship between form and content. In the world of architecture, form is not only made from brick, glass and wood. Form means something. When a material is processed with sufficient technical skill and insi...... perspectives. You can read the chapters in any order you like – from the beginning, end or the middle. There is no correct order. The project is methodologically inductive: the more essays you read, the broader your knowledge of tectonics get....

  16. In Silico Approach to Support that p-Nitrophenol Monooxygenase from Arthrobacter sp. Strain JS443 Catalyzes the Initial Two Sequential Monooxygenations.

    Science.gov (United States)

    Kallubai, Monika; Amineni, Umamaheswari; Mallavarapu, Megharaj; Kadiyala, Venkateswarlu

    2015-06-01

    p-Nitrophenol (PNP), used primarily for manufacturing pesticides and dyes, has been recognized as a priority environmental pollutant. It is therefore important to reduce the input of this toxicant into the environment and to establish approaches for its removal from the contaminated sites. PNP monooxygenase, a novel enzyme from Gram-positive bacteria like Arthrobacter sp. and Bacillus sp., that comprises two components, a flavoprotein reductase and an oxygenase, catalyzes the initial two sequential monooxygenations to convert PNP to trihydroxybenzene. Accurate and reliable prediction of this enzyme-substrate interactions and binding affinity are of vital importance in understanding these catalytic mechanisms of the two sequential reactions. As crystal structure of the enzyme has not yet been published, we built a homology model for PNP monooxygenase using crystallized chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100 (3HWC) as the template. The model was assessed for its reliability using PROCHECK, ERRAT and ProSA. Molecular docking of the physiological substrates, PNP and 4-nitrocatechol (4-NC), was carried out using Glide v5.7 implemented in Maestro v9.2, and the binding energies were calculated to substantiate the prediction. Docking complexes formed by molecular level interactions of PNP monooxygenase-PNP/4-NC without or with the cofactors, FAD and NADH, showed good correlation with the established experimental evidence that the two-component PNP monooxygenase catalyzes both the hydroxylation of PNP and the oxidative release of nitrite from 4-NC in B. sphaericus JS905. Furthermore, molecular dynamics simulations performed for docking complexes using Desmond v3.0 showed stable nature of the interactions as well.

  17. Making Mathematical Induction Meaningful

    Science.gov (United States)

    Hirsch, Christian R.

    1976-01-01

    The author argues that the "least number principle" (well-ordering for positive integers) is more intuitively acceptable to high school students than mathematical induction which can be derived from it. (SD)

  18. Thermal kinetic inductance detector

    Energy Technology Data Exchange (ETDEWEB)

    Cecil, Thomas; Gades, Lisa; Miceli, Antonio; Quaranta, Orlando

    2016-12-20

    A microcalorimeter for radiation detection that uses superconducting kinetic inductance resonators as the thermometers. The detector is frequency-multiplexed which enables detector systems with a large number of pixels.

  19. Induction heating coupler

    Science.gov (United States)

    Fox, Robert L. (Inventor); Copeland, Carl E. (Inventor); Swaim, Robert J. (Inventor); Coultrip, Robert H. (Inventor); Johnston, David F. (Inventor); Phillips, W. Morris (Inventor); Johnson, Samuel D. (Inventor); Dinkins, James R. (Inventor); Buckley, John D. (Inventor)

    1994-01-01

    An induction heating device includes a handle having a hollow interior and two opposite ends, a wrist connected to one end of the handle, a U-shaped pole piece having two spaced apart ends, a tank circuit including an induction coil wrapped around the pole piece and a capacitor connected to the induction coil, a head connected to the wrist and including a housing for receiving the U-shaped pole piece, the two spaced apart ends of the pole piece extending outwardely beyond the housing, and a power source connected to the tank circuit. When the tank circuit is energized and a susceptor is placed in juxtaposition to the ends of the U-shaped pole piece, the susceptor is heated by induction heating due to magnetic flux passing between the two ends of the pole piece.

  20. Unification By Induction

    CERN Document Server

    Adewole, A I A

    2001-01-01

    We show that the problem of unifying electromagnetism with gravity has an elegant solution in classical physics through the phenomenon of induction. By studying the way that induction leads to the formation of electromagnetic fields, we identify the classical field equations which the unified field must satisfy and a corresponding set of constitutive equations for the medium sustaining the field. The unification problem is then reduced to the problem of finding the exact form of these constitutive equations for different media by experiments.

  1. Induction of the Proepicardium.

    Science.gov (United States)

    Maya-Ramos, Lisandro; Cleland, James; Bressan, Michael; Mikawa, Takashi

    2013-09-01

    The proepicardium is a transient extracardiac embryonic tissue that gives rise to the epicardium and a number of coronary vascular cell lineages. This important extracardiac tissue develops through multiple steps of inductive events, from specification of multiple cell lineages to morphogenesis. This article will review our current understanding of inductive events involved in patterning of the proepicardium precursor field, specification of cell types within the proepicardium, and their extension and attachment to the heart.

  2. Induction of the Proepicardium

    Directory of Open Access Journals (Sweden)

    Takashi Mikawa

    2013-07-01

    Full Text Available The proepicardium is a transient extracardiac embryonic tissue that gives rise to the epicardium and a number of coronary vascular cell lineages. This important extracardiac tissue develops through multiple steps of inductive events, from specification of multiple cell lineages to morphogenesis. This article will review our current understanding of inductive events involved in patterning of the proepicardium precursor field, specification of cell types within the proepicardium and their extension and attachment to the heart.

  3. Diophantine Correct Open Induction

    CERN Document Server

    Raffer, Sidney

    2010-01-01

    We give an induction-free axiom system for diophantine correct open induction. We relate the problem of whether a finitely generated ring of Puiseux polynomials is diophantine correct to a problem about the value-distribution of a tuple of semialgebraic functions with integer arguments. We use this result, and a theorem of Bergelson and Leibman on generalized polynomials, to identify a class of diophantine correct subrings of the field of descending Puiseux series with real coefficients.

  4. Induction Heating Systems

    Science.gov (United States)

    1991-01-01

    Induction heating technology, a magnetic non-deforming process, was developed by Langley researchers to join plastic and composite components in space. Under NASA license, Inductron Corporation uses the process to produce induction heating systems and equipment for numerous applications. The Torobonder, a portable system, comes with a number of interchangeable heads for aircraft repair. Other developments are the E Heating Head, the Toroid Joining Gun, and the Torobrazer. These products perform bonding applications more quickly, safely and efficiently than previous methods.

  5. Homology Modeling and Molecular Docking Analysis of Streptomyces peucetius CYP125A4 as C26 Monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Won; Lee, Na Rae; Oh, Tae Jin [SunMoon University, Asan (Korea, Republic of); Lee, Ji Hun [Physico-Chemical Assay Team, R and D, CELLTRION, Inc., Incheon (Korea, Republic of)

    2012-06-15

    Among 23 cytochrome P450s, CYP125A4 was proposed as a putative monooxygenase based on the high level of amino acid sequence homology (54% identity and 75% similarity) with the well characterized C27-steroid Mycobacterium tuberculosis CYP125A1. Utilizing MTBCYP125A1 as a template, homology modeling of SPCYP125A4 was conducted by Accelrys Discovery Studio 3.1 software. The modeled SPCYP125A4 structure with lowest energy value was subsequently assessed for its stereochemical quality and side-chain environment. The final model was generated by showing its active site through the molecular dynamics. The docking of steroids showed broad specificity of SPCYP125A4 with different orientation of ligand within active site facing the heme. One poses of C27-steroid with C26 facing the heme with distance of 3.734 A from the Fe were predominant

  6. Enantioselective Benzylic Hydroxylation Catalysed by P450 Monooxygenases: Characterisation of a P450cam Mutant Library and Molecular Modelling.

    Science.gov (United States)

    Eichler, Anja; Gricman, Łukasz; Herter, Susanne; Kelly, Paul P; Turner, Nicholas J; Pleiss, Jürgen; Flitsch, Sabine L

    2016-03-02

    Cytochrome P450 monooxygenases can catalyse the stereoselective C-H activation of a very broad range of substrates. Prediction and control of enantioselectivity of this enzyme class is of great interest for the synthesis of high-value chiral molecules. Here we have used a combination of molecular dynamics simulations and experimental screening to study the enantioselectivity of a library of active-site mutants of chimeric P450cam-RhFRed towards the benzylic hydroxylation of structurally related regioisomers of ethylmethylbenzene. Small variations either in substrate structure or in enzyme active site architecture were shown to lead to dramatic changes in enantioselectivity; this was broadly in agreement with computational predictions. In addition to validating computational approaches, these studies have provided us with a deeper understanding of effects that might control stereoselectivity in these biooxidation reactions.

  7. Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Plants synthesize the osmoprotectant glycine betaine (GB) via choline→betaine aldehyde→glycine betaine[1]. Two enzymes are involved in the pathway, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). A full length CMO cDNA (1,643bp) was cloned from Amaranthus tricolor. The open reading frame encoded a 442-amino acid polypeptide, which showed 69% identity with CMOs in Spinacia oleracea L. And Beta vulgaris L. DNA gel blot analysis indicated the presence of one copy of CMO gene in the A. Tricolor genome. The expressions of CMO and BADH proteins in A.tricolor leaves significantly increased under salinization, drought and heat stress (42℃), as determined by immunoblot analysis, but did not respond to cold stress (4℃), or exogenous ABA application. The increase of GB content in leaves was parallel to CMO and BADH contents.

  8. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase.

    Science.gov (United States)

    Pierce, Brian C; Agger, Jane Wittrup; Wichmann, Jesper; Meyer, Anne S

    2017-03-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of substrates, including both soy spent flake, a by-product of the soy food industry, and soy spent flake pretreated with sodium hydroxide. Products from enzymatic treatments were analyzed using mass spectrometry and high performance anion exchange chromatography. We demonstrate that TrCel61A is capable of oxidizing cellulose from both pretreated soy spent flake and phosphoric acid swollen cellulose, oxidizing at both the C1 and C4 positions. In addition, we show that the oxidative activity of TrCel61A displays a synergistic effect capable of boosting endoglucanase activity, and thereby substrate depolymerization of soy cellulose, by 27%. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. The TMAO-Producing Enzyme Flavin-Containing Monooxygenase 3 Regulates Obesity and the Beiging of White Adipose Tissue

    Directory of Open Access Journals (Sweden)

    Rebecca C. Schugar

    2017-06-01

    Full Text Available Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here, we demonstrate that the gut microbiota-initiated trimethylamine N-oxide (TMAO-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme flavin-containing monooxygenase 3 (FMO3 conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue.

  10. Characterization of in vitro metabolites of methylenedioxypyrovalerone (MDPV): An N-oxide metabolite formation mediated by flavin monooxygenase.

    Science.gov (United States)

    Kim, In Sook; Rehman, Shaheed Ur; Choi, Min Sun; Jang, Moonhee; Yang, Wonkyung; Kim, Eunmi; Yoo, Hye Hyun

    2016-11-30

    Methylenedioxypyrovalerone (MDPV) has emerged in recent years as a recreational substance with psychostimulant properties. In this study, in vitro metabolites of MDPV were characterized based on liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/QTOF MS). MDPV was incubated with human liver microsomes, human recombinant cDNA-expressed cytochrome P450 enzymes and flavin monooxygenase (FMO). MDPV was metabolized to yield eight metabolites (M1-M8) with major metabolic reactions such as demethylenation and oxidation. Among them, M6 was assigned as an N-oxide metabolite. FMO was found to be a principal enzyme responsible for the formation of M6; FMO1 and FMO3 were the main enzymes involved in N-oxidation of MDPV. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

    Science.gov (United States)

    Lee, Won-Heong; Park, Eun-Hee; Kim, Myoung-Dong

    2014-12-28

    Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

  12. Simultaneous biocatalyst production and Baeyer-Villiger oxidation for bioconversion of cyclohexanone by recombinant Escherichia coli expressing cyclohexanone monooxygenase.

    Science.gov (United States)

    Lee, Won-Heong; Park, Yong-Cheol; Lee, Dae-Hee; Park, Kyungmoon; Seo, Jin-Ho

    2005-01-01

    Cyclohexanone monooxygenase (CHMO) catalyzing Baeyer-Villiger oxidation converts cyclic ketones into optically pure lactones, which have been used as building blocks in organic synthesis. A recombinant Escherichia coli BL21(DE3)/pMM4 expressing CHMO originated from Acinetobacter sp. NCIB 9871 was used to produce epsilon-caprolactone through a simultaneous biocatalyst production and Baeyer-Villiger oxidation (SPO) process. A fed-batch process was designed to obtain high cell density for improving production of epsilon-caprolactone. The fed-batch SPO process gave the best results, 10.2 g/L of epsilon-caprolactone and 0.34 g/(L.h) of productivity, corresponding to a 10.5- and 3.4-fold enhancement compared with those of the batch SPO, respectively.

  13. Lytic polysaccharide monooxygenases: a crystallographer’s view on a new class of biomass-degrading enzymes

    Science.gov (United States)

    Frandsen, Kristian E. H.; Lo Leggio, Leila

    2016-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future. PMID:27840684

  14. Hydroxylation of Longiborneol by a Clm2-Encoded CYP450 Monooxygenase to Produce Culmorin in Fusarium graminearum.

    Science.gov (United States)

    Bahadoor, Adilah; Schneiderman, Danielle; Gemmill, Larissa; Bosnich, Whynn; Blackwell, Barbara; Melanson, Jeremy E; McRae, Garnet; Harris, Linda J

    2016-01-22

    A second structural gene required for culmorin biosynthesis in the plant pathogen Fusarium graminearum is described. Clm2 encodes a regio- and stereoselective cytochrome P450 monooxygenase for C-11 of longiborneol (1). Clm2 gene disruptants were grown in liquid culture and assessed for culmorin production via HPLC-evaporative light scattering detection. The analysis indicated a complete loss of culmorin (2) from the liquid culture of the ΔClm2 mutants. Culmorin production resumed in a ΔClm2 complementation experiment. A detailed analysis of the secondary metabolites extracted from the large-scale liquid culture of disruptant ΔClm2D20 revealed five new natural products: 3-hydroxylongiborneol (3), 5-hydroxylongiborneol (4), 12-hydroxylongiborneol (5), 15-hydroxylongiborneol (6), and 11-epi-acetylculmorin (7). The structures of the new compounds were elucidated by a combination of HRMS, 1D and 2D NMR, and X-ray crystallography.

  15. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    Science.gov (United States)

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L

    2003-02-01

    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

  16. Mechanism of the 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from Pseudomonas putida S16.

    Science.gov (United States)

    Yu, Hao; Hausinger, Robert P; Tang, Hong-Zhi; Xu, Ping

    2014-10-17

    6-Hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB), a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, catalyzes pyridine-ring β-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine. Here, we generated His6-tagged HspB in Escherichia coli, characterized the properties of the recombinant enzyme, and investigated its mechanism of catalysis. In contrast to conclusions reported previously, the second product of the HspB reaction was shown to be succinate, with isotope labeling experiments providing direct evidence that the newly introduced oxygen atom of succinate is derived from H2O. Phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB. The reduction of HspB (with maxima at 375 and 460 nm, and a shoulder at 485 nm) by NADH was followed by stopped-flow spectroscopy, and the rate constant for reduction was shown to be stimulated by HSP. Reduced HspB reacts with oxygen to form a C(4a)-(hydro)peroxyflavin intermediate with an absorbance maximum at ∼400 nm within the first few milliseconds before converting to the oxidized flavoenzyme species. The formed C(4a)-hydroperoxyflavin intermediate reacts with HSP to form an intermediate that hydrolyzes to the products 2,5-dihydroxypyridine and succinate. The investigation on the catalytic mechanism of a flavoprotein pyridine-ring β-position hydroxylase provides useful information for the biosynthesis of pyridine derivatives.

  17. Coordinated production and utilization of FADH2 by NAD(P)H-flavin oxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase.

    Science.gov (United States)

    Louie, Tai Man; Xie, X Sunney; Xun, Luying

    2003-06-24

    4-Hydroxyphenylacetate (4HPA) 3-monooxygenase (HpaB) is a reduced flavin adenine dinucleotide (FADH(2)) utilizing monooxygenase. Its cosubstrate, FADH(2), is supplied by HpaC, an NAD(P)H-flavin oxidoreductase. Because HpaB is the first enzyme for 4HPA metabolism, FADH(2) production and utilization become a major metabolic event when Escherichia coli W grows on 4HPA. An important question is how FADH(2) is produced and used, as FADH(2) is unstable in the presence of free O(2). One solution is metabolic channeling by forming a transitory HpaB-HpaC complex. However, our in vivo and in vitro data failed to support the interaction. Further investigation pointed to an alternative scheme for HpaB to sequester FADH(2). The intracellular HpaB concentration was about 122 microM in 4HPA-growing cells, much higher than the total intracellular FAD concentration, and HpaB had a high affinity for FADH(2) (K(d) of 70 nM), suggesting that most FADH(2) is bound to HpaB in vivo. The HpaB-bound FADH(2) was either used to rapidly oxidize 4HPA or slowly oxidized by O(2) to FAD and H(2)O(2) in the absence of 4HPA. Thus, HpaB's high intracellular concentration, its high affinity for FADH(2), its property of protecting bound FADH(2) in the absence of 4HPA, and its ability to rapidly use FADH(2) to oxidize 4HPA when 4HPA is available can coordinate FADH(2) production and utilization by HpaB and HpaC in vivo. This type of coordination, in responding to demand, for production and utilization of labile metabolites has not been reported to date.

  18. Socialization of New Teachers: Does Induction Matter?

    Science.gov (United States)

    Nasser-Abu Alhija, Fadia; Fresko, Barbara

    2010-01-01

    The present paper focuses on new teachers' satisfaction with their first year of teaching from the perspective of socialization. The relationship between satisfaction with socialization and teacher background, school environment, placement, and induction variables was examined. Data were collected from 243 Israeli beginning teachers by means of…

  19. Junctional communication of embryonic cells after induction

    Institute of Scientific and Technical Information of China (English)

    ZengMibai; JiangWansu

    1990-01-01

    Cell couplings before and after neural induction in embryos of Cynops orientalis were studied by means of single cell injection of Lucifer Yellow.Differences both in incidence and the extent of cell couplings were demonstrated.Results of cell couplings were correlated with electron microscopic observations of freeze-etching replicas.

  20. 14 CFR 121.283 - Induction system ice prevention.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Induction system ice prevention. 121.283 Section 121.283 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... Induction system ice prevention. A means for preventing the malfunctioning of each engine due to...

  1. 14 CFR 125.181 - Induction system ice prevention.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Induction system ice prevention. 125.181 Section 125.181 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... Requirements § 125.181 Induction system ice prevention. A means for preventing the malfunctioning of...

  2. Symmetric Structure of Induction Motor Systems

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    In this paper, symmetric structure of induction motor system in stationary αβ0 coordinates is studied bythe geometric approach. The results show that the system possesses symmetry (G, θ, Ф) and infinitesimal symme-try. Under certain conditions, the system can be transformed into a form possessing state-space symmetry (G, Ф)and infinitesimal state-space symmetry by means of state feedback and input coordinate base transform. The resultscan be extended to the fifth order induction motor system fed by hysteresis-band current-controlled PWM inverter.

  3. A Novel Induction Algorithm for DM

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    DM usually means an efficient knowledge discovery from database, and the immune algorithm is a biological theory-based and global searching algorithm. A novel induction algorithm is proposed here which integrates a power of individual immunity and an evolutionary mechanism of population. This algorithm does not take great care of discovering some classifying information, but unknown knowledge or a predication on higher level rules. Theoretical analysis and simulations both show that this algorithm is prone to the stabilization of a population and the improvement of entire capability, and also keeping a high degree of preciseness during the rule induction.

  4. nanoSQUID operation using kinetic rather than magnetic induction.

    Science.gov (United States)

    McCaughan, Adam N; Zhao, Qingyuan; Berggren, Karl K

    2016-06-14

    We report on a method of nanoSQUID modulation which uses kinetic inductance rather than magnetic inductance to manip-ulate the internal fluxoid state. We produced modulation using injected current rather than an applied magnetic field. Using this injected current, we were able to observe the triangle-wave shaped modulation of the device critical current which was periodic according to the London fluxoid quantization condition. The measurement results also confirmed that the fluxoid state inside a superconducting loop can be manipulated using primarily kinetic inductance. By using primarily kinetic inductance rather than magnetic inductance, the size of the coupling inductor was reduced by a factor of 10. As a result, this approach may provide a means to reduce the size of SQUID-based superconducting electronics. Additionally, this method provides a convenient way to perform kinetic inductance characterizations of superconducting thin films.

  5. Refining Inductive Types

    CERN Document Server

    Atkey, Robert; Ghani, Neil

    2012-01-01

    Dependently typed programming languages allow sophisticated properties of data to be expressed within the type system. Of particular use in dependently typed programming are indexed types that refine data by computationally useful information. For example, the N-indexed type of vectors refines lists by their lengths. Other data types may be refined in similar ways, but programmers must produce purpose-specific refinements on an ad hoc basis, developers must anticipate which refinements to include in libraries, and implementations must often store redundant information about data and their refinements. In this paper we show how to generically derive inductive characterisations of refinements of inductive types, and argue that these characterisations can alleviate some of the aforementioned difficulties associated with ad hoc refinements. Our characterisations also ensure that standard techniques for programming with and reasoning about inductive types are applicable to refinements, and that refinements can the...

  6. Both FMNH2 and FADH2 can be utilized by the dibenzothiophene monooxygenase from a desulfurizing bacterium Mycobacterium goodii X7B.

    Science.gov (United States)

    Li, Jingchen; Feng, Jinhui; Li, Qian; Ma, Cuiqing; Yu, Bo; Gao, Chao; Wu, Geng; Xu, Ping

    2009-05-01

    To investigate the flavin utilization by dibenzothiophene monooxygenase (DszC), DszC of a desulfurizing bacterium Mycobacterium goodii X7B was purified from the recombinant Escherichia coli. It was shown to be able to utilize either FMNH(2) or FADH(2) when coupled with a flavin reductase that reduces either FMN or FAD. Sequence analysis indicated that DszC was similar to the C(2) component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, which can use both FADH(2) and FMNH(2) as substrates. Both flavins at high concentrations could inhibit the activity of DszC due to autocatalytic oxidation of reduced flavins. The results suggest that DszC should be reclassified as an FMNH(2) and FADH(2) both-utilizing monooxygenase component and the flavins should be controlled at properly reduced levels to obtain optimal biodesulfurization results.

  7. Supersonic induction plasma jet modeling

    Energy Technology Data Exchange (ETDEWEB)

    Selezneva, S.E. E-mail: svetlana2@hermes.usherbS_Selezneva2@hermes.usherb; Boulos, M.I

    2001-06-01

    Numerical simulations have been applied to study the argon plasma flow downstream of the induction plasma torch. It is shown that by means of the convergent-divergent nozzle adjustment and chamber pressure reduction, a supersonic plasma jet can be obtained. We investigate the supersonic and a more traditional subsonic plasma jets impinging onto a normal substrate. Comparing to the subsonic jet, the supersonic one is narrower and much faster. Near-substrate velocity and temperature boundary layers are thinner, so the heat flux near the stagnation point is higher in the supersonic jet. The supersonic plasma jet is characterized by the electron overpopulation and the domination of the recombination over the dissociation, resulting into the heating of the electron gas. Because of these processes, the supersonic induction plasma permits to separate spatially different functions (dissociation and ionization, transport and deposition) and to optimize each of them. The considered configuration can be advantageous in some industrial applications, such as plasma-assisted chemical vapor deposition of diamond and polymer-like films and in plasma spraying of nanoscaled powders.

  8. Modeling Induction Motor Imbalances

    DEFF Research Database (Denmark)

    Armah, Kabenla; Jouffroy, Jerome; Duggen, Lars

    2016-01-01

    This paper gives a study into the development of a generalized model for a three-phase induction motor that offers flexibility of simulating balanced and unbalanced parameter scenarios. By analyzing the interaction of forces within the motor, we achieve our main objective of deriving the system...

  9. Learning and Inductive Inference

    Science.gov (United States)

    1982-07-01

    is also a paradigm example of induction as discussed by philosophers ( Carnap , 1950). Data compression is of practical value for storage and...IBelser, A. 0. Hlolzman, and A. Kent (Eds.., Eneyclopedia of computer science and technology (Vol. II). New York: Marcel Dekker, 24 51. " Carnap , R. 1950

  10. Iteration, Not Induction

    Science.gov (United States)

    Dobbs, David E.

    2009-01-01

    The main purpose of this note is to present and justify proof via iteration as an intuitive, creative and empowering method that is often available and preferable as an alternative to proofs via either mathematical induction or the well-ordering principle. The method of iteration depends only on the fact that any strictly decreasing sequence of…

  11. Teaching Mathematical Induction I.

    Science.gov (United States)

    Dubinsky, Ed

    1986-01-01

    A novel approach to teaching mathematical induction was used, based on a Piagetian theory of learning abstract mathematical concepts in which the learner uses reflective abstraction to construct new schemas out of old ones. Computer experiences are used to induce students to make the appropriate reflective abstractions. (MNS)

  12. Using Continuity Induction

    Science.gov (United States)

    Hathaway, Dan

    2011-01-01

    Here is a technique for proving the fundamental theorems of analysis that provides a unified way to pass from local properties to global properties on the real line, just as ordinary induction passes from local implication (if true for "k", the theorem is true for "k" + 1) to a global conclusion in the natural numbers.

  13. Training in Inductive Reasoning.

    Science.gov (United States)

    Tomic, Welko

    This study investigated the effects of K. J. Klauer's (1989) inductive reasoning training program of teaching children. Effects of training and the range of transfer of the training were assessed. The subjects were 34 third-grade Dutch children of average ability, matched on age, sex, and IQ. Children from the training condition (N=17) received…

  14. Developmental changes of cytochrome P450 dependent monooxygenase functions after transplantation of fetal liver tissue suspension into spleens of adult syngenic rats.

    Science.gov (United States)

    Lupp, A; Trautmann, A K; Krausse, T; Klinger, W

    1998-06-01

    Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher 344 inbred rats. Animals were sacrificed at 3 days, 1, 2, 4 weeks, and 2, 4 and 6 months after transplantation and cytochrome P450 (P450) dependent monooxygenase functions in spleen and liver 9000 g supernatants were assessed by measuring three model reactions for different P450 subtypes: ethoxyresorufin O-deethylation (EROD; mainly 1A), ethoxycoumarin O-deethylation (ECOD; predominantly 1A, 2A, 2B) and ethylmorphine N-demethylation (END; mainly 3A). Values of transplant recipients were compared to those of sham operated and age matched control rats. Spleen weights were significantly higher in transplanted rats, compared to controls or sham operated animals, but there was no influence of the transplants within the spleens on liver weights. With fetal livers at the 21st day of gestation, the day of transplantation, a weak EROD and ECOD, but no END activity was seen. Spleens of controls or sham operated animals displayed nearly no P450 mediated monooxygenase functions. In the explant containing spleens a significant and increasing EROD activity was found from 4 weeks after surgery on and an ECOD activity already 2 weeks after transplantation. END was only slightly enhanced at 6 months after surgery. The livers of all three groups of rats displayed normal EROD, ECOD and END activities. Transplantation of fetal liver tissue suspensions into the spleens did not influence the P450 dependent monooxygenase functions within the livers of the animals. From these results it can be concluded that intrasplenically transplanted liver cells originating from syngenic fetal liver tissue suspensions proliferate and differentiate within the host organs. They display P450 dependent monooxygenase functions with some developmental changes during the observed time period of 6 months.

  15. Alpha-subunit positions methionine 180 and glutamate 214 of Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase influence catalysis.

    Science.gov (United States)

    Vardar, Gönül; Wood, Thomas K

    2005-02-01

    Alpha-subunit position M180 of toluene-o-xylene monooxygenase influences the regiospecific oxidation of aromatics (e.g., from o-cresol, M180H forms 3-methylcatechol, methylhydroquinone, and 4-methylresorcinol, whereas the wild type forms only 3-methylcatechol). Position E214 influences the rate of reaction (e.g., E214G increases p-nitrophenol oxidation 15-fold) by controlling substrate entrance and product efflux as a gate residue.

  16. In vitro characterization of an enzymatic redox cascade composed of an alcohol dehydrogenase, an enoate reductases and a Baeyer-Villiger monooxygenase.

    Science.gov (United States)

    Oberleitner, Nikolin; Peters, Christin; Rudroff, Florian; Bornscheuer, Uwe T; Mihovilovic, Marko D

    2014-12-20

    An artificial enzyme cascade composed of an alcohol dehydrogenase, an enoate reductase and a Baeyer-Villiger monooxygenase was investigated in vitro to gain deeper mechanistic insights and understand the assets and drawbacks of this multi-step biocatalysis. Several substrates composed of different structural motifs were examined and provided access to functionalized chiral compounds in high yields (up to >99%) and optical purities (up to >99%). Hence, the applicability of the presented enzymatic cascade was exploited for the synthesis of biorenewable polyesters.

  17. Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase.

    Science.gov (United States)

    Xun, L; Sandvik, E R

    2000-02-01

    4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH(2))-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH(2) in vitro, HpaB was able to use FADH(2) and O(2) for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH(2) for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH(2)-utilizing monooxygenase. FADH(2) generated by Fre was rapidly oxidized by O(2) to form H(2)O(2) in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH(2) and transitorily protected it from rapid autoxidation by O(2). When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O(2) for FADH(2) utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH(2) produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH(2) was autoxidized by O(2), causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH(2)-utilizing monooxygenase that uses FADH(2) as a substrate rather than as a cofactor.

  18. Comparative analysis of the conventional and novel pmo (particulate methane monooxygenase) operons from methylocystis strain SC2.

    Science.gov (United States)

    Ricke, Peter; Erkel, Christoph; Kube, Michael; Reinhardt, Richard; Liesack, Werner

    2004-05-01

    In addition to the conventional pmoA gene (pmoA1) encoding the active site polypeptide of particulate methane monooxygenase, a novel pmoA gene copy (pmoA2) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the pmoA1 and pmoA2 gene copies in the type II MOB Methylocystis strain SC2 are each part of a complete pmoCAB gene cluster (pmoCAB1, pmoCAB2). A bacterial artificial chromosome (BAC) library of strain SC2 genomic DNA was constructed, and BAC clones carrying either pmoCAB1 or pmoCAB2 were identified. Comparative sequence analysis showed that these two gene clusters exhibit low levels of identity at both the DNA level (67.4 to 70.9%) and the derived protein level (59.3 to 65.6%). In contrast, the secondary structures predicted for PmoCAB1 and PmoCAB2, as well as the derived transmembrane-spanning regions, are nearly identical. This suggests that PmoCAB2 is, like PmoCAB1, a highly hydrophobic, membrane-associated protein. A total of 190 of the 203 amino acid residues representing a highly conserved consensus sequence of the currently known PmoCAB1 and AmoCAB sequence types could be identified in PmoCAB2. The amoCAB gene cluster encodes ammonia monooxygenase and is evolutionarily related to pmoCAB. Analysis of a set of amino acid residues that allowed differentiation between conventional PmoA and AmoA provided further support for the hypothesis that pmoCAB2 encodes a functional equivalent of PmoCAB1. In experiments in which we used 5' rapid amplification of cDNA ends we identified transcriptional start sites 320 and 177 bp upstream of pmoC1 and pmoC2, respectively. Immediately upstream of the transcriptional start sites of both pmoCAB1 and pmoCAB2, sequence motifs similar to Escherichia coli sigma(70) promoters were identified.

  19. The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

    Science.gov (United States)

    Ouafik, L H; Stoffers, D A; Campbell, T A; Johnson, R C; Bloomquist, B T; Mains, R E; Eipper, B A

    1992-10-01

    Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along

  20. Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23.

    Science.gov (United States)

    Boonmak, Chanita; Takahashi, Yasunori; Morikawa, Masaaki

    2014-05-01

    An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 °C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladAαB23, ladAβB23, and ladB B23, on its chromosome, whose protein products share significant amino acid sequence identities, 49.8, 34.4, and 22.7 %, respectively, with that of ladA alkane monooxygenase gene found on a plasmid of Geobacillus thermodetrificans NG 80-2. Each of the three genes, ladAαB23, ladAβB23, and ladB B23, was heterologously expressed individually in an alkB1 deletion mutant strain, Pseudomonas fluorescens KOB2Δ1. It was found that all three genes were functional in P. fluorescens KOB2Δ1, and partially restored alkane degradation activity. In this study, we suggest that G. thermoleovorans B23 utilizes multiple LadA-type alkane monooxygenases for the degradation of a broad range of alkanes.

  1. Decision Procedures for Proving Inductive Theorems without Induction

    OpenAIRE

    Aoto, Takahito; Stratulat, Sorin

    2014-01-01

    International audience; Automated inductive reasoning for term rewriting has been extensively studied in the literature. Classes of equations and term rewriting systems (TRSs) with decidable inductive validity have been identified and used to automatize the inductive reasoning. We give procedures for deciding the inductive validity of equations in some standard TRSs on natural numbers and lists. Contrary to previous decidability results, our procedures can automatically decide without involvi...

  2. Creativity and Memory: Effects of an Episodic-Specificity Induction on Divergent Thinking.

    Science.gov (United States)

    Madore, Kevin P; Addis, Donna Rose; Schacter, Daniel L

    2015-09-01

    People produce more episodic details when imagining future events and solving means-end problems after receiving an episodic-specificity induction-brief training in recollecting details of a recent event-than after receiving a control induction not focused on episodic retrieval. Here we show for the first time that an episodic-specificity induction also enhances divergent creative thinking. In Experiment 1, participants exhibited a selective boost on a divergent-thinking task (generating unusual uses of common objects) after a specificity induction compared with a control induction; by contrast, performance following the two inductions was similar on an object association task thought to involve little divergent thinking. In Experiment 2, we replicated the specificity-induction effect on divergent thinking using a different control induction, and also found that participants performed similarly on a convergent-thinking task following the two inductions. These experiments provide novel evidence that episodic memory is involved in divergent creative thinking.

  3. Measurement of Self-Inductance.

    Science.gov (United States)

    Mak, S. Y.; Tao, P. K.

    1988-01-01

    Discusses four different methods for measuring self-inductance based on the definition of inductance, the alternative definition, phase difference and LC resonance. Provides circuit diagrams and typical oscilloscope traces. (YP)

  4. Saturation mutagenesis of Bradyrhizobium sp. BTAi1 toluene 4-monooxygenase at alpha-subunit residues proline 101, proline 103, and histidine 214 for regiospecific oxidation of aromatics.

    Science.gov (United States)

    Yanık-Yıldırım, K Cansu; Vardar-Schara, Gönül

    2014-11-01

    A novel toluene monooxygenase (TMO) six-gene cluster from Bradyrhizobium sp. BTAi1 having an overall 35, 36, and 38 % protein similarity with toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, and toluene-para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, respectively, was cloned and expressed in Escherichia coli TG1, and its potential activity was investigated for aromatic hydroxylation and trichloroethylene (TCE) degradation. The natural substrate toluene was hydroxylated to p-cresol, indicating that the new toluene monooxygenase (T4MO·BTAi1) acts as a para hydroxylating enzyme, similar to T4MO and TpMO. Some shifts in regiospecific hydroxylations were observed compared to the other wild-type TMOs. For example, wild-type T4MO·BTAi1 formed catechol (88 %) and hydroquinone (12 %) from phenol, whereas all the other wild-type TMOs were reported to form only catechol. Furthermore, it was discovered that TG1 cells expressing wild-type T4MO·BTAi1 mineralized TCE at a rate of 0.67 ± 0.10 nmol Cl(-)/h/mg protein. Saturation and site directed mutagenesis were used to generate eight variants of T4MO·BTAi1 at alpha-subunit positions P101, P103, and H214: P101T/P103A, P101S, P101N/P103T, P101V, P103T, P101V/P103T, H214G, and H214G/D278N; by testing the substrates phenol, nitrobenzene, and naphthalene, positions P101 and P103 were found to influence the regiospecific oxidation of aromatics. For example, compared to wild type, variant P103T produced four fold more m-nitrophenol from nitrobenzene as well as produced mainly resorcinol (60 %) from phenol whereas wild-type T4MO·BTAi1 did not. Similarly, variants P101T/P103A and P101S synthesized more 2-naphthol and 2.3-fold and 1.6-fold less 1-naphthol from naphthalene, respectively.

  5. Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance.

    Science.gov (United States)

    Liu, Simu; Bartnikas, Lisa M; Volko, Sigrid M; Ausubel, Frederick M; Tang, Dingzhong

    2016-01-01

    Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

  6. Isolation of Homogeneous Polysaccharide Monooxygenases from Fungal Sources and Investigation of Their Synergism with Cellulases when Acting on Cellulose.

    Science.gov (United States)

    Bulakhov, A G; Gusakov, A V; Chekushina, A V; Satrutdinov, A D; Koshelev, A V; Matys, V Yu; Sinitsyn, A P

    2016-05-01

    Lytic polysaccharide monooxygenases (PMO) discovered several years ago are enzymes classified as oxidoreductases. In nature, they participate in microbial degradation of cellulose together with cellulases that belong to the hydrolytic type of enzymes (class of hydrolases). Three PMO from ascomycetes - Thielavia terrestris, Trichoderma reesei, and Myceliophthora thermophila - were isolated and purified to homogeneous state using various types of chromatography. The first two enzymes are recombinant proteins heterologously expressed by the Penicillium verruculosum fungus, while the third is a native PMO secreted by M. thermophila. When acting on microcrystalline cellulose, all these PMOs displayed synergism with the cellulase complex of the P. verruculosum fungus. Replacing 10% of cellulases (by protein concentration) with PMO in the presence of 6.25 mM gallic acid or 2.5 µM of cellobiose dehydrogenase from M. thermophila, used as electron donors for PMO, resulted in the 17-31% increase in the yield of reducing sugars after 24-48 h of the enzymatic reaction.

  7. Inhibition of the Flavin-Dependent Monooxygenase Siderophore A (SidA) Blocks Siderophore Biosynthesis and Aspergillus fumigatus Growth.

    Science.gov (United States)

    Martín Del Campo, Julia S; Vogelaar, Nancy; Tolani, Karishma; Kizjakina, Karina; Harich, Kim; Sobrado, Pablo

    2016-11-18

    Aspergillus fumigatus is an opportunistic fungal pathogen and the most common causative agent of fatal invasive mycoses. The flavin-dependent monooxygenase siderophore A (SidA) catalyzes the oxygen and NADPH dependent hydroxylation of l-ornithine (l-Orn) to N(5)-l-hydroxyornithine in the biosynthetic pathway of hydroxamate-containing siderophores in A. fumigatus. Deletion of the gene that codes for SidA has shown that it is essential in establishing infection in mice models. Here, a fluorescence polarization high-throughput assay was used to screen a 2320 compound library for inhibitors of SidA. Celastrol, a natural quinone methide, was identified as a noncompetitive inhibitor of SidA with a MIC value of 2 μM. Docking experiments suggest that celastrol binds across the NADPH and l-Orn pocket. Celastrol prevents A. fumigatus growth in blood agar. The addition of purified ferric-siderophore abolished the inhibitory effect of celastrol. Thus, celastrol inhibits A. fumigatus growth by blocking siderophore biosynthesis through SidA inhibiton.

  8. ELONGATED UPPERMOST INTERNODE encodes a cytochrome P450 monooxygenase that epoxidizes gibberellins in a novel deactivation reaction in rice.

    Science.gov (United States)

    Zhu, Yongyou; Nomura, Takahito; Xu, Yonghan; Zhang, Yingying; Peng, Yu; Mao, Bizeng; Hanada, Atsushi; Zhou, Haicheng; Wang, Renxiao; Li, Peijin; Zhu, Xudong; Mander, Lewis N; Kamiya, Yuji; Yamaguchi, Shinjiro; He, Zuhua

    2006-02-01

    The recessive tall rice (Oryza sativa) mutant elongated uppermost internode (eui) is morphologically normal until its final internode elongates drastically at the heading stage. The stage-specific developmental effect of the eui mutation has been used in the breeding of hybrid rice to improve the performance of heading in male sterile cultivars. We found that the eui mutant accumulated exceptionally large amounts of biologically active gibberellins (GAs) in the uppermost internode. Map-based cloning revealed that the Eui gene encodes a previously uncharacterized cytochrome P450 monooxygenase, CYP714D1. Using heterologous expression in yeast, we found that EUI catalyzed 16alpha,17-epoxidation of non-13-hydroxylated GAs. Consistent with the tall and dwarfed phenotypes of the eui mutant and Eui-overexpressing transgenic plants, respectively, 16alpha,17-epoxidation reduced the biological activity of GA(4) in rice, demonstrating that EUI functions as a GA-deactivating enzyme. Expression of Eui appeared tightly regulated during plant development, in agreement with the stage-specific eui phenotypes. These results indicate the existence of an unrecognized pathway for GA deactivation by EUI during the growth of wild-type internodes. The identification of Eui as a GA catabolism gene provides additional evidence that the GA metabolism pathway is a useful target for increasing the agronomic value of crops.

  9. Rescue of white egg 1 mutant by introduction of the wild-type Bombyx kynurenine 3-monooxygenase gene

    Institute of Scientific and Technical Information of China (English)

    GUO-XING QUAN; ISAO KOBAYASHI; KATSURA KOJIMA; KEIRO UCHINO; TOSHIO KANDA; HIDEKI SEZUTSU; TORU SHIMADA; TOSHIKI TAMURA

    2007-01-01

    In silkworms, the white egg 1 (w-1) mutant, which is characterized by white eyes and white eggs, is deficient in Bombyx kynurenine 3-monooxygenase (KMO) activity. To investigate whether the w-1 mutant phenotype is rescued by introducing the wild-type KMO gene, we constructed transgenic silkworms with the wild-type Bombyx KMO gene under the control of either the cytoplasmic actin gene promoter (A3KMO) or the native KMO gene promoter (KKMO). We created two transgenic lines with A3KMO and one line with KKMO constructs. The eyes of adults in these lines were brown, and the eggs laid by the transgenic females were also brown. Reverse transcription-polymerase chain reaction(RT-PCR) analysis showed that the A3KMO silkworm lines expressed the transcript in the mid-gut, fat bodies, and Malpighian tubules. The KKMO line expressed the transcript only in the fat bodies and Malpighian tubules. The intensity of eye and egg color in the transgenic lines was proportional to the KMO expression level. Interestingly, transgenic larvae with the A3KMO construct had a light brown larval cuticle, but the KKMO line did not. These results indicate that the wild-type KMO gene can be used as a marker gene for visually screening transgenic silkworms.

  10. Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserina.

    Science.gov (United States)

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

  11. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway1[OPEN

    Science.gov (United States)

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  12. Identification of dimethylamine monooxygenase in marine bacteria reveals a metabolic bottleneck in the methylated amine degradation pathway.

    Science.gov (United States)

    Lidbury, Ian; Mausz, Michaela A; Scanlan, David J; Chen, Yin

    2017-07-01

    Methylated amines (MAs) are ubiquitous in the marine environment and their subsequent flux into the atmosphere can result in the formation of aerosols and ultimately cloud condensation nuclei. Therefore, these compounds have a potentially important role in climate regulation. Using Ruegeria pomeroyi as a model, we identified the genes encoding dimethylamine (DMA) monooxygenase (dmmABC) and demonstrate that this enzyme degrades DMA to monomethylamine (MMA). Although only dmmABC are required for enzyme activity in recombinant Escherichia coli, we found that an additional gene, dmmD, was required for the growth of R. pomeroyi on MAs. The dmmDABC genes are absent from the genomes of multiple marine bacteria, including all representatives of the cosmopolitan SAR11 clade. Consequently, the abundance of dmmDABC in marine metagenomes was substantially lower than the genes required for other metabolic steps of the MA degradation pathway. Thus, there is a genetic and potential metabolic bottleneck in the marine MA degradation pathway. Our data provide an explanation for the observation that DMA-derived secondary organic aerosols (SOAs) are among the most abundant SOAs detected in fine marine particles over the North and Tropical Atlantic Ocean.

  13. Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s.

    Science.gov (United States)

    Parvez, Mohammad; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Kgosiemang, Ipeleng Kopano Rosinah; Bamal, Hans Denis; Pagadala, Nataraj Sekhar; Xie, Ting; Yang, Haoran; Chen, Hengye; Theron, Chrispian William; Monyaki, Richie; Raselemane, Seiso Caiphus; Salewe, Vuyani; Mongale, Bogadi Lorato; Matowane, Retshedisitswe Godfrey; Abdalla, Sara Mohamed Hasaan; Booi, Wool Isaac; van Wyk, Mari; Olivier, Dedré; Boucher, Charlotte E; Nelson, David R; Tuszynski, Jack A; Blackburn, Jonathan Michael; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Chen, Wanping; Syed, Khajamohiddin

    2016-09-12

    Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria -42; fungi -19; plant -28; animal -22; plant and animal -1 and common P450 family -1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study's results offer new understanding of the dynamic structural nature of P450s.

  14. Sparteine monooxygenase in brain and liver: Identified by the dopamine uptake blocker ( sup 3 H)GBR-12935

    Energy Technology Data Exchange (ETDEWEB)

    Kalow, W.; Tyndale, R.F.; Niznik, H.B.; Inaba, T. (Univ. of Toronto, Ontario (Canada))

    1990-02-26

    P450IID6 (human sparteine monooxygenase) metabolizes many drugs including neuroleptics, antidepressants, and beta-blockers. The P450IID6 exists in human, bovine, rat and canine brains, but in very low quantities causing methodological difficulties in its assessment. Work with ({sup 3}H)GBR-12935; 1-(2-(diphenylmethoxy) ethyl)-4-(3-phenyl propyl) piperazine has shown that it binds a neuronal/hepatic protein with high affinity ({approximately}7nM) and a rank order of inhibitory potency suggesting that the binding protein is cytochrome P450IID6. The binding was used to predict that d-amphetamine and methamphetamine would interact with P450IID6. Inhibition studies indicated that these compounds were competitive inhibitors of P450IID6. Haloperidol (HAL) and it's metabolite hydroxy-haloperidol (RHAL) are both competitive inhibitors of P450IID6 activity and were found to inhibit ({sup 3}H)GBR-12935 binding. K{sub i} values of twelve compounds (known to interact with the DA transporter or P450IID6) for ({sup 3}H)GRB-12935 binding and P450IID6 activity. The techniques are now available for measurements of cytochrome P450IID6 in healthy and diseased brain/liver tissue using radio-receptor binding assay techniques with ({sup 3}H)GBR-12935.

  15. Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.

    Science.gov (United States)

    Abdelwahab, Heba; Robinson, Reeder; Rodriguez, Pedro; Adly, Camelia; El-Sohaimy, Sohby; Sobrado, Pablo

    2016-09-15

    l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

  16. Cloning and expressing DBT (dibenzothiophene) monooxygenase gene(dszC) from Rhodococcus sp.DS-3 in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    MA Ting; LI Shanshan; LI Guoqiang; WANG Renjing; LIANG Fenglai; LIU Rulin

    2006-01-01

    Dibenzothiophene (DBT) monooxygenase (DszC)catalysis,the first and also the key step in the microbial DBT desulfurization,is the conversion of DBT to DBT sulfone (DBTO2).In this study,dszC of a DBT-desulfiaizing bacterium Rhodococcus sp.DS-3 was cloned by PCR.The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank.The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E.coli BL21 strain.The expression amount of DszC was about 20% of total supernatant at low temperature.The soluble DszC in the supematant was purified by Ni2+ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity.Only one band was detected by Western-blotting,which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3,paC5) and Rhodococcus sp.DS-3.The activity of purified DszC was 0.36 U.DszC can utilize the organic compound such as DBT and methyl-DBT,hut not DBT derivates such as DBF,which has no sulfur or inorganic sulfur.

  17. Intermediate Q from soluble methane monooxygenase hydroxylates the mechanistic substrate probe norcarane: evidence for a stepwise reaction.

    Science.gov (United States)

    Brazeau, B J; Austin, R N; Tarr, C; Groves, J T; Lipscomb, J D

    2001-12-05

    Norcarane is a valuable mechanistic probe for enzyme-catalyzed hydrocarbon oxidation reactions because different products or product distributions result from concerted, radical, and cation based reactions. Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b catalyzes the oxidation of norcarane to afford 3-hydroxymethylcyclohexene and 3-cycloheptenol, compounds characteristic of radical and cationic intermediates, respectively, in addition to 2- and 3-norcaranols. Past single turnover transient kinetic studies have identified several optically distinct intermediates from the catalytic cycle of the hydroxylase component of sMMO. Thus, the reaction between norcarane and key reaction intermediates can be directly monitored. The presence of norcarane increases the rate of decay of only one intermediate, the high-valent bis-mu-oxo Fe(IV)(2) cluster-containing species compound Q, showing that it is responsible for the majority of the oxidation chemistry. The observation of products from both radical and cationic intermediates from norcarane oxidation catalyzed by sMMO is consistent with a mechanism in which an initial substrate radical intermediate is formed by hydrogen atom abstraction. This intermediate then undergoes either oxygen rebound, intramolecular rearrangement followed by oxygen rebound, or loss of a second electron to yield a cationic intermediate to which OH(-) is transferred. The estimated lower limit of 20 ps for the lifetime of the putative radical intermediate is in accord with values determined from previous studies of sterically hindered sMMO probes.

  18. Mutation of the glucosinolate biosynthesis enzyme cytochrome P450 83A1 monooxygenase increases camalexin accumulation and powdery mildew resistance

    Directory of Open Access Journals (Sweden)

    Simu eLiu

    2016-03-01

    Full Text Available Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1, which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

  19. Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6.

    Directory of Open Access Journals (Sweden)

    Alexandre Ismail

    2016-01-01

    Full Text Available Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6, a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations or completely (a G248R-L382E double-mutation blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.

  20. Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6.

    Science.gov (United States)

    Ismail, Alexandre; Leroux, Vincent; Smadja, Myriam; Gonzalez, Lucie; Lombard, Murielle; Pierrel, Fabien; Mellot-Draznieks, Caroline; Fontecave, Marc

    2016-01-01

    Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis.

  1. Mathematical Induction: A Pedagogical Discussion.

    Science.gov (United States)

    Ernest, Paul

    1984-01-01

    Discusses the nature of mathematical induction and what constitutes a correct proof by this method and common misconceptions of induction with recommendations for their remediation. The topic is also analyzed into behavioral skills and subjected to a conceptual analysis. Criteria for analyzing and evaluating textbook treatment of induction are…

  2. Inductive Reasoning: A Training Approach

    Science.gov (United States)

    Klauer, Karl Josef; Phye, Gary D.

    2008-01-01

    Researchers have examined inductive reasoning to identify different cognitive processes when participants deal with inductive problems. This article presents a prescriptive theory of inductive reasoning that identifies cognitive processing using a procedural strategy for making comparisons. It is hypothesized that training in the use of the…

  3. Induction motor control

    Science.gov (United States)

    Hansen, Irving G.

    Electromechanical actuators developed to date have commonly ultilized permanent magnet (PM) synchronous motors. More recently switched reluctance (SR) motors have been advocated due to their robust characteristics. Implications of work which utilized induction motors and advanced control techniques are discussed. When induction motors are operated from an energy source capable of controlling voltages and frequencies independently, drive characteristics are obtained which are superior to either PM or SR motors. By synthesizing the machine frequency from a high-frequency carrier (nominally 20 kHz), high efficiencies, low distortion, and rapid torque response are available. At this time multiple horsepower machine drives were demonstrated, and work is on-going to develop a 20 hp average, 40 hp peak class of aerospace actuators. This effort is based upon high-frequency power distribution and management techniques developed by NASA for Space Station Freedom.

  4. Inductive Supervised Quantum Learning

    Science.gov (United States)

    Monràs, Alex; Sentís, Gael; Wittek, Peter

    2017-05-01

    In supervised learning, an inductive learning algorithm extracts general rules from observed training instances, then the rules are applied to test instances. We show that this splitting of training and application arises naturally, in the classical setting, from a simple independence requirement with a physical interpretation of being nonsignaling. Thus, two seemingly different definitions of inductive learning happen to coincide. This follows from the properties of classical information that break down in the quantum setup. We prove a quantum de Finetti theorem for quantum channels, which shows that in the quantum case, the equivalence holds in the asymptotic setting, that is, for large numbers of test instances. This reveals a natural analogy between classical learning protocols and their quantum counterparts, justifying a similar treatment, and allowing us to inquire about standard elements in computational learning theory, such as structural risk minimization and sample complexity.

  5. Induction motor control

    Science.gov (United States)

    Hansen, Irving G.

    1990-01-01

    Electromechanical actuators developed to date have commonly ultilized permanent magnet (PM) synchronous motors. More recently switched reluctance (SR) motors have been advocated due to their robust characteristics. Implications of work which utilized induction motors and advanced control techniques are discussed. When induction motors are operated from an energy source capable of controlling voltages and frequencies independently, drive characteristics are obtained which are superior to either PM or SR motors. By synthesizing the machine frequency from a high-frequency carrier (nominally 20 kHz), high efficiencies, low distortion, and rapid torque response are available. At this time multiple horsepower machine drives were demonstrated, and work is on-going to develop a 20 hp average, 40 hp peak class of aerospace actuators. This effort is based upon high-frequency power distribution and management techniques developed by NASA for Space Station Freedom.

  6. Inductively commutated coilguns

    Energy Technology Data Exchange (ETDEWEB)

    Mongeau, P.P. (EML Research, Inc., Hudson, MA (US))

    1991-01-01

    In this paper the concept and relevance of power factor is presented in regards to high performance launchers. As the scale of launchers grows and as efforts to improve efficiency continue power factor considerations will become crucial in engineering design and ultimate launcher performance limits. The use of motion induced commutation to improve the power factor are discussed. Various approaches to inductive commutation are presented, including: the brush-commutated 9 MJ Coilgun, the solid state-switched coilgun and the quenchgun.

  7. Inductively commutated coilguns

    Science.gov (United States)

    Mongeau, Peter P.

    1991-01-01

    The concept and relevance of power factor is presented in the context of high-performance launchers. As the scale of launchers grows and efforts to improve efficiency continue, power factor considerations will become crucial in engineering design and ultimate launcher performance limits. The use of motion-induced commutation to improve the power factor are discussed. Various approaches to inductive commutation are presented, including the brush-commutated 9-MJ coilgun, the solid state-switched coilgun, and the quenchgun.

  8. The Principle of Uniformitarianism and the Problem of Induction in Geology--A Geology Student's Search for Certainty

    Science.gov (United States)

    Serra, Sandro

    1978-01-01

    The argument presented here, against the principle of uniformitarianism as a means of transforming an inductive argument into a deductive one, is based on the fact that this principle is itself established by inductive reasoning. (MDR)

  9. A taxonomy of inductive problems.

    Science.gov (United States)

    Kemp, Charles; Jern, Alan

    2014-02-01

    Inductive inferences about objects, features, categories, and relations have been studied for many years, but there are few attempts to chart the range of inductive problems that humans are able to solve. We present a taxonomy of inductive problems that helps to clarify the relationships between familiar inductive problems such as generalization, categorization, and identification, and that introduces new inductive problems for psychological investigation. Our taxonomy is founded on the idea that semantic knowledge is organized into systems of objects, features, categories, and relations, and we attempt to characterize all of the inductive problems that can arise when these systems are partially observed. Recent studies have begun to address some of the new problems in our taxonomy, and future work should aim to develop unified theories of inductive reasoning that explain how people solve all of the problems in the taxonomy.

  10. Toward a unified chromatic induction model.

    Science.gov (United States)

    Otazu, Xavier; Parraga, C Alejandro; Vanrell, Maria

    2010-10-01

    In a previous work (X. Otazu, M. Vanrell, & C. A. Párraga, 2008b), we showed how several brightness induction effects can be predicted using a simple multiresolution wavelet model (BIWaM). Here we present a new model for chromatic induction processes (termed Chromatic Induction Wavelet Model or CIWaM), which is also implemented on a multiresolution framework and based on similar assumptions related to the spatial frequency and the contrast surround energy of the stimulus. The CIWaM can be interpreted as a very simple extension of the BIWaM to the chromatic channels, which in our case are defined in the MacLeod-Boynton (lsY) color space. This new model allows us to unify both chromatic assimilation and chromatic contrast effects in a single mathematical formulation. The predictions of the CIWaM were tested by means of several color and brightness induction experiments, which showed an acceptable agreement between model predictions and psychophysical data.

  11. Inductively Coupled Augmented Railgun

    CERN Document Server

    Bahder, Thomas B

    2011-01-01

    We derive the non-linear dynamical equations for an augmented electromagnetic railgun, whose augmentation circuit is inductively coupled to the gun circuit. We solve these differential equations numerically using example parameter values. We find a complicated interaction between the augmentation circuit, gun circuit, and mechanical degrees of freedom, leading to a complicated optimization problem. For certain values of parameters, we find that an augmented electromagnetic railgun has an armature kinetic energy that is 42% larger than the same railgun with no augmentation circuit. Optimizing the parameters may lead to further increase in performance.

  12. Low inductance gas switching.

    Energy Technology Data Exchange (ETDEWEB)

    Chavez, Ray; Harjes, Henry Charles III; Wallace, Zachariah; Elizondo, Juan E.

    2007-10-01

    The laser trigger switch (LTS) is a key component in ZR-type pulsed power systems. In ZR, the pulse rise time through the LTS is > 200 ns and additional stages of pulse compression are required to achieve the desired <100 ns rise time. The inductance of the LTS ({approx}500nH) in large part determines the energy transfer time through the switch and there is much to be gained in improving system performance and reducing system costs by reducing this inductance. The current path through the cascade section of the ZR LTS is at a diameter of {approx} 6-inches which is certainly not optimal from an inductance point of view. The LTS connects components of much greater diameter (typically 4-5 feet). In this LDRD the viability of switch concepts in which the diameter of cascade section is greatly increased have been investigated. The key technical question to be answered was, will the desired multi-channel behavior be maintained in a cascade section of larger diameter. This LDRD proceeded in 2 distinct phases. The original plan for the LDRD was to develop a promising switch concept and then design, build, and test a moderate scale switch which would demonstrate the key features of the concept. In phase I, a switch concept which meet all electrical design criteria and had a calculated inductance of 150 nH was developed. A 1.5 MV test switch was designed and fabrication was initiated. The LDRD was then redirected due to budgetary concerns. The fabrication of the switch was halted and the focus of the LDRD was shifted to small scale experiments designed to answer the key technical question concerning multi-channel behavior. In phase II, the Multi-channel switch test bed (MCST) was designed and constructed. The purpose of MCST was to provide a versatile, fast turn around facility for the study the multi-channel electrical breakdown behavior of a ZR type cascade switch gap in a parameter space near that of a ZR LTS. Parameter scans on source impedance, gap tilt, gap spacing and

  13. Induction motor starting current

    Energy Technology Data Exchange (ETDEWEB)

    Arneaud, J.M.; Langman, R.A. [Tasmania Univ., Hobart, TAS (Australia)

    1995-12-31

    Large errors may occur if leakage path saturation is neglected when reduced-voltage test results are used to predict the direct-on-line starting current of induction motors. The results of applying three existing and two new methods for starting current prediction are compared with test data from 52 motors. A quantitative assessment is made of the probable reduction in error that would be achieved by increasing the number of available sets of reduced-voltage, locked rotor test results or by including slot design data. Guidelines are given for selecting an appropriate predictive method. (author). 4 tabs., 1 fig., 6 refs.

  14. Protein engineering of toluene 4-monooxygenase of Pseudomonas mendocina KR1 for synthesizing 4-nitrocatechol from nitrobenzene.

    Science.gov (United States)

    Fishman, Ayelet; Tao, Ying; Bentley, William E; Wood, Thomas K

    2004-09-20

    After discovering that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes nitrobenzene to 4-nitrocatechol, albeit at a very low rate, this reaction was improved using directed evolution and saturation mutagenesis. Screening 550 colonies from a random mutagenesis library generated by error-prone PCR of tmoAB using Escherichia coli TG1/pBS(Kan)T4MO on agar plates containing nitrobenzene led to the discovery of nitrocatechol-producing mutants. One mutant, NB1, contained six amino acid substitutions (TmoA Y22N, I84Y, S95T, I100S, S400C; TmoB D79N). It was believed that position I100 of the alpha subunit of the hydroxylase (TmoA) is the most significant for the change in substrate reactivity due to previous results in our lab with a similar enzyme, toluene ortho-monooxygenase of Burkholderia cepacia G4. Saturation mutagenesis at this position resulted in the generation of two more nitrocatechol mutants, I100A and I100S; the rate of 4-nitrocatechol formation by I100A was more than 16 times higher than that of wild-type T4MO at 200 microM nitrobenzene (0.13 +/- 0.01 vs. 0.008 +/- 0.001 nmol/min.mg protein). HPLC and mass spectrometry analysis revealed that variants NB1, I100A, and I100S produce 4-nitrocatechol via m-nitrophenol, while the wild-type produces primarily p-nitrophenol and negligible amounts of nitrocatechol. Relative to wild-type T4MO, whole cells expressing variant I100A convert nitrobenzene into m-nitrophenol with a Vmax of 0.61 +/- 0.037 vs. 0.16 +/- 0.071 nmol/min.mg protein and convert m-nitrophenol into nitrocatechol with a Vmax of 3.93 +/- 0.26 vs. 0.58 +/- 0.033 nmol/min.mg protein. Hence, the regiospecificity of nitrobenzene oxidation was changed by the random mutagenesis, and this led to a significant increase in 4-nitrocatechol production. The regiospecificity of toluene oxidation was also altered, and all of the mutants produced 20% m-cresol and 80% p-cresol, while the wild-type produces 96% p-cresol. Interestingly, the rate of

  15. Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Baldwin, J.; Voegtli, W.C.; Khidekel, N.; Moënne-Loccoz, P.; Krebs, C.; Ley, B.A.; Huynh, B.H.; Loehr, T.M.; Rosenzweig, A.C.; Bollinger, Jr., J.M. (NWU)

    2010-03-05

    The outcome of O{sub 2} activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine){sub 2}-(carboxylate){sub 4} ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a {mu}-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H{sub peroxo}) in O{sub 2} activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the 'extra' electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the {mu}-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122 and which converts very slowly (t{sub 1/2} {approx} 7 h) upon incubation at 0 C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone {epsilon}-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with {sup 16}O{sub 2} or {sub 18}O{sub 2} show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O{sub 2}. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Moessbauer and EXAFS characterization of

  16. Theory-based Bayesian models of inductive learning and reasoning.

    Science.gov (United States)

    Tenenbaum, Joshua B; Griffiths, Thomas L; Kemp, Charles

    2006-07-01

    Inductive inference allows humans to make powerful generalizations from sparse data when learning about word meanings, unobserved properties, causal relationships, and many other aspects of the world. Traditional accounts of induction emphasize either the power of statistical learning, or the importance of strong constraints from structured domain knowledge, intuitive theories or schemas. We argue that both components are necessary to explain the nature, use and acquisition of human knowledge, and we introduce a theory-based Bayesian framework for modeling inductive learning and reasoning as statistical inferences over structured knowledge representations.

  17. The dance of meaning

    DEFF Research Database (Denmark)

    Rasmussen, Ole Elstrup

    2005-01-01

    competence, qualifications, sense making, reasoning, meaning, intentionality, interpersonal relationship......competence, qualifications, sense making, reasoning, meaning, intentionality, interpersonal relationship...

  18. Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1

    Science.gov (United States)

    Koh, Sung-Cheol; Bowman, John P.; Sayler, Gary S.

    1993-01-01

    A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in Methylosinus trichosporium OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 551 ± 27 and 321 ± 16 nmol h-1 mg of protein -1, respectively. The maximal TCE degradation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 2,325 ± 260 and 995 ± 160 nmol h-1 mg of protein-1, respectively. The substrate affinity of 68-1 sMMO to naphthalene (Km, 70 ± 4 μM) and TCE (Km, 225 ± 13 μM), however, was comparatively lower than that of the sMMO of OB3b, which had affinities of 40 ± 3 and 126 ± 8 μM, respectively. Genomic DNA slot and Southern blot analyses with an sMMO gene probe from Methylosinus trichosporium OB3b showed that the sMMO genes of 68-1 have little genetic homology to those of OB3b. This result may indicate the evolutionary diversification of the sMMOs. Images PMID:16348920

  19. Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA.

    Science.gov (United States)

    Robinson, Reeder; Franceschini, Stefano; Fedkenheuer, Michael; Rodriguez, Pedro J; Ellerbrock, Jacob; Romero, Elvira; Echandi, Maria Paulina; Martin Del Campo, Julia S; Sobrado, Pablo

    2014-04-01

    Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP⁺ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Genetic Variant in Flavin-Containing Monooxygenase 3 Alters Lipid Metabolism in Laying Hens in a Diet-Specific Manner

    Science.gov (United States)

    Wang, Jing; Long, Cheng; Zhang, Haijun; Zhang, Yanan; Wang, Hao; Yue, Hongyuan; Wang, Xiaocui; Wu, Shugeng; Qi, Guanghai

    2016-01-01

    Genetic variant T329S in flavin-containing monooxygenase 3 (FMO3) impairs trimethylamine (TMA) metabolism in birds. The TMA metabolism that under complex genetic and dietary regulation, closely linked to cardiovascular disease risk. We determined whether the genetic defects in TMA metabolism may change other metabolic traits in birds, determined whether the genetic effects depend on diets, and to identify genes or gene pathways that underlie the metabolic alteration induced by genetic and diet factors. We used hens genotyped as FMO3 c.984 A>T as well as those with the homozygous normal genotype. For each genotype, hens were provided with either a corn-soybean meal basal diets (SM), which contains lower levels of TMA precursor, or the basal diets supplemented with 21% of rapeseed meal (RM), which contains higher levels of TMA precursor. An integrative analysis of metabolomic and transcriptomic was used to explore the metabolic patterns of FMO3 genetic variant in hens that were fed the two defined diets. In birds that consumed SM diets, the T329S mutation increased levels of plasma TMA and lipids, FMO3 mRNA levels, and the expression of genes involved in long chain polyunsaturated fatty acid biosynthesis. In birds that consumed RM diets, the T329S mutation induced fishy odor syndrome, a repression in LXR pathway and a reciprocal change in lipid metabolism. Variations in TMA and lipid metabolism were linked to the genetic variant in FMO3 in a diet-specific manner, which suggest FMO3 functions in TMA metabolism and lipid homeostasis. LXR pathway and polyunsaturated fatty acid metabolism are two possible mechanisms of FMO3 action in response to dietary TMA precursor. PMID:27877090

  1. Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.

    Science.gov (United States)

    Cafaro, Valeria; Scognamiglio, Roberta; Viggiani, Ambra; Izzo, Viviana; Passaro, Irene; Notomista, Eugenio; Piaz, Fabrizio Dal; Amoresano, Angela; Casbarra, Annarita; Pucci, Piero; Di Donato, Alberto

    2002-11-01

    This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.

  2. A cytochrome P450 monooxygenase commonly used for negative selection in transgenic plants causes growth anomalies by disrupting brassinosteroid signaling

    Directory of Open Access Journals (Sweden)

    Manivasagam Sindhu

    2011-04-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases form a large superfamily of enzymes that catalyze diverse reactions. The P450SU1 gene from the soil bacteria Streptomyces griseolus encodes CYP105A1 which acts on various substrates including sulfonylurea herbicides, vitamin D, coumarins, and based on the work presented here, brassinosteroids. P450SU1 is used as a negative-selection marker in plants because CYP105A1 converts the relatively benign sulfonyl urea pro-herbicide R7402 into a highly phytotoxic product. Consistent with its use for negative selection, transgenic Arabidopsis plants were generated with P450SU1 situated between recognition sequences for FLP recombinase from yeast to select for recombinase-mediated excision. However, unexpected and prominent developmental aberrations resembling those described for mutants defective in brassinosteroid signaling were observed in many of the lines. Results The phenotypes of the most affected lines included severe stunting, leaf curling, darkened leaves characteristic of anthocyanin accumulation, delayed transition to flowering, low pollen and seed yields, and delayed senescence. Phenotype severity correlated with P450SU1 transcript abundance, but not with transcript abundance of other experimental genes, strongly implicating CYP105A1 as responsible for the defects. Germination and seedling growth of transgenic and control lines in the presence and absence of 24-epibrassinolide indicated that CYP105A1 disrupts brassinosteroid signaling, most likely by inactivating brassinosteroids. Conclusions Despite prior use of this gene as a genetic tool, deleterious growth in the absence of R7402 has not been elaborated. We show that this gene can cause aberrant growth by disrupting brassinosteroid signaling and affecting homeostasis.

  3. The metabolomics of (+/-)-arecoline 1-oxide in the mouse and its formation by human flavin-containing monooxygenases.

    Science.gov (United States)

    Giri, Sarbani; Krausz, Kristopher W; Idle, Jeffrey R; Gonzalez, Frank J

    2007-02-15

    The alkaloid arecoline is a main constituent of areca nuts that are chewed by approximately 600 million persons worldwide. A principal metabolite of arecoline is arecoline 1-oxide whose metabolism has been poorly studied. To redress this, synthetic (+/-)-arecoline 1-oxide was administered to mice (20mg/kg p.o.) and a metabolomic study performed on 0-12h urine using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) with multivariate data analysis. A total of 16 mass/retention time pairs yielded 13 metabolites of (+/-)-arecoline 1-oxide, most of them novel. Identity of metabolites was confirmed by tandem mass spectrometry. The principal pathways of metabolism of (+/-)-arecoline 1-oxide were mercapturic acid formation, with catabolism to mercaptan and methylmercaptan metabolites, apparent CC double-bond reduction, carboxylic acid reduction to the aldehyde (a novel pathway in mammals), N-oxide reduction, and de-esterification. Relative percentages of metabolites were determined directly from the metabolomic data. Approximately, 50% of the urinary metabolites corresponded to unchanged (+/-)-arecoline 1-oxide, 25% to other N-oxide metabolites, while approximately, 30% corresponded to mercapturic acids or their metabolites. Many metabolites, principally mercapturic acids and their derivatives, were excreted as diastereomers that could be resolved by UPLC-TOFMS. Arecoline was converted to arecoline 1-oxide in vitro by human flavin-containing monooxygenases FMO1 (K(M): 13.6+/-4.9muM; V(MAX): 0.114+/-0.01nmolmin(-1)microg(-1) protein) and FMO3 (K(M): 44.5+/-8.0microM; V(MAX): 0.014+/-0.001nmolmin(-1)microg(-1) protein), but not by FMO5 or any of 11 human cytochromes P450. This report underscores the power of metabolomics in drug metabolite mining.

  4. The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus.

    Science.gov (United States)

    Táncsics, András; Benedek, Tibor; Szoboszlay, Sándor; Veres, Péter G; Farkas, Milán; Máthé, István; Márialigeti, Károly; Kukolya, József; Lányi, Szabolcs; Kriszt, Balázs

    2015-02-01

    Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus. Copyright © 2014 Elsevier GmbH. All rights reserved.

  5. Possible Peroxo State of the Dicopper Site of Particulate Methane Monooxygenase from Combined Quantum Mechanics and Molecular Mechanics Calculations.

    Science.gov (United States)

    Itoyama, Shuhei; Doitomi, Kazuki; Kamachi, Takashi; Shiota, Yoshihito; Yoshizawa, Kazunari

    2016-03-21

    Enzymatic methane hydroxylation is proposed to efficiently occur at the dinuclear copper site of particulate methane monooxygenase (pMMO), which is an integral membrane metalloenzyme in methanotrophic bacteria. The resting state and a possible peroxo state of the dicopper active site of pMMO are discussed by using combined quantum mechanics and molecular mechanics calculations on the basis of reported X-ray crystal structures of the resting state of pMMO by Rosenzweig and co-workers. The dicopper site has a unique structure, in which one copper is coordinated by two histidine imidazoles and another is chelated by a histidine imidazole and primary amine of an N-terminal histidine. The resting state of the dicopper site is assignable to the mixed-valent Cu(I)Cu(II) state from a computed Cu-Cu distance of 2.62 Å from calculations at the B3LYP-D/TZVP level of theory. A μ-η(2):η(2)-peroxo-Cu(II)2 structure similar to those of hemocyanin and tyrosinase is reasonably obtained by using the resting state structure and dioxygen. Computed Cu-Cu and O-O distances are 3.63 and 1.46 Å, respectively, in the open-shell singlet state. Structural features of the dicopper peroxo species of pMMO are compared with those of hemocyanin and tyrosinase and synthetic dicopper model compounds. Optical features of the μ-η(2):η(2)-peroxo-Cu(II)2 state are calculated and analyzed with TD-DFT calculations.

  6. Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme.

    Science.gov (United States)

    Bonnemaison, Mathilde L; Bäck, Nils; Duffy, Megan E; Ralle, Martina; Mains, Richard E; Eipper, Betty A

    2015-08-28

    The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme*

    Science.gov (United States)

    Bonnemaison, Mathilde L.; Bäck, Nils; Duffy, Megan E.; Ralle, Martina; Mains, Richard E.; Eipper, Betty A.

    2015-01-01

    The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised. PMID:26170456

  8. Cooperation between MEF2 and PPARγ in human intestinal β,β-carotene 15,15'-monooxygenase gene expression

    Directory of Open Access Journals (Sweden)

    Yan Bingfang

    2006-02-01

    Full Text Available Abstract Background Vitamin A and its derivatives, the retinoids, are essential for normal embryonic development and maintenance of cell differentiation. β, β-carotene 15,15'-monooxygenase 1 (BCMO1 catalyzes the central cleavage of β-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A. However, human and various rodent species show markedly different efficiencies in intestinal BCMO1-mediated carotene to retinoid conversion. The aim of this study is to identify potentially human-specific regulatory control mechanisms of BCMO1 gene expression. Results We identified and functionally characterized the human BCMO1 promoter sequence and determined the transcriptional regulation of the BCMO1 gene in a BCMO1 expressing human intestinal cell line, TC-7. Several functional transcription factor-binding sites were identified in the human promoter that are absent in the mouse BCMO1 promoter. We demonstrate that the proximal promoter sequence, nt -190 to +35, confers basal transcriptional activity of the human BCMO1 gene. Site-directed mutagenesis of the myocyte enhancer factor 2 (MEF2 and peroxisome proliferator-activated receptor (PPAR binding elements resulted in decreased basal promoter activity. Mutation of both promoter elements abrogated the expression of intestinal cell BCMO1. Electrophoretic mobility shift and supershift assays and transcription factor co-expression in TC-7 cells showed MEF2C and PPARγ bind to their respective DNA elements and synergistically transactivate BCMO1 expression. Conclusion We demonstrate that human intestinal cell BCMO1 expression is dependent on the functional cooperation between PPARγ and MEF2 isoforms. The findings suggest that the interaction between MEF2 and PPAR factors may provide a molecular basis for interspecies differences in the transcriptional regulation of the BCMO1 gene.

  9. Creativity and Memory: Effects of an Episodic Specificity Induction on Divergent Thinking

    Science.gov (United States)

    Madore, Kevin P.; Addis, Donna Rose; Schacter, Daniel L.

    2015-01-01

    After receiving an episodic specificity induction - brief training in recollecting details of a recent event - people produce more episodic details when imagining future events and solving means-end problems than after receiving a control induction not focused on episodic retrieval. Here we show for the first time that an episodic specificity induction also enhances divergent creative thinking. In Experiment 1, participants exhibited a selective boost on a divergent thinking task that involves generating unusual uses of common objects after a specificity induction compared with a control induction; by contrast, performance was similar on an object association task thought to involve little divergent thinking. In Experiment 2, we replicated the specificity induction effect on divergent thinking using a different control induction, and also found that participants performed similarly on a convergent thinking task following both inductions. These experiments provide novel evidence that episodic memory is involved in divergent creative thinking. PMID:26205963

  10. Pulsed inductive HF laser

    Energy Technology Data Exchange (ETDEWEB)

    Razhev, A M; Kargapol' tsev, E S [Institute of Laser Physics, Siberian Branch, Russian Academy of Sciences, Novosibirsk (Russian Federation); Churkin, D S; Demchuk, S V [Novosibirsk State University, Novosibirsk (Russian Federation)

    2016-03-31

    We report the results of experimentally investigated dependences of temporal, spectral and spatial characteristics of an inductive HF-laser generation on the pump conditions. Gas mixtures H{sub 2} – F{sub 2}(NF{sub 3} or SF6{sub 6}) and He(Ne) – H{sub 2} – F{sub 2}(NF{sub 3} or SF{sub 6}) were used as active media. The FWHM pulse duration reached 0.42 μs. This value corresponded to a pulsed power of 45 kW. For the first time, the emission spectrum of an inductive HF laser was investigated, which consisted of seven groups of bands with centres around the wavelengths of 2732, 2736, 2739, 2835, 2837, 2893 and 2913 nm. The cross section profile of the laser beam was a ring with a diameter of about 20 mm and width of about 5 mm. Parameters of laser operation in the repetitively pulsed regime were sufficiently stable. The amplitude instability of light pulses was no greater than 5% – 6%. (lasers)

  11. Identification of the Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase regulatory gene (touR) and of its cognate promoter.

    Science.gov (United States)

    Arenghi, F L; Pinti, M; Galli, E; Barbieri, P

    1999-09-01

    Toluene-o-xylene monooxygenase is an enzymatic complex, encoded by the touABCDEF genes, responsible for the early stages of toluene and o-xylene degradation in Pseudomonas stutzeri OX1. In order to identify the loci involved in the transcriptional regulation of the tou gene cluster, deletion analysis and complementation studies were carried out with Pseudomonas putida PaW340 as a heterologous host harboring pFB1112, a plasmid that allowed regulated expression, inducible by toluene and o-xylene and their corresponding phenols, of the toluene-o-xylene monooxygenase. A locus encoding a positive regulator, designated touR, was mapped downstream from the tou gene cluster. TouR was found to be similar to transcriptional activators of aromatic compound catabolic pathways belonging to the NtrC family and, in particular, to DmpR (83% similarity), which controls phenol catabolism. By using a touA-C2,3O fusion reporter system and by primer extension analysis, a TouR cognate promoter (P(ToMO)) was mapped, which showed the typical -24 TGGC, -12 TTGC sequences characteristic of sigma(54)-dependent promoters and putative upstream activating sequences. By using the reporter system described, we found that TouR responds to mono- and dimethylphenols, but not the corresponding methylbenzenes. In this respect, the regulation of the P. stutzeri system differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effectors. Northern analyses indicated low transcription levels of tou structural genes in the absence of inducers. Basal toluene-o-xylene monooxygenase activity may thus transform these compounds to phenols, which then trigger the TouR-mediated response.

  12. The plmS2-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803

    OpenAIRE

    Mohini S. Ghatge; Reynolds, Kevin A.

    2005-01-01

    Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS2 open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Ki...

  13. A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

    OpenAIRE

    KOJIMA, Yuka; Várnai, Anikó; Ishida, Takuya; Sunagawa, Naoki; Petrovic, Dejan M.; Igarashi, Kiyohiko; Jellison, Jody; GOODELL, BARRY; Alfredsen, Gry; Westereng, Bjørge; Vincent G H Eijsink; Yoshida, Makoto

    2016-01-01

    ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and...

  14. Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23

    OpenAIRE

    2014-01-01

    An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 A degrees C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladA alpha(B23), ladA beta(B23), and ladB (B23), on its chromosome, whose protein products sh...

  15. Vector control of induction machines

    CERN Document Server

    Robyns, Benoit

    2012-01-01

    After a brief introduction to the main law of physics and fundamental concepts inherent in electromechanical conversion, ""Vector Control of Induction Machines"" introduces the standard mathematical models for induction machines - whichever rotor technology is used - as well as several squirrel-cage induction machine vector-control strategies. The use of causal ordering graphs allows systematization of the design stage, as well as standardization of the structure of control devices. ""Vector Control of Induction Machines"" suggests a unique approach aimed at reducing parameter sensitivity for

  16. Induction Linac Pulsers

    Energy Technology Data Exchange (ETDEWEB)

    Faltens, Andris

    2011-01-07

    The pulsers used in most of the induction linacs evolved from the very large body of work that was done in the U.S. and Great Britain during the development of the pulsed magnetron for radar. The radar modulators started at {approx}100 kW and reached >10 MW by 1945. A typical pulse length was 1 {mu}s at a repetition rate of 1,000 pps. A very comprehensive account of the modulator development is Pulse Generators by Lebacqz and Glasoe, one of the Radiation Laboratory Series. There are many permutations of possible modulators, two of the choices being tube type and line type. In earlier notes I wrote that technically the vacuum tube pulser met all of our induction linac needs, in the sense that a number of tubes, in series and parallel if required, could produce our pulses, regulate their voltage, be useable in feed-forward correctors, and provide a low source impedance. At a lower speed, an FET array is similar, and we have obtained and tested a large array capable of >10 MW switching. A modulator with an electronically controlled output only needs a capacitor for energy storage and in a switched mode can transfer the energy from the capacitor to the load at high efficiency. Driving a full size Astron induction core and a simulated resistive 'beam load' we achieved >50% efficiency. These electronically controlled output pulses can produce the pulses we desire but are not used because of their high cost. The second choice, the line type pulser, visually comprises a closing switch and a distributed or a lumped element transmission line. The typical switch cannot open or stop conducting after the desired pulse has been produced, and consequently all of the initially stored energy is dissipated. This approximately halves the efficiency, and the original cost estimating program LIACEP used this factor of two, even though our circuits are usually worse, and even though our inveterate optimists often omit it. The 'missing' energy is that which is

  17. Construction of Meaning: Film.

    Science.gov (United States)

    Pryluck, Calvin

    1995-01-01

    Notes that film has no clear set of rules, unlike all languages, which are deductive systems interpreted according to clear sets of rules. Suggests that film is an inductive system whose interpretation is based on a general understanding of events depicted as modified by production variables such as lighting, camera angles, and the context of…

  18. Cooling by Thermodynamic Induction

    Science.gov (United States)

    Patitsas, S. N.

    2017-03-01

    A method is described for cooling conductive channels to below ambient temperature. The thermodynamic induction principle dictates that the electrically biased channel will cool if the electrical conductance decreases with temperature. The extent of this cooling is calculated in detail for both cases of ballistic and conventional transport with specific calculations for carbon nanotubes and conventional metals, followed by discussions for semiconductors, graphene, and metal-insulator transition systems. A theorem is established for ballistic transport stating that net cooling is not possible. For conventional transport, net cooling is possible over a broad temperature range, with the range being size-dependent. A temperature clamping scheme for establishing a metastable nonequilibrium stationary state is detailed and followed with discussion of possible applications to on-chip thermoelectric cooling in integrated circuitry and quantum computer systems.

  19. Cooling by Thermodynamic Induction

    Science.gov (United States)

    Patitsas, S. N.

    2016-11-01

    A method is described for cooling conductive channels to below ambient temperature. The thermodynamic induction principle dictates that the electrically biased channel will cool if the electrical conductance decreases with temperature. The extent of this cooling is calculated in detail for both cases of ballistic and conventional transport with specific calculations for carbon nanotubes and conventional metals, followed by discussions for semiconductors, graphene, and metal-insulator transition systems. A theorem is established for ballistic transport stating that net cooling is not possible. For conventional transport, net cooling is possible over a broad temperature range, with the range being size-dependent. A temperature clamping scheme for establishing a metastable nonequilibrium stationary state is detailed and followed with discussion of possible applications to on-chip thermoelectric cooling in integrated circuitry and quantum computer systems.

  20. Kinetic inductance magnetometer.

    Science.gov (United States)

    Luomahaara, Juho; Vesterinen, Visa; Grönberg, Leif; Hassel, Juha

    2014-09-10

    Sensing ultra-low magnetic fields has various applications in the fields of science, medicine and industry. There is a growing need for a sensor that can be operated in ambient environments where magnetic shielding is limited or magnetic field manipulation is involved. To this end, here we demonstrate a new magnetometer with high sensitivity and wide dynamic range. The device is based on the current nonlinearity of superconducting material stemming from kinetic inductance. A further benefit of our approach is of extreme simplicity: the device is fabricated from a single layer of niobium nitride. Moreover, radio frequency multiplexing techniques can be applied, enabling the simultaneous readout of multiple sensors, for example, in biomagnetic measurements requiring data from large sensor arrays.

  1. Bayesian Causal Induction

    CERN Document Server

    Ortega, Pedro A

    2011-01-01

    Discovering causal relationships is a hard task, often hindered by the need for intervention, and often requiring large amounts of data to resolve statistical uncertainty. However, humans quickly arrive at useful causal relationships. One possible reason is that humans use strong prior knowledge; and rather than encoding hard causal relationships, they encode beliefs over causal structures, allowing for sound generalization from the observations they obtain from directly acting in the world. In this work we propose a Bayesian approach to causal induction which allows modeling beliefs over multiple causal hypotheses and predicting the behavior of the world under causal interventions. We then illustrate how this method extracts causal information from data containing interventions and observations.

  2. Inductive Position Sensor

    Science.gov (United States)

    Youngquist, Robert C. (Inventor); Simmons, Stephen M. (Inventor)

    2015-01-01

    An inductive position sensor uses three parallel inductors, each of which has an axial core that is an independent magnetic structure. A first support couples first and second inductors and separate them by a fixed distance. A second support coupled to a third inductor disposed between the first and second inductors. The first support and second support are configured for relative movement as distance changes from the third inductor to each of the first and second inductors. An oscillating current is supplied to the first and second inductors. A device measures a phase component of a source voltage generating the oscillating current and a phase component of voltage induced in the third inductor when the oscillating current is supplied to the first and second inductors such that the phase component of the voltage induced overlaps the phase component of the source voltage.

  3. Electromagnetic induction studies

    Science.gov (United States)

    Hermance, J. F.

    1983-04-01

    Recent developments in electromagnetic induction studies of the lithosphere and the asthenosphere are reviewed. Attention is given to geoelectrical studies of active tectonic areas in terms of the major zones of crustal extension, the basin and range province along western regions of North America, and the Rio Grande rift. Studies have also been performed of tectonic activity around Iceland, the Salton Trough and Cerro Prieto, and the subduction zones of the Cascade Mountains volcanic belt, where magnetotelluric and geomagnetic variation studies have been done. Geomagnetic variations experiments have been reported in the Central Appalachians, and submarine electromagnetic studies along the Juan de Fuca ridge. Controlled source electromagnetic and dc resistivity investigations have been carried out in Nevada, Hawaii, and in the Adirondacks Mountains. Laboratory examinations on the conductivity of representative materials over a broad range of temperature, pressure, and chemistry are described.

  4. Inductive Electron Heating Revisited

    Science.gov (United States)

    Tuszewski, M.

    1996-11-01

    Inductively Coupled Plasmas (ICPs) have been studied for over a century. Recently, ICPs have been rediscovered by the multi-billion dollar semiconductor industry as an important class of high-density, low-pressure plasma sources suitable for the manufacture of next-generation integrated circuits. Present low-pressure ICP development is among the most active areas of plasma research. However, this development remains largely empirical, a prohibitively expensive approach for upcoming 300-mm diameter wafers. Hence, there is an urgent need for basic ICP plasma physics research, including experimental characterization and predictive numerical modeling. Inductive radio frequency (rf) power absorption is fundamental to the ICP electron heating and the resulting plasma transport but remains poorly understood. For example, recent experimental measurements and supporting fluid calculationsfootnote M. Tuszewski, Phys. Rev. Lett. 77 in press (1996) on a commercial deposition tool prototype show that the induced rf magnetic fields in the source can cause an order of magnitude reduction in plasma conductivity and in electron heating power density. In some cases, the rf fields penetrate through the entire volume of the ICP discharges while existing models that neglect the induced rf magnetic fields predict rf absorption in a thin skin layer near the plasma surface. The rf magnetic fields also cause more subtle changes in the plasma density and in the electron temperature spatial distributions. These data will be presented and the role of basic research in the applied world of semiconductor manufacturing will be discussed. ^*This research was conducted under the auspices of the U.S. DOE, supported by funds provided by the University of California for discretionary research by Los Alamos National Laboratory.

  5. The membrane-associated monooxygenase in the butane-oxidizing Gram-positive bacterium Nocardioides sp. strain CF8 is a novel member of the AMO/PMO family.

    Science.gov (United States)

    Sayavedra-Soto, Luis A; Hamamura, Natsuko; Liu, Chih-Wen; Kimbrel, Jeffrey A; Chang, Jeff H; Arp, Daniel J

    2011-06-01

    The Gram-positive bacterium Nocardioides sp. strain CF8 uses a membrane-associated monooxygenase (pBMO) to grow on butane. The nucleotide sequences of the genes encoding this novel monooxygenase were revealed through analysis of a de novo assembled draft genome sequence determined by high-throughput sequencing of the whole genome. The pBMO genes were in a similar arrangement to the genes for ammonia monooxygenase (AMO) from the ammonia-oxidizing bacteria and for particulate methane monooxygenase (pMMO) from the methane-oxidizing bacteria. The pBMO genes likely constitute an operon in the order bmoC, bmoA and bmoB. The nucleotide sequence was less than 50% similar to the genes for AMO and pMMO. The operon for pBMO was confirmed to be a single copy in the genome by Southern and computational analyses. In an incubation on butane the increase of transcriptional activity of the pBmoA gene was congruent with the increase of pBMO activity and suggested correspondence between gene expression and the utilization of butane. Phylogenetic comparison revealed distant but significant similarity of all three pBMO subunits to homologous members of the AMO/pMMO family and indicated that the pBMO represents a deeply branching third lineage of this group of particulate monooxygenases. No other bmoCAB-like genes were found to cluster with pBMO lineage in phylogenetic analysis by database searches including genomic and metagenomic sequence databases. pBMO is the first example of the AMO/pMMO-like monooxygenase from Gram-positive bacteria showing similarities to proteobacterial pMMO and AMO sequences. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  6. Graph-based inductive reasoning.

    Science.gov (United States)

    Boumans, Marcel

    2016-10-01

    This article discusses methods of inductive inferences that are methods of visualizations designed in such a way that the "eye" can be employed as a reliable tool for judgment. The term "eye" is used as a stand-in for visual cognition and perceptual processing. In this paper "meaningfulness" has a particular meaning, namely accuracy, which is closeness to truth. Accuracy consists of precision and unbiasedness. Precision is dealt with by statistical methods, but for unbiasedness one needs expert judgment. The common view at the beginning of the twentieth century was to make the most efficient use of this kind of judgment by representing the data in shapes and forms in such a way that the "eye" can function as a reliable judge to reduce bias. The need for judgment of the "eye" is even more necessary when the background conditions of the observations are heterogeneous. Statistical procedures require a certain minimal level of homogeneity, but the "eye" does not. The "eye" is an adequate tool for assessing topological similarities when, due to heterogeneity of the data, metric assessment is not possible. In fact, graphical assessments precedes measurement, or to put it more forcefully, the graphic method is a necessary prerequisite for measurement.

  7. Making meaning(s with cover designs

    Directory of Open Access Journals (Sweden)

    Yvonne Reed

    2011-08-01

    Full Text Available In this article Halliday’s systemic functional grammar, Kress and Van Leeuwen’s grammar of visual design and Bernstein’s work on pedagogic discourse are drawn on in order to analyze some of the ways in which meanings are made and some meanings privileged over others in the designing, both visual and discursive, of the covers of three course books prepared for teacher education programmes in South Africa. The primary aim of this analysis is to understand what meanings are offered to readers as entrants into or as established members of communities of practice in the teaching profession and the academy.

  8. A Student Teamwork Induction Protocol

    Science.gov (United States)

    Kamau, Caroline; Spong, Abigail

    2015-01-01

    Faulty group processes have harmful effects on performance but there is little research about intervention protocols to pre-empt them in higher education. This naturalistic experiment compared a control cohort with an inducted cohort. The inducted cohort attended a workshop, consultations, elected a leader and used tools (a group log and group…

  9. Sampling Assumptions in Inductive Generalization

    Science.gov (United States)

    Navarro, Daniel J.; Dry, Matthew J.; Lee, Michael D.

    2012-01-01

    Inductive generalization, where people go beyond the data provided, is a basic cognitive capability, and it underpins theoretical accounts of learning, categorization, and decision making. To complete the inductive leap needed for generalization, people must make a key "sampling" assumption about how the available data were generated.…

  10. From Inductive Reasoning to Proof

    Science.gov (United States)

    Yopp, David A.

    2009-01-01

    Mathematical proof is an expression of deductive reasoning (drawing conclusions from previous assertions). However, it is often inductive reasoning (conclusions drawn on the basis of examples) that helps learners form their deductive arguments, or proof. In addition, not all inductive arguments generate more formal arguments. This article draws a…

  11. Non-inductive current drive

    NARCIS (Netherlands)

    Westerhof, E.

    2012-01-01

    This lecture addresses the various ways of non-inductive current generation. In particular, the topics covered include the bootstrap current, RF current drive, neutral beam current drive, alternative methods, and possible synergies between different ways of non-inductive current generation.

  12. NON-INDUCTIVE CURRENT DRIVE

    NARCIS (Netherlands)

    Westerhof, E.

    2010-01-01

    This lecture addresses the various ways of non-inductive current generation. In particular, the topics covered include the bootstrap current, RF current drive, neutral beam current drive, alternative methods, and possible synergies between different ways of non-inductive current generation.

  13. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens.

    Directory of Open Access Journals (Sweden)

    Lehlohonolo Benedict Qhanya

    Full Text Available Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s, heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence. Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea, Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala, revealed the presence of numerous putative P450s ranging from 267 (A. mellea to 14 (M. osmundae. Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

  14. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens.

    Science.gov (United States)

    Qhanya, Lehlohonolo Benedict; Matowane, Godfrey; Chen, Wanping; Sun, Yuxin; Letsimo, Elizabeth Mpholoseng; Parvez, Mohammad; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Syed, Khajamohiddin

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

  15. The lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Otoikhian, Adenike [Oregon Health & Sciences University; Barry, Amanda N. [Los Alamos National Laboratory; Mayfield, Mary [Oregon Health & Science University; Nilges, Mark [Illinois EPR Center; Huang, Yiping [Johns Hopkins University; Lutsenko, Svetlana [Johns Hopkins University; Blackburn, Ninian [Oregon Health & Science University

    2012-05-14

    Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper translocating ATPase (ATP7A or ATP7B) but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1, 15N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased towards 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met while at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.

  16. Systematic identification and evolutionary analysis of catalytically versatile cytochrome p450 monooxygenase families enriched in model basidiomycete fungi.

    Directory of Open Access Journals (Sweden)

    Khajamohiddin Syed

    Full Text Available Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin's theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families, CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant

  17. Diversity of flavin-binding monooxygenase genes (almA) in marine bacteria capable of degradation long-chain alkanes.

    Science.gov (United States)

    Wang, Wanpeng; Shao, Zongze

    2012-06-01

    Many bacteria have been reported as degraders of long-chain (LC) n-alkanes, but the mechanism is poorly understood. Flavin-binding monooxygenase (AlmA) was recently found to be involved in LC-alkane degradation in bacteria of the Acinetobacter and Alcanivorax genera. However, the diversity of this gene and the role it plays in other bacteria remains unclear. In this study, we surveyed the diversity of almA in marine bacteria and in bacteria found in oil-enrichment communities. To identify the presence of this gene, a pair of degenerate PCR primers were was designed based on conserved motifs of the almA gene sequences in public databases. Using this approach, we identified diverse almA genes in the hydrocarbon-degrading bacteria and in bacterial communities from the surface seawater of the Xiamen coastal area, the South China Sea, the Indian Ocean, and the Atlantic Ocean. As a result, almA was positively detected in 35 isolates belonging to four genera, and a total of 39 different almA sequences were obtained. Five isolates were confirmed to harbor two to three almA genes. From the Xiamen coastal area and the Atlantic Ocean oil-enrichment communities, a total of 60 different almA sequences were obtained. These sequences mainly formed two clusters in the phylogenetic tree, named Class I and Class II, and these shared 45-56% identity at the amino acid level. Class I contained 11 sequences from bacteria represented by the Salinisphaera and Parvibaculum genera. Class II was larger and more diverse, and it was composed of 88 sequences from Proteobacteria, Gram-negative bacteria, and the enriched bacterial communities. These communities were represented by the Alcanivorax and Marinobacter genera, which are the two most popular genera hosting the almA gene. AlmA was also detected across a wide geographical range, as determined by the origin of the bacterial host. Our results demonstrate the diversity of almA and confirm its high rate of occurrence in hydrocarbon

  18. Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase.

    Science.gov (United States)

    Chauhan, Shefali; Hosseinzadeh, Parisa; Lu, Yi; Blackburn, Ninian J

    2016-04-01

    Peptidylglycine monooxygenase (PHM) is a dicopper enzyme that plays a vital role in the amidation of glycine-extended pro-peptides. One of the crucial aspects of its chemistry is the transfer of two electrons from an electron-storing and -transferring site (CuH) to the oxygen binding site and catalytic center (CuM) over a distance of 11 Å during one catalytic turnover event. Here we present our studies of the first electron transfer (ET) step (reductive phase) in wild-type (WT) PHM as well as its variants. Stopped flow was used to record the reduction kinetic traces using the chromophoric agent N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) as the reductant. The reduction was found to be biphasic in the WT PHM with an initial fast phase (17.2 s(-1)) followed by a much slower phase (0.46 s(-1)). We were able to ascribe the fast and slow phase to the CuH and CuM sites, respectively, by making use of the H242A and H107AH108A mutants that contain only the CuH site and CuM site, respectively. In the absence of substrate, the redox potentials determined by cyclic voltammetry were 270 mV (CuH site) and -15 mV (CuM site), but binding of substrate (Ac-YVG) was found to alter both potentials so that they converged to a common value of 83 mV. Substrate binding also accelerated the slow reductive phase by ~10-fold, an effect that could be explained at least partially by the equalization of the reduction potential of the copper centers. Studies of H108A showed that the ET to the CuM site is blocked, highlighting the role of the H108 ligand as a component of the reductive ET pathway. Strikingly, the rate of reduction of the H172A variant was unaffected despite the rate of catalysis being 3 orders of magnitude slower than that of the WT PHM. These studies strongly indicate that the reductive phase and catalytic phase ET pathways are different and suggest a bifurcated ET pathway in PHM. We propose that H172 and Y79 form part of an alternate pathway for the catalytic phase

  19. Misoprostol for induction of labor.

    Science.gov (United States)

    Stephenson, Megan L; Wing, Deborah A

    2015-10-01

    Labor-induction rates have increased considerably in the United States as well as around the world. With up to half of all induced labors requiring cervical ripening, prostaglandins have been utilized to increase induction success and achieve vaginal delivery. Misoprostol, a synthetic prostaglandin E1 analog has the ability to mimic the changes of spontaneous labor and has been used off label for over 30 years as a labor-induction agent. In the following article, cervical ripening and induction of labor with misoprostol will be discussed. The risks and benefits of misoprostol for ripening and induction and routes of administration will be reviewed, as well as future directions and new developments for its use.

  20. Induction: The Early Years of Teaching.

    Science.gov (United States)

    Guyton, Edi; Davis, Douglas; Colarusso, Ron; Grainger, Barbara

    2002-01-01

    Addresses the need for induction programs for beginning teachers and discusses the theoretical and research support for them. Develops the notion of induction as a community responsibility and describes a university-school induction consortium, a university induction program, an induction program at a middle school, and the role of the principal…

  1. Induction: The Early Years of Teaching.

    Science.gov (United States)

    Guyton, Edi; Davis, Douglas; Colarusso, Ron; Grainger, Barbara

    2002-01-01

    Addresses the need for induction programs for beginning teachers and discusses the theoretical and research support for them. Develops the notion of induction as a community responsibility and describes a university-school induction consortium, a university induction program, an induction program at a middle school, and the role of the principal…

  2. Discrimination of the prochiral hydrogens at the C-2 position of n-alkanes by the methane/ammonia monooxygenase family proteins.

    Science.gov (United States)

    Miyaji, Akimitsu; Miyoshi, Teppei; Motokura, Ken; Baba, Toshihide

    2015-08-14

    The selectivity of ammonia monooxygenase from Nitrosomonas europaea (AMO-Ne) for the oxidation of C4-C8n-alkanes to the corresponding alcohol isomers was examined to show the ability of AMO-Ne to recognize the n-alkane orientation within the catalytic site. AMO-Ne in whole cells produces 1- and 2-alcohols from C4-C8n-alkanes, and the regioselectivity is dependent on the length of the carbon chain. 2-Alcohols produced from C4-C7n-alkanes were predominantly either the R- or S-enantiomers, while 2-octanol produced from n-octane was racemic. These results indicate that AMO-Ne can discriminate between the prochiral hydrogens at the C-2 position, with the degree of discrimination varying according to the n-alkane. Compared to the particulate methane monooxygenase (pMMO) of Methylococcus capsulatus (Bath) and that of Methylosinus trichosporium OB3b, AMO-Ne showed a distinct ability to discriminate between the orientation of n-butane and n-pentane in the catalytic site.

  3. Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries.

    Science.gov (United States)

    Bouhajja, E; McGuire, M; Liles, M R; Bataille, G; Agathos, S N; George, I F

    2017-01-01

    The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.

  4. para-Nitrophenol 4-monooxygenase and hydroxyquinol 1,2-dioxygenase catalyze sequential transformation of 4-nitrocatechol in Pseudomonas sp. strain WBC-3.

    Science.gov (United States)

    Wei, Min; Zhang, Jun-Jie; Liu, Hong; Zhou, Ning-Yi

    2010-11-01

    Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy. PnpA (PNP 4-monooxygenase) and PnpB (para-benzoquinone reductase) were shown to be involved in the initial steps of PNP catabolism via hydroquinone. We demonstrated here that PnpA also catalyzed monooxygenation of 4-nitrocatechol (4-NC) to hydroxyquinol, probably via hydroxyquinone. It was the first time that a single-component PNP monooxygenase has been shown to catalyze this conversion. PnpG encoded by a gene located in the PNP degradation cluster was purified as a His-tagged protein and identified as a hydroxyquinol dioxygenase catalyzing a ring-cleavage reaction of hydroxyquinol. Although all the genes necessary for 4-NC metabolism seemed to be present in the PNP degradation cluster in strain WBC-3, it was unable to grow on 4-NC as a sole source of carbon, nitrogen and energy. This was apparently due to the substrate's inability to trigger the expression of genes involved in degradation. Nevertheless, strain WBC-3 could completely degrade both PNP and 4-NC when PNP was used as the inducer, demonstrating its potential in bioremediation of the environment polluted by both 4-NC and PNP.

  5. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Gohain, Neelakshi [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany); Thomashow, Linda S. [Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430 (United States); USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, Pullman, Washington 99164-6430 (United States); Mavrodi, Dmitri V. [Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430 (United States); Blankenfeldt, Wulf, E-mail: wulf.blankenfeldt@mpi-dortmund.mpg.de [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)

    2006-10-01

    PhzS, an FAD-dependent monooxygenase that catalyzes a reaction involved in the biosynthesis of the virulence factor pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and seleno-l-methionine-labelled crystals is reported. The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystals belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 Å, α = γ = 90, β = 110.5°. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 Å. Seleno-l-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 Å, α = γ = 90, β = 110.5° and the diffraction limit is 2.7 Å.

  6. Exploring inductive risk case studies of values in science

    CERN Document Server

    Richards, Ted

    2017-01-01

    Science is the most reliable means available for understanding the world around us and our place in it. But, since science draws conclusions based on limited empirical evidence, there is always a chance that a scientific inference will be incorrect. That chance, known as inductive risk, is endemic to science. Though inductive risk has always been present in scientific practice, the role of values in responding to it has only recently gained extensive attention from philosophers, scientists, and policy-makers. Exploring Inductive Risk brings together a set of eleven concrete case studies with the goals of illustrating the pervasiveness of inductive risk, assisting scientists and policymakers in responding to it, and moving theoretical discussions of this phenomenon forward. The case studies range over a wide variety of scientific contexts, including the drug approval process, high energy particle physics, dual-use research, climate science, research on gender disparities in employment, clinical trials, and to...

  7. Internalized Meaning Factualism

    DEFF Research Database (Denmark)

    Hohwy, Jakob

    2006-01-01

    are defended by, e.g., Robert Brandom, Paul Horwich and Huw Price), that promises to solve the problems. According to this position, the problem of meaning can be solved by prohibiting an external perspective on meaning constituting properties. The idea is that if we stick to a perspective on meaning......The normative character of meaning creates deep problems for the attempt to give a reductive explanation of the constitution of meaning. I identify and critically examine an increasingly popular Carnap-style position, which I call Internalized Meaning Factualism (versions of which I argue...... that is internal to meaning discourse, then we can preserve the normativity of meaning and yet locate meaning in the natural world. I develop a generic motivation for this position, but argue that, since this motivation is consistent with the Ramsey-Carnap-Lewis-Jackson reductionist strategy, internalized meaning...

  8. Inductively generating Euler diagrams.

    Science.gov (United States)

    Stapleton, Gem; Rodgers, Peter; Howse, John; Zhang, Leishi

    2011-01-01

    Euler diagrams have a wide variety of uses, from information visualization to logical reasoning. In all of their application areas, the ability to automatically layout Euler diagrams brings considerable benefits. In this paper, we present a novel approach to Euler diagram generation. We develop certain graphs associated with Euler diagrams in order to allow curves to be added by finding cycles in these graphs. This permits us to build Euler diagrams inductively, adding one curve at a time. Our technique is adaptable, allowing the easy specification, and enforcement, of sets of well-formedness conditions; we present a series of results that identify properties of cycles that correspond to the well-formedness conditions. This improves upon other contributions toward the automated generation of Euler diagrams which implicitly assume some fixed set of well-formedness conditions must hold. In addition, unlike most of these other generation methods, our technique allows any abstract description to be drawn as an Euler diagram. To establish the utility of the approach, a prototype implementation has been developed.

  9. Influence of Induction Machine and Mechanism Parameters on Starting Transient Processes in Case of Constant Load Conditions

    Directory of Open Access Journals (Sweden)

    Dimitar Spirov

    2005-10-01

    Full Text Available Two-phase induction machine dynamic model in a coordinate system which rotates at synchronous speed and one-mass dynamic model of mechanism driven in relative units describing transient processes when starting an induction machine in case of constant load conditions are developed.The influence of equivalent circuit parameters of induction machine and mechanism parameters on impact currents and torques and starting time of common used induction machines is studied by means of design of experiment method.

  10. Antibody induction versus corticosteroid induction for liver transplant recipients

    DEFF Research Database (Denmark)

    Penninga, Luit; Wettergren, André; Wilson, Colin H

    2014-01-01

    BACKGROUND: Liver transplantation is an established treatment option for end-stage liver failure. To date, no consensus has been reached on the use of immunosuppressive T-cell specific antibody induction compared with corticosteroid induction of immunosuppression after liver transplantation....... OBJECTIVES: To assess the benefits and harms of T-cell specific antibody induction versus corticosteroid induction for prevention of acute rejection in liver transplant recipients. SEARCH METHODS: We searched The Cochrane Hepato-Biliary Group Controlled Trials Register, the Cochrane Central Register...... of Controlled Trials (CENTRAL), MEDLINE, EMBASE, Science Citation Index Expanded, and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) on 30 September 2013 together with reference checking, citation searching, contact with trial authors and pharmaceutical companies...

  11. Geometric Mean--What Does It Mean?

    Science.gov (United States)

    Kalder, Robin S.

    2012-01-01

    The National Council of Teachers of Mathematics and numerous mathematics educators promote the combination of conceptual understanding and procedural learning in the successful instruction of mathematics. Despite this, when geometric mean is taught in a typical American geometry class, it is taught as a process only despite the many connections…

  12. Optimal control of induction heating processes

    CERN Document Server

    Rapoport, Edgar

    2006-01-01

    This book introduces new approaches to solving optimal control problems in induction heating process applications. Optimal Control of Induction Heating Processes demonstrates how to apply and use new optimization techniques for different types of induction heating installations. Focusing on practical methods for solving real engineering optimization problems, the text features a variety of specific optimization examples for induction heater modes and designs, particularly those used in industrial applications. The book describes basic physical phenomena in induction heating and induction

  13. Overview of Bearingless Induction Motors

    Directory of Open Access Journals (Sweden)

    Xiaodong Sun

    2014-01-01

    Full Text Available Bearingless induction motors combining functions of both torque generation and noncontact magnetic suspension together have attracted more and more attention in the past decades due to their definite advantages of compactness, simple structure, less maintenance, no wear particles, high rotational speed, and so forth. This paper overviews the key technologies of the bearingless induction motors, with emphasis on motor topologies, mathematical models, and control strategies. Particularly, in the control issues, the vector control, independent control, direct torque control, nonlinear decoupling control, sensorless control, and so forth are investigated. In addition, several possible development trends of the bearingless induction motors are also discussed.

  14. Induction of formaldehyde contact sensitivity

    DEFF Research Database (Denmark)

    Andersen, Klaus Ejner; Boman, A; Vølund, A

    1985-01-01

    intradermal induction with 0.65% formaldehyde; in Stockholm it was 84% after induction with 0.34%. The data from the two laboratories could be described by parallel displaced dose response curves suggesting that the guinea pig strain used in Stockholm was significantly more susceptible to formaldehyde than......, and formaldehyde 1% and 0.1% was used for challenge. The incidence of contact sensitivity depended on the intradermal, but not on the topical induction dose. Statistical analyses showed a non-monotonous (non-linear) dose response relationship. The estimated maximal sensitization rate in Copenhagen was 80% after...

  15. Comparative Study of Oral and Vaginal Misoprostol for Induction of Labour, Maternal and Foetal Outcome

    OpenAIRE

    Komala, Kambhampati; Reddy, Meherlatha; Quadri, Iqbal Jehan; B., Suneetha; V., Ramya

    2013-01-01

    Background: Misoprostol is a new promising agent for cervical ripening and induction of labour .The ideal dose, route and frequency of administration of misoprostol are still under investigation. Although, vaginal application of misoprostol has been validated as a reasonable mean of induction, there is a patient resistance to digital examination and there is a risk of ascending infection. For this reason, oral administration of misoprostol for cervical ripening and labour induction has been t...

  16. Means on scattered compacta

    Directory of Open Access Journals (Sweden)

    Banakh T.

    2014-01-01

    Full Text Available We prove that a separable Hausdor_ topological space X containing a cocountable subset homeomorphic to [0, ω1] admits no separately continuous mean operation and no diagonally continuous n-mean for n ≥ 2.

  17. Functional interpretation and inductive definitions

    CERN Document Server

    Avigad, Jeremy

    2008-01-01

    Extending G\\"odel's \\emph{Dialectica} interpretation, we provide a functional interpretation of classical theories of positive arithmetic inductive definitions, reducing them to theories of finite-type functionals defined using transfinite recursion on well-founded trees.

  18. Mathematical Induction in the Classroom

    Science.gov (United States)

    Avital, Shmuel; Hansen, Rodney T.

    1976-01-01

    This paper discusses the process of investigating a mathematical situation, making conjectures, formulating an equation, then using mathematical induction to show the result is valid. Several examples appropriate for secondary school level are given. (DT)

  19. A Note on Mathematical Induction

    Science.gov (United States)

    Brumfiel, Charles

    1974-01-01

    A classic problem involving the "misuse" of mathematical induction is presented. The error in the "proof" is then exposed. The generalization of this problem is presented as a false theorem which should serve to highlight the error. (LS)

  20. Adjustable Induction-Heating Coil

    Science.gov (United States)

    Ellis, Rod; Bartolotta, Paul

    1990-01-01

    Improved design for induction-heating work coil facilitates optimization of heating in different metal specimens. Three segments adjusted independently to obtain desired distribution of temperature. Reduces time needed to achieve required temperature profiles.

  1. Induction heating coupler and annealer

    Science.gov (United States)

    Fox, Robert L. (Inventor); Johnson, Samuel D. (Inventor); Copeland, Carl E. (Inventor); Coultrip, Robert H. (Inventor); Phillips, W. Morris (Inventor); Johnston, David F. (Inventor); Swaim, Robert J. (Inventor); Dinkins, James R. (Inventor)

    1994-01-01

    An induction heating device includes a handle having a hollow interior and two opposite ends, a wrist connected to one end of the handle, a U-shaped pole piece having- two spaced apart ends, a tank circuit including an induction coil wrapped around the pole piece and a capacitor connected to the induction coil, a head connected to the wrist and including a housing for receiving the U-shaped pole piece, the two spaced apart ends of the pole piece extending outwardly beyond the housing, and a power source connected to the tank circuit. When the tank circuit is energized and a susceptor is placed in juxtaposition to the ends of the U-shaped pole piece, the susceptor is heated by induction heating due to a magnetic flux passing between the two ends of the pole piece.

  2. ORGANIZATIONAL DEVELOPMENT ACTION: INDUCTION PROGRAM

    National Research Council Canada - National Science Library

    José G. VARGAS-HERNÁNDEZ

    2016-01-01

    .... Also, an induction program is presented as an intervention proposal of organization development, on the premise that this program is a first step in the solving of the existing low productivity problem...

  3. Inductance and hypergeometric functions. II

    DEFF Research Database (Denmark)

    Karlsson, Per W.

    2000-01-01

    A previously obtained integral for the self-inductance of a solenoid is further transformed. The resulting formula involves double Kampé de Fériet functions which are analytic continuations rather than power series.......A previously obtained integral for the self-inductance of a solenoid is further transformed. The resulting formula involves double Kampé de Fériet functions which are analytic continuations rather than power series....

  4. Automated Discovery of Inductive Theorems

    OpenAIRE

    McCasland, Roy; Bundy, Alan; Serge, Autexier

    2007-01-01

    Inductive mathematical theorems have, as a rule, historically been quite difficult to prove – both for mathematics students and for auto- mated theorem provers. That said, there has been considerable progress over the past several years, within the automated reasoning community, towards proving some of these theorems. However, little work has been done thus far towards automatically discovering them. In this paper we present our methods of discovering (as well as proving) inductive theorems, ...

  5. Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues.

    Science.gov (United States)

    Liu, L; Massey, T E

    1992-04-01

    In the present investigation, we have examined the role of lipoxygenases in the bioactivation of aflatoxin B1 (AFB1) in hepatic and extrahepatic tissues. The enzyme activities were evaluated by determining [3H]AFB1-DNA adduct formation. The results demonstrated that both purified soybean lipoxygenase and guinea-pig tissue cytosolic lipoxygenases were able to activate AFB1 to form [3H]AFB1-DNA adduct(s). The reaction was completely inhibited by nordihydroguaiaretic acid (NDGA, 0.1 mM), a lipoxygenase inhibitor and an antioxidant, but not by indomethacin (0.1 mM), an inhibitor of prostaglandin H synthase (PHS), indicating that this reaction is associated with lipoxygenase activity, and/or is involved in a peroxyl radical process. While purified lipoxygenase showed arachidonic acid (AA)-dependent properties, the omission of AA did not diminish guinea-pig tissue cytosolic [3H]AFB1-DNA adduct formation, possibly because AA was released from lipid particles by AFB1. Within the range of hemoglobin (Hb) concentrations found in lung, kidney and liver cytosols (1.4-11.1 microM) and microsomes (0-0.5 microM), neither pure Hb, nor Hb of cytosols or microsomes from whole blood caused detectable AA-dependent AFB1-DNA binding. This indicates that Hb, as a contaminant with quasi-lipoxygenase activity, did not contribute to AFB1 activation attributed to guinea-pig tissue lipoxygenases. [3H]AFB1 concentrations at half-maximal DNA binding rate of pulmonary cytochrome P450 monooxygenases (P450) and lipoxygenases were similar, though P450 had a much higher maximum DNA binding rate. Pulmonary microsomal PHS activity for AFB1 activation was too low for its half-maximal binding concentrations of [3H]AFB1 and maximum rate to be accurately determined. In kidney, maximum rates for lipoxygenase, PHS and P450 were similar, whereas half-maximal binding concentrations for reactions by lipoxygenase and P450 were lower compared to that of PHS. The half-maximal binding concentration of hepatic

  6. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Directory of Open Access Journals (Sweden)

    Pandey Sona

    2010-11-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases (P450s catalyze oxidation of various substrates using oxygen and NAD(PH. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an

  7. Inductive System Monitors Tasks

    Science.gov (United States)

    2008-01-01

    The Inductive Monitoring System (IMS) software developed at Ames Research Center uses artificial intelligence and data mining techniques to build system-monitoring knowledge bases from archived or simulated sensor data. This information is then used to detect unusual or anomalous behavior that may indicate an impending system failure. Currently helping analyze data from systems that help fly and maintain the space shuttle and the International Space Station (ISS), the IMS has also been employed by data classes are then used to build a monitoring knowledge base. In real time, IMS performs monitoring functions: determining and displaying the degree of deviation from nominal performance. IMS trend analyses can detect conditions that may indicate a failure or required system maintenance. The development of IMS was motivated by the difficulty of producing detailed diagnostic models of some system components due to complexity or unavailability of design information. Successful applications have ranged from real-time monitoring of aircraft engine and control systems to anomaly detection in space shuttle and ISS data. IMS was used on shuttle missions STS-121, STS-115, and STS-116 to search the Wing Leading Edge Impact Detection System (WLEIDS) data for signs of possible damaging impacts during launch. It independently verified findings of the WLEIDS Mission Evaluation Room (MER) analysts and indicated additional points of interest that were subsequently investigated by the MER team. In support of the Exploration Systems Mission Directorate, IMS is being deployed as an anomaly detection tool on ISS mission control consoles in the Johnson Space Center Mission Operations Directorate. IMS has been trained to detect faults in the ISS Control Moment Gyroscope (CMG) systems. In laboratory tests, it has already detected several minor anomalies in real-time CMG data. When tested on archived data, IMS was able to detect precursors of the CMG1 failure nearly 15 hours in advance of

  8. Agency, Context and Meaning

    DEFF Research Database (Denmark)

    Folkmann, Mads Nygaard

    2014-01-01

    history, 2) the question of context in and of design, i.e. which contexts give meaning to design; this question calls for interpretive models of cultural analysis of the circuit of design in acknowledging phases and aspects of production, mediation and consumption, and 3) the question of the meaning...... of meaning formulation and cultural contexts and, by this, contest design. In reflecting the foundational ground of design in terms of its agency, contexts and meaning constituents, design and its questioning of meaning can be critically reframed....

  9. Meaning in mathematics education

    CERN Document Server

    Valero, Paola; Hoyles, Celia; Skovsmose, Ole

    2005-01-01

    What does it mean to know mathematics? How does meaning in mathematics education connect to common sense or to the meaning of mathematics itself? How are meanings constructed and communicated and what are the dilemmas related to these processes? There are many answers to these questions, some of which might appear to be contradictory. Thus understanding the complexity of meaning in mathematics education is a matter of huge importance. There are twin directions in which discussions have developed - theoretical and practical - and this book seeks to move the debate forward along both dimensions while seeking to relate them where appropriate. A discussion of meaning can start from a theoretical examination of mathematics and how mathematicians over time have made sense of their work. However, from a more practical perspective, anybody involved in teaching mathematics is faced with the need to orchestrate the myriad of meanings derived from multiple sources that students develop of mathematical knowledge.

  10. "Maternal serum Dehydroepiandrosterone Sulfate levels and successful labor induction "

    Directory of Open Access Journals (Sweden)

    Modares Gilani M

    2003-06-01

    Full Text Available To evaluate the maternal serum dehydroepiandrosterone sulfate level as a factor associated with the outcome of labor induction. Venous blood was collected from 45 women at the initiation of labor induction. Pregnancies complicated by maternal corticosteriod use, anterpartum chorioamnionitis, or cesarean delivery for indications other than arrest disorders, were excluded from analysis. In 42 women meeting inclusion criteria, induction followed established protocol. Serum dehydroepiandrosterone sulfate levels were measured by radioimmunoassay and correlated with the outcome of each induction attempt. A successful result was defined as progression to active labor. The welch approximate t-test, Mann-Whitney test, Fisher exact test, simple regression, and multiple regression were used for statistical analysis, with P<0.05 considered to be significant. The mean (±standard error dehydroepiandrosterone sulfate level was higher in women who progressed to active labor (n=25 than in those with unsuccessful attempts (n=17, (48,63±6.53 µg/dl versus 26.86±5.17 mg/dl, respectively; p= 0.03. Compared with women with dehydroepiandrosterone sulfate levels above 60 µg/dl, women with lower levels had an unsuccessful induction odds ratio (OR of 6.92 (95% confidence interval 1.74, 32.52, p= 0.01. The OR increased as dehydroepiandrosterone sulfate levels decreased. Dehydroepiandrosterone sulfate may be an important factor in successful labor induction.

  11. Acoustic analysis of an induction motor with viscoelastic bearing supports

    NARCIS (Netherlands)

    Tillema, H.G.; Wensing, J.A.

    2002-01-01

    The demand for silent bearing applications has resulted in the development of an effective damping layer between the outer ring of a rolling bearing and the surrounding structure. By means of numerical modeling using both FEM and BEM techniques an induction motor for household appliances is analyzed

  12. Complex Model of Induction Heating - Demounting of Shaft Flange

    Directory of Open Access Journals (Sweden)

    Milan Krasl

    2004-01-01

    Full Text Available This paper deals with exemplary solutions of one specifies problem of induction heating. This problem was partly determined by means of numerical method as a light-coupling problem. Numerical solutions were calculated with the help of professional programs (Ansys, Fluent, QuickField. Results of the solution and comparison with measurements are discussed in conclusion.

  13. Predictors of successful induction of labour at a tertiary obstetric ...

    African Journals Online (AJOL)

    Predictors of successful induction of labour at a tertiary obstetric service in Southwest ... Study Design, Setting and Patients: This was a retrospective, descriptive, ... The mean duration of the process was 12.0 ± 6.6 hours with misoprostol, ...

  14. Antibody induction versus placebo, no induction, or another type of antibody induction for liver transplant recipients

    DEFF Research Database (Denmark)

    Penninga, Luit; Wettergren, André; Wilson, Colin H

    2014-01-01

    BACKGROUND: Liver transplantation is an established treatment option for end-stage liver failure. To date, no consensus has been reached on the use of immunosuppressive T-cell antibody induction for preventing rejection after liver transplantation. OBJECTIVES: To assess the benefits and harms...... of immunosuppressive T-cell specific antibody induction compared with placebo, no induction, or another type of T-cell specific antibody induction for prevention of acute rejection in liver transplant recipients. SEARCH METHODS: We searched The Cochrane Hepato-Biliary Group Controlled Trials Register, the Cochrane...... Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, Science Citation Index Expanded, and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) until September 2013. SELECTION CRITERIA: Randomised clinical trials assessing immunosuppression with T...

  15. Electromagnetic induction in non-uniform domains

    CERN Document Server

    Giesecke, A; Luddens, F; Stefani, F; Gerbeth, G; Léorat, J; Guermond, J -L

    2010-01-01

    Kinematic simulations of the induction equation are carried out for different setups suitable for the von-K\\'arm\\'an-Sodium (VKS) dynamo experiment. Material properties of the flow driving impellers are considered by means of high conducting and high permeability disks that are present in a cylindrical volume filled with a conducting fluid. Two entirely different numerical codes are mutually validated by showing quantitative agreement on Ohmic decay and kinematic dynamo problems using various configurations and physical parameters. Field geometry and growth rates are strongly modified by the material properties of the disks even if the high permeability/high conductivity material is localized within a quite thin region. In contrast the influence of external boundary conditions remains small. Utilizing a VKS like mean fluid flow and high permeability disks yields a reduction of the critical magnetic Reynolds number for the onset of dynamo action of the simplest non-axisymmetric field mode. However this decreas...

  16. Secondary School Students' Understanding of Mathematical Induction: Structural Characteristics and the Process of Proof Construction

    Science.gov (United States)

    Palla, Marina; Potari, Despina; Spyrou, Panagiotis

    2012-01-01

    In this study, we investigate the meaning students attribute to the structure of mathematical induction (MI) and the process of proof construction using mathematical induction in the context of a geometric recursion problem. Two hundred and thirteen 17-year-old students of an upper secondary school in Greece participated in the study. Students'…

  17. An inductive conductivity meter for monitoring the salinity of dialysis water

    DEFF Research Database (Denmark)

    Diamond, J.M.

    1970-01-01

    An inductive conductivity meter is described, especially adapted as a salinity monitor for dialysis water. Salinity are given. The principal problems of the inductive conductivity meter result from the low conductivity of electrolytes. The weak coupling due to the electrolyte means that stray...

  18. X-ray absorption spectroscopic studies of the dinuclear iron center in methane monooxygenase and the sulfure and chlorine centers in photographic materials

    Energy Technology Data Exchange (ETDEWEB)

    DeWitt, J.G.

    1992-12-01

    The dinuclear iron center of the hydroxylase component of soluble methane monooxygenase (MMO) from Methylococcus capsulatus and Methylosinus trichosporiwn has been studied by X-ray absorption spectroscopy. Analysis of the Fe K-edge EXAFS revealed that the first shell coordination of the Fe(HI)Fe(IH) oxidized state of the hydroxylase from M. capsulatus consists of approximately 6 N and 0 atoms at an average distance of 2.04 {Angstrom}. The Fe-Fe distance was determined to be 3.4 {Angstrom}. No evidence for the presence of a short oxo bridge in the iron center of the oxidized hydroxylase was found, suggesting that the active site of MMO is significantly different from the active sites of the dinuclear iron proteins hemery and ribonucleotide reductase. In addition, the results of the first shell fits suggest that there are more oxygen than nitrogen donor ligands.

  19. X-ray absorption spectroscopic studies of the dinuclear iron center in methane monooxygenase and the sulfure and chlorine centers in photographic materials

    Energy Technology Data Exchange (ETDEWEB)

    DeWitt, J.G.

    1992-12-01

    The dinuclear iron center of the hydroxylase component of soluble methane monooxygenase (MMO) from Methylococcus capsulatus and Methylosinus trichosporiwn has been studied by X-ray absorption spectroscopy. Analysis of the Fe K-edge EXAFS revealed that the first shell coordination of the Fe(HI)Fe(IH) oxidized state of the hydroxylase from M. capsulatus consists of approximately 6 N and 0 atoms at an average distance of 2.04 [Angstrom]. The Fe-Fe distance was determined to be 3.4 [Angstrom]. No evidence for the presence of a short oxo bridge in the iron center of the oxidized hydroxylase was found, suggesting that the active site of MMO is significantly different from the active sites of the dinuclear iron proteins hemery and ribonucleotide reductase. In addition, the results of the first shell fits suggest that there are more oxygen than nitrogen donor ligands.

  20. Alkanesulfonate degradation by novel strains of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and Rhodococcus sp., and evidence for an ethanesulfonate monooxygenase in A. xylosoxidans strain AE4.

    Science.gov (United States)

    Erdlenbruch, B N; Kelly, D P; Murrell, J C

    2001-12-01

    Novel isolates of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and a Rhodococcus sp. are described. These grew with short-chain alkanesulfonates as their sole source of carbon and energy. T. wratislaviensis strain SB2 grew well with C(3)-C(6) linear alkanesulfonates, isethionate and taurine, Rhodococcus sp. strain CB1 used C(3)-C(10) linear alkanesulfonates, taurine and cysteate, but neither strain grew with ethanesulfonate. In contrast, A. xylosoxidans strain AE4 grew well with ethanesulfonate, making it the first bacterium to be described which can grow with this compound. It also grew with unsubstituted C(3)-C(5) alkanesulfonates and isethionate. Hydrolysis was excluded as a mechanism for alkanesulfonate metabolism in these strains; and evidence is given for a diversity of uptake and desulfonatase systems. We provide evidence for an initial monooxygenase-dependent desulfonation in the metabolism of ethanesulfonate and propanesulfonate by A. xylosoxidans strain AE4.

  1. Conversion of α-chitin substrates with varying particle size and crystallinity reveals substrate preferences of the chitinases and lytic polysaccharide monooxygenase of Serratia marcescens.

    Science.gov (United States)

    Nakagawa, Yuko S; Eijsink, Vincent G H; Totani, Kazuhide; Vaaje-Kolstad, Gustav

    2013-11-20

    Industrial depolymerization of chitinous biomass generally requires numerous steps and the use of deleterious substances. Enzymatic methods provide an alternative, but fundamental knowledge that could direct potential development of industrial enzyme cocktails is scarce. We have studied the contribution of monocomponent chitinases (ChiA, -B, and -C) and the lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens on depolymerization of α-chitin substrates with varying particle size and crystallinity that were generated using a converge mill. For all chitinases activity was positively correlated to a decline in particle size and crystallinity. Especially ChiC, the only nonprocessive endochitinase from the S. marcescens chitinolytic machinery, benefited from mechanical pretreatment. Combining the chitinases revealed clear synergies for all substrates tested. CBP21, the chitin-active LPMO from S. marcescens, increased solubilization of substrates with high degrees of crystallinity when combined with each of the three chitinases, but this synergy was reduced upon decline in crystallinity.

  2. Estimation of physical parameters in induction motors

    DEFF Research Database (Denmark)

    Børsting, H.; Knudsen, Morten; Rasmussen, Henrik

    1994-01-01

    Parameter estimation in induction motors is a field of great interest, because accurate models are needed for robust dynamic control of induction motors......Parameter estimation in induction motors is a field of great interest, because accurate models are needed for robust dynamic control of induction motors...

  3. Kinetic inductance measured in a superconducting wire

    Science.gov (United States)

    Meservey, R. H.; Tedrow, P. M.

    1970-01-01

    Ultrasensitive technique to measure kinetic inductance has test specimen included as part of the inductance of a tank circuit of a tunnel diode oscillator. Frequency counter measures shift in frequency of oscillator, caused by changes in inductance. Frequency shift in tank circuit is proportional to change in kinetic inductance

  4. On the internal structures of inductive types

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The paper investigates the internal structures of hereditary inductive types in logical type theory. By defining a bisimulation equality on the inhabitants of each hereditary inductive type, one is able to show that the inhabitants of a hereditary inductive type satisfy the basic properties of sets. A hereditary inductive type can therefore be conceived as a universe of sets.

  5. On generic representation of implicit induction procedures

    NARCIS (Netherlands)

    Naidich, D.

    1996-01-01

    We develop a generic representation of implicit induction proof procedures within the cover set induction framework. Our work further develops the approach of cover set induction on propositional orderings. We show that in order to represent a substantially wide range of implicit induction procedure

  6. Music, Meaning and Culture

    Directory of Open Access Journals (Sweden)

    Richard Widdess

    2012-09-01

    Full Text Available This paper situates musical meaning in culture, addressing music as social symbol and as ongoing process of meaning creation. Three examples of non- Western musical practice are used to illustrate the embedding of musical meaning in cultural context. The performance of an Australian Aboriginal song is shown to exemplify the interdependence of song style and social structure as a matrix for the emergence of cultural meanings; an example of North Indian performance is adduced to demonstrate the multi-layered nature of meaning as embodied in musical performance; and an example of collective festival performance from Nepal illustrates ways in which the structure of musical performance can mirror local cultural forms. Each of the three examples lends weight to the idea that music's meanings are often non-linguistic and reflect foundational schemas that are specific to the cultures from the musics are drawn.

  7. Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast.

    Science.gov (United States)

    Jeon, Hyunwoo; Durairaj, Pradeepraj; Lee, Dowoo; Ahsan, Md Murshidul; Yun, Hyungdon

    2016-12-28

    Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at 30°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

  8. Cavity residue leucine 95 and channel residues glutamine 204, aspartic acid 211, and phenylalanine 269 of toluene o-xylene monooxygenase influence catalysis.

    Science.gov (United States)

    Kurt, Cansu; Sönmez, Burcu; Vardar, Nurcan; Yanık-Yıldırım, K Cansu; Vardar-Schara, Gönül

    2016-09-01

    Structural analysis of toluene-o-xylene monooxygenase (ToMO) hydroxylase revealed the presence of three hydrophobic cavities, a channel, and a pore leading from the protein surface to the active site. Here, saturation mutagenesis was used to investigate the catalytic roles of alpha-subunit (TouA) second cavity residue L95 and TouA channel residues Q204, D211, and F269. By testing the substrates toluene, phenol, nitrobenzene, and/or naphthalene, these positions were found to influence the catalytic activity of ToMO. Several regiospecific variants were identified from TouA positions Q204, F269, and L95. For example, TouA variant Q204H had the regiospecificity of nitrobenzene changed significantly from 30 to 61 % p-nitrophenol. Interestingly, a combination of mutations at Q204H and A106V altered the regiospecificity of nitrobenzene back to 27 % p-nitrophenol. TouA variants F269Y, F269P, Q204E, and L95D improved the meta-hydroxylating capability of nitrobenzene by producing 87, 85, 82, and 77 % m-nitrophenol, respectively. For naphthalene oxidation, TouA variants F269V, Q204A, Q204S/S222N, and F269T had the regiospecificity changed from 16 to 9, 10, 23, and 25 % 2-naphthol, respectively. Here, two additional TouA residues, S222 and A106, were also identified that may have important roles in catalysis. Most of the isolated variants from D211 remained active, whereas having a hydrophobic residue at this position appeared to diminish the catalytic activity toward naphthalene. The mutational effects on the ToMO regiospecificity described here suggest that it is possible to further fine tune and engineer the reactivity of multicomponent diiron monooxygenases toward different substrates at positions that are relatively distant from the active site.

  9. Mannitol Stress Directs Flavonoid Metabolism toward Synthesis of Flavones via Differential Regulation of Two Cytochrome P450 Monooxygenases in Coleus forskohlii.

    Science.gov (United States)

    Awasthi, Praveen; Gupta, Ajai Prakash; Bedi, Yashbir S; Vishwakarma, Ram A; Gandhi, Sumit G

    2016-01-01

    Cytochrome P450 monooxygenases (CYP450s) are known to play important roles in biosynthesis of all secondary metabolites, including flavonoids. Despite this, few CYP450s have been functionally characterized in model plants and roles of fewer CYP450s are known in non-model, medicinal, and aromatic plants. Our study in Coleus forskohlii indicates that flavone synthase (CYP93B) and flavonoid 3' monooxygenase (CYP706C) are key enzymes positioned at a metabolic junction, to execute the biosynthesis of different sub-classes of flavonoids (flavones, flavonol, anthocynanin, isoflavones etc.) from a common precursor. Such branch points are favored targets for artificially modulating the metabolic flux toward specific metabolites, through genetic manipulation or use of elicitors that differentially impact the expression of branch point genes. Genkwanin, the only flavone reported from C. forskohlii, is known to possess anti-inflammatory activity. It is biosynthesized from the general flavonoid precursor: naringenin. Two differentially expressed cytochrome P450 genes (CfCYP93B, CfCYP706C), exhibiting maximum expression in leaf tissues, were isolated from C. forskohlii. Mannitol treatment resulted in increased expression of CfCYP93B and decrease in expression of CfCYP706C. Metabolite quantification data showed that genkwanin content increased and anthocyanin levels decreased in response to mannitol treatment. Alignment, phylogenetic analysis, modeling, and molecular docking analysis of protein sequences suggested that CfCYP93B may be involved in conversion of naringenin to flavones (possibly genkwanin via apigenin), while CfCYP706C may act on common precursors of flavonoid metabolism and channel the substrate toward production of flavonols or anthocynanins. Decrease in expression of CfCYP706C and increase in accumulation of genkwanin suggested that mannitol treatment may possibly lead to accumulation of genkwanin via suppression of a competitive branch of flavonoids in C

  10. Effects of Quinizarin and Five Synthesized Derivatives on Fifth Larval Instar Midgut Ecdysone 20-Monooxygenase Activity of the Tobacco Hornworm Manduca sexta

    Directory of Open Access Journals (Sweden)

    Christopher A. Drummond

    2014-01-01

    Full Text Available The plant allelochemical, quinizarin (1,4-dihydroxy-9,10-anthraquinone, and five anthraquinones that were synthesized from quinizarin, namely, 1,4-anthraquinone; 2-hydroxy-1,4-anthraquinone; 2-methoxy-1,4-anthraquinone; 9-hydroxy-1,4-anthraquinone; and 9-methoxy-1,4-anthraquinone, were assessed as to their effects on the essential, P450-dependent ecdysone 20-monooxygenase system of the insect model Manduca sexta (tobacco hornworm. This steroid hydroxylase converts the arthropod molting hormone, ecdysone, to the physiologically required 20-hydroxyecdysone form. M. sexta fifth larval instar midgut homogenates were incubated with increasing concentrations (10−8 to 10−3 M of each of the six anthraquinones followed by ecdysone 20-monooxygenase assessments using a radioenzymological assay. Four of the five anthraquinones exhibited I50’s of about 4×10-6 to 6×10-2 M. The most effective inhibitors were 2-methoxy-1,4-anthraquinone and 1,4-anthraquinone followed by 9-hydroxy-1,4 anthraquinone and 9-methoxy-1,4-anthraquinone. At lower concentrations the latter anthraquinone stimulated E20M activity. Quinizarin was less inhibitory and 2-hydroxy-1,4-anthraquinone was essentially without effect. Significantly, these studies make evident for the first time that anthraquinones can affect insect E20M activity, and thus insect endocrine regulation and development, and that a relationship between anthraquinone structure and effectiveness is apparent. These studies represent the first demonstrations of anthraquinones affecting any steroid hydroxylase system.

  11. Tuning the specificity of the recombinant multicomponent toluene o-xylene monooxygenase from Pseudomonas sp. strain OX1 for the biosynthesis of tyrosol from 2-phenylethanol.

    Science.gov (United States)

    Notomista, Eugenio; Scognamiglio, Roberta; Troncone, Luca; Donadio, Giuliana; Pezzella, Alessandro; Di Donato, Alberto; Izzo, Viviana

    2011-08-01

    Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene o-xylene monooxygenase (ToMO) is a BMM isolated from Pseudomonas sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of Escherichia coli strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.

  12. Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:FAD oxidoreductase (TftC) of Burkholderia cepacia AC1100.

    Science.gov (United States)

    Webb, Brian N; Ballinger, Jordan W; Kim, Eunjung; Belchik, Sara M; Lam, Ka-Sum; Youn, Buhyun; Nissen, Mark S; Xun, Luying; Kang, Chulhee

    2010-01-15

    Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH(2)-dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH(2). We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 A resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH(2)-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH(2) is generated by TftC, and TftD uses FADH(2) to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH(2) between TftC and TftD without direct physical interaction between the two enzymes.

  13. Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxidoreductase (TftC) of Burkholderia cepacia AC1100.

    Science.gov (United States)

    Gisi, Michelle R; Xun, Luying

    2003-05-01

    Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH(2))-utilizing monooxygenase, and FADH(2) was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH(2) and retarded its rapid oxidation by O(2). A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH(2)-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.

  14. Integrated one-pot enrichment and immobilization of styrene monooxygenase (StyA) using SEPABEAD EC-EA and EC-Q1A anion-exchange carriers.

    Science.gov (United States)

    Ruinatscha, Reto; Karande, Rohan; Buehler, Katja; Schmid, Andreas

    2011-07-18

    A straightforward one-pot procedure combining enrichment and immobilization of recombinantely expressed FADH₂ dependent styrene monooxygenase (StyA) directly from Escherichia coli cell extracts was investigated. Sepabeads EC-EA and EC-Q1A anion-exchange carriers were employed to non-covalently adsorb StyA from the cell extracts depending on basic parameters such as varying initial protein concentrations and pH. The protein fraction of the cell extract contained around 25% StyA. At low initial protein concentrations (2.5 mg mL⁻¹) and pH 6, the enzyme could be enriched up to 52.4% on Sepabeads EC-EA and up to 46.0% on Sepabeads EC-Q1A, accounting for an almost complete StyA adsorption from the cell extracts. Higher initial protein concentrations were necessary to exploit the high loading capacity of the beads. At 20 mg mL⁻¹, up to 37.6% of the theoretical bead loading capacity could be utilized for StyA binding using Sepabeads EC-EA, and 34.0% using Sepabeads EC-Q1A. For both carriers, protein leakage under reaction conditions could be reduced to less than 2%. During assays, the FADH₂ cofactor necessary for StyA activity was supplied by the NADH-FAD reductase component styrene monooxygenase B (StyB). StyA immobilized on Sepabeads EC-Q1A displayed twice as high styrene epoxidation rates (0.2 U mg(StyA)⁻¹) as compared to Sepabeads EC-EA. This activity could be increased to 0.7 U mg(StyA)⁻¹ by co-immobilizing StyB on Sepabeads EC-Q1A, which corresponds to 33% of the soluble StyA activity.

  15. Parallel k-means++

    Energy Technology Data Exchange (ETDEWEB)

    2017-04-04

    A parallelization of the k-means++ seed selection algorithm on three distinct hardware platforms: GPU, multicore CPU, and multithreaded architecture. K-means++ was developed by David Arthur and Sergei Vassilvitskii in 2007 as an extension of the k-means data clustering technique. These algorithms allow people to cluster multidimensional data, by attempting to minimize the mean distance of data points within a cluster. K-means++ improved upon traditional k-means by using a more intelligent approach to selecting the initial seeds for the clustering process. While k-means++ has become a popular alternative to traditional k-means clustering, little work has been done to parallelize this technique. We have developed original C++ code for parallelizing the algorithm on three unique hardware architectures: GPU using NVidia's CUDA/Thrust framework, multicore CPU using OpenMP, and the Cray XMT multithreaded architecture. By parallelizing the process for these platforms, we are able to perform k-means++ clustering much more quickly than it could be done before.

  16. Cardiovascular and respiratory responses during musical mood induction.

    Science.gov (United States)

    Etzel, Joset A; Johnsen, Erica L; Dickerson, Julie; Tranel, Daniel; Adolphs, Ralph

    2006-07-01

    Music is used to induce moods in experimental settings as well as for therapeutic purposes. Prior studies suggest that subjects listening to certain types of music experience strong moods and show physiological responses associated with the induced emotions. We hypothesized that cardiovascular and respiratory patterns could discriminate moods induced via music. 18 healthy subjects listened to 12 music clips, four each to induce happiness, sadness, and fear, while cardiovascular and respiratory responses were recorded using an electrocardiogram and chest strain-gauge belt. After each clip subjects completed a questionnaire. Subjects consistently reported experiencing the targeted mood, suggesting successful mood induction. Cardiovascular activity was measured by calculating time domain measures and heart rate changes during each clip. Respiratory activity was measured by total, inspiration, and expiration lengths as well as changes in mean respiration rate during each clip. Evaluation of individuals' patterns and mixed-model analyses were performed. Contrary to expectations, the time domain measures of subjects' cardiovascular responses did not vary significantly between the induced moods, although a heart rate deceleration was found during the sadness inductions and acceleration during the fear inductions. The time domain respiratory measures varied with clip type: the mean breath length was longest for the sad induction, intermediate during fear, and shortest during the happiness induction. However, analysis using normalized least mean squares adaptive filters to measure time correlation indicated that much of this difference may be attributable to entrainment of respiration to characteristics of the music which varied between the stimuli. Our findings point to the difficulty in detecting psychophysiological correlates of mood induction, and further suggest that part of this difficulty may arise from failure to differentiate it from tempo-related contributions

  17. Meaning in life and intuition.

    Science.gov (United States)

    Heintzelman, Samantha J; King, Laura A

    2016-03-01

    Three correlational studies and 2 experiments examined the association between meaning in life (MIL) and reliance on intuitive information processing. In Studies 1-3 (total N = 5,079), Faith in Intuition (FI) scale and MIL were correlated positively, controlling for religiosity, positive mood, self-esteem, basic need satisfaction, and need for cognition. Two experiments manipulated processing style. In Study 4 (N = 614), participants were randomly assigned to complete the Cognitive Reflection Task (CRT; Fredrick, 2005) either immediately before (reflective/low intuitive mindset condition) or immediately after (control condition) rating MIL. Condition did not affect MIL. However, low MIL rated before the CRT predicted superior performance and greater time spent on the task. The association between reflection and MIL was curvilinear, such that MIL was strongly negatively related to CRT performance particularly at low levels of MIL. In Study 5 (N = 804), intuitive or reflective mindsets were induced and FI and MIL were measured. Induced processing style study did not affect MIL. However, those high in MIL were more responsive to the intuitive mindset induction. The relationship between FI and MIL was curvilinear (in this and the correlational studies), with intuitive processing being strongly positively related to MIL particularly at higher levels of MIL. Although often considered in the context of conscious reflection, MIL shares a positive relationship with reliance on gut feelings, and high MIL may facilitate reliance on those feelings. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  18. Induction, Space and Positive Ethics : Induction, Space, Ethics

    OpenAIRE

    Kirsh, Marvin

    2008-01-01

    4 pages, 0 figures, 1 reference; International audience; One may purport that ones' awareness of space for scientific purposes comes about from a potential awareness of its' absence that is derived from times when ones attention is not focused on it. Yet simply one might extract the notion that space and entailed properties of it are elemental —i.e. conceptually non reducible and that from which all emanates. The words non-ethical induction, entailing the existence of ethical induction, if co...

  19. Flexible heating head for induction heating

    Science.gov (United States)

    Fox, Robert L. (Inventor); Johnson, Samuel D. (Inventor); Coultrip, Robert H. (Inventor); Phillips, W. Morris (Inventor)

    1993-01-01

    An induction heating head includes a length of wire having first and second opposite ends and being wound in a flat spiral shape to form an induction coil, a capacitor connected to the first and second ends of the wire, the induction coil and capacitor defining a tank circuit, and a flexible, elastomeric body molded to encase the induction coil. When a susceptor is placed in juxtaposition to the body, and the tank circuit is powered, the susceptor is inductively heated.

  20. Cyclic and Inductive Calculi are equivalent

    CERN Document Server

    Voicu, Razvan

    2011-01-01

    Brotherston and Simpson [citation] have formalized and investigated cyclic reasoning, reaching the important conclusion that it is at least as powerful as inductive reasoning (specifically, they showed that each inductive proof can be translated into a cyclic proof). We add to their investigation by proving the converse of this result, namely that each inductive proof can be translated into an inductive one. This, in effect, establishes the equivalence between first order cyclic and inductive calculi.

  1. Cyclic and Inductive Calculi are equivalent

    OpenAIRE

    Razvan VOICU; Li, Mengran

    2011-01-01

    Brotherston and Simpson [citation] have formalized and investigated cyclic reasoning, reaching the important conclusion that it is at least as powerful as inductive reasoning (specifically, they showed that each inductive proof can be translated into a cyclic proof). We add to their investigation by proving the converse of this result, namely that each inductive proof can be translated into an inductive one. This, in effect, establishes the equivalence between first order cyclic and inductive...

  2. Induction of labour.

    Science.gov (United States)

    Leduc, Dean; Biringer, Anne; Lee, Lily; Dy, Jessica

    2013-09-01

    Objectif : Analyser la littérature la plus récente afin de formuler des recommandations factuelles à l’intention des fournisseurs de soins obstétricaux au sujet du déclenchement du travail. Options : Mise en œuvre d’un déclenchement du travail dans le cadre d’une grossesse. Issues : Chronologie et méthode appropriées pour ce qui est du déclenchement, mode d’accouchement approprié et issues maternelles et périnatales optimales. Résultats : La littérature publiée a été récupérée par l’intermédiaire de recherches menées dans PubMed, CINAHL et The Cochrane Library en 2010, au moyen d’un vocabulaire contrôlé (p. ex. « labour », « induced », « labour induction », « cervical ripening ») et de mots clés (p. ex. « induce », « induction », « augmentation ») appropriés. Les résultats ont été restreints aux analyses systématiques, aux essais comparatifs randomisés / essais cliniques comparatifs et aux études observationnelles. Aucune restriction n’a été appliquée en matière de date ou de langue. Les recherches ont été mises à jour de façon régulière et intégrées à la directive clinique jusqu’à la fin de 2010. La littérature grise (non publiée) a été identifiée par l’intermédiaire de recherches menées dans les sites Web d’organismes s’intéressant à l’évaluation des technologies dans le domaine de la santé et d’organismes connexes, dans des collections de directives cliniques, dans des registres d’essais cliniques et auprès de sociétés de spécialité médicale nationales et internationales. Valeurs : La qualité des résultats est évaluée au moyen des critères décrits dans le rapport du Groupe d’étude canadien sur les soins de santé préventifs (Tableau 1). Validation : Les données, les déclarations sommaires et les recommandations que contient la présente directive clinique ont été vérifiées en les comparant à celles de directives

  3. 38 CFR 21.282 - Effective date of induction into a rehabilitation program; retroactive induction.

    Science.gov (United States)

    2010-07-01

    ... induction into a rehabilitation program; retroactive induction. 21.282 Section 21.282 Pensions, Bonuses, and... Vocational Rehabilitation and Employment Under 38 U.S.C. Chapter 31 Induction into A Rehabilitation Program § 21.282 Effective date of induction into a rehabilitation program; retroactive induction. (a)...

  4. Mean field games

    KAUST Repository

    Gomes, Diogo A.

    2014-01-06

    In this talk we will report on new results concerning the existence of smooth solutions for time dependent mean-field games. This new result is established through a combination of various tools including several a-priori estimates for time-dependent mean-field games combined with new techniques for the regularity of Hamilton-Jacobi equations.

  5. Processing Film, Processing Meaning

    Science.gov (United States)

    Perkowski, Lisa M.

    2015-01-01

    Adolescents are at a ripe age to make meaning and think abstractly (Kerlavage, 1998); yet, they are not "born knowing how to get ideas into materials, or how materials can be manipulated to shape ideas and meaning" (Burton, 2012, p. 14). Adolescents need guidance in understanding abstract concepts, and art teachers play an important role…

  6. Induction Motor with Switchable Number of Poles and Toroidal Winding

    Directory of Open Access Journals (Sweden)

    MUNTEANU, A.

    2011-05-01

    Full Text Available This paper presents a study of an induction motor provided with toroidal stator winding. The ring-type coils offer a higher versatility in obtaining a different number of pole pairs by means of delta/star and series/parallel connections respectively. As consequence, the developed torque can vary within large limits and the motor can be utilized for applications that require, for example, high load torque values for a short time. The study involves experimental tests and FEM simulation for an induction machine with three configurations of pole pairs. The conclusions attest the superiority of the toroidal winding for certain applications such as electric vehicles or lifting machines.

  7. The Meaning of Happiness in Consumer Research

    DEFF Research Database (Denmark)

    Brandi Sørensen, Elin; Uth Thomsen, Thyra

    2016-01-01

    In this study we investigate the meaning of happiness in a consumption context. We employ an inductive approach and present the results of an exploratory pilot study with eight consumers. The study is based on a Multi-Sensory-Sculpting (MSS) procedure in which we asked consumers to build sculptures...... that represent consumer happiness. Following the MSS guidelines, consumers were interviewed about the meanings of their sculpture in order to elicit embodied cognition about the topic at hand. In this paper we present the meanings of consumer happiness in the participants‟ accounts and discuss implications...... for consumer research. Further, we discuss the applicability of the MSS-procedure to the topic of consumer happiness, and how to optimize it for later studies on consumer happiness....

  8. The Meaning of Happiness in Consumer Research

    DEFF Research Database (Denmark)

    Brandi Sørensen, Elin; Uth Thomsen, Thyra

    2016-01-01

    In this study we investigate the meaning of happiness in a consumption context. We employ an inductive approach and present the results of an exploratory pilot study with eight consumers. The study is based on a Multi-Sensory-Sculpting (MSS) procedure in which we asked consumers to build sculptures...... that represent consumer happiness. Following the MSS guidelines, consumers were interviewed about the meanings of their sculpture in order to elicit embodied cognition about the topic at hand. In this paper we present the meanings of consumer happiness in the participants‟ accounts and discuss implications...... for consumer research. Further, we discuss the applicability of the MSS-procedure to the topic of consumer happiness, and how to optimize it for later studies on consumer happiness....

  9. Induction interview form in EDH

    CERN Document Server

    Information technology Department, AIS (Administrative Information Services) Group

    2007-01-01

    As part of the efforts to rationalise administrative procedures, the IT and HR Departments have developed a new EDH form for induction interviews, which can be accessed using the link below. In accordance with Administrative Circular No. 2 ('Recruitment, Appointment and possible developments regarding the contractual position of Staff Members', Rev. 3), the work and training objectives to be achieved during the probation period shall be specified in writing to all new staff members during an induction interview. The interview shall take place between the new staff member and his supervisor within six weeks of his taking up his duties at the latest. https://edh.cern.ch/Document/MAPS/Induction1) (or from the EDH desktop, by clicking on 'Other Tasks' and going to the 'HR & Training' heading) Please note that this form is to be used exclusively for new staff members. A separate EDH form will be developed for fellows.Information technology Department, AIS (Administrative Information Services) Group Human Re...

  10. Induction interview form in EDH

    CERN Document Server

    Information technology Department, AIS (Administrative Information Services) Group,

    2007-01-01

    As part of the efforts to rationalise administrative procedures, the IT and HR Departments have developed a new EDH form for induction interviews, which can be accessed using the link below. In accordance with Administrative Circular No. 2 ('Recruitment, Appointment and possible developments regarding the contractual position of Staff Members', Rev. 3), the work and training objectives to be achieved during the probation period shall be specified in writing to all new staff members during an induction interview. The interview shall take place between the new staff member and his supervisor within six weeks of him taking up his duties at the latest. https://edh.cern.ch/Document/MAPS/Induction (or from the EDH desktop, by clicking on 'Other Tasks' and going to the 'HR & Training' heading) Please note that this form is to be used exclusively for new staff members. A separate EDH form will be developed for fellows. Information technology Department, AIS (Administrative Information Services) Group Human...

  11. Is there a future for ovulation induction in the current era of assisted reproduction?

    NARCIS (Netherlands)

    E.J. van Santbrink (Evert); B.C.J.M. Fauser (Bart)

    2003-01-01

    textabstractThe clinical use of medical induction of ovulation in normogonadotrophic anovulatory women (WHO II), including polycystic ovary syndrome, is increasingly questioned. However, we believe that this treatment modality still represents a highly effective means of fertility

  12. Situational and Structural Variation in Youth Perceptions of Maternal Guilt Induction.

    Science.gov (United States)

    Rote, Wendy M; Smetana, Judith G

    2017-08-14

    Parental induction of empathy-related guilt plays an important role in children's moral development. However, guilt induction can also be psychologically controlling and detrimental for youth adjustment. This study provided a more nuanced view of parental guilt induction by examining how the nature of a child's misdeed and the structure and content of the parental guilt inductive statement impact children's perceptions of it. Using hypothetical vignettes, this study experimentally examined the impact of the type (domain) of child behavior, highlighted victim, and focus of parental criticism on 156 children's and early and middle adolescents' (age: Ms = 8.82, 12.11, and 15.84 years) perceptions of maternal guilt induction. Attributions of guilt and shame increased most for younger children, when mothers focused on indirect harm to themselves about personal issues, and when mothers criticized their child as a person (shame only). Youth evaluated guilt induction least positively for personal issues and when mothers criticized the child's personality while focusing on indirect harm to themselves. With age, youth were less accepting of maternal guilt induction and more likely to endorse negative and parent-centered intentions, especially for personal issues. Older youth also drew less distinction between guilt induction over multifaceted and personal issues. Guilt induction over moral issues was generally perceived most positively. Additional interactions also emerged. These findings suggest that the meaning and effects of guilt induction on children's development may depend on the way in which it is enacted. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  13. Closed inductively coupled plasma cell

    Science.gov (United States)

    Manning, Thomas J.; Palmer, Byron A.; Hof, Douglas E.

    1990-01-01

    A closed inductively coupled plasma cell generates a relatively high power, low noise plasma for use in spectroscopic studies. A variety of gases can be selected to form the plasma to minimize spectroscopic interference and to provide a electron density and temperature range for the sample to be analyzed. Grounded conductors are placed at the tube ends and axially displaced from the inductive coil, whereby the resulting electromagnetic field acts to elongate the plasma in the tube. Sample materials can be injected in the plasma to be excited for spectroscopy.

  14. Scalable K-Means++

    CERN Document Server

    Bahmani, Bahman; Vattani, Andrea; Kumar, Ravi; Vassilvitskii, Sergei

    2012-01-01

    Over half a century old and showing no signs of aging, k-means remains one of the most popular data processing algorithms. As is well-known, a proper initialization of k-means is crucial for obtaining a good final solution. The recently proposed k-means++ initialization algorithm achieves this, obtaining an initial set of centers that is provably close to the optimum solution. A major downside of the k-means++ is its inherent sequential nature, which limits its applicability to massive data: one must make k passes over the data to find a good initial set of centers. In this work we show how to drastically reduce the number of passes needed to obtain, in parallel, a good initialization. This is unlike prevailing efforts on parallelizing k-means that have mostly focused on the post-initialization phases of k-means. We prove that our proposed initialization algorithm k-means|| obtains a nearly optimal solution after a logarithmic number of passes, and then show that in practice a constant number of passes suffic...

  15. Meaning in Work Life

    DEFF Research Database (Denmark)

    Ravn, Ib

    meaningful is still lacking (Harpaz & Fu, 2002), as are definitions of key terms. Here, I define meaning as the experience that something fits into a wider context or coheres within a larger whole.     I conceptualize meaning in work life as deriving from four factors, each of which helps us contribute...... to something larger than ourselves (Seligman, 2002) through our work. Thus, a person experiences meaning in work life when she... A. is able to use her signature strengths at work (Peterson and Seligman, 2004), B. makes an important contribution to the workplace (Drucker, 1999), C. participates in a productive...

  16. Mean-periodic functions

    Directory of Open Access Journals (Sweden)

    Carlos A. Berenstein

    1980-01-01

    Full Text Available We show that any mean-periodic function f can be represented in terms of exponential-polynomial solutions of the same convolution equation f satisfies, i.e., u∗f=0(μ∈E′(ℝn. This extends to n-variables the work of L. Schwartz on mean-periodicity and also extends L. Ehrenpreis' work on partial differential equations with constant coefficients to arbitrary convolutors. We also answer a number of open questions about mean-periodic functions of one variable. The basic ingredient is our work on interpolation by entire functions in one and several complex variables.

  17. Is membrane sweeping beneficial at the initiation of labor induction?

    Science.gov (United States)

    Al-Harmi, Jehad; Chibber, Rachana; Fouda, Mohamed; Mohammed, Zeinab K; El-Saleh, Eyad; Tasneem, Asiya

    2015-07-01

    To determine whether cervical membrane sweeping during labor induction is beneficial. Outcomes of labor after induction in pregnant women at term were compared in a randomized trial. Women were assigned to having their membranes "swept" or "not swept" at the initiation of labor induction. We recruited a total of 870 women of which 70 were excluded. There were 400 nullipara (Group A) [198 "swept", 202 "not swept"] and 400 multiparas (Group B) (201 "swept" and 199 "not swept"]. Among group A who received intravaginal prostaglandin (PG) E2, those who had simultaneous sweeping had significantly shorter mean induction-labor interval (12.9 ± 1.3 versus 16.2 ± 1.1 hours, p = 0.046), lower mean dose of oxytocin (6.6 ± 0.6 versus 10.11 ± 1.4 mU/minute, p = 0.01), and increased normal delivery rates (vaginal delivery 82.8% versus 58.6%, p = 0.01). Sweeping also had a favorable effect on nulliparas who had ARM and received oxytocin alone (mean induction-labor interval 5.9 ± 2.9 versus 10.9 ± 2.6 hours p = 0.04, mean maximum dose of oxytocin 9.8 ± 1.1 versus 15.2 ± 1.1 mU/min, p = 0.01). These results were restricted to women with unfavorable cervix in Group A those who had membrane sweeping. Membrane sweeping, has beneficial effects on labor and delivery, which is limited to nulliparas with unfavorable cervix requiring PGE2 or Oxytocin alone.

  18. What conceptual spaces can do for Carnap's late inductive logic.

    Science.gov (United States)

    Sznajder, Marta

    2016-04-01

    In the last published account of his late inductive logic, the Basic System of Inductive Logic, Rudolf Carnap introduced a new element to the systems of inductive logic, namely the so-called attribute spaces. These geometrical structures model the meanings of the predicates of the object language and have a similar structure as the conceptual spaces employed by cognitive scientists like Peter Gärdenfors. I show how the development of the theory of conceptual spaces helps us to see the addition of attribute spaces as a step forward in explicating the concept of confirmation. I discuss the differences and similarities of the two theories and investigate the possibilities for developing further connections. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. MISOPROSTOL FOR INDUCTION OF LABOR IN TERM PREGNANCY

    Directory of Open Access Journals (Sweden)

    Gede Angga Permana AW

    2014-01-01

    Full Text Available Induction of labor refers to the process whereby uterine contractions are initiated bymedical or surgical means before the onset of spontaneous labor. Several studies haveshown that continuous intravenous infusion of oxytocin is less efficient, particularlywhen there are unfavorable cervical conditions, leading frequently to a cesarean section,because of induction failure. Misoprostol is a cervical modifying agent and laborinductor. The typical effect of a single dose of oral misoprostol is an increase in uterinetonus. Sustained plasma level of misoprostol is required for the development of regularcontractions appear. The bioavailability of vaginal misoprostol also greater compared tooral, sublingual and rectal administration. Using misoprostol for cervical ripening andlabor induction represented a 47% reduction in the risk of having a cesarean section(risk ratio = 0.53. In cases of unfavorable cervix condition, the use of misoprostolcould produce several beneficial effects compared to other inductor such as oxytocin

  20. Meaning in couples relationships.

    Directory of Open Access Journals (Sweden)

    Rodrigues T.F.

    2014-09-01

    Full Text Available Based on psycholinguistics and L. Vygotsky’s (2007 theories on sign, meaning and sense categories, as later discussed by A. Leontiev (2004, 2009, we present a case study that focuses on the intricacies of a love relationship for a woman who remained in a painful marriage. Interview material is presented in a Relational-Historical Psychology theoretical framework to provide central categories of meaning and sense. This is understood as a privileged method for apprehending the uniqueness of a human being. To segment the qualitative material, we used the “Analysis of the Nuclei of Meanings for the Apprehension of the Constitution of Sense,” by Aguiar and Ozella (2006, 2013. This approach seeks to discriminate the meanings and senses that constitute the content of a speech sample.

  1. Soils - Mean Permeability

    Data.gov (United States)

    Kansas Data Access and Support Center — This digital spatial data set provides information on the magnitude and spatial pattern of depth-weighted, mean soil permeability throughout the State of Kansas. The...

  2. Mean nuclear volume

    DEFF Research Database (Denmark)

    Mogensen, O.; Sørensen, Flemming Brandt; Bichel, P.

    1999-01-01

    We evaluated the following nine parameters with respect to their prognostic value in females with endometrial cancer: four stereologic parameters [mean nuclear volume (MNV), nuclear volume fraction, nuclear index and mitotic index], the immunohistochemical expression of cancer antigen (CA125...

  3. Stats means business

    CERN Document Server

    Buglear, John

    2010-01-01

    Stats Means Business is an introductory textbook written for Business, Hospitality and Tourism students who take modules on Statistics or Quantitative research methods. Recognising that most users of this book will have limited if any grounding in the subject, this book minimises technical language, provides clear definition of key terms, and gives emphasis to interpretation rather than technique.Stats Means Business enables readers to:appreciate the importance of statistical analysis in business, hospitality and tourism understand statis

  4. Comparison of sevoflurane with isoflurane for rapid mask induction in midazolam and butorphanol-sedated dogs.

    Science.gov (United States)

    Mutoh, T; Kojima, K; Takao, K; Nishimura, R; Sasaki, N

    2001-05-01

    Rapid mask induction can be a useful induction technique for veterinary patients, although it is often accompanied by exaggerated excitement responses in unpremedicated animals (Mutoh et al.: Jpn. J. Vet. Anesth. Surg. 26, 109-116; J. Vet. Med. Sci. 57, 1007-1013; J. Vet. Med. Sci. 57, 1121-1124; 1995). The aim of this study was to compare sevoflurane with isoflurane for rapid mask induction in six dogs sedated by a combination of midazolam (0.1 mg/kg) and butorphanol (0.2 mg/kg). Induction with sevoflurane (5%, 2.4 minimum alveolar concentration [MAC]) in O2 resulted in shorter time to loss of the palpebral reflex, negative tail clamp response, and successful intubation than with isoflurane (3%, 2.4 MAC) in O2. There were no changes in heart rate or mean arterial blood pressure during induction with sevoflurane, whereas an increase in heart rate was observed in dogs induced with isoflurane. A decrease in respiratory rate compared with the pre-induction rate was observed during induction, and associated mild respiratory acidosis, characterized by an increase in arterial PCO2, was measured at the end of the induction period in both induction groups. None of the animals had episodes of induction-related complications. These results suggest that both sevoflurane and isoflurane produce a smooth onset of induction in midazolam and butorphanol-sedated dogs. Sevoflurane is a more suitable for rapid mask induction than isoflurane since it provides faster induction associated with a lower blood/gas partition coefficient.

  5. THE CARDIOVASCULAR EFFECTS OF MIDAZOLAM CO-INDUCTION TO PROPOFOL FOR INDUCTION IN GERIATRIC PATIENTS

    Directory of Open Access Journals (Sweden)

    Kaushal

    2014-10-01

    Full Text Available : BACKGROUND: The aim of the study was to investigate whether a small dose of midazolam and lessening the propofol dosage could prevent the cardiovascular change at tracheal intubation for induction in geriatric patients. METHODS: ninety patients over 65 (ASA physical status 1, 2 scheduled for elective surgery received general anaesthesia with fentanyl and propofol or midazolam. Patients in group P (n= 45 were induced with 0.9% NaCl 0.03 ml /kg, propofol 1.2 mg/kg and fentanyl. Patients in group MP (n= 45 were induced with midazolam 0.03 mg/ kg, propofol 0.8 mg/kg and fentanyl. The time taken to reach loss of consciousness (LOC. After LOC 0.5 mg/kg of atracurium was given and tracheal intubation was performed. The mean blood pressure (MBP and heart rate (HR were recorded were induction as the base value, before intubation, immediately post intubation and 3 minutes after intubation. RESULT: compared with the base values, MBP at before intubation and # minutes after intubation was significantly decreased in group P and group MP (P <0.05. compared with group P, the decrease of MBP was significantly less at before intubation, immediately after intubation and 3 minutes after intubation in group MP (P<0.05. The time taken to reach LOC was significantly decreased in group MP compared with that in group P (P<0.05. There was no significant difference of HR at any time between the two groups. CONCLUSION: co-induction with midazolam and propofol could prevent a marked BP decrease at tracheal intubation for induction in geriatric patients.

  6. Teaching the Photoelectric Effect Inductively

    Science.gov (United States)

    Sokolowski, Andrzej

    2013-01-01

    Research has shown that students have difficulty understanding the underlying process of the photoelectric effect. Thus, this study sought to utilize an inductively situated lesson for teaching the photoelectric effect, hypothesizing that this type of enquiry would help learners delve deeper into the principles of the phenomenon and provide a…

  7. Experiments with an Induction Cooker

    Science.gov (United States)

    Zilavy, Peter

    2009-01-01

    The induction cooker is a common appliance nowadays. How does it work? Why is it not possible to use aluminium utensils with it? What experiments can be carried out with it (at different levels) and not only in physics lessons? Searching for the answers to these and other questions is the purpose of this article. (Contains 5 figures.)

  8. Direct-reading inductance meter

    Science.gov (United States)

    Kolbly, R. B.

    1977-01-01

    Meter indicates from 30 nH to 3 micro H. Reference inductor of 15 micro H is made by winding 50 turns of Number 26 Formvar wire on Micrometal type 50-2 (or equivalent) core. Circuit eliminates requirement for complex instrument compensation prior to taking coil inductance measurement and thus is as easy to operate as common ohmmeter.

  9. Low-inductance bus lines

    Science.gov (United States)

    Kernick, A.

    1977-01-01

    Laminated bus strips and bifilar litz cable connectors for high-power rectifiers, thrisistors, and transistors provide low inductance and eliminate electromagnetic interference in high-power circuits. These techniques offer significant cost advantages because of ease of assembly and consistent high quality of product. Effectiveness makes general usage in static power conversion likely.

  10. Mathematical Induction: Deductive Logic Perspective

    Science.gov (United States)

    Dogan, Hamide

    2016-01-01

    Many studies mentioned the deductive nature of Mathematical Induction (MI) proofs but almost all fell short in explaining its potential role in the formation of the misconceptions reported in the literature. This paper is the first of its kind looking at the misconceptions from the perspective of the abstract of the deductive logic from one's…

  11. Dirac Induction for loop groups

    NARCIS (Netherlands)

    Posthuma, H.

    2011-01-01

    Using a coset version of the cubic Dirac operators for affine Lie algebras, we give an algebraic construction of the Dirac induction homomorphism for loop group representations. With this, we prove a homogeneous generalization of the Weyl-Kac character formula and show compatibility with Dirac induc

  12. Plasma-heating by induction

    Science.gov (United States)

    Harrington, K.; Thorpe, M. L.

    1969-01-01

    Induction-heated plasma torch operates with an input of 1 Mw of direct current of which 71 percent is transferred to the plasma and the remainder is consumed by electrical losses in the system. Continuous operation of the torch should be possible for as long as 5,000 hours.

  13. Inductive logic on conceptual spaces

    NARCIS (Netherlands)

    Sznajder, Marta

    2017-01-01

    This thesis investigates the question of how conceptual frameworks influence inductive reasoning. A conceptual framework is a collection of concepts used for a particular purpose; we can think of it as a semantic environment in which observations, or evidence, are recorded, and beliefs are formed an

  14. Teaching Inductive Reasoning with Puzzles

    Science.gov (United States)

    Wanko, Jeffrey J.

    2017-01-01

    Working with language-independent logic structures can help students develop both inductive and deductive reasoning skills. The Japanese publisher Nikoli (with resources available both in print and online) produces a treasure trove of language-independent logic puzzles. The Nikoli print resources are mostly in Japanese, creating the extra…

  15. Oral misoprostol for induction of labour.

    Science.gov (United States)

    Alfirevic, Zarko; Aflaifel, Nasreen; Weeks, Andrew

    2014-06-13

    achieved in 24 hours, uterine hyperstimulation with fetal heart rate (FHR) changes, and caesarean section were highly heterogenous - for uterine hyperstimulation with FHR changes this was related to dosage with lower rates in those with lower doses of oral misoprostol. However, there were fewer babies born with a low Apgar score in the oral group (RR 0.60, 95% CI 0.44 to 0.82; 19 trials; 4009 babies) and a decrease in postpartum haemorrhage (RR 0.57, 95% CI 0.34 to 0.95; 10 trials; 1478 women). However, the oral misoprostol group had an increase in meconium-stained liquor (RR 1.22, 95% CI 1.03 to 1.44; 24 trials; 3634 women). Oral misoprostol as an induction agent is effective at achieving vaginal birth. It is more effective than placebo, as effective as vaginal misoprostol and results in fewer caesarean sections than vaginal dinoprostone or oxytocin.Where misoprostol remains unlicensed for the induction of labour, many practitioners will prefer to use a licensed product like dinoprostone. If using oral misoprostol, the evidence suggests that the dose should be 20 to 25 mcg in solution. Given that safety is the primary concern, the evidence supports the use of oral regimens over vaginal regimens. This is especially important in situations where the risk of ascending infection is high and the lack of staff means that women cannot be intensely monitored.

  16. Butterfly-valve inductive orientation detector

    Science.gov (United States)

    Garrett, Steven

    1980-04-01

    Relative changes of inductance ΔL/L of a single layer coil surrounding a thin electrically conducting disk which can rotate about an axis perpendicular to the coil axis are studied experimentally as a means of measuring angular displacements. ΔL/L is found to be a strong function of disk diameter and is weakly dependent on the ratio of disk thickness to electromagnetic skin depth when this ratio is of the order unity. Values of ΔL/L as a function of disk diameter are given for lead, brass and copper. Detection sensitivities using a resonant tank circuit or an astatic transformer are given in terms of ΔL/L and it is shown that sensitivities of the order of 10-3 to 10-4 deg are practical. Application of this system to the Rayleigh disk and cryogenic environments are emphasized and an expression for the magnetic torque due to detection currents is given.

  17. Evaluative Meaning in Translation

    Directory of Open Access Journals (Sweden)

    Primož Jurko

    2015-12-01

    Full Text Available Evaluative meaning is frequently understood as a form of connotation, pertaining to single lexical items. However, the term has recently been applied also to longer stretches of text, spanning several words. The paper generally deals with the latter aspect and provides a corpus-driven analysis of several Slovene and English phrases that may appear semantically neutral, yet turn out to be heavily non-neutral in terms of their semantic properties. After a short introduction of the main approaches to the topic, including its dilemmas, the paper focuses on the L1-into-L2 translation of such strings, with particular emphasis on proper rendering of the evaluative meaning. While further research is warranted into the matter, the work presented here attests to the complex and ubiquitous nature of the Sinclairian extended unit of meaning.

  18. Meaning of Fractions

    Science.gov (United States)

    Dewi, D. A. K.; Suryadi, D.; Suratno, T.; Mulyana, E.; Kurniawan, H.

    2017-02-01

    Introducing fractions is identical to divide an object. Suppose we divide the apple into two parts. One divided into two parts, the question arises whether one part can be called a half or not. Based on this activity, how can students give meaning to fractions. This study aims at designing a different fractions lesson by applying Didactical Design Research. In doing so, we undertook several research phases: 1) thinking what is fractions and why students should learn this concept; 2) designing didactical situation based on identified learning obstacles; and 3) reflecting retrospectively on the lesson design and its implementation as to redesign the fractions lesson. Our analysis revealed that most students held epistemological obstacles in giving meaning of fractions because they only know fractions as numbers that have numerator and denominator. By positioning ourselves as students, we discuss the ideal design to help students in constructing the meaning of fractions.

  19. Computing meaning v.4

    CERN Document Server

    Bunt, Harry; Pulman, Stephen

    2013-01-01

    This book is a collection of papers by leading researchers in computational semantics. It presents a state-of-the-art overview of recent and current research in computational semantics, including descriptions of new methods for constructing and improving resources for semantic computation, such as WordNet, VerbNet, and semantically annotated corpora. It also presents new statistical methods in semantic computation, such as the application of distributional semantics in the compositional calculation of sentence meanings. Computing the meaning of sentences, texts, and spoken or texted dialogue i

  20. Mean Field ICA

    DEFF Research Database (Denmark)

    Petersen, Kaare Brandt

    2006-01-01

    This thesis describes investigations and improvements of a technique for Independent Component Analysis (ICA), called "Mean Field ICA". The main focus of the thesis is the optimization part of the algorithm, the so-called "EM algorithm". Using different approaches it is demonstrated that the EM...... Gradient Recipe is applicable to a wide selection of models. Furthermore, the Mean Field ICA model is extended to incorporate ltering over time in a so-called "convolutive ICA" model. Finally, by using mixture of Gaussians as source priors, the generative and ltering approach to ICA is compared...

  1. Intersubjective meaning making

    DEFF Research Database (Denmark)

    Davidsen, Jacob

    of single-touch screen interaction among 8-9 year-old children presented here, shows that while the constraints of single-touch screens does not support equality of interaction at the verbal and the physical level, there seems to be an intersubjective learning outcome. More precisely, the constraints...... of single-touch screens offer support for intersubjective meaning making in its ability of constraining the interaction. By presenting a short embodied interaction analysis of 22 seconds of collaboration, I illustrate how an embodied interaction perspective on intersubjective meaning making can tell...... a different story about touch-screen supported collaborative learning....

  2. Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for oxidizing nitrobenzene to 3-nitrocatechol, 4-nitrocatechol, and nitrohydroquinone.

    Science.gov (United States)

    Vardar, Gönül; Ryu, Kang; Wood, Thomas K

    2005-01-26

    Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 was found to oxidize nitrobenzene (NB) to form m-nitrophenol (m-NP, 72%) and p-NP (28%) with an initial rate of 0.098 and 0.031 nmol/(min mg protein), respectively. It was also discovered that wild-type ToMO forms 4-nitrocatechol (4-NC) from m-NP and p-NP with an initial rate of 0.15 and 0.0082 nmol/(min mg protein), respectively, and 3-NC (12%) and nitrohydroquinone (NHQ, 88%) from o-NP with an initial rate of 0.11 and 0.8 nmol/(min mg protein), respectively. To increase the oxidation rate and alter the oxidation regiospecificity of nitro aromatics as well as to study the role of the active site residues I100, Q141, T201, and F205 of the alpha hydroxylase fragment of ToMO (TouA), DNA shuffling and saturation mutagenesis were used to generate random mutants. The mutants were initially identified by screening via a rapid agar plate assay and then were further examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC). Several mutants with higher rates of activities and with different regiospecificities were identified; for example, Escherichia coli TG1 cells expressing either TouA mutant M180T/E284G or E214G/D312N/M399V produce 4-NC 4.5- and 20-fold faster than wild-type ToMO (0.037 and 0.16 nmol/min mg protein from p-NP, respectively). TouA mutant A107T/E214A had the regiospecificity of NB changed significantly from 28% to 79% p-NP. From 200 microM NB, TouA variants A101T/M114T, A110T/E392D, M180T/E284G, and E214G/D312N/M399V produce 4-NC whereas wild-type ToMO does not. From m-NP, TouA mutant I100Q produces 4-NC (37%) and NHQ (63%), whereas wild-type ToMO produces only 4-NC (100%). Variant A107T/E214A acts like a para enzyme and forms p-cresol as the major product (93%) from toluene with enhanced activity (2.3-fold), whereas wild-type ToMO forms 32%, 21%, and 47% of o-, m-, and p-cresol, respectively. Hence, the non-specific ToMO was converted into a regiospecific enzyme

  3. Programming with Inductive and Co-Inductive Types

    Science.gov (United States)

    1992-01-27

    Lecture Notes in Computer Science 389, eds. D. H. Pitt, D. E. Rydeheard, P. Dybjer, A. M. Pitts, and A. Poign6...University of Edinburgh. 1987. [5] Thierry Coquand and Christine Paulin. Inductively defined types. In COLOG-88, Lecture Notes in Computer Science 417, eds...Computer Science, Lecture Notes in Computer Science 283, eds. D. H. Pitt, A. Poing6, and D. E. Rydeheard, pages 140-157. September 1987. [9] Tatsuya

  4. Induction generator-induction motor wind-powered pumping system

    Energy Technology Data Exchange (ETDEWEB)

    Miranda, M.S.; Lyra, R.O.C.; Silva, S.R. [CPDEE - UFMG, Belo Horizonte (Brazil)

    1997-12-31

    The energy storage matter plays an important role in wind-electric conversion systems for isolated applications. Having that in mind, two different approaches can be basically considered: either the immediate conversion of the generated electric energy, as in a water pumping system or electric energy storage for later use, as in a battery charging system. Due to some features such as no need of an external reactive power source and, sometimes, a gearbox, permanent-magnet synchronous generators have been broadly used in low rated power isolated systems. Despite that, system performance can be affected when the generator is feeding an inductive load (e.g., an induction motor) under variable-speed-variable-frequency operational conditions. Since there is no effective flux control, motor overload may occur at high wind speeds. Thus, good system performance can be obtained through additional control devices which may increase system cost. Although being rugged and cheap, induction machines always work as a reactive power drain; therefore, they demand an external reactive power source. Considering that, reactive static compensators appear as an attractive alternative to the cost x performance problem. In addition to that, different control strategies can be used so that system performance can be improved.

  5. Flux Tracking Control of Induction Motors

    Institute of Scientific and Technical Information of China (English)

    LanLin; XiaowuMu; ChunxiaBu

    2004-01-01

    This paper deals with flux tracking control of induction motors. Firstly,we analyze convergency of non-homogeneous linear time-varying systems and a sufficient condition is given. Finally, the flux regulator of induction motors is discussed.

  6. Inductance Scaling of a Helicoil Using ALEGRA

    Science.gov (United States)

    2013-05-01

    5066 The inductance scaling of several helicoil configurations are investigated using the March 2011 release of the Sandia magnetohydrodynamics (MHD...inductance scaling of several helicoil configurations are investigated using the March 2011 release of the Sandia magnetohydrodynamics (MHD) code

  7. Induction of anesthesia in coronary artery bypass graft surgery: the hemodynamic and analgesic effects of ketamine

    Directory of Open Access Journals (Sweden)

    Elif Basagan-Mogo

    2010-01-01

    Full Text Available OBJECTIVE: The aim of this prospective, randomized study was to evaluate the hemodynamic and analgesic effects of ketamine by comparing it with propofol starting at the induction of anesthesia until the end of sternotomy in patients undergoing coronary artery bypass grafting surgery. INTRODUCTION: Anesthetic induction and maintenance may induce myocardial ischemia in patients with coronary artery disease. A primary goal in the anesthesia of patients undergoing coronary artery bypass grafting surgery is both the attenuation of sympathetic responses to noxious stimuli and the prevention of hypotension. METHODS: Thirty patients undergoing coronary artery bypass grafting surgery were randomized to receive either ketamine 2 mg.kg-1 (Group K or propofol 0.5 mg.kg-1 (Group P during induction of anesthesia. Patients also received standardized doses of midazolam, fentanyl, and rocuronium in the induction sequence. The duration of anesthesia from induction to skin incision and sternotomy, as well as the supplemental doses of fentanyl and sevoflurane, were recorded. Heart rate, mean arterial pressure, central venous pressure, pulmonary arterial pressure, pulmonary capillary wedge pressure, cardiac index, systemic and pulmonary vascular resistance indices, stroke work index, and left and right ventricular stroke work indices were obtained before induction of anesthesia; one minute after induction; one, three, five, and ten minutes after intubation; one minute after skin incision; and at one minute after sternotomy. RESULTS: There were significant changes in the measured and calculated hemodynamic variables when compared to their values before induction. One minute after induction, mean arterial pressure and the systemic vascular resistance index decreased significantly in group P (p<0.01. CONCLUSION: There were no differences between groups in the consumption of sevoflurane or in the use of additional fentanyl. The combination of ketamine, midazolam, and

  8. The meaning of talent

    Directory of Open Access Journals (Sweden)

    Raquel Lorenzo

    2006-01-01

    Full Text Available The knowledge society gives a remarkable role to human potentialities into organizations. For this reason has emerged talent management. But it is necessary to know its meaning to put on practice programs. In this paper, the author analyzes different points of views about this topic.

  9. Engagement Means Everyone

    Science.gov (United States)

    Patton, Carol

    2012-01-01

    Employee engagement is not just HR's responsibility. While HR is responsible for the process of measuring and driving engagement, improving it is actually everyone's responsibility. And that means reducing the barriers to productivity to drive business performance. Training departments can play a pivotal role. Their job is to enhance curriculum or…

  10. Meaning in Context

    DEFF Research Database (Denmark)

    Christiansen, Henning; Dahl, Veronica

    2005-01-01

    A model for context-dependent natural language semantics is proposed and formalized in terms of possible worlds. The meaning of a sentence depends on context and at the same time affects that context representing the knowledge about the world collected from a discourse. The model fits well...

  11. Conversations across Meaning Variance

    Science.gov (United States)

    Cordero, Alberto

    2013-01-01

    Progressive interpretations of scientific theories have long been denounced as naive, because of the inescapability of meaning variance. The charge reportedly applies to recent realist moves that focus on theory-parts rather than whole theories. This paper considers the question of what "theory-parts" of epistemic significance (if any) relevantly…

  12. What does 'regionalisation' mean?

    DEFF Research Database (Denmark)

    Hegland, Troels Jacob; Ounanian, Kristen; Raakjær, Jesper

    2012-01-01

    . To draw implications for policy, we explore these perceptions and seek, by means of interviews and a survey of participants in the Regional Advisory Councils with hands-on experience in regional cooperation in European fisheries management, to flesh out the question whether regionalisation is seen a good...

  13. The Problem of Meaning.

    Science.gov (United States)

    Barrell, Arthur Rex

    1970-01-01

    The study of semantics, the study of meaning, promises more complete communicative thought transfer if several problem areas can be solved or at least generally agreed upon. The problem of exact definition of words arises from the fact that no one work is the exact equivalent of another. However, the study of many languages forces the mind to…

  14. The meaning of curvature

    DEFF Research Database (Denmark)

    diffusion to volume growth. We are e.g. interested in obtaining precise bounds for mean exit times for Brownian motions and for isoperimetric inequalities. One way to obtain such bounds are via curvature controlled comparison with corresponding values in constant curvature spaces and in other tailor-made so...

  15. PR Means Positive Results.

    Science.gov (United States)

    Foree, Marilyn

    1992-01-01

    Describes the advantages of public relations to a school music program. Suggests that, to succeed, a music program must have the support of school administrators, teachers, parents, and students alike. Recommends means of attracting support from the four groups. Stresses the importance of effective local publicity. (SG)

  16. Meaning = Information + Evolution

    CERN Document Server

    Rovelli, Carlo

    2016-01-01

    Notions like meaning, signal, intentionality, are difficult to relate to a physical word. I study a purely physical definition of "meaningful information", from which these notions can be derived. It is inspired by a model recently illustrated by Kolchinsky and Wolpert, and improves on Dretske classic work on the relation between knowledge and information. I discuss what makes a physical process into a "signal".

  17. Phenomenology and Meaning Attribution

    African Journals Online (AJOL)

    ... may be both confusing and disorienting to academic and clinical practitioners who ... questions of students of phenomenology often are the most appropriate ... Meaning attribution is the psychological study of the ... a best-seller with my publisher. But, except for ..... perhaps listening to music, having a chat, and things are ...

  18. The Measurement of Meaning.

    Science.gov (United States)

    Osgood, Charles E.; And Others

    The problem of measuring meaning viewed strictly as a psychological conception via the application of semantic differential theory is presented in this study. The first half of the book discusses theoretical background, logic of semantic measurement, basic factor analysis research, and a critical scientific evaluation of measuring procedures. The…

  19. Engagement Means Everyone

    Science.gov (United States)

    Patton, Carol

    2012-01-01

    Employee engagement is not just HR's responsibility. While HR is responsible for the process of measuring and driving engagement, improving it is actually everyone's responsibility. And that means reducing the barriers to productivity to drive business performance. Training departments can play a pivotal role. Their job is to enhance curriculum or…

  20. CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes.

    Science.gov (United States)

    Syed, Khajamohiddin; Porollo, Aleksey; Lam, Ying Wai; Grimmett, Paul E; Yadav, Jagjit S

    2013-04-01

    Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons, albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).

  1. X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase.

    Science.gov (United States)

    Sazinsky, Matthew H; Dunten, Pete W; McCormick, Michael S; DiDonato, Alberto; Lippard, Stephen J

    2006-12-26

    Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket

  2. Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph.

    Science.gov (United States)

    Fox, B G; Froland, W A; Dege, J E; Lipscomb, J D

    1989-06-15

    Methane monooxygenase has been purified from the Type II methanotroph Methylosinus trichosporium OB3b. As observed for methane monooxygenase isolated from Type I methanotrophs, three protein components are required: a 39.7-kDa NADH reductase containing 1 mol each of FAD and a [2Fe-2S] cluster, a 15.8-kDa protein factor termed component B that contains no metals or cofactors, and a 245-kDa hydroxylase which appears to contain an oxo- or hydroxo-bridged binuclear iron cluster. Through the use of stabilizing reagents, the hydroxylase is obtained in high yield and exhibits a specific activity 8-25-fold greater than reported for previous preparations. The component B and reductase exhibit 1.5- and 4-fold greater specific activity, respectively. Quantitation of the hydroxylase oxo-bridged cluster using EPR and Mössbauer spectroscopies reveals that the highest specific activity preparations (approximately 1700 nmol/min/mg) contain approximately 2 clusters/mol. In contrast, hydroxylase preparations exhibiting a wide range of specific activities below 500 nmol/min/mg contain approximately 1 cluster/mol on average. Efficient turnover coupled to NADH oxidation requires all three protein components. However, both alkanes and alkenes are hydroxylated by the chemically reduced hydroxylase under single turnover conditions in the absence of component B and the reductase. Neither of these components catalyzes hydroxylation individually nor do they significantly affect the yield of hydroxylated product from the chemically reduced hydroxylase. Hydroxylase reduced only to the mixed valent [Fe(II).Fe(III)] state is unreactive toward O2 and yields little hydroxylated product on single turnover. This suggests that the catalytically active species is the fully reduced form. The data presented here provide the first evidence based on catalysis that the site of the monooxygenation reaction is located on the hydroxylase. It thus appears likely that the oxo-bridged iron cluster is capable of

  3. Indução do crescimento ovariano em Aegla uruguayana (Crustacea, Anomura, Aeglidae mediante a incorporação de neuroreguladores ao alimento Induction of ovarian growth in Aegla uruguayana (Anomura, Aeglidae by means of neuroregulators incorporated to food

    Directory of Open Access Journals (Sweden)

    Daniela da S. Castiglioni

    2009-09-01

    grupos experimentais.The stimulatory effect of the spiperone and naloxone on the ovarian growth was evaluated in females of Aegla uruguayana Schmitt, 1942, being that these neuroregulators were incorporated to food and administrated at a dose of 10-8 mol/animal to each session food. Adult females were sampled with nets in a stream near the municipality of Salto, Province of Buenos Aires, Argentina. At the beginning of the experiment, 10 females were randomly selected, sacrificed, weighed and their ovaries were quickly dissected and weighed to serve as the initial control for evaluating the degree of ovarian growth (gonadosomatic index -GI. Others females (30 were divided in three experimental groups - (a control: females fed on control pellets composed by fish food - 34% protein and 43% protein; (b spiperone: females fed on pellets enriched with the dopaminegic antagonist spiperone; (c naloxone: females fed on pellets enriched with the enkephalinergic antagonist naloxone. After seven week of experiment the females were sacrificed and evaluated the GI. The lipids and cholesterol levels of ovaries and hepatopancreas were quantified. Naloxone produced a significant increase of lipids levels in both ovaries and hepatopancreas in relation to control group. Spiperone caused significant increase of lipids levels at the gonads and hepatopancreas and cholesterol in hepatopancreas when compared with the control. The lipids levels were significantly lower in hemolymph of the females that were fed with pellets with spiperone and higher at the females treated with naloxone when compared to females that were fed only fish food. The spiperone and naloxone when inhibited the effect of the dopamine and endogenous opioids, probably caused the secretion of the gonad stimulating hormone and the inhibition of the gonad inhibiting hormone, therefore induction of the ovarian development. Such hypothesis can be strengthened for the increases of gonadosomatic indices in these experimental groups.

  4. Structured Statistical Models of Inductive Reasoning

    Science.gov (United States)

    Kemp, Charles; Tenenbaum, Joshua B.

    2009-01-01

    Everyday inductive inferences are often guided by rich background knowledge. Formal models of induction should aim to incorporate this knowledge and should explain how different kinds of knowledge lead to the distinctive patterns of reasoning found in different inductive contexts. This article presents a Bayesian framework that attempts to meet…

  5. Online Monitoring of Induction Motors

    Energy Technology Data Exchange (ETDEWEB)

    McJunkin, Timothy R. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Agarwal, Vivek [Idaho National Lab. (INL), Idaho Falls, ID (United States); Lybeck, Nancy Jean [Idaho National Lab. (INL), Idaho Falls, ID (United States)

    2016-01-01

    The online monitoring of active components project, under the Advanced Instrumentation, Information, and Control Technologies Pathway of the Light Water Reactor Sustainability Program, researched diagnostic and prognostic models for alternating current induction motors (IM). Idaho National Laboratory (INL) worked with the Electric Power Research Institute (EPRI) to augment and revise the fault signatures previously implemented in the Asset Fault Signature Database of EPRI’s Fleet Wide Prognostic and Health Management (FW PHM) Suite software. Induction Motor diagnostic models were researched using the experimental data collected by Idaho State University. Prognostic models were explored in the set of literature and through a limited experiment with 40HP to seek the Remaining Useful Life Database of the FW PHM Suite.

  6. Teaching the photoelectric effect inductively

    Science.gov (United States)

    Sokolowski, Andrzej

    2013-01-01

    Research has shown that students have difficulty understanding the underlying process of the photoelectric effect. Thus, this study sought to utilize an inductively situated lesson for teaching the photoelectric effect, hypothesizing that this type of enquiry would help learners delve deeper into the principles of the phenomenon and provide a better background for its quantification. The lesson was conducted with a group of fifteen high-school physics students and a computerized simulated experiment was utilized as the medium for instruction. Students’ responses to a qualitative question about the role of a battery in the photoelectric circuit supported the hypothesis that the lesson was a valuable learning experience. This paper presents an outline for the inductively reasoned learning process.

  7. Induction studies with satellite data

    DEFF Research Database (Denmark)

    Olsen, Nils

    1999-01-01

    analysis of the geomagnetic field is performed, and the Q-response, which is the transfer function between the internal (induced) and the external (inducing) expansion coefficients is determined for a specific frequency. In the second approach, known as the geomagnetic depth sounding method, the C....... This paper reviews and discusses the possibilities for induction studies using high-precision magnetic measurements from low-altitude satellites. The different methods and various transfer functions are presented, with special emphasis on the differences in analysing data from ground stations and from...... satellites. The results of several induction studies with scalar satellite data (from the POGO satellites) and with vector data (from the Magsat mission) demonstrate the ability to probe the Earth's conductivity from space. However, compared to the results obtained with ground data the satellite results...

  8. The performance of induction levitators

    Science.gov (United States)

    Eastham, J. F.; Rodger, D.

    1984-09-01

    The present investigation is concerned with the performance of induction levitators, which are employed in vehicles for contactless transport systems, utilizing magnetic levitation (Maglev). A small model (38 cm long) of an induction levitator is shown. The armature consists of a laminated 'u' shaped iron yoke. Around the limbs of the yoke are wound two primary excitation coils carrying single phase 50 Hz current. Eddy currents, induced in the conducting secondary, produce a force of repulsion between secondary and yoke. A lateral stabilizing force can also be obtained. A description is presented of a study of the characteristics of these forces. Attention is given of a finite element model and the application of the Galerkin weighted residual technique, experimental and calculated results, and a design study of two single phase levitators for a 50 tonne Maglev vehicle.

  9. Inductive shearing of drilling pipe

    Energy Technology Data Exchange (ETDEWEB)

    Ludtka, Gerard M.; Wilgen, John; Kisner, Roger; Mcintyre, Timothy

    2016-04-19

    Induction shearing may be used to cut a drillpipe at an undersea well. Electromagnetic rings may be built into a blow-out preventer (BOP) at the seafloor. The electromagnetic rings create a magnetic field through the drillpipe and may transfer sufficient energy to change the state of the metal drillpipe to shear the drillpipe. After shearing the drillpipe, the drillpipe may be sealed to prevent further leakage of well contents.

  10. Induction and Mutually Obstructing Equilibria

    OpenAIRE

    Hahn, Dorothea; Liu, Mario

    2004-01-01

    A unified, consistent and simple view of the Faraday law of induction is presented, which consists of two points: discriminating the lab- from the rest-frame electric field and understanding it is the impossibility for both fields to vanish simultaneously, which generates and maintains the circular current. A number of illustrative examples are considered, including a mechano-electric pendulum to exhibit periodic and reversible conversion between electrical and mechanical energy.

  11. Inductive, Analogical, and Communicative Generalization

    Directory of Open Access Journals (Sweden)

    Adri Smaling

    2003-03-01

    Full Text Available Three forms of inductive generalization - statistical generalization, variation-based generalization and theory-carried generalization - are insufficient concerning case-to-case generalization, which is a form of analogical generalization. The quality of case-to-case generalization needs to be reinforced by setting up explicit analogical argumentation. To evaluate analogical argumentation six criteria are discussed. Good analogical reasoning is an indispensable support to forms of communicative generalization - receptive and responsive (participative generalization — as well as exemplary generalization.

  12. Inductive shearing of drilling pipe

    Science.gov (United States)

    Ludtka, Gerard M.; Wilgen, John; Kisner, Roger; Mcintyre, Timothy

    2016-04-19

    Induction shearing may be used to cut a drillpipe at an undersea well. Electromagnetic rings may be built into a blow-out preventer (BOP) at the seafloor. The electromagnetic rings create a magnetic field through the drillpipe and may transfer sufficient energy to change the state of the metal drillpipe to shear the drillpipe. After shearing the drillpipe, the drillpipe may be sealed to prevent further leakage of well contents.

  13. The law of electromagnetic induction

    Directory of Open Access Journals (Sweden)

    V.J. Kutkovetskyy

    2014-09-01

    Full Text Available Mathematical models of the electromagnetic induction law which do not take into account Faraday’s restrictions are not in full accordance with the physical phenomenon and so they are not laws. Their incomplete correspondence with real devices results in such "paradoxes" as unlimited magnetic field of unipolar generators, infinite sizes of inductors for DC and AC machines modeled, and so on.

  14. First-Year Undergraduate Induction: Who Attends and How Important Is Induction for First Year Attainment?

    Science.gov (United States)

    Murtagh, S.; Ridley, A.; Frings, D.; Kerr-Pertic, S.

    2017-01-01

    The first year of study in higher education is a time of major transition for students. While the importance of induction has been widely demonstrated, there is evidence to suggest that not all students benefit equally from participation in induction. This study examined attendance rates at induction, the relationship between induction attendance…

  15. Negotiating meaning through artefacts

    DEFF Research Database (Denmark)

    Tavella, Elena

    2015-01-01

    This research contributes to the domain of strategy making, specifically to unpacking the complexity of sociomateriality in strategy discourse. Scholars have emphasized the potential of artefacts to enhance sensemaking during strategizing. However there is a lack of insight into how artefacts...... and conversational aspects are linked at the micro‑level of discourse, also how artefacts and sensemaking shape one another. This research addresses this gap by empirically analyzing strategy discourse within a facilitated modelling workshop. Considering strategizing as a socially constructed activity, the author...... analyzes a workshop transcript to assess the extent to which stakeholders’ appropriation of artefacts supports them in engaging in negotiation of meaning with action implications. Moreover, how artefacts and negotiation of meaning shape one another is identified. The data suggest that appropriating...

  16. Means of Transport

    DEFF Research Database (Denmark)

    Balle, Søren Hattesen

    2004-01-01

    The car is probably one of the most central signifiers of modern scientific and technological progress. Not only did its introduction in early 20th century America quite literally move scientific progress into the street, it also provided its increasing number of users with new and technologically...... improved means of fast physical movement from one place to another. Yet, the impact that the technology of automobility came to have was not of a limited materialistic nature. As many literary and cultural critics have noted, car technology extended its influence into the field of imaginative transport...... as a means of physical and spiritual transport is thus consistently embedded in a less idealized context, where its technological machinery is foregrounded. In conclusion, the paper argues that Williams’s thematic treatment of the car in his writings may be read as a modern poet’s meta-poetic gloss on poetry...

  17. Feeling, Meaning, and Intentionality

    DEFF Research Database (Denmark)

    Bundgaard, Peer

    2014-01-01

    This article addresses the phenomenology of aesthetic experience. It first, critically, considers one of the most influential approaches to the psychophysics of aesthetic perception, viz. neuroaesthetics. Hereafter, it outlines constitutive tenets of aesthetic perception in terms of a particular...... relative to its object and the tools for meaning-making specific to that object, and not relative to the feeling (of beauty) it may elicit. Finally, I sketch the import this fact may have on a research program in empirical aesthetics....

  18. The Meaning of Mandela

    DEFF Research Database (Denmark)

    Krabbe, Julia Suárez

    2017-01-01

    Book Reviews The Meaning of Mandela: A Broader Canvas Sabelo J. Ndlovu-Gatsheni, The Decolonial Mandela: Peace, Justice and the Politics of Life (Oxford and New York, Berghahn Books, 2016), 186 pp., hardback, £64.00, US$90.00, ISBN 978-1-78533-118-3; paperback, £19.00, US$27.95, ISBN 978-1-78533-......-1-78533-296-8; e-book, ISBN 978-1-79533-119-0...

  19. Weighted Mean Impact Analysis

    OpenAIRE

    Burger, Svenja

    2015-01-01

    Linear regression is a popular tool that is often applied to biometric and epidemiological data. It relies on strong model assumptions that are rarely satisfied. To overcome this difficulty, Brannath and Scharpenberg (2014) proposed a new population based interpretation of linear regression coefficients. The idea is to quantify how much the unconditional mean of the dependent variable Y can be changed by changing the distribution of the independent variable X. The maximum change is called "me...

  20. Meaning and significance of

    Directory of Open Access Journals (Sweden)

    Ph D Student Roman Mihaela

    2011-05-01

    Full Text Available The concept of "public accountability" is a challenge for political science as a new concept in this area in full debate and developement ,both in theory and practice. This paper is a theoretical approach of displaying some definitions, relevant meanings and significance odf the concept in political science. The importance of this concept is that although originally it was used as a tool to improve effectiveness and eficiency of public governance, it has gradually become a purpose it itself. "Accountability" has become an image of good governance first in the United States of America then in the European Union.Nevertheless,the concept is vaguely defined and provides ambiguous images of good governance.This paper begins with the presentation of some general meanings of the concept as they emerge from specialized dictionaries and ancyclopaedies and continues with the meanings developed in political science. The concept of "public accontability" is rooted in economics and management literature,becoming increasingly relevant in today's political science both in theory and discourse as well as in practice in formulating and evaluating public policies. A first conclusin that emerges from, the analysis of the evolution of this term is that it requires a conceptual clarification in political science. A clear definition will then enable an appropriate model of proving the system of public accountability in formulating and assessing public policies, in order to implement a system of assessment and monitoring thereof.