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Sample records for protoporphyrinogen oxidase inhibitor

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

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

    2003-10-01

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

  2. Generation of Resistance to the Diphenyl Ether Herbicide, Oxyfluorfen, via Expression of the Bacillus subtilis Protoporphyrinogen Oxidase Gene in Transgenic Tobacco Plants.

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    Choi, K W; Han, O; Lee, H J; Yun, Y C; Moon, Y H; Kim, M; Kuk, Y I; Han, S U; Guh, J O

    1998-01-01

    In an effort to develop transgenic plants resistant to diphenyl ether herbicides, we introduced the protoporphyrinogen oxidase (EC 1.3.3.4) gene of Bacillus subtilis into tobacco plants. The results from a Northern analysis and leaf disc assay indicate that the expression of the B. subtilis protoporphyrinogen oxidase gene under the cauliflower mosaic virus 35S promoter generated resistance to the diphenyl ether herbicide, oxyfluorfen, in transgenic tobacco plants.

  3. Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase.

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    Warabi, E; Usui, K; Tanaka, Y; Matsumoto, H

    2001-08-01

    The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.

  4. Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

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    Birchfield, N B; Casida, J E

    1996-01-01

    Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

  5. Herbicidal and antioxidant responses of transgenic rice overexpressing Myxococcus xanthus protoporphyrinogen oxidase.

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    Jung, Sunyo; Back, Kyoungwhan

    2005-05-01

    We analyzed the herbicidal and antioxidant defense responses of transgenic rice plants that overexpressed the Myxococcus xanthus protoporphyrinogen oxidase gene. Leaf squares of the wild-type incubated with oxyfluorfen were characterized by necrotic leaf lesions and increases in conductivity and malonyldialdehyde levels, whereas transgenic lines M4 and M7 did not show any change with up to 100 microM oxyfluorfen. The wild-type had decreased F(v)/F(m) and produced a high level of H(2)O(2) at 18 h after foliar application of oxyfluorfen, whereas transgenic lines M4 and M7 were unaffected. In response to oxyfluorfen, violaxanthin, beta-carotene, and chlorophylls (Chls) decreased in wild-type plants, whereas antheraxanthin and zeaxanthin increased. Only a slight decline in Chls was observed in transgenic lines at 48 h after oxyfluorfen treatment. Noticeable increases of cytosolic Cu/Zn-superoxide dismutase, peroxidase isozymes 1 and 2, and catalase were observed after at 48 h of oxyfluorfen treatment in the wild-type. Non-enzymatic antioxidants appeared to respond faster to oxyfluorfen-induced photodynamic stress than did enzymatic antioxidants. Protective responses for the detoxification of active oxygen species were induced to counteract photodynamic stress in oxyfluorfen-treated, wild-type plants. However, oxyfluorfen-treated, transgenic plants suffered less oxidative stress, confirming increased herbicidal resistance resulted from dual expression of M. xanthus Protox in chloroplasts and mitochondria.

  6. Eucalyptus ESTs corresponding to the protoporphyrinogen IX oxidase enzyme related to the synthesis of heme, chlorophyll, and to the action of herbicides

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    Edivaldo Domingues Velini

    2005-01-01

    Full Text Available This work was aimed at locating Eucalyptus ESTs corresponding to the PROTOX or PPO enzyme (Protoporphyrinogen IX oxidase, E.C. 1.3.3.4 directly related to resistance to herbicides that promote oxidative stress, changing the functionality of this enzyme. PROTOX, which is the site of action of diphenyl-ether (oxyfluorfen, lactofen, fomesafen, oxadiazole (oxadiazon and oxadiargyl, and aryl triazolinone (sulfentrazone and carfentrazone herbicides, acts on the synthesis route of porphyrins which is associated with the production of chlorophyll a, catalases, and peroxidases. One cluster and one single read were located, with e-values better than e-70, associated to PROTOX. The alignment results between amino acid sequences indicated that this enzyme is adequately represented in the ESTs database of the FORESTs project.

  7. Design and syntheses of novel N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione and N-(benzothiazol-5-yl)isoindoline-1,3-dione as potent protoporphyrinogen oxidase inhibitors.

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    Jiang, Li-Li; Zuo, Yang; Wang, Zhi-Fang; Tan, Yin; Wu, Qiong-You; Xi, Zhen; Yang, Guang-Fu

    2011-06-08

    Discovery of protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors has been one of the hottest research areas in the field of herbicide development for many years. As a continuation of our research work on the development of new PPO-inhibiting herbicides, a series of novel N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-diones (1a-p) and N-(benzothiazol-5-yl)isoindoline-1,3-diones (2a-h) were designed and synthesized according to the ring-closing strategy of two ortho-substituents. The bioassay results indicated that some newly synthesized compounds exhibited higher PPO inhibition activity than the control of sulfentrazone. Compound 1a, S-(5-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-6-fluorobenzothiazol-2-yl) O-methyl carbonothioate, was identified as the most potent inhibitor with k(i) value of 0.08 μM, about 9 times higher than that of sulfentrazone (k(i) = 0.72 μM). Further green house assay showed that compound 1b, methyl 2-((5-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-6-fluorobenzothiazol-2-yl)thio)acetate, exhibited herbicidal activity comparable to that of sulfentrazone even at a concentration of 37.5 g ai/ha. In addition, among six tested crops, wheat exhibited high tolerance to compound 1b even at a dosage of 300 g ai/ha. These results indicated that compound 1b might have the potential to be developed as a new herbicide for weed control of wheat field.

  8. Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

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    Lee, H J; Lee, S B; Chung, J S; Han, S U; Han, O; Guh, J O; Jeon, J S; An, G; Back, K

    2000-06-01

    Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.

  9. Monoamine Oxidase Inhibitors (MAOIs)

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    ... health-medications/index.shtml. Accessed May 16, 2016. Hirsch M, et al. Monoamine oxidase inhibitors (MAOIs) for ... www.uptodate.com/home. Accessed May 16, 2016. Hirsch M, et al. Discontinuing antidepressant medications in adults. ...

  10. The increasing role of monoamine oxidase type B inhibitors in Parkinson's disease therapy.

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    Elmer, Lawrence W; Bertoni, John M

    2008-11-01

    The role of monoamine oxidase type B inhibitors in the treatment of Parkinson's disease has expanded with the new monoamine oxidase B inhibitor rasagiline and a new formulation, selegiline oral disintegrating tablets. As primary therapy in early disease monoamine oxidase B inhibitors reduce motor disability and delay the need for levodopa. In more advanced disease requiring levodopa, adjunctive monoamine oxidase B inhibitors reduce 'off' time and may improve gait and freezing. Rasagiline and selegiline oral disintegrating tablets may reduce the safety risks associated with the amfetamine and methamfetamine metabolites of conventional oral selegiline while retaining or improving therapeutic efficacy. Articles were identified by searches of PubMed and searches on the Internet and reviewed. All articles and other referenced materials were retrieved using the keywords 'Parkinson's disease', 'treatment' and 'monoamine oxidase B inhibitor' and were published between 1960 and 2007, with older references selected for historical significance. Only papers published in English were reviewed. Accumulating data support the use of monoamine oxidase B inhibitors as monotherapy for early and mild Parkinson's disease and as adjunctive therapy for more advanced Parkinson's disease with levodopa-associated motor fluctuations. The recently released monoamine oxidase B inhibitor rasagiline and a new formulation, selegiline oral disintegrating tablets, have potential advantages over conventional oral selegiline.

  11. Effects of a diphenyl-ether herbicide, oxyfluorfen, on human BFU-E/CFU-E development and haemoglobin synthesis.

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    Rio, B; Parent-Massin, D; Lautraite, S; Hoellinger, H

    1997-02-01

    The diphenyl-ether herbicides exert their phytotoxic activity by preventing chlorophyll formation in plants as a result of inhibition of protoporphyrinogen oxidase. This enzyme is the last step of the common pathway for chlorophyll and haem biosynthesis. The aim of this work is to determine whether herbicide inhibitors of plant protoporphyrinogen oxidase could act on the human protoporphyrinogen oxidase involved in haemoglobin synthesis and cause heamatologic diseases. Human erythroblastic progenitors (BFU-E/CFU-E: Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid) were exposed to oxyfluorfen, a diphenyl-ether herbicide in the presence of erythropoietin, and the haematoxicity evaluated in vitro by scoring the development of BFU-E/CFU-E colonies after 7 and 14 days of culture. The toxic effect on differentiation has been evaluated using four criteria: morphology, total protein, total porphyrin, and haemoglobin content. The study of BFU-E/CFU-E proliferation and differentiation showed a cytotoxic effect of oxyfluorfen only at very high concentrations. In contrast, haemoglobin synthesis can be inhibited by concentration of oxyfluorfen (10(-4) M) that have no adverse effect on cellular proliferation.

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

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    Barbaro Michela

    2013-01-01

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

  13. Monoamine oxidase inhibitors from Gentiana lutea.

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    Haraguchi, Hiroyuki; Tanaka, Yasumasa; Kabbash, Amal; Fujioka, Toshihiro; Ishizu, Takashi; Yagi, Akira

    2004-08-01

    Three monoamine oxidase (MAO) inhibitors were isolated from Gentiana lutea. Their structures were elucidated to be 3-3''linked-(2'-hydroxy-4-O-isoprenylchalcone)-(2'''-hydroxy-4''-O-isoprenyldihydrochalcone) (1), 2-methoxy-3-(1,1'-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one and 5-hydroxyflavanone. These compounds, and the hydrolysis product of 1, displayed competitive inhibitory properties against MAO-B which was more effective than MAO-A.

  14. Dual inhibitors of cholinesterases and monoamine oxidases for Alzheimer's disease.

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    Knez, Damijan; Sova, Matej; Košak, Urban; Gobec, Stanislav

    2017-05-01

    Accumulating evidence indicates a solid relationship between several enzymes and Alzheimer's disease. Cholinesterases and monoamine oxidases are closely associated with the disease symptomatology and progression and have been tackled simultaneously using several multifunctional ligands. This design strategy offers great chances to alter the course of Alzheimer's disease, in addition to alleviation of the symptoms. More than 15 years of research has led to the identification of various dual cholinesterase/monoamine oxidase inhibitors, while some showing positive outcomes in clinical trials, thus giving rise to additional research efforts in the field. The aim of this review is to provide an update on the novel dual inhibitors identified recently and to shed light on their therapeutic potential.

  15. 2-acetylphenol analogs as potent reversible monoamine oxidase inhibitors

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    Legoabe LJ

    2015-07-01

    Full Text Available Lesetja J Legoabe,1 Anél Petzer,1 Jacobus P Petzer1,21Centre of Excellence for Pharmaceutical Sciences, 2Department of Pharmaceutical Chemistry, School of Pharmacy, North-West University, Potchefstroom, South AfricaAbstract: Based on a previous report that substituted 2-acetylphenols may be promising leads for the design of novel monoamine oxidase (MAO inhibitors, a series of C5-substituted 2-acetylphenol analogs (15 and related compounds (two were synthesized and evaluated as inhibitors of human MAO-A and MAO-B. Generally, the study compounds exhibited inhibitory activities against both MAO-A and MAO-B, with selectivity for the B isoform. Among the compounds evaluated, seven compounds exhibited IC50 values <0.01 µM for MAO-B inhibition, with the most selective compound being 17,000-fold selective for MAO-B over the MAO-A isoform. Analyses of the structure–activity relationships for MAO inhibition show that substitution on the C5 position of the 2-acetylphenol moiety is a requirement for MAO-B inhibition, and the benzyloxy substituent is particularly favorable in this regard. This study concludes that C5-substituted 2-acetylphenol analogs are potent and selective MAO-B inhibitors, appropriate for the design of therapies for neurodegenerative disorders such as Parkinson’s disease.Keywords: monoamine oxidase, MAO, inhibition, 2-acetylphenol, structure–activity relationship

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

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

    2009-07-01

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

  17. Monoamine Oxidase B Inhibitors in Parkinson's Disease.

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    Dezsi, Livia; Vecsei, Laszlo

    2017-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder with a prevalence increasing with age. Oxidative stress and glutamate toxicity are involved in its pathomechanism. There are still many unmet needs of PD patients, including the alleviation of motor fluctuations and dyskinesias, and the development of therapies with neuroprotective potential. To give an overview of the pharmacological properties, the efficacy and safety of the monoamine oxidase B (MAO-B) inhibitors in the treatment of PD, with special focus on the results of randomized clinical trials. A literature search was conducted in PubMed for 'PD treatment', 'MAO-B inhibitors', 'selegiline', 'rasagiline', 'safinamide' and 'clinical trials' with 'MAO-B inhibitors' in 'Parkinson' disease'. MAO-B inhibitors have a favorable pharmacokinetic profile, improve the dopamine deficient state and may have neuroprotective properties. Safinamide exhibits an anti-glutamatergic effect as well. When applied as monotherapy, MAO-B inhibitors provide a modest, but significant improvement of motor function and delay the need for levodopa. Rasagiline and safinamide were proven safe and effective when added to a dopamine agonist in early PD. As add-on to levodopa, MAO-B inhibitors significantly reduced off-time and were comparable in efficacy to COMT inhibitors. Improvements were achieved as regards certain non-motor symptoms as well. Due to the efficacy shown in clinical trials and their favorable side-effect profile, MAO-B inhibitors are valuable drugs in the treatment of PD. They are recommended as monotherapy in the early stages of the disease and as add-on therapy to levodopa in advanced PD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Indanones as high-potency reversible inhibitors of monoamine oxidase.

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    Mostert, Samantha; Petzer, Anél; Petzer, Jacobus P

    2015-05-01

    Recent reports document that α-tetralone (3,4-dihydro-2H-naphthalen-1-one) is an appropriate scaffold for the design of high-potency monoamine oxidase (MAO) inhibitors. Based on the structural similarity between α-tetralone and 1-indanone, the present study involved synthesis of 34 1-indanone and related indane derivatives as potential inhibitors of recombinant human MAO-A and MAO-B. The results show that C6-substituted indanones are particularly potent and selective MAO-B inhibitors, with IC50 values ranging from 0.001 to 0.030 μM. C5-Substituted indanone and indane derivatives are comparatively weaker MAO-B inhibitors. Although the 1-indanone and indane derivatives are selective inhibitors of the MAO-B isoform, a number of homologues are also potent MAO-A inhibitors, with three homologues possessing IC50 values 1-indanone as a reversible MAO inhibitor with a competitive mode of inhibition. It may be concluded that 1-indanones are promising leads for the design of therapies for neurodegenerative and neuropsychiatric disorders such as Parkinson's disease and depression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Structure-Activity Relationship Analysis of 3-phenylcoumarin-Based Monoamine Oxidase B Inhibitors

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    Rauhamäki, Sanna; Postila, Pekka A.; Niinivehmas, Sanna; Kortet, Sami; Schildt, Emmi; Pasanen, Mira; Manivannan, Elangovan; Ahinko, Mira; Koskimies, Pasi; Nyberg, Niina; Huuskonen, Pasi; Multamäki, Elina; Pasanen, Markku; Juvonen, Risto O.; Raunio, Hannu; Huuskonen, Juhani; Pentikäinen, Olli T.

    2018-03-01

    Monoamine oxidase B (MAO-B) catalyzes deamination of monoamines such as neurotransmitters dopamine and norepinephrine. Accordingly, small-molecule MAO-B inhibitors potentially alleviate the symptoms of dopamine-linked neuropathologies such as depression or Parkinson’s disease. Coumarin with a functionalized 3-phenyl ring system is a promising scaffold for building potent MAO-B inhibitors. Here, a vast set of 3-phenylcoumarin derivatives was designed using virtual combinatorial chemistry or rationally de novo and synthesized using microwave chemistry. The derivatives inhibited the MAO-B at 100 nM - 1 µM. The IC50 value of the most potent derivative 1 was 56 nM. A docking-based structure-activity relationship analysis summarizes the atom-level determinants of the MAO-B inhibition by the derivatives. Finally, the cross-reactivity of the derivatives was tested against monoamine oxidase A and a specific subset of enzymes linked to estradiol metabolism, known to have coumarin-based inhibitors. Overall, the results indicate that the 3-phenylcoumarins, especially derivative 1, present unique pharmacological features worth considering in future drug development.

  20. Electroconvulsive therapy in patients taking monoamine oxidase inhibitors.

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    Dolenc, Tamara J; Habl, Samar S; Barnes, Roxann D; Rasmussen, Keith G

    2004-12-01

    Concerns have been expressed regarding the use of general anesthesia for electroconvulsive therapy (ECT) in patients taking monoamine oxidase inhibitors (MAOIs). We review the published literature and present 4 new cases and conclude that there is no evidence of a dangerous interaction between ECT and MAOI use. In general, a cautious approach would be to discontinue MAOIs before ECT if the medication has not been helpful; however, there is no need for a washout interval before starting ECT. Furthermore, if there is otherwise a reason for continuing the MAOI, it can be continued during index ECT or initiated during maintenance ECT.

  1. Efficacy, safety, and patient preference of monoamine oxidase B inhibitors in the treatment of Parkinson's disease

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    Bradley J Robottom

    2011-01-01

    Full Text Available Bradley J RobottomDepartment of Neurology, University of Maryland School of Medicine, Baltimore, MD, USAAbstract: Parkinson's disease (PD is the second most common neurodegenerative disease and the most treatable. Treatment of PD is symptomatic and generally focuses on the replacement or augmentation of levodopa. A number of options are available for treatment, both in monotherapy of early PD and to treat complications of advanced PD. This review focuses on rasagiline and selegiline, two medications that belong to a class of antiparkinsonian drugs called monoamine oxidase B (MAO-B inhibitors. Topics covered in the review include mechanism of action, efficacy in early and advanced PD, effects on disability, the controversy regarding disease modification, safety, and patient preference for MAO-B inhibitors.Keywords: monoamine oxidase inhibitors, rasagiline, selegiline, Parkinson's disease, efficacy, safety

  2. Electronegativity of aromatic amines as a basis for the development of ground state inhibitors of lysyl oxidase

    International Nuclear Information System (INIS)

    Williamson, P.R.; Kagan, H.M.

    1987-01-01

    Benzylamine derivatives containing para substituents of differing electronegativity as well as isomers of aminomethylpyridine have been assessed for their substrate and inhibitor potentials toward lysyl oxidase. Substituted benzylamines with increasingly electronegative para substituents had the lowest KI values and thus were the most effective inhibitors of the oxidation of elastin by lysyl oxidase. The kcat values for these compounds as substrates of lysyl oxidase were also reduced with increasingly electronegative para substituents. Both the Dkcat and D(kcat/Km) kinetic isotope effects decreased with increasingly electronegative p-substituents in [alpha, alpha'- 2 H]benzylamines. In contrast, there was no Dkcat solvent isotope effect with [ 2 H] H 2 O while the D(kcat/Km) solvent isotope effect tended to increase with increasingly electronegative p-substituents. These results are consistent with the stabilization of an enzyme-generated substrate carbanion and the retardation of substrate oxidation by electronegative substituents. Such ground state stabilization can result in compounds with increased potential for the inhibition of the oxidation of protein substrates of lysyl oxidase

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

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    Vaikari, Vijaya Pooja; Kota, Rajesh; Chen, Kevin; Yeh, Tzu-Shao; Jhaveri, Niyati; Groshen, Susan L.; Olenyuk, Bogdan Z.; Chen, Thomas C.; Hofman, Florence M.; Shih, Jean C.

    2016-01-01

    Glioblastoma (GBM) is an aggressive brain tumor which is currently treated with temozolomide (TMZ). Tumors usually become resistant to TMZ and recur; no effective therapy is then available. Monoamine Oxidase A (MAO A) oxidizes monoamine neurotransmitters resulting in reactive oxygen species which cause cancer. This study shows that MAO A expression is increased in human glioma tissues and cell lines. MAO A inhibitors, clorgyline or the near-infrared-dye MHI-148 conjugated to clorgyline (NMI), were cytotoxic for glioma and decreased invasion in vitro. Using the intracranial TMZ-resistant glioma model, clorgyline or NMI alone or in combination with low-dose TMZ reduced tumor growth and increased animal survival. NMI was localized specifically to the tumor. Immunocytochemistry studies showed that the MAO A inhibitor reduced proliferation, microvessel density and invasion, and increased macrophage infiltration. In conclusion, we have identified MAO A inhibitors as potential novel stand-alone drugs or as combination therapy with low dose TMZ for drug-resistant gliomas. NMI can also be used as a non-invasive imaging tool. Thus has a dual function for both therapy and diagnosis. PMID:26871599

  4. Molecular phylogeny and intricate evolutionary history of the three isofunctional enzymes involved in the oxidation of protoporphyrinogen IX.

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    Kobayashi, Koichi; Masuda, Tatsuru; Tajima, Naoyuki; Wada, Hajime; Sato, Naoki

    2014-08-01

    Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. The amine oxidase inhibitor phenelzine limits lipogenesis in adipocytes without inhibiting insulin action on glucose uptake.

    Science.gov (United States)

    Carpéné, Christian; Grès, Sandra; Rascalou, Simon

    2013-06-01

    The antidepressant phenelzine is a monoamine oxidase inhibitor known to inhibit various other enzymes, among them semicarbazide-sensitive amine oxidase (currently named primary amine oxidase: SSAO/PrAO), absent from neurones but abundant in adipocytes. It has been reported that phenelzine inhibits adipocyte differentiation of cultured preadipocytes. To further explore the involved mechanisms, our aim was to study in vitro the acute effects of phenelzine on de novo lipogenesis in mature fat cells. Therefore, glucose uptake and incorporation into lipid were measured in mouse adipocytes in response to phenelzine, other hydrazine-based SSAO/PrAO-inhibitors, and reference agents. None of the inhibitors was able to impair the sevenfold activation of 2-deoxyglucose uptake induced by insulin. Phenelzine did not hamper the effect of lower doses of insulin. However, insulin-stimulated glucose incorporation into lipids was dose-dependently inhibited by phenelzine and pentamidine, but not by semicarbazide or BTT2052. In contrast, all these SSAO/PrAO inhibitors abolished the transport and lipogenesis stimulation induced by benzylamine. These data indicate that phenelzine does not inhibit glucose transport, the first step of lipogenesis, but inhibits at 100 μM the intracellular triacylglycerol assembly, consistently with its long-term anti-adipogenic effect and such rapid action was not found with all the hydrazine derivatives tested. Therefore, the alterations of body weight control consecutive to the use of this antidepressant drug might be not only related to central effects on food intake/energy expenditure, but could also depend on its direct action in adipocytes. Nonetheless, phenelzine antilipogenic action is not merely dependent on SSAO/PrAO inhibition.

  6. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  7. Structure–Activity Relationship of Xanthones as Inhibitors of Xanthine Oxidase

    Directory of Open Access Journals (Sweden)

    Ling-Yun Zhou

    2018-02-01

    Full Text Available Polygala plants contain a large number of xanthones with good physiological activities. In our previous work, 18 xanthones were isolated from Polygala crotalarioides. Extented study of the chemical composition of the other species Polygala sibirica led to the separation of two new xanthones—3-hydroxy-1,2,6,7,8-pentamethoxy xanthone (A and 6-O-β-d-glucopyranosyl-1,7-dimethoxy xanthone (C—together with 14 known xanthones. Among them, some xanthones have a certain xanthine oxidase (XO inhibitory activity. Furthemore, 14 xanthones as XO inhibitors were selected to develop three-dimensional quantitative structure–activity relationship (3D-QSAR using comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA models. The CoMFA model predicted a q2 value of 0.613 and an r2 value of 0.997. The best CoMSIA model predicted a q2 value of 0.608 and an r2 value of 0.997 based on a combination of steric, electrostatic, and hydrophobic effects. The analysis of the contour maps from each model provided insight into the structural requirements for the development of more active XO inhibitors.

  8. Discovery and biological evaluation of some (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety as potent xanthine oxidase inhibitors.

    Science.gov (United States)

    Zhang, Ting-Jian; Li, Song-Ye; Yuan, Wei-Yan; Wu, Qing-Xia; Wang, Lin; Yang, Su; Sun, Qi; Meng, Fan-Hao

    2017-02-15

    A series of (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety were synthesized and identified as novel xanthine oxidase inhibitors. Among them, the most promising compounds 1h and 1k were obtained with IC 50 values of 0.6μM and 0.8μM, respectively, which were more than 10-fold potent compared with allopurinol. The Lineweaver-Burk plot revealed that compound 1h acted as a mixed-type xanthine oxidase inhibitor. SAR analysis showed that the benzaldehyde moiety played a more important role than the anthraquinone moiety for inhibition potency. The basis of significant inhibition of xanthine oxidase by 1h was rationalized by molecular modeling studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Apocynin: Chemical and Biophysical Properties of a NADPH Oxidase Inhibitor

    Directory of Open Access Journals (Sweden)

    Valdecir F. Ximenes

    2013-03-01

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

  10. Inhibitors of NADPH oxidase decrease endotoxin mediated induction of inducible nitric oxide expression in mouse macrophages

    Czech Academy of Sciences Publication Activity Database

    Krejčová, Daniela; Okénková, Kateřina; Konopka, Roman; Lojek, Antonín; Kubala, Lukáš

    2007-01-01

    Roč. 101, č. 14 (2007), s203-s204 E-ISSN 1213-7103. [Mezioborová česko-slovenská toxikologická konference /12./. Praha, 11.06.2007-13.06.2007] R&D Projects: GA ČR(CZ) GA524/06/1197 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : lipopolysaccharide * inhibitors of NADPH oxidase * macrophage s Subject RIV: BO - Biophysics

  11. Efficacy, safety, and patient preference of monoamine oxidase B inhibitors in the treatment of Parkinson's disease.

    Science.gov (United States)

    Robottom, Bradley J

    2011-01-20

    Parkinson's disease (PD) is the second most common neurodegenerative disease and the most treatable. Treatment of PD is symptomatic and generally focuses on the replacement or augmentation of levodopa. A number of options are available for treatment, both in monotherapy of early PD and to treat complications of advanced PD. This review focuses on rasagiline and selegiline, two medications that belong to a class of antiparkinsonian drugs called monoamine oxidase B (MAO-B) inhibitors. Topics covered in the review include mechanism of action, efficacy in early and advanced PD, effects on disability, the controversy regarding disease modification, safety, and patient preference for MAO-B inhibitors.

  12. Flavonoid glycosides isolated from unique legume plant extracts as novel inhibitors of xanthine oxidase.

    Directory of Open Access Journals (Sweden)

    Chrysoula Spanou

    Full Text Available Legumes and the polyphenolic compounds present in them have gained a lot of interest due to their beneficial health implications. Dietary polyphenolic compounds, especially flavonoids, exert antioxidant properties and are potent inhibitors of xanthine oxidase (XO activity. XO is the main contributor of free radicals during exercise but it is also involved in pathogenesis of several diseases such as vascular disorders, cancer and gout. In order to discover new natural, dietary XO inhibitors, some polyphenolic fractions and pure compounds isolated from two legume plant extracts were tested for their effects on XO activity. The fractions isolated from both Vicia faba and Lotus edulis plant extracts were potent inhibitors of XO with IC(50 values range from 40-135 µg/mL and 55-260 µg/mL, respectively. All the pure polyphenolic compounds inhibited XO and their K(i values ranged from 13-767 µM. Ten of the compounds followed the non competitive inhibitory model whereas one of them was a competitive inhibitor. These findings indicate that flavonoid isolates from legume plant extracts are novel, natural XO inhibitors. Their mode of action is under investigation in order to examine their potential in drug design for diseases related to overwhelming XO action.

  13. Amperometric biosensor based on prussian blue and nafion modified screen-printed electrode for screening of potential xanthine oxidase inhibitors from medicinal plants.

    Science.gov (United States)

    El Harrad, Loubna; Amine, Aziz

    2016-04-01

    A simple and sensitive amperometric biosensor was developed for the screening of potential xanthine oxidase inhibitors from medicinal plants. This biosensor was prepared by immobilization of xanthine oxidase on the surface of prussian blue modified screen-printed electrodes using nafion and glutaraldehyde. The developed biosensor showed a linear amperometric response at an applied potential of +0.05 V toward the detection of hypoxanthine from 5 μM to 45 μM with a detection limit of 0.4 μM (S/N=3) and its sensitivity was found to be 600 mA M(-1) cm(-2). In addition, the biosensor exhibited a good storage stability. The inhibition of xanthine oxidase by allopurinol was studied under the optimized conditions. The linear range of allopurinol concentration is obtained up to 2.5 μM with an estimated 50% of inhibitionI50=1.8 μM. The developed biosensor was successfully applied to the screening of xanthine oxidase inhibitors from 13 medicinal plants belonging to different families. Indeed, Moroccan people traditionally use these plants as infusion for the treatment of gout and its related symptoms. For this purpose, water extracts obtained from the infusion of these plants were used for the experiments. In this work, 13 extracts were assayed and several of them demonstrated xanthine oxidase inhibitory effect, with an inhibition greater than 50% compared to spectrophotometry measurements that only few extracts showed an inhibition greater than 50%. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase.

    Science.gov (United States)

    Nisler, Jaroslav; Kopečný, David; Končitíková, Radka; Zatloukal, Marek; Bazgier, Václav; Berka, Karel; Zalabák, David; Briozzo, Pierre; Strnad, Miroslav; Spíchal, Lukáš

    2016-09-01

    Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies. Cytokinin hormones regulate a wide range of essential processes in plants. Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, TDZ), formerly registered as a cotton defoliant, is a well known inhibitor of cytokinin oxidase/dehydrogenase (CKX), an enzyme catalyzing the degradation of cytokinins. TDZ thus increases the lifetime of cytokinins and their effects in plants. We used in silico modeling to design, synthesize and characterize twenty new TDZ derivatives with improved inhibitory properties. Two compounds, namely 1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl)phenyl]-3-(1,2,3-thiadiazol-5-yl)urea (HETDZ), displayed up to 15-fold lower IC 50 values compared with TDZ for AtCKX2 from Arabidopsis thaliana and ZmCKX1 and ZmCKX4a from Zea mays. Binding modes of 3FMTDZ and HETDZ were analyzed by X-ray crystallography. Crystal structure complexes, solved at 2.0 Å resolution, revealed that HETDZ and 3FMTDZ bound differently in the active site of ZmCKX4a: the thiadiazolyl ring of 3FMTDZ was positioned over the isoalloxazine ring of FAD, whereas that of HETDZ had the opposite orientation, pointing toward the entrance of the active site. The compounds were further tested for cytokinin activity in several cytokinin bioassays. We suggest that the combination of simple synthesis, lowered cytokinin activity, and enhanced inhibitory effects on CKX isoforms, makes 3FMTDZ and HETDZ suitable candidates for in vivo studies.

  15. Antidepressant-like effects of the xanthine oxidase enzyme inhibitor allopurinol in rats. A comparison with fluoxetine.

    Science.gov (United States)

    Gürbüz Özgür, Börte; Aksu, Hatice; Birincioğlu, Mustafa; Dost, Turhan

    2015-11-01

    Allopurinol is a xanthine oxidase enzyme inhibitor that is widely used for the treatment of hyperuricemia and gout. The activity of tryptophan 2,3-dioxygenase, which metabolizes tryptophan (TRP), is decreased by xanthine oxidase inhibitors, causing TRP levels in the body to be increased. Increases in TRP levels in the brain might have antidepressant effects. The purpose of this study is to evaluate the antidepressant effects of allopurinol compared to those of fluoxetine, which is a proven antidepressant. Thirty-two Wistar albino male rats were divided into four groups (control, 10mg/kg fluoxetine, 50mg/kg allopurinol, 50mg/kg allopurinol+10 mg/kg fluoxetine; n=8 per group), and forced swimming tests were performed before and after 14days of drug administration. Serotonin, 5-hydroxyindolacetic acid and uric acid levels were measured in blood samples after the final treatment. When allopurinol and fluoxetine were administered separately, a decrease in the duration of immobility and an increased duration of swimming were observed in the forced swimming test. The results showed similar antidepressant efficacies between allopurinol and fluoxetine. However, we found no statistically significant difference in the antidepressant effect of the combined therapy versus single drug therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Cardiovascular activity of rasagiline, a selective and potent inhibitor of mitochondrial monoamine oxidase B: comparison with selegiline

    OpenAIRE

    Abassi, Zaid A; Binah, Ofer; Youdim, Moussa B H

    2004-01-01

    Selegiline is used for treating Parkinson's disease. Despite its efficacy, the clinical use of selegiline in combination with L-dihydroxphenylalanine in Parkinsonian patients is hampered by cardiovascular complications, such as hypotension. This study was designed to compare in rats the cardiovascular effects of selegiline and rasagiline, their metabolites L-methamphetamine and aminoindan (TVP-136), respectively, and the second rasagiline metabolite non-monoamine oxidase (MAO) inhibitor TVP-1...

  17. Role of pH in oxidase variability of Aeromonas hydrophila.

    OpenAIRE

    Hunt, L K; Overman, T L; Otero, R B

    1981-01-01

    Some strains of Aeromonas hydrophila may be oxidase negative or only weakly oxidase positive by the Kovacs method taken from the surface of a differential medium, such as MacConkey agar. Six strains of A. hydrophila, two oxidase variable, one oxidase constant, and three weakly oxidase positive on MacConkey agar, were studied to determine the cause of oxidase variability. The bacteriostatic dyes in MacConkey agar were considered possible inhibitors of the oxidase reaction. The concentration of...

  18. The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress

    International Nuclear Information System (INIS)

    Riganti, Chiara; Costamagna, Costanzo; Doublier, Sophie; Miraglia, Erica; Polimeni, Manuela; Bosia, Amalia; Ghigo, Dario

    2008-01-01

    We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-κB and decreased intracellular level of its inhibitor IkBα. These effects, accompanied by increased production of H 2 O 2 , were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-κB activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed

  19. Efficacy, safety, and patient preference of monoamine oxidase B inhibitors in the treatment of Parkinson’s disease

    Science.gov (United States)

    Robottom, Bradley J

    2011-01-01

    Parkinson’s disease (PD) is the second most common neurodegenerative disease and the most treatable. Treatment of PD is symptomatic and generally focuses on the replacement or augmentation of levodopa. A number of options are available for treatment, both in monotherapy of early PD and to treat complications of advanced PD. This review focuses on rasagiline and selegiline, two medications that belong to a class of antiparkinsonian drugs called monoamine oxidase B (MAO-B) inhibitors. Topics covered in the review include mechanism of action, efficacy in early and advanced PD, effects on disability, the controversy regarding disease modification, safety, and patient preference for MAO-B inhibitors. PMID:21423589

  20. Rho-kinase inhibitor and nicotinamide adenine dinucleotide phosphate oxidase inhibitor prevent impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats.

    Science.gov (United States)

    Iida, Hiroki; Iida, Mami; Takenaka, Motoyasu; Fukuoka, Naokazu; Dohi, Shuji

    2008-06-01

    We previously reported that acute cigarette smoking can cause a dysfunction of endothelium-dependent vasodilation in cerebral vessels, and that blocking the angiotensin II (Ang II) type 1 (AT1) receptor with valsartan prevented this impairment. Our aim was to investigate the effects of a Rho-kinase inhibitor (fasudil) and a Nicotinamide Adenine Dinucleotide PHosphate (NADPH) oxidase inhibitor (apocynin) on smoking-induced endothelial dysfunction in cerebral arterioles. In Sprague-Dawley rats, we used a closed cranial window preparation to measure changes in pial vessel diameters following topical acetylcholine (ACh) before smoking. After one-minute smoking, we again examined the arteriolar responses to ACh. Finally, after intravenous fasudil or apocynin pre-treatment we re-examined the vasodilator responses to topical ACh (before and after cigarette smoking). Under control conditions, cerebral arterioles were dose-dependently dilated by topical ACh (10(-6) M and 10(-5) M). One hour after a one-minute smoking (1 mg-nicotine cigarette), 10(-5) M ACh constricted cerebral arterioles. However, one hour after a one-minute smoking, 10(-5) M ACh dilated cerebral pial arteries both in the fasudil pre-treatment and the apocynin pre-treatment groups, responses that were significantly different from those obtained without fasudil or apocynin pre-treatment. Thus, inhibition of Rho-kinase and NADPH oxidase activities may prevent the above smoking-induced impairment of endothelium-dependent vasodilation.

  1. Monoamine oxidase inhibitory activity in tobacco particulate matter: Are harman and norharman the only physiologically relevant inhibitors?

    Science.gov (United States)

    Truman, Penelope; Grounds, Peter; Brennan, Katharine A

    2017-03-01

    Monoamine oxidase inhibition is significant in smokers, but it is still unclear how the inhibition that is seen in the brains and bodies of smokers is brought about. Our aim was to test the contribution of the harman and norharman in tobacco smoke to MAO-A inhibition from tobacco smoke preparations, as part of a re-examination of harman and norharman as the cause of the inhibition of MAO-A inhibition in the brain. Tobacco smoke particulate matter and cigarette smoke particulate matter were prepared and the amounts of harman and norharman measured. The results were compared with the total monoamine oxidase-A inhibitory activity. At a nicotine concentration of 0.6μM (a "physiological" concentration in blood) the total monoamine oxidase-A inhibitory activity measured in these samples was sufficient to inhibit the enzyme by approximately 10%. Of this inhibitory activity, only a small proportion of the total was found to be due to harman and norharman. These results show that harman and norharman provide only a moderate contribution to the total monoamine oxidase-A inhibitory activity of tobacco smoke, perhaps under 10%. This suggests that other inhibitors (either known or unknown) may be more significant contributors to total inhibitory activity than has yet been established, and deserve closer examination. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. High throughput Screening to Identify Natural Human Monoamine Oxidase B Inhibitors

    Science.gov (United States)

    Mazzio, E; Deiab, S; Park, K; Soliman, KFA

    2012-01-01

    Age-related increase in monoamine oxidase B (MAO-B) may contribute to CNS neurodegenerative diseases. Moreover, MAO-B inhibitors are used in the treatment of idiopathic Parkinson disease as preliminary monotherapy or adjunct therapy with L-dopa. To date, meager natural sources of MAO-B inhibitors have been identified, and the relative strength, potency and rank of many plants relative to standard drugs such as Selegiline (L-deprenyl, Eldepryl) are not known. In this work, we developed and utilized a high throughput enzyme microarray format to screen and evaluate 905 natural product extracts (0.025–.7 mg/ml) to inhibit human MAO-B derived from BTI-TN-5B1-4 cells infected with recombinant baculovirus. The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis-matrix assisted laser desorption ionization-time-of-flight-tandem mass spectroscopy, and enzyme activity was confirmed by [1] substrate conversion (3-mM benzylamine) to H202 and [2] benzaldehyde. Of the 905 natural extracts tested, the lowest IC50s [Comfrey, Bringraj, Skullcap, Kava-kava, Wild Indigo, Gentian and Green Tea. In conclusion, the data reflect relative potency information by rank of commonly used herbs and plants that contain human MAO-B inhibitory properties in their natural form. PMID:22887993

  3. Modulation of NADPH oxidase activity by known uraemic retention solutes.

    Science.gov (United States)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera; Cohen, Gerald; Schaefer, Mandy; Boehringer, Falko; Tepel, Martin; Kunkel, Desiree; Zidek, Walter; Jankowski, Joachim

    2014-08-01

    Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

  4. A multiple treatment comparison meta-analysis of monoamine oxidase type B inhibitors for Parkinson's disease.

    Science.gov (United States)

    Binde, C D; Tvete, I F; Gåsemyr, J; Natvig, B; Klemp, M

    2018-05-30

    To the best of our knowledge, there are no systematic reviews or meta-analyses that compare rasagiline, selegiline and safinamide. Therefore, we aimed to perform a drug class review comparing all available monoamine oxidase type B (MAO-B) inhibitors in a multiple treatment comparison. We performed a systematic literature search to identify randomized controlled trials assessing the efficacy of MAO-B inhibitors in patients with Parkinson's disease. MAO-B inhibitors were evaluated either as monotherapy or in combination with levodopa or dopamine agonists. Endpoints of interest were change in the Unified Parkinson's Disease Rating Scale (UPDRS) score and serious adverse events. We estimated the relative effect of each MAO-B inhibitor versus the comparator drug by creating three networks of direct and indirect comparisons. For each of the networks, we considered a joint model. The systematic literature search and study selection process identified 27 publications eligible for our three network analyses. We found the relative effects of rasagiline, safinamide and selegiline treatment given alone and compared to placebo in a model without explanatory variables to be 1.560 (1.409, 1.734), 1.449 (0.873, 2.413) and 1.532 (1.337, 1.757) respectively. We also found all MAO-B inhibitors to be efficient when given together with levodopa. When ranking the MAO-B inhibitors given in combination with levodopa, selegiline was the most effective and rasagiline was the second best. All of the included MAO-B inhibitors were effective compared to placebo when given as monotherapy. Combination therapy with MAO-B inhibitors and levodopa showed that all three MAO-B inhibitors were effective compared to placebo, but selegiline was the most effective drug. © 2018 The British Pharmacological Society.

  5. Effects of co-administration of dietary sodium arsenite and an NADPH oxidase inhibitor on the rat bladder epithelium

    International Nuclear Information System (INIS)

    Suzuki, Shugo; Arnold, Lora L.; Pennington, Karen L.; Kakiuchi-Kiyota, Satoko; Cohen, Samuel M.

    2009-01-01

    Arsenite (As III ), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. Reactive oxygen species (ROS) can be important factors for carcinogenesis and tumor progression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is known to produce intracellular ROS, therefore, we investigated the ability of apocynin (acetovanillone), an NADPH oxidase inhibitor, to inhibit the cytotoxicity and regenerative cell proliferation of arsenic in vitro and in vivo. Apocynin had similar effects in reducing the cytotoxicity of As III and dimethylarsinous acid (DMA III ) in rat urothelial cells in vitro. When tested at the same concentrations as apocynin, other antioxidants, such as L-ascorbate and N-acetylcysteine, did not inhibit As III -induced cytotoxicity but they were more effective at inhibiting DMA III -induced cytotoxicity compared with apocynin. In vivo, female rats were treated for 3 weeks with 100 ppm As III . Immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that apocynin reduced oxidative stress partially induced by As III treatment on rat urothelium, and significantly reduced the cytotoxicity of superficial cells detected by scanning electron microscopy (SEM). However, based on the incidence of simple hyperplasia and the bromodeoxyuridine (BrdU) labeling index, apocynin did not inhibit As III -induced urothelial cell proliferation. These data suggest that the NADPH oxidase inhibitor, apocynin, may have the ability to partially inhibit arsenic-induced oxidative stress and cytotoxicity of the rat bladder epithelium in vitro and in vivo. However, apocynin did not inhibit the regenerative cell proliferation induced by arsenite in a short-term study.

  6. Positron emitter labeled enzyme inhibitors

    International Nuclear Information System (INIS)

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-01-01

    This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

  7. Attenuation of MPTP-induced dopaminergic neurotoxicity by TV3326, a cholinesterase-monoamine oxidase inhibitor.

    Science.gov (United States)

    Sagi, Yotam; Weinstock, Marta; Youdim, Moussa B H

    2003-07-01

    (R)-[(N-propargyl-(3R) aminoindan-5-yl) ethyl methyl carbamate] (TV3326) is a novel cholinesterase and brain-selective monoamine oxidase (MAO)-A/-B inhibitor. It was developed for the treatment of dementia co-morbid with extra pyramidal disorders (parkinsonism), and depression. On chronic treatment in mice it attenuated striatal dopamine depletion induced by MPTP and prevented the reduction in striatal tyrosine hydroxylase activity, like selective B and non-selective MAO inhibitors. TV3326 preferentially inhibits MAO-B in the striatum and hippocampus, and the degree of MAO-B inhibition correlates with the prevention of MPTP-induced dopamine depletion. Complete inhibition of MAO-B is not necessary for full protection from MPTP neurotoxicity. Unlike that seen after treatment with other MAO-A and -B inhibitors, recovery of striatal and hippocampal MAO-A and -B activities from inhibition by TV3326 did not show first-order kinetics. This has been attributed to the generation of a number of metabolites by TV3326 that cause differential inhibition of these enzymes. Inhibition of brain MAO-A and -B by TV3326 resulted in significant elevations of dopamine, noradrenaline and serotonin in the striatum and hippocampus. This may explain its antidepressant-like activity, resembling that of moclobemide in the forced-swim test in rats.

  8. Monamine oxidase inhibitors: current and emerging agents for Parkinson disease.

    Science.gov (United States)

    Fernandez, Hubert H; Chen, Jack J

    2007-01-01

    Monoamine oxidase type B (MAO-B) is the predominant isoform responsible for the metabolic breakdown of dopamine in the brain. Selective inhibition of brain MAO-B results in elevation of synaptosomal dopamine concentrations. Data have been reported regarding the selective MAO-B inhibitors, rasagiline and selegiline, for the symptomatic treatment of Parkinson disease (PD). Selegiline has demonstrated efficacy as monotherapy in patients with early PD (Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism study), but evidence of selegiline efficacy as adjunctive treatment in levodopa-treated PD patients with motor fluctuations is equivocal. A new formulation of selegiline (Zydis selegiline) has been evaluated in 2 small, placebo-controlled studies as adjunctive therapy to levodopa. The Zydis formulation allows pregastric absorption of selegiline, minimizing first-pass metabolism, and thereby increasing selegiline bioavailability and reducing the concentration of amphetamine metabolites. Rasagiline is a selective, second-generation, irreversible MAO-B inhibitor, with at least 5 times the potency of selegiline in vitro and in animal models. Rasagiline has demonstrated efficacy in 1 large, randomized, double-blind, placebo-controlled trial (TVP-1012 in Early Monotherapy for Parkinson's Disease Outpatients) as initial monotherapy in patients with early PD, and in 2 large, controlled trials (Parkinson's Rasagiline: Efficacy and Safety in the Treatment of "Off," Lasting Effect in Adjunct Therapy With Rasagiline Given Once Daily) as adjunctive treatment in levodopa-treated PD patients with motor fluctuations. Unlike selegiline, rasagiline is an aminoindan derivative with no amphetamine metabolites. A randomized clinical trial is underway to confirm preclinical and preliminary clinical data suggesting rasagiline has disease-modifying effects.

  9. Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study.

    Science.gov (United States)

    Kopecný, David; Briozzo, Pierre; Popelková, Hana; Sebela, Marek; Koncitíková, Radka; Spíchal, Lukás; Nisler, Jaroslav; Madzak, Catherine; Frébort, Ivo; Laloue, Michel; Houba-Hérin, Nicole

    2010-08-01

    Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme-inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N'-phenylurea (CPPU) was solved. CPPU binds in a planar conformation and competes for the same binding site with natural substrates like N(6)-(2-isopentenyl)adenine (iP) and zeatin (Z). Nitrogens at the urea backbone are hydrogen bonded to the putative active site base Asp169. Subsequently, site-directed mutagenesis of L492 and E381 residues involved in the inhibitor binding was performed. The crystal structures of L492A mutant in a complex with CPPU and N-(2-chloro-pyridin-4-yl)-N'-benzylurea (CPBU) were solved and confirm the importance of a stacking interaction between the 2-chloro-4-pyridinyl ring of the inhibitor and the isoalloxazine ring of the FAD cofactor. Amino derivatives like N-(2-amino-pyridin-4-yl)-N'-phenylurea (APPU) inhibited ZmCKO1 more efficiently than CPPU, as opposed to the inhibition of E381A/S mutants, emphasizing the importance of this residue for inhibitor binding. As highly specific CKO inhibitors without undesired side effects are of major interest for physiological studies, all studied compounds were further analyzed for cytokinin activity in the Amaranthus bioassay and for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4. By contrast to CPPU itself, APPU and several benzylureas bind only negligibly to the receptors and exhibit weak cytokinin activity. Copyright 2010 Elsevier Masson SAS. All rights reserved.

  10. Rasagiline (TVP-1012): a new selective monoamine oxidase inhibitor for Parkinson's disease.

    Science.gov (United States)

    Guay, David R P

    2006-12-01

    This article reviews the chemistry, pharmacodynamics, pharmacokinetics, clinical efficacy, tolerability, drug-interaction potential, indications, dosing, and potential role of rasagiline mesylate, a new selective monoamine oxidase (MAO) type B (MAO-B) inhibitor, in the treatment of Parkinson's disease. A MEDLINE/PUBMED search (1986 through September 2006) was conducted to identify studies involving rasagiline written in English. Additional references were obtained from the bibliographies of these studies. All studies evaluating any aspect of rasagiline, including in vitro, in vivo (animal), and human studies, were reviewed. Rasagiline mesylate was developed with the goal of producing a selective MAO-B inhibitor that is not metabolized to (presumed) toxic metabolites (eg, amphetamine and methamphetamine, which are byproducts of the metabolism of selegiline, another selective MAO-B inhibitor). In vitro and in vivo data have confirmed the drug's selectivity for MAO-B. Rasagiline is almost completely eliminated by oxidative metabolism (catalyzed by cytochrome P-450 [CYP] isozyme 1A2) followed by renal excretion of conjugated parent compound and metabolites. Drug clearance is sufficiently slow to allow once-daily dosing. Several studies have documented its efficacy as monotherapy for early-stage disease and as adjunctive therapy in L-dopa recipients with motor fluctuations. As monotherapy, rasagiline is well tolerated with an adverse-effect profile similar to that of placebo. As adjunctive therapy, it exhibits the expected adverse effects of dopamine excess, which can be ameliorated by reducing the L-dopa dosage. CYP1A2 inhibitors slow the elimination of rasagiline and mandate dosage reduction. Hepatic impairment has an analogous effect. The recommended dosage regimens for monotherapy and adjunctive therapy are 1 and 0.5 mg PO QD, respectively. Despite the well-documented selectivity of rasagiline, the manufacturer recommends virtually all of the dietary (vis

  11. Herbicide-induced experimental variegate porphyria in mice: tissue porphyrinogen accumulation and response to porphyrogenic drugs.

    Science.gov (United States)

    Krijt, J; Stranska, P; Maruna, P; Vokurka, M; Sanitrak, J

    1997-01-01

    Administration of oxadiazon or oxyfluorfen (1000 ppm in the diet) to male BALB/c mice for 9 days resulted in experimental porphyria, resembling the acute phase of human variegate porphyria. Urinary concentrations of 5-aminolevulinic acid and porphobilinogen reached 1500 and 3000 mumol/L, respectively. Both herbicides caused a decrease of protoporphyrinogen oxidase activity in liver and kidney. Brain protoporphyrinogen oxidase activity was not altered. Liver and kidney porphyrin content increased to 11 and 17 nmol/g, respectively (control mice, 2 nmol/g). Over 50% of liver and kidney porphyrins were in the reduced (porphyrinogen) form. Bile of oxadiazon-treated mice contained 700 nmol/mL of protoporphyrinogen (control mice, 15 nmol/mL). Porphyrin content of the trigeminal nerve increased from 1 nmol/g in control animals to 11 nmol/g in oxadiazon-treated animals, suggesting a possible contribution of peripheral nerve porphyrins to porphyric neuropathy. Mice treated with 125 ppm of oxadiazon in the diet for 9 days excreted moderately elevated levels of porphobilinogen in urine (control mice, less than 50 mumol/L; treated mice, 330 mumol/L). Administration of phenobarbital or phenytoin (single injections on days 7, 8, and 9) increased the urinary porphobilinogen concentration to 3500 mumol/L. This response to porphyrogenic drugs resembles the response observed in human acute porphyrias.

  12. High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott (CNRS-UMR); (WU-MED)

    2012-05-24

    Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC{sub 50} in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 {angstrom} resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the {approx}100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

  13. The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress

    International Nuclear Information System (INIS)

    Riganti, Chiara; Costamagna, Costanzo; Bosia, Amalia; Ghigo, Dario

    2006-01-01

    Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H 2 O 2 concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution

  14. Synthesis of suicide inhibitors of monoamine oxidase: carbon-11 labeled clorgyline, L-deprenyl and D-deprenyl

    International Nuclear Information System (INIS)

    MacGregor, R.R.; Fowler, J.S.; Wolf, A.P.; Halldin, C.; Langstroem, B.

    1988-01-01

    The suicide inhibitors of monoamine oxidase type A and B, clorgyline and L-deprenyl have been labeled with carbon-11 by [ 11 C]methylation of the norbases with [ 11 C]H 3 I. The less active enantiomer of deprenyl (D-deprenyl) was also labeled using this procedure. The synthesis time was 35 minutes, the radiochemical yield was 25-40% and the specific activity was 0.8-2.0 Ci/μmol (calculated to EOB). Procedures for synthesis of the precursor norbases as well as the synthesis of unlabeled clorgyline, L-deprenyl and D-deprenyl are given. (author)

  15. Crystal structures of monoamine oxidase B in complex with four inhibitors of the N-propargylaminoindan class.

    Science.gov (United States)

    Binda, Claudia; Hubálek, Frantisek; Li, Min; Herzig, Yaacov; Sterling, Jeffrey; Edmondson, Dale E; Mattevi, Andrea

    2004-03-25

    Monoamine oxidase B (MAO B) is an outer mitochondrial membrane enzyme that catalyzes the oxidation of arylalkylamine neurotransmitters. The crystal structures of MAO B in complex with four of the N-propargylaminoindan class of MAO covalent inhibitors (rasagiline, N-propargyl-1(S)-aminoindan, 6-hydroxy-N-propargyl-1(R)-aminoindan, and N-methyl-N-propargyl-1(R)-aminoindan) have been determined at a resolution of better than 2.1 A. Rasagiline, 6-hydroxy-N-propargyl-1(R)-aminoindan, and N-methyl-N-propargyl-1(R)-aminoindan adopt essentially the same conformation with the extended propargyl chain covalently bound to the flavin and the indan ring located in the rear of the substrate cavity. N-Propargyl-1(S)-aminoindan binds with the indan ring in a flipped conformation with respect to the other inhibitors, which causes a slight movement of the Tyr326 side chain. Four ordered water molecules are an integral part of the active site and establish H-bond interactions to the inhibitor atoms. These structural studies may guide future drug design to improve selectivity and efficacy by introducing appropriate substituents on the rasagiline molecular scaffold.

  16. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by 14C putrescine method

    International Nuclear Information System (INIS)

    Fogel, W.A.; Bieganski, T.; Wozniak, J.; Maslinski, C.

    1978-01-01

    The Δ 1 pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi 14 C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation Δ 1 pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced Δ 1 pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on Δ 1 pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme. (author)

  17. NADPH oxidases as novel pharmacologic targets against influenza A virus infection.

    Science.gov (United States)

    Vlahos, Ross; Selemidis, Stavros

    2014-12-01

    Influenza A viruses represent a major global health care challenge, with imminent pandemics, emerging antiviral resistance, and long lag times for vaccine development, raising a pressing need for novel pharmacologic strategies that ideally target the pathology irrespective of the infecting strain. Reactive oxygen species (ROS) pervade all facets of cell biology with both detrimental and protective properties. Indeed, there is compelling evidence that activation of the NADPH oxidase 2 (NOX2) isoform of the NADPH oxidase family of ROS-producing enzymes promotes lung oxidative stress, inflammation, injury, and dysfunction resulting from influenza A viruses of low to high pathogenicity, as well as impeding virus clearance. By contrast, the dual oxidase isoforms produce ROS that provide vital protective antiviral effects for the host. In this review, we propose that inhibitors of NOX2 are better alternatives than broad-spectrum antioxidant approaches for treatment of influenza pathologies, for which clinical efficacy may have been limited owing to poor bioavailability and inadvertent removal of beneficial ROS. Finally, we briefly describe the current suite of NADPH oxidase inhibitors and the molecular features of the NADPH oxidase enzymes that could be exploited by drug discovery for development of more specific and novel inhibitors to prevent or treat disease caused by influenza. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by /sup 14/C putrescine method

    Energy Technology Data Exchange (ETDEWEB)

    Fogel, W A [Polish Academy of Sciences, Cracow (Poland). Inst. of Pharmacology; Bieganski, T; Wozniak, J; Maslinski, C

    1978-01-01

    The ..delta../sup 1/ pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi /sup 14/C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation ..delta../sup 1/ pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced ..delta../sup 1/ pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on ..delta../sup 1/ pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme.

  19. Synthesis and in vitro Evaluation of 2-heteroarylidene-1-tetralone Derivatives as Monoamine Oxidase Inhibitors.

    Science.gov (United States)

    Amakali, Klaudia T; Legoabe, Lesetja J; Petzer, Anél; Petzer, Jacobus P

    2018-05-14

    The present study investigates the human monoamine oxidase (MAO) inhibition properties of a series of twelve 2-heteroarylidene-1-tetralone derivatives. Also included are related cyclohexylmethylidene, cyclopentylmethylidene and benzylidene substituted 1-tetralones. These compounds are related to the 2-benzylidene-1-indanone class of compounds which has previously been shown to inhibit the MAOs, with specificity for the MAO-B isoform. The target compounds were synthesised by the Claisen-Schmidt condensation between 7-methoxy-1-tetralone or 1-tetralone, and various aldehydes, under acid (hydrochloric acid) or base (potassium hydroxide) catalysis. The results of the MAO inhibition studies showed that the 2-heteroarylidene-1-tetralone and related derivatives are in most instances more selective inhibitors of the MAO-B isoform compared to MAO-A. (2E)-2-Benzylidene-7-methoxy-3,4-dihydronaphthalen-1(2 H)-one (IC 50 =0.707 μM) was found to be the most potent MAO-B inhibitor, while the most potent MAO-A inhibitor was (2E)-2-[(2-chloropyridin-3-yl)methylidene]-7-methoxy-3,4-dihydronaphthalen-1(2 H)-one (IC 50 =1.37 μM). The effect of the heteroaromatic substituent on MAO-B inhibition activity, in decreasing order was found to be: cyclohexyl, phenyl>thiophene>pyridine, furane, pyrrole, cyclopentyl. This study concludes that, although some 2-heteroarylidene-1-tetralone derivatives are good potency MAO inhibitors, in general their inhibition potencies, particularly for MAO-B, are lower than structurally related chalcones and 1-indanone derivatives that were previously studied. © Georg Thieme Verlag KG Stuttgart · New York.

  20. Modulation of NADPH oxidase activity by known uraemic retention solutes

    DEFF Research Database (Denmark)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera

    2014-01-01

    chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. RESULTS: Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized......BACKGROUND: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased...... inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. METHODS: Mononuclear leucocytes...

  1. 77 FR 41081 - Sulfentrazone; Pesticide Tolerances

    Science.gov (United States)

    2012-07-12

    ... of the data supporting the petition, EPA has modified the tolerance levels for some commodities and... information in support of this action. EPA has sufficient data to assess the hazards of and to make a... target of sulfentrazone. Protoporphyrinogen oxidase inhibition in the mammalian species may result in...

  2. 76 FR 76304 - Saflufenacil; Pesticide Tolerances

    Science.gov (United States)

    2011-12-07

    ... response to these comments is discussed in Unit IV.C. Based upon review of the data supporting the petition..., EPA has reviewed the available scientific data and other relevant information in support of this... hematopoietic system as the target organ of saflufenacil. Protoporphyrinogen oxidase inhibition in the mammalian...

  3. Genomic sequencing of uric acid metabolizing and clearing genes in relationship to xanthine oxidase inhibitor dose.

    Science.gov (United States)

    Carroll, Matthew B; Smith, Derek M; Shaak, Thomas L

    2017-03-01

    It remains unclear why the dose of xanthine oxidase inhibitors (XOI) allopurinol or febuxostat varies among patients though they reach similar serum uric acid (SUA) goal. We pursued genomic sequencing of XOI metabolism and clearance genes to identify single-nucleotide polymorphisms (SNPs) relate to differences in XOI dose. Subjects with a diagnosis of Gout based on the 1977 American College of Rheumatology Classification Criteria for the disorder, who were on stable doses of a XOI, and who were at their goal SUA level, were enrolled. The primary outcome was relationship between SNPs in any of these genes to XOI dose. The secondary outcome was relationship between SNPs and change in pre- and post-treatment SUA. We enrolled 100 subjects. The average patient age was 68.6 ± 10.6 years old. Over 80% were men and 77% were Caucasian. One SNP was associated with a higher XOI dose: rs75995567 (p = 0.031). Two SNPs were associated with 300 mg daily of allopurinol: rs11678615 (p = 0.022) and rs3731722 on Aldehyde Oxidase (AO) (His1297Arg) (p = 0.001). Two SNPs were associated with a lower dose of allopurinol: rs1884725 (p = 0.033) and rs34650714 (p = 0.006). For the secondary outcome, rs13415401 was the only SNP related to a smaller mean SUA change. Ten SNPs were identified with a larger change in SUA. Though multiple SNPs were identified in the primary and secondary outcomes of this study, rs3731722 is known to alter catalytic function for some aldehyde oxidase substrates.

  4. Identification of aldehyde oxidase 1 and aldehyde oxidase homologue 1 as dioxin-inducible genes

    International Nuclear Information System (INIS)

    Rivera, Steven P.; Choi, Hyun Ho; Chapman, Brett; Whitekus, Michael J.; Terao, Mineko; Garattini, Enrico; Hankinson, Oliver

    2005-01-01

    Aldehyde oxidases are a family of highly related molybdo-flavoenzymes acting upon a variety of compounds of industrial and medical importance. We have identified aldehyde oxidase 1 (AOX1) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) inducible gene in the mouse hepatoma cell line Hepa-1. AOX1 mRNA levels were not increased by dioxin in mutant derivatives of the Hepa-1 cell line lacking either functional aryl hydrocarbon receptor (AHR) or aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, thus demonstrating that transcriptional induction of AOX1 in response to dioxin occurs through the AHR pathway. Dioxin induction of AOX1 mRNA was also observed in mouse liver. In addition, levels of AOX1 protein as well as those of aldehyde oxidase homologue 1 (AOH1), a recently identified homolog of AOX1, were elevated in mouse liver in response to dioxin. Employing an aldehyde oxidase specific substrate, AOX1/AOH1 activity was shown to be induced by dioxin in mouse liver. This activity was inhibited by a known inhibitor of aldehyde oxidases, and eliminated by including tungstate in the mouse diet, which is known to lead to inactivation of molybdoflavoenzymes, thus confirming that the enzymatic activity was attributable to AOX1/AOH1. Our observations thus identify two additional xenobiotic metabolizing enzymes induced by dioxin

  5. Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Nisler, Jaroslav; Kopečný, D.; Končitíková, R.; Zatloukal, Marek; Bazgier, Václav; Berka, K.; Zalabák, D.; Briozzo, P.; Strnad, Miroslav; Spíchal, Lukáš

    2016-01-01

    Roč. 92, 1-2 (2016), s. 235-248 ISSN 0167-4412 R&D Projects: GA MŠk(CZ) LO1204; GA ČR GA15-22322S Institutional support: RVO:61389030 Keywords : Cytokinin oxidase/dehydrogenase * Crystal structure * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.356, year: 2016

  6. Gene cloning and characterization of NADH oxidase from ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... potent inhibitors of NADH oxidases, silver nitrate and potassium cyanide did not show any significant ... anaerobes, a class of organisms that have not been ... DNA and amino acid sequence analyses were performed using.

  7. Aristolochic acid and its derivatives as inhibitors of snake venom L-amino acid oxidase.

    Science.gov (United States)

    Bhattacharjee, Payel; Bera, Indrani; Chakraborty, Subhamoy; Ghoshal, Nanda; Bhattacharyya, Debasish

    2017-11-01

    Snake venom L-amino acid oxidase (LAAO) exerts toxicity by inducing hemorrhage, pneumorrhagia, pulmonary edema, cardiac edema, liver cell necrosis etc. Being well conserved, inhibitors of the enzyme may be synthesized using the template of the substrate, substrate binding site and features of the catalytic site of the enzyme. Previous findings showed that aristolochic acid (AA), a major constituent of Aristolochia indica, inhibits Russell's viper venom LAAO enzyme activity since, AA interacts with DNA and causes genotoxicity, derivatives of this compound were synthesized by replacing the nitro group to reduce toxicity while retaining the inhibitory potency. The interactions of AA and its derivatives with LAAO were followed by inhibition kinetics and surface plasmon resonance. Similar interactions with DNA were followed by absorption spectroscopy and atomic force microscopy. LAAO-induced cytotoxicity was evaluated by generation of reactive oxygen species (ROS), cell viability assays, confocal and epifluorescence microscopy. The hydroxyl (AA-OH) and chloro (AA-Cl) derivatives acted as inhibitors of LAAO but did not interact with DNA. The derivatives significantly reduced LAAO-induced ROS generation and cytotoxicity in human embryonic kidney (HEK 293) and hepatoma (HepG2) cell lines. Confocal images indicated that AA, AA-OH and AA-Cl interfered with the binding of LAAO to the cell membrane. AA-OH and AA-Cl significantly inhibited LAAO activity and reduced LAAO-induced cytotoxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Combination monoamine oxidase inhibitor and beta-blocker treatment of migraine, with anxiety and depression.

    Science.gov (United States)

    Merikangas, K R; Merikangas, J R

    1995-11-01

    This paper presents the results of a study comparing the effectiveness of a beta-adrenergic blocking agent, atenolol, a monoamine oxidase inhibitor (MAO-I), phenelzine, and the combination in treatment of 61 adults with migraine headache. The goals of the study are (1) to investigate the safety of concomitant treatment of migraine with beta-blockers and phenelzine, (2) to assess whether orthostatic hypertension and other side effects would be relieved, and (3) to compare the results of this open trial of phenelzine to those of a previous study using similar methods. Phenelzine was associated with a large decrease in the frequency and severity of migraine attacks. Anxiety and depression were also reduced by phenelzine both alone, and in combination with a beta-blocker. The results show that the combination of MAO-I's and beta-blockers can be administered safely, and can lead to the reduction in the side effects with either drug alone.

  9. Comparative investigation to see the efficacy of polyphenol oxidase inhibitors parallel to sulphite for control of enzymatic browning in guava juice

    International Nuclear Information System (INIS)

    Zai, M.N.K.A.

    2008-01-01

    Comparative study was conducted to determine the efficacy of polyphenol oxidase inhibitors to control browning in guava juice/concentrates parallel to sulfite. Although sulfites are highly effective to control the browning but these have shown adverse effect on health particularly to those who have pulmonary disorder like asthma. Filtration with useful filter aid and juicing agents are found to be helpful in removing browning residue particulates fractions to extend the shelf life of the juice. Non-enzymatic browning can be prevented by cold blanching. (author)

  10. Human monoamine oxidase is inhibited by tobacco smoke: β-carboline alkaloids act as potent and reversible inhibitors

    International Nuclear Information System (INIS)

    Herraiz, Tomas; Chaparro, Carolina

    2005-01-01

    Monoamine oxidase (MAO) is a mitochondrial outer-membrane flavoenzyme involved in brain and peripheral oxidative catabolism of neurotransmitters and xenobiotic amines, including neurotoxic amines, and a well-known target for antidepressant and neuroprotective drugs. Recently, positron emission tomography imaging has shown that smokers have a much lower activity of peripheral and brain MAO-A (30%) and -B (40%) isozymes compared to non-smokers. This MAO inhibition results from a pharmacological effect of smoke, but little is known about its mechanism. Working with mainstream smoke collected from commercial cigarettes we confirmed that cigarette smoke is a potent inhibitor of human MAO-A and -B isozymes. MAO inhibition was partly reversible, competitive for MAO-A, and a mixed-type inhibition for MAO-B. Two β-carboline alkaloids, norharman (β-carboline) and harman (1-methyl-β-carboline), were identified by GC-MS, quantified, and isolated from the mainstream smoke by solid phase extraction and HPLC. Kinetics analysis revealed that β-carbolines from cigarette smoke were competitive, reversible, and potent inhibitors of MAO enzymes. Norharman was an inhibitor of MAO-A (K i = 1.2 ± 0.18 μM) and MAO-B (K i = 1.12 ± 0.19 μM), and harman of MAO-A (K i = 55.54 ± 5.3 nM). β-Carboline alkaloids are psychopharmacologically active compounds that may occur endogenously in human tissues, including the brain. These results suggest that β-carboline alkaloids from cigarette smoke acting as potent reversible inhibitors of MAO enzymes may contribute to the MAO-reduced activity produced by tobacco smoke in smokers. The presence of MAO inhibitors in smoke like β-carbolines and others may help us to understand some of the purported neuropharmacological effects associated with smoking

  11. The new inhibitor of monoamine oxidase, M30, has a neuroprotective effect against dexamethasone-induced brain cell apoptosis

    Directory of Open Access Journals (Sweden)

    Shakevia Johnson

    2010-11-01

    Full Text Available Stress detrimentally affects the brain and body and can lead to or be accompanied by depression. Although stress and depression may contribute to each other, the exact molecular mechanism underlying the effects is unclear. However, there is a correlation between stress and an increase in glucocorticoid secretion which causes a subsequent increase in monoamine oxidase (MAO activity during stress. Consequently, MAO inhibitors have been used as traditional antidepressant drugs. Cellular treatment with the synthetic glucocorticoid, dexamethasone (a cellular stressor, has been reported to markedly increase both MAO A and MAO B catalytic activities, as well as apoptosis. This study compares the neuroprotective abilities of M30 (a new generation inhibitor of both MAO A and MAO B with rasagiline (Azilect®, another new MAO B inhibitor and selegiline (Deprenyl®, a traditional MAO B inhibitor in the prevention of dexamethasone-induced brain cell death and MAO activity in human neuroblastoma cells, SH-SY5Y. M30 demonstrated the highest inhibitory effect on MAO A; however, M30 showed the lowest inhibitory effect on MAO B enzymatic activity in comparison to rasagiline and selegiline. Although, M30 exhibited the greatest neuroprotective effect by decreasing cell death rates and apoptotic DNA damage compared to rasagiline and selegiline, these neuroprotective effects of M30 were, overall, similar to rasagiline. Summarily, M30 has a generally greater impact on neuroprotection than the MAO B inhibitors, selegiline and rasagiline. Our results suggest that M30 may have great potential in alleviating disorders involving increases in both MAO A and MAO B, such as stress-induced disorders.

  12. [Substrate-inhibitory analysis of monoamine oxidase from hepatopancreas of the octopus Bathypolypus arcticus].

    Science.gov (United States)

    Basova, I N; Iagodina, O V

    2012-01-01

    Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, beta-phenylethylamine, while, on the other hand, deaminates histamine and does not deaminate putrescine--classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs of the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine--irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.

  13. Phosphorescent inner filter effect-based sensing of xanthine oxidase and its inhibitors with Mn-doped ZnS quantum dots.

    Science.gov (United States)

    Tang, Dandan; Zhang, Jinyi; Zhou, Rongxin; Xie, Ya-Ni; Hou, Xiandeng; Xu, Kailai; Wu, Peng

    2018-05-10

    Overexpression and crystallization of uric acid have been recognized as the course of hyperuricemia and gout, which is produced via xanthine oxidase (XOD)-catalyzed oxidation of xanthine. Therefore, the medicinal therapy of hyperuricemia and gout is majorly based on the inhibition of the XOD enzymatic pathway. The spectroscopic nature of xanthine and uric acid, namely both absorption (near the ultraviolet region) and emission (non-fluorescent) characteristics, hinders optical assay development for XOD analysis. Therefore, the state-of-the-art analysis of XOD and the screening of XOD inhibitors are majorly based on chromatography. Here, we found the near ultraviolet absorption of uric acid overlapped well with the absorption of a large bandgap semiconductor quantum dots, ZnS. On the other hand, the intrinsic weak fluorescence of ZnS QDs can be substantially improved via transition metal ion doping. Therefore, herein, we developed an inner filter effect-based assay for XOD analysis and inhibitor screening with Mn-doped ZnS QDs. The phosphorescence of Mn-doped ZnS QDs could be quenched by uric acid generated from xanthine catabolism by XOD, leading to the phosphorescence turn-off detection of XOD with a limit of detection (3σ) of 0.02 U L-1. Furthermore, the existence of XOD inhibitors could inhibit the XOD enzymatic reaction, resulting in weakened phosphorescence quenching. Therefore, the proposed assay could also be explored for the facile screening analysis of XOD inhibitors, which is important for the potential medicinal therapy of hyperuricemia and gout.

  14. Potato and Mushroom Polyphenol Oxidase Activities Are Differently Modulated by Natural Plant Extracts

    NARCIS (Netherlands)

    Kuijpers, T.F.M.; Herk, van T.; Vincken, J.P.; Janssen, R.H.; Narh, D.L.; Berkel, van W.J.H.; Gruppen, H.

    2014-01-01

    Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the

  15. Functional heterogeneity of NADPH oxidase-mediated contractions to endothelin with vascular aging.

    Science.gov (United States)

    Meyer, Matthias R; Barton, Matthias; Prossnitz, Eric R

    2014-11-24

    Aging, a physiological process and main risk factor for cardiovascular and renal diseases, is associated with endothelial cell dysfunction partly resulting from NADPH oxidase-dependent oxidative stress. Because increased formation of endothelium-derived endothelin-1 (ET-1) may contribute to vascular aging, we studied the role of NADPH oxidase function in age-dependent contractions to ET-1. Renal arteries and abdominal aortas from young and old C57BL6 mice (4 and 24 months of age) were prepared for isometric force measurements. Contractions to ET-1 (0.1-100 nmol/L) were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat (3 μmol/L). To exclude age-dependent differential effects of NO bioactivity between vascular beds, all experiments were conducted in the presence of the NO synthase inhibitor L-NAME (300 μmol/L). In young animals, ET-1-induced contractions were 6-fold stronger in the renal artery than in the aorta (prenal artery and aorta, respectively (pAging had no effect on NADPH oxidase-dependent and -independent contractions to ET-1 in the renal artery. In contrast, contractions to ET-1 were markedly reduced in the aged aorta (5-fold, page-dependent heterogeneity of NADPH oxidase-mediated vascular contractions to ET-1, demonstrating an inherent resistance to functional changes in the renal artery but not in the aorta with aging. Thus, local activity of NADPH oxidase differentially modulates responses to ET-1 with aging in distinct vascular beds. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Controversies in Neurology: why monoamine oxidase B inhibitors could be a good choice for the initial treatment of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Reichmann Heinz

    2011-09-01

    Full Text Available Abstract Background Early initiation of pharmacotherapy in Parkinson's disease (PD is nowadays widely advocated by experts since the delay of treatment has shown to be associated with a significant deterioration of health related quality of life in affected patients. Due to marked advances in PD treatment during the last decades, physicians are nowadays fortunately equipped with a variety of substances that can effectively ameliorate emerging motor symptoms of the disease, among them levodopa, dopamine agonists and monoamine oxidase type B (MAO-B inhibitors. Despite numerous drug intervention trials in early PD, there is however still ongoing controversy among neurologists which substance to use for the initial treatment of the disease. Discussion In multiple studies, MAO-B inhibitors, such as selegiline and rasagiline, have shown to provide mild symptomatic effects, delay the need for levodopa, and to reduce the incidence of motor fluctuations. Although their symptomatic efficacy is inferior compared to dopamine agonists and levodopa, MAO-B inhibitors undoubtedly have fewer side effects and are easy to administer. In contrary to their competitors, MAO-B inhibitors may furthermore offer a chance for disease modification, which so far remains a major unmet need in the management of PD and eventually makes them ideal candidates for the early treatment of the disease. Summary MAO-B inhibitors may constitute a preferable therapeutic option for early PD, mainly due to their favourable safety profile and their putative neuroprotective capabilities. Since the symptomatic effects of MAO-B inhibitors are comparatively mild, dopamine agonists and levodopa should however be considered for initial treatment in those PD patients, in whom robust and immediate symptomatic relief needs to be prioritized.

  17. Controversies in neurology: why monoamine oxidase B inhibitors could be a good choice for the initial treatment of Parkinson's disease.

    Science.gov (United States)

    Löhle, Matthias; Reichmann, Heinz

    2011-09-22

    Early initiation of pharmacotherapy in Parkinson's disease (PD) is nowadays widely advocated by experts since the delay of treatment has shown to be associated with a significant deterioration of health related quality of life in affected patients. Due to marked advances in PD treatment during the last decades, physicians are nowadays fortunately equipped with a variety of substances that can effectively ameliorate emerging motor symptoms of the disease, among them levodopa, dopamine agonists and monoamine oxidase type B (MAO-B) inhibitors. Despite numerous drug intervention trials in early PD, there is however still ongoing controversy among neurologists which substance to use for the initial treatment of the disease. In multiple studies, MAO-B inhibitors, such as selegiline and rasagiline, have shown to provide mild symptomatic effects, delay the need for levodopa, and to reduce the incidence of motor fluctuations. Although their symptomatic efficacy is inferior compared to dopamine agonists and levodopa, MAO-B inhibitors undoubtedly have fewer side effects and are easy to administer. In contrary to their competitors, MAO-B inhibitors may furthermore offer a chance for disease modification, which so far remains a major unmet need in the management of PD and eventually makes them ideal candidates for the early treatment of the disease. MAO-B inhibitors may constitute a preferable therapeutic option for early PD, mainly due to their favourable safety profile and their putative neuroprotective capabilities. Since the symptomatic effects of MAO-B inhibitors are comparatively mild, dopamine agonists and levodopa should however be considered for initial treatment in those PD patients, in whom robust and immediate symptomatic relief needs to be prioritized.

  18. Effect of a heme oxygenase-1 inducer on NADPH oxidase ...

    African Journals Online (AJOL)

    Effect of a heme oxygenase-1 inducer on NADPH oxidase expression in ... and immunohistochemistry of hepatic NOX1 and NOX4 were investigated in week 4. ... (HO-1 inhibitor) administration caused upregulation of NOX gene expression ...

  19. Azure B, a metabolite of methylene blue, is a high-potency, reversible inhibitor of monoamine oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Petzer, Anél, E-mail: 12264954@nwu.ac.za [Unit for Drug Research and Development, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520 (South Africa); Harvey, Brian H. [Division of Pharmacology, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520 (South Africa); Wegener, Gregers [Centre for Psychiatric Research, Aarhus University Hospital-Risskov, Skovagervej 2, 8240 Risskov (Denmark); Petzer, Jacobus P. [Division of Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520 (South Africa)

    2012-02-01

    Methylene blue (MB) has been shown to act at multiple cellular and molecular targets and as a result possesses diverse medical applications. Among these is a high potency reversible inhibition of monoamine oxidase A (MAO-A) that may, at least in part, underlie its adverse effects but also its psycho- and neuromodulatory actions. MB is metabolized to yield N-demethylated products of which azure B, the monodemethyl species, is the major metabolite. Similar to MB, azure B also displays a variety of biological activities and may therefore contribute to the pharmacological profile of MB. Based on these observations, the present study examines the interactions of azure B with recombinant human MAO-A and -B. The results show that azure B is a potent MAO-A inhibitor (IC{sub 50} = 11 nM), approximately 6-fold more potent than is MB (IC{sub 50} = 70 nM) under identical conditions. Measurements of the time-dependency of inhibition suggest that the interaction of azure B with MAO-A is reversible. Azure B also reversibly inhibits the MAO-B isozyme with an IC{sub 50} value of 968 nM. These results suggest that azure B may be a hitherto under recognized contributor to the pharmacology and toxicology of MB by blocking central and peripheral MAO-A activity and as such needs to be considered during its use in humans and animals. Highlights: ► Methylene blue (MB) is a known potent MAO-A inhibitor. ► Azure B, the major metabolite of MB, is more potent as a MAO-A inhibitor. ► Azure B may be a contributor to the CNS pharmacology and toxicology of MB.

  20. Low-Dose Dextromethorphan, a NADPH Oxidase Inhibitor, Reduces Blood Pressure and Enhances Vascular Protection in Experimental Hypertension

    Science.gov (United States)

    Wu, Tao-Cheng; Chao, Chih-Yu; Lin, Shing-Jong; Chen, Jaw-Wen

    2012-01-01

    Background Vascular oxidative stress may be increased with age and aggravate endothelial dysfunction and vascular injury in hypertension. This study aimed to investigate the effects of dextromethorphan (DM), a NADPH oxidase inhibitor, either alone or in combination treatment, on blood pressure (BP) and vascular protection in aged spontaneous hypertensive rats (SHRs). Methodology/Principal Findings Eighteen-week-old WKY rats and SHRs were housed for 2 weeks. SHRs were randomly assigned to one of the 12 groups: untreated; DM monotherapy with 1, 5 or 25 mg/kg/day; amlodipine (AM, a calcium channel blocker) monotherapy with 1 or 5 mg/kg/day; and combination therapy of DM 1, 5 or 25 mg/kg/day with AM 1 or 5 mg/kg/day individually for 4 weeks. The in vitro effects of DM were also examined. In SHRs, AM monotherapy dose-dependently reduced arterial systolic BP. DM in various doses significantly and similarly reduced arterial systolic BP. Combination of DM with AM gave additive effects on BP reduction. DM, either alone or in combination with AM, improved aortic endothelial function indicated by ex vivo acetylcholine-induced relaxation. The combination of low-dose DM with AM gave most significant inhibition on aortic wall thickness in SHRs. Plasma total antioxidant status was significantly increased by all the therapies except for the combination of high-dose DM with high-dose AM. Serum nitrite and nitrate level was significantly reduced by AM but not by DM or the combination of DM with AM. Furthermore, in vitro treatment with DM reduced angiotensin II-induced reactive oxygen species and NADPH oxidase activation in human aortic endothelial cells. Conclusions/Significance Treatment of DM reduced BP and enhanced vascular protection probably by inhibiting vascular NADPH oxidase in aged hypertensive animals with or without AM treatment. It provides the potential rationale to a novel combination treatment with low-dose DM and AM in clinical hypertension. PMID:23049937

  1. Low-dose dextromethorphan, a NADPH oxidase inhibitor, reduces blood pressure and enhances vascular protection in experimental hypertension.

    Directory of Open Access Journals (Sweden)

    Tao-Cheng Wu

    Full Text Available BACKGROUND: Vascular oxidative stress may be increased with age and aggravate endothelial dysfunction and vascular injury in hypertension. This study aimed to investigate the effects of dextromethorphan (DM, a NADPH oxidase inhibitor, either alone or in combination treatment, on blood pressure (BP and vascular protection in aged spontaneous hypertensive rats (SHRs. METHODOLOGY/PRINCIPAL FINDINGS: Eighteen-week-old WKY rats and SHRs were housed for 2 weeks. SHRs were randomly assigned to one of the 12 groups: untreated; DM monotherapy with 1, 5 or 25 mg/kg/day; amlodipine (AM, a calcium channel blocker monotherapy with 1 or 5 mg/kg/day; and combination therapy of DM 1, 5 or 25 mg/kg/day with AM 1 or 5 mg/kg/day individually for 4 weeks. The in vitro effects of DM were also examined. In SHRs, AM monotherapy dose-dependently reduced arterial systolic BP. DM in various doses significantly and similarly reduced arterial systolic BP. Combination of DM with AM gave additive effects on BP reduction. DM, either alone or in combination with AM, improved aortic endothelial function indicated by ex vivo acetylcholine-induced relaxation. The combination of low-dose DM with AM gave most significant inhibition on aortic wall thickness in SHRs. Plasma total antioxidant status was significantly increased by all the therapies except for the combination of high-dose DM with high-dose AM. Serum nitrite and nitrate level was significantly reduced by AM but not by DM or the combination of DM with AM. Furthermore, in vitro treatment with DM reduced angiotensin II-induced reactive oxygen species and NADPH oxidase activation in human aortic endothelial cells. CONCLUSIONS/SIGNIFICANCE: Treatment of DM reduced BP and enhanced vascular protection probably by inhibiting vascular NADPH oxidase in aged hypertensive animals with or without AM treatment. It provides the potential rationale to a novel combination treatment with low-dose DM and AM in clinical hypertension.

  2. Xanthine oxidase inhibitors from Archidendron clypearia (Jack.) I.C. Nielsen: Results from systematic screening of Vietnamese medicinal plants

    Institute of Scientific and Technical Information of China (English)

    Nguyen Thuy Duong; Pham Duc Vinh; Phuong Thien Thuong; Nguyen Thi Hoai; Le Nguyen Thanh; Tran The Bach; Nguyen Hai Nam; Nguyen Hoang Anh

    2017-01-01

    Objective:To screen Vietnamese medicinal plants for xanthine oxidase (XO) inhibitory activity and to isolate XO inhibitor(s) from the most active plant.Methods: The plants materials were extracted by methanol. The active plant materials were fractionated using different organic solvents, includingn-hexane, ethyl acetate, andn-butanol. Bioassay-guided fractionation and column chromatography were used to isolate compounds. The compounds structures were elucidated by analysis of spectroscopic data, including IR, MS, and NMR. Results:Three hundreds and eleven methanol extracts (CME) belonging to 301 Vietnamese herbs were screened for XO inhibitory activity. Among these plants, 57 extracts displayed XO inhibitory activity at 100 μg/mL with inhibition rates of over 50%. The extracts of Archidendron clypearia,Smilax poilanei,Linociera ramiflora and Passiflora foetida exhibited the greatest potency with IC50 values below 30 μg/mL. Chemical study performed on the extract ofArchidendron clypearia resulted in the isolation of six compounds, including 1-octacosanol, docosenoic acid, daucosterol, methyl gallate, quercitrin and (?)-7-O-galloyltricetiflavan. The compound (?)-7-O-galloyltricetiflavan showed the most potent XO inhibitory activity with an IC50 value of 25.5 μmol/L.Conclusions:From this investigation, four Vietnamese medicinal plants were identified to have XO inhibitory effects with IC50 values of the methanol extracts below 30 μg/mL. Compound (?)-7-O-galloyltricetiflavan was identified as an XO inhibitor from Archidendron clypearia with IC50 value of 25.5 μmol/L.

  3. High-performance liquid chromatography coupled with post-column dual-bioactivity assay for simultaneous screening of xanthine oxidase inhibitors and free radical scavengers from complex mixture.

    Science.gov (United States)

    Li, D Q; Zhao, J; Li, S P

    2014-06-06

    Xanthine oxidase (XO) can catalyze hypoxanthine and xanthine to generate uric acid and reactive oxygen species (ROS), including superoxide anion radical (O₂(•-)) and hydrogen peroxide. XO inhibitors and free radical scavengers are beneficial to the treatment of gout and many related diseases. In the present study, an on-line high-performance liquid chromatography (HPLC) coupled with post-column dual-bioactivity assay was established and successfully applied to simultaneously screening of XO inhibitors and free radical scavengers from a complex mixture, Oroxylum indicum extract. The integrated system of HPLC separation, bioactivity screening and mass spectrometry identification was proved to be simple and effective for rapid and sensitive screening of individual bioactive compounds in complex mixtures. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

    Czech Academy of Sciences Publication Activity Database

    Kopečný, D.; Šebela, M.; Briozzo, P.; Spíchal, Lukáš; Houba-Hérin, N.; Mašek, V.; Joly, N.; Madzak, C.; Anzenbacher, P.; Laloue, M.

    2008-01-01

    Roč. 380, č. 5 (2008), s. 886-899 ISSN 0022-2836 R&D Projects: GA ČR(CZ) GP522/08/P113 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinin oxidase/dehydrogenase * cytokinin signaling * protein structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.146, year: 2008

  5. Functional mechanism of neuroprotection by inhibitors of type B monoamine oxidase in Parkinson's disease.

    Science.gov (United States)

    Naoi, Makoto; Maruyama, Wakako

    2009-08-01

    Neuroprotective therapy has been proposed for age-related neurodegenerative disorders, including Parkinson's disease. Inhibitors of type B monoamine oxidase (MAOB-Is), rasagiline and (-)deprenyl, are the most promising candidate neuroprotective drugs. Clinical trials of rasagiline in patients with Parkinson's disease suggest that rasagiline may have some disease-modifying effects. Results using animal and cellular models have proved that the MAOB-Is protect neurons by the intervention of 'intrinsic' mitochondrial apoptotic cascade and the induction of prosurvival antiapoptotic Bcl-2 and neurotrophic factors. Rasagiline-related MAOB-Is prevent mitochondrial permeability transition induced by various insults and activation of subsequent apoptotic cascades: cytochrome c release, casapase activation, and condensation and fragmentation of nuclear DNA. MAOB-Is increase transcription of prosurvival genes through activating the nuclear transcription factor-(NF) system. Rasagiline increases the protein and mRNA levels of GDNF in dopaminergic SH-SY5Y cells, whereas (-)deprenyl increases those of BDNF. Systemic administration of (-)deprenyl and rasagiline increases these neurotrophic factors in the cerebrospinal fluid from patients with Parkinson's disease and nonhuman primates. This review presents recent advances in our understanding of the neuroprotection offered by MAOB-Is and possible evaluation of neuroprotective efficacy in clinical samples is discussed.

  6. Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

    Science.gov (United States)

    Löw, Hans; Crane, Frederick L; Morré, D James

    2012-11-01

    The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases: in vitro studies on antioxidant activity, prevention of lipid peroxide formation and monoamine oxidase inhibition.

    Science.gov (United States)

    Zheng, Hailin; Gal, Shunit; Weiner, Lev M; Bar-Am, Orit; Warshawsky, Abraham; Fridkin, Mati; Youdim, Moussa B H

    2005-10-01

    Iron-dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)-B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinson's disease, Alzheimer's disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO-B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO-B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO-B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron-dependent lipid peroxidation in rat brain homogenates with IC50 values (12-16 microM), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 microM were able to attenuate cell death induced by serum deprivation and by 6-hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti-Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non-selective MAO-A and MAO-B inhibitor (IC50 < 0.1 microM). However, HLA20 was more selective for MAO-B but had poor MAO inhibition, with an IC50 value of 64.2 microM. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.

  8. Monoamine oxidase inhibitors l-deprenyl and clorgyline protect nonmalignant human cells from ionising radiation and chemotherapy toxicity.

    LENUS (Irish Health Repository)

    Seymour, C B

    2003-11-17

    l-Deprenyl (R-(-)-deprenyl, selegiline) is an inhibitor of monoamine oxidase-B (MAO-B) that is known to protect nerve cells from a variety of chemical and physical insults. As apoptosis is a common mechanism of radiation-induced cell death, the effect of l-deprenyl on the survival of cultured cells and tissue explants was studied following exposure to gamma radiation. The results obtained were compared with the effects of the less-selective MAO-B inhibitor pargyline and the MAO-A inhibitor clorgyline. l-Deprenyl at a concentration of 10(-9) M protected the nontumorigenic cell line (HaCaT) and normal human urothelial explants from the effects of cobalt-60 gamma radiation, but did not protect tumorigenic human cell lines HaCaT-ras, HPV-transfected human keratinocytes (HPV-G cells), or PC3. Human bladder carcinoma explants were not protected. Clorgyline showed a smaller protective effect of normal cells, whereas pargyline had no effect. Radiation-induced delayed effects (genomic instability measured as delayed cell death) were prevented in normal cells by l-deprenyl but, interestingly, deprenyl appeared to increase the amount of delayed death in the tumorigenic cell lines. Studies using l-deprenyl prior to the exposure of nonmalignant cells to cisplatin showed that cell death due to this agent was also reduced. Treatment of cultures of nontumorigenic cells with l-deprenyl or clorgyline significantly increased the levels of the protein Bcl-2 following irradiation, but there was no such effect on the already-elevated levels of this protein in the tumour samples. Since the Bcl-2 has been shown to be an inhibitor of apoptosis or programmed cell death, this would imply that the protective effects of l-deprenyl and clorgyline involve activation of antiapoptotic pathways within the normal cell. This hypothesis is supported by data showing reduced levels of apoptosis in HaCAT cells and in normal bladder explant cultures following treatment with l-deprenyl.

  9. Effect of Soy Sauce on Serum Uric Acid Levels in Hyperuricemic Rats and Identification of Flazin as a Potent Xanthine Oxidase Inhibitor.

    Science.gov (United States)

    Li, Huipin; Zhao, Mouming; Su, Guowan; Lin, Lianzhu; Wang, Yong

    2016-06-15

    This is the first report on the ability of soy sauce to effectively reduce the serum uric acid levels and xanthine oxidase (XOD) activities of hyperuricemic rats. Soy sauce was partitioned sequentially into ethyl acetate and water fractions. The ethyl acetate fraction with strong XOD inhibition effect was purified further. On the basis of xanthine oxidase inhibitory (XOI) activity-guided purification, nine compounds including 3,4-dihydroxy ethyl cinnamate, diisobutyl terephthalate, harman, daidzein, flazin, catechol, thymine, genistein, and uracil were obtained. It was the first time that 3,4-dihydroxy ethyl cinnamate and diisobutyl terephthalate had been identified from soy sauce. Flazin with hydroxymethyl furan ketone group at C-1 and carboxyl at C-3 exhibited the strongest XOI activity (IC50 = 0.51 ± 0.05 mM). According to fluorescence quenching and molecular docking experiments, flazin could enter into the catalytic center of XOD to interact with Lys1045, Gln1194, and Arg912 mainly by hydrophobic forces and hydrogen bonds. Flazin, catechol, and genistein not only were potent XOD inhibitors but also held certain antioxidant activities. According to ADME (absorption, distribution, metabolism, and excretion) simulation in silico, flazin had good oral bioavailability in vivo.

  10. Characterization and Thermodynamic Relationship of Three Polymorphs of a Xanthine Oxidase Inhibitor, Febuxostat.

    Science.gov (United States)

    Patel, Jinish; Jagia, Moksh; Bansal, Arvind Kumar; Patel, Sarsvatkumar

    2015-11-01

    Febuxostat (FXT), a xanthine oxidase inhibitor, is an interesting and unique molecule, which exhibits extensive polymorphism, with over 15 polymorphic forms reported to date. The primary purpose of the study was to characterize the three polymorphic forms with respect to their thermodynamic quantities and establish thermodynamic relationship between them. The polymorphs were characterized by thermal and powder X-ray diffraction methods. Three different methods were used to calculate the transition temperatures (Ttr) and thereby their thermodynamic relationships. Although the first and second method used calorimetric data (melting point and heat of fusion), the third method employed the use of configurational free energy phase diagram. The onset melting points of three polymorphic forms were found to be 482.89 ± 0.37 K for form I, 476.30 ± 1.21 K for form II, and 474.19 ± 0.11 K for form III. Moreover, the powder X-ray diffraction patterns for each form were also unique. The polymorphic pair of form I and II and of form I and III was found to be enantiotropic, whereas pair of form II and III was monotropic. Besides the relative thermodynamic aspects (free energy differences, enthalpy, entropy contributions) using different methods, the pharmaceutical implications and phase transformation aspects have also been covered. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  11. Radio-isotopic determination of platelet monoamine oxidase and regulation of its activity by an indigenous drug

    International Nuclear Information System (INIS)

    Dubey, G.P.; Srivastava, V.K.; Agrawal, A.; Udupa, K.N.

    1988-01-01

    Platelet monoamine oxidase is a mitochondrial enzyme taking part in the deamination reaction of total catecholamine. Recent studies of monoamine oxidase inhibitors have gained its importance in the control of variety of psychosomatic disorders like mental depression, arterial hypertension and anxiety neurosis. 30 apparently normal individuals and 42 diagnosed cases of essential hypertension were selected for the present study. The platelet monoamine oxidase activity was measured by using 14 C-tryptamine bisuccinate. Comparatively low activity of platelet monoamine oxidase was noticed in hypertension cases than in the normal. After oral administration of an indigenous drug 'Geriforte' for three months, a significant rise in platelet monoamine oxidase activity was noticed in hypertension cases. It can be concluded that this indigenous formulation has the capacity to regulate the monoamine oxidase activity, as such, it may provide an alternative remedy in the management of psychosomatic disorders. (author). 11 refs

  12. A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites

    DEFF Research Database (Denmark)

    Dang, Pham My-Chan; Stensballe, Allan; Boussetta, Tarek

    2006-01-01

    mass spectrometry to show that GM-CSF and TNF-alpha induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM......Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem...

  13. Imaging Monoamine Oxidase in the Human Brain

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-11-10

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets.

  14. Imaging Monoamine Oxidase in the Human Brain

    International Nuclear Information System (INIS)

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-01-01

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets

  15. Xanthine oxidase inhibitors from Archidendron clypearia (Jack.) I.C. Nielsen: Results from systematic screening of Vietnamese medicinal plants.

    Science.gov (United States)

    Duong, Nguyen Thuy; Vinh, Pham Duc; Thuong, Phuong Thien; Hoai, Nguyen Thi; Thanh, Le Nguyen; Bach, Tran The; Nam, Nguyen Hai; Anh, Nguyen Hoang

    2017-06-01

    To screen Vietnamese medicinal plants for xanthine oxidase (XO) inhibitory activity and to isolate XO inhibitor(s) from the most active plant. The plants materials were extracted by methanol. The active plant materials were fractionated using different organic solvents, including n-hexane, ethyl acetate, and n-butanol. Bioassay-guided fractionation and column chromatography were used to isolate compounds. The compounds structures were elucidated by analysis of spectroscopic data, including IR, MS, and NMR. Three hundreds and eleven methanol extracts (CME) belonging to 301 Vietnamese herbs were screened for XO inhibitory activity. Among these plants, 57 extracts displayed XO inhibitory activity at 100 μg/mL with inhibition rates of over 50%. The extracts of Archidendron clypearia (A. clypearia), Smilax poilanei, Linociera ramiflora and Passiflora foetida exhibited the greatest potency with IC 50 values below 30 μg/mL. Chemical study performed on the extract of A. clypearia resulted in the isolation of six compounds, including 1-octacosanol, docosenoic acid, daucosterol, methyl gallate, quercitrin and (-)-7-O-galloyltricetiflavan. The compound (-)-7-O-galloyltricetiflavan showed the most potent XO inhibitory activity with an IC 50 value of 25.5 μmol/L. From this investigation, four Vietnamese medicinal plants were identified to have XO inhibitory effects with IC 50 values of the methanol extracts below 30 μg/mL. Compound (-)-7-O- galloyltricetiflavan was identified as an XO inhibitor from A. clypearia with IC 50 value of 25.5 μmol/L. Copyright © 2017 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.

  16. Validation of tautomeric and protomeric binding modes by free energy calculations. A case study for the structure based optimization of d-amino acid oxidase inhibitors

    Science.gov (United States)

    Orgován, Zoltán; Ferenczy, György G.; Steinbrecher, Thomas; Szilágyi, Bence; Bajusz, Dávid; Keserű, György M.

    2018-02-01

    Optimization of fragment size d-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

  17. [Effect of inducers and inhibitors of mixed function oxidases on body resistance to endotoxins of gram-negative bacteria].

    Science.gov (United States)

    Liniuchev, M N; Zubik, T M; Kovelenov, A Iu; Bulyko, V I; Sergeev, V V

    1989-06-01

    Experimental typhoid intoxication in white mice leads to the inhibition of microsomal oxidation in the liver, which is manifested by the prolongation of hexenal-induced sleep and a decrease in the toxic action of parathion. Phenobarbital, capable of inducing oxidases with mixed function (OMF), enhances the process of the detoxification of endotoxin injected into the animals, which is manifested by the increase of its LD50. Soluble levomycetin succinate, widely used for the treatment of typhoid-paratyphoid infections, is a powerful inhibitor of OMF (as shown by the hexenal test). Benzonal, the analog of phenobarbital, removes the inhibitory effect of the antibiotic. Experimental studies carried out in the course of this investigation make it possible to substantiate the clinical trial of these preparations (OMF inducers) used in the complex therapy of typhoid-paratyphoid infections for the stimulation of natural detoxification mechanisms of the body. Benzonal is the preparation of choice for use in clinical practice.

  18. ASIC-like currents in freshly isolated cerebral artery smooth muscle cells are inhibited by endogenous oxidase activity.

    Science.gov (United States)

    Chung, Wen-Shuo; Farley, Jerry M; Drummond, Heather A

    2011-01-01

    The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 μM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase. Copyright © 2011 S. Karger AG, Basel.

  19. Depletion of rat cortical norepinephrine and the inhibition of [3H]norepinephrine uptake by xylamine does not require monoamine oxidase activity

    International Nuclear Information System (INIS)

    Dudley, M.W.

    1988-01-01

    Inhibition of monoamine oxidase A through pretreatment of rats with clorgyline or the pro-drug MDL 72,394 did not block the amine-depleting action of xylamine. Xylamine treatment resulted in a loss of approximately 60% of the control level of norepinephrine in the cerebral cortex. A 1-hr pretreatment, but not a 24-hr pretreatment, with the monoamine oxidase B inhibitor, L-deprenyl, prevented the depletion of norepinephrine by xylamine. In addition, pretreatment with MDL 72,974, a monoamine oxidase B inhibitor without amine-releasing or uptake - inhibiting effects, did not prevent cortical norepinephrine levels. Inhibition of monoamine oxidase by either MDL 72,974 or MDL 72,394 did not prevent the inhibition of [ 3 H]norepinephrine uptake into rat cortical synaptosomes by xylamine. These data indicate that monoamine oxidase does not mediate the amine-releasing or uptake inhibiting properties of xylamine. The protection afforded by L-deprenyl following a 1-hr pretreatment most probably was due to accumulation of its metabolite, L-amphetamine, which would inhibit the uptake carrier. A functional carrier is required for depletion since desipramine administered 1 hr prior to xylamine, was also able to prevent depletion of norepinephrine

  20. Multipotent cholinesterase/monoamine oxidase inhibitors for the treatment of Alzheimer's disease: design, synthesis, biochemical evaluation, ADMET, molecular modeling, and QSAR analysis of novel donepezil-pyridyl hybrids.

    Science.gov (United States)

    Bautista-Aguilera, Oscar M; Esteban, Gerard; Chioua, Mourad; Nikolic, Katarina; Agbaba, Danica; Moraleda, Ignacio; Iriepa, Isabel; Soriano, Elena; Samadi, Abdelouahid; Unzeta, Mercedes; Marco-Contelles, José

    2014-01-01

    The design, synthesis, and biochemical evaluation of donepezil-pyridyl hybrids (DPHs) as multipotent cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors for the potential treatment of Alzheimer's disease (AD) is reported. The 3D-quantitative structure-activity relationship study was used to define 3D-pharmacophores for inhibition of MAO A/B, acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) enzymes and to design DPHs as novel multi-target drug candidates with potential impact in the therapy of AD. DPH14 (Electrophorus electricus AChE [EeAChE]: half maximal inhibitory concentration [IC50] =1.1±0.3 nM; equine butyrylcholinesterase [eqBuChE]: IC50 =600±80 nM) was 318-fold more potent for the inhibition of AChE, and 1.3-fold less potent for the inhibition of BuChE than the reference compound ASS234. DPH14 is a potent human recombinant BuChE (hBuChE) inhibitor, in the same range as DPH12 or DPH16, but 13.1-fold less potent than DPH15 for the inhibition of human recombinant AChE (hAChE). Compared with donepezil, DPH14 is almost equipotent for the inhibition of hAChE, and 8.8-fold more potent for hBuChE. Concerning human monoamine oxidase (hMAO) A inhibition, only DPH9 and 5 proved active, compound DPH9 being the most potent (IC50 [MAO A] =5,700±2,100 nM). For hMAO B, only DPHs 13 and 14 were moderate inhibitors, and compound DPH14 was the most potent (IC50 [MAO B] =3,950±940 nM). Molecular modeling of inhibitor DPH14 within EeAChE showed a binding mode with an extended conformation, interacting simultaneously with both catalytic and peripheral sites of EeAChE thanks to a linker of appropriate length. Absortion, distribution, metabolism, excretion and toxicity analysis showed that structures lacking phenyl-substituent show better druglikeness profiles; in particular, DPHs13-15 showed the most suitable absortion, distribution, metabolism, excretion and toxicity properties. Novel donepezil-pyridyl hybrid DPH14 is a potent, moderately selective h

  1. On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase.

    Directory of Open Access Journals (Sweden)

    Patrick Schaub

    Full Text Available CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40 hydrocarbon substrate.

  2. Structure-Based Design and Optimization of Multitarget-Directed 2H-Chromen-2-one Derivatives as Potent Inhibitors of Monoamine Oxidase B and Cholinesterases.

    Science.gov (United States)

    Farina, Roberta; Pisani, Leonardo; Catto, Marco; Nicolotti, Orazio; Gadaleta, Domenico; Denora, Nunzio; Soto-Otero, Ramon; Mendez-Alvarez, Estefania; Passos, Carolina S; Muncipinto, Giovanni; Altomare, Cosimo D; Nurisso, Alessandra; Carrupt, Pierre-Alain; Carotti, Angelo

    2015-07-23

    The multifactorial nature of Alzheimer's disease calls for the development of multitarget agents addressing key pathogenic processes. To this end, by following a docking-assisted hybridization strategy, a number of aminocoumarins were designed, prepared, and tested as monoamine oxidases (MAOs) and acetyl- and butyryl-cholinesterase (AChE and BChE) inhibitors. Highly flexible N-benzyl-N-alkyloxy coumarins 2-12 showed good inhibitory activities at MAO-B, AChE, and BChE but low selectivity. More rigid inhibitors, bearing meta- and para-xylyl linkers, displayed good inhibitory activities and high MAO-B selectivity. Compounds 21, 24, 37, and 39, the last two featuring an improved hydrophilic/lipophilic balance, exhibited excellent activity profiles with nanomolar inhibitory potency toward hMAO-B, high hMAO-B over hMAO-A selectivity and submicromolar potency at hAChE. Cell-based assays of BBB permeation, neurotoxicity, and neuroprotection supported the potential of compound 37 as a BBB-permeant neuroprotective agent against H2O2-induced oxidative stress with poor interaction as P-gp substrate and very low cytotoxicity.

  3. Quinolinic Carboxylic Acid Derivatives as Potential Multi-target Compounds for Neurodegeneration: Monoamine Oxidase and Cholinesterase Inhibition.

    Science.gov (United States)

    Khan, Nehal A; Khan, Imtiaz; Abid, Syed M A; Zaib, Sumera; Ibrar, Aliya; Andleeb, Hina; Hameed, Shahid; Iqbal, Jamshed

    2018-01-01

    Parkinson's disease (PD), a debilitating and progressive disorder, is among the most challenging and devastating neurodegenerative diseases predominantly affecting the people over 60 years of age. To confront PD, an advanced and operational strategy is to design single chemical functionality able to control more than one target instantaneously. In this endeavor, for the exploration of new and efficient inhibitors of Parkinson's disease, we synthesized a series of quinoline carboxylic acids (3a-j) and evaluated their in vitro monoamine oxidase and cholinesterase inhibitory activities. The molecular docking and in silico studies of the most potent inhibitors were performed to identify the probable binding modes in the active site of the monoamine oxidase enzymes. Moreover, molecular properties were calculated to evaluate the druglikeness of the compounds. The biological evaluation results revealed that the tested compounds were highly potent against monoamine oxidase (A & B), 3c targeted both the isoforms of MAO with IC50 values of 0.51 ± 0.12 and 0.51 ± 0.03 µM, respectively. The tested compounds also demonstrated high and completely selective inhibitory action against acetylcholinesterase (AChE) with IC50 values ranging from 4.36 to 89.24 µM. Among the examined derivatives, 3i was recognized as the most potent inhibitor of AChE with an IC50 value of 4.36 ± 0.12 ±µM. The compounds appear to be promising inhibitors and could be used for the future development of drugs targeting neurodegenerative disorders. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. Sildenafil promotes eNOS activation and inhibits NADPH oxidase in the transgenic sickle cell mouse penis.

    Science.gov (United States)

    Musicki, Biljana; Bivalacqua, Trinity J; Champion, Hunter C; Burnett, Arthur L

    2014-02-01

    Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Continuous treatment with sildenafil reversed (P penis. Sildenafil treatment of WT mice did not affect any of these parameters. Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. © 2013 International Society for Sexual Medicine.

  5. Modification of the effects of 5-methoxy-N,N-dimethyltryptamine on exploratory behavior in rats by monoamine oxidase inhibitors

    Science.gov (United States)

    Halberstadt, Adam L.; Buell, Mahalah R.; Masten, Virginia L.; Risbrough, Victoria B.; Geyer, Mark A.

    2010-01-01

    RATIONALE The hallucinogenic tea known as ayahuasca is made from a combination of psychoactive plants that contribute the active components N,N-dimethyltryptamine (DMT) and 5-methoxy-DMT (5-MeO-DMT), as well as the monoamine oxidase (MAO) inhibitors (MAOIs) harmine and harmaline for oral activity. OBJECTIVE The present study examined the effects of 5-MeO-DMT in combination with MAOIs in rats using the Behavioral Pattern Monitor (BPM), which enables analyses of patterns of locomotor activity and exploration. Interaction studies using the serotonin (5-HT)1A antagonist WAY-100635 (1.0 mg/kg) and the 5-HT2A antagonist MDL 11,939 (1.0 mg/kg) were also performed to assess the respective contributions of these receptors to the behavioral effects of 5-MeO-DMT in MAOI-treated animals. RESULTS 5-MeO-DMT (0.01, 0.1, and 1.0 mg/kg) decreased locomotor activity and investigatory behavior. In rats pretreated with a behaviorally inactive dose of harmaline (0.1 mg/kg), 1.0 mg/kg 5-MeO-DMT had biphasic effects on locomotor activity, initially reducing locomotion and then increasing activity as time progressed. The ability of harmaline to shift 5-MeO-DMT to a biphasic locomotor pattern was shared by the selective MAOA inhibitor clorgyline, whereas the selective MAOB inhibitor (−)-deprenyl was ineffective. The late hyperactivity induced by the combination of 1.0 mg/kg 5-MeO-DMT and 0.3 mg/kg clorgyline was blocked by pretreatment with MDL 11,939. Pretreatment with WAY-100635 failed to attenuate either the early hypoactivity or the late hyperactivity. CONCLUSIONS The ability of harmaline to modify the behavioral effects of 5-MeO-DMT is mediated by inhibition of MAOA. Further, 5-HT2A receptors are responsible for the late hyperactivity induced by 5-MeO-DMT in the presence of MAOA inhibitors. PMID:18604652

  6. New pyrrole inhibitors of monoamine oxidase: synthesis, biological evaluation, and structural determinants of MAO-A and MAO-B selectivity.

    Science.gov (United States)

    La Regina, Giuseppe; Silvestri, Romano; Artico, Marino; Lavecchia, Antonio; Novellino, Ettore; Befani, Olivia; Turini, Paola; Agostinelli, Enzo

    2007-03-08

    A series of new pyrrole derivatives have been synthesized and evaluated for their monoamine oxidase (MAO) A and B inhibitory activity and selectivity. N-Methyl,N-(benzyl),N-(pyrrol-2-ylmethyl)amine (7) and N-(2-benzyl),N-(1-methylpyrrol-2-ylmethyl)amine (18) were the most selective MAO-B (7, SI = 0.0057) and MAO-A (18, SI = 12500) inhibitors, respectively. Docking and molecular dynamics simulations gave structural insights into the MAO-A and MAO-B selectivity. Compound 18 forms an H-bond with Gln215 through its protonated amino group into the MAO-A binding site. This H-bond is absent in the 7/MAO-A complex. In contrast, compound 7 places its phenyl ring into an aromatic cage of the MAO-B binding pocket, where it forms charge-transfer interactions. The slightly different binding pose of 18 into the MAO-B active site seems to be forced by a bulkier Tyr residue, which replaces a smaller Ile residue present in MAO-A.

  7. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase.

    Science.gov (United States)

    White, Caroline N; Figtree, Gemma A; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Rasmussen, Helge H

    2009-04-01

    The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

  8. Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites.

    Science.gov (United States)

    Kallio, A; Jänne, J

    1983-01-01

    Treatment of mice bearing L1210 leukaemia with 2-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a profound depletion of putrescine and spermidine in the tumour cells. Sequential combination of methylglyoxal bis(guanylhydrazone), an inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), with difluoromethylornithine largely reversed the polyamine depletion and led to a marked accumulation of cadaverine in the tumour cells. Experiments carried out with the combination of difluoromethylornithine and aminoguanidine, a potent inhibitor of diamine oxidase (EC 1.4.3.6), indicated that the methylglyoxal bis(guanylhydrazone)-induced reversal of polyamine depletion was mediated by the known inhibition of diamine oxidase by the diguanidine. In spite of the normalization of the tumour cell polyamine pattern upon administration of methylglyoxal bis(guanylhydrazone) to difluoromethylornithine-treated animals, the combination of these two drugs produced a growth-inhibitory effect not achievable with either of the compounds alone. PMID:6411077

  9. Reversible Inhibitors of Monoamine Oxidase-A (RIMAs): Robust, Reversible Inhibition of Human Brain MAO-A by CX157

    Science.gov (United States)

    Fowler, Joanna S; Logan, Jean; Azzaro, Albert J; Fielding, Robert M; Zhu, Wei; Poshusta, Amy K; Burch, Daniel; Brand, Barry; Free, James; Asgharnejad, Mahnaz; Wang, Gene-Jack; Telang, Frank; Hubbard, Barbara; Jayne, Millard; King, Payton; Carter, Pauline; Carter, Scott; Xu, Youwen; Shea, Colleen; Muench, Lisa; Alexoff, David; Shumay, Elena; Schueller, Michael; Warner, Donald; Apelskog-Torres, Karen

    2010-01-01

    Reversible inhibitors of monoamine oxidase-A (RIMA) inhibit the breakdown of three major neurotransmitters, serotonin, norepinephrine and dopamine, offering a multi-neurotransmitter strategy for the treatment of depression. CX157 (3-fluoro-7-(2,2,2-trifluoroethoxy)phenoxathiin-10,10-dioxide) is a RIMA, which is currently in development for the treatment of major depressive disorder. We examined the degree and reversibility of the inhibition of brain monoamine oxidase-A (MAO-A) and plasma CX157 levels at different times after oral dosing to establish a dosing paradigm for future clinical efficacy studies, and to determine whether plasma CX157 levels reflect the degree of brain MAO-A inhibition. Brain MAO-A levels were measured with positron emission tomography (PET) imaging and [11C]clorgyline in 15 normal men after oral dosing of CX157 (20–80 mg). PET imaging was conducted after single and repeated doses of CX157 over a 24-h time course. We found that 60 and 80 mg doses of CX157 produced a robust dose-related inhibition (47–72%) of [11C]clorgyline binding to brain MAO-A at 2 h after administration and that brain MAO-A recovered completely by 24 h post drug. Plasma CX157 concentration was highly correlated with the inhibition of brain MAO-A (EC50: 19.3 ng/ml). Thus, CX157 is the first agent in the RIMA class with documented reversible inhibition of human brain MAO-A, supporting its classification as a RIMA, and the first RIMA with observed plasma levels that can serve as a biomarker for the degree of brain MAO-A inhibition. These data were used to establish the dosing regimen for a current clinical efficacy trial with CX157. PMID:19890267

  10. The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

    Science.gov (United States)

    Morrison, Ryan D.; Blobaum, Anna L.; Byers, Frank W.; Santomango, Tammy S.; Bridges, Thomas M.; Stec, Donald; Brewer, Katrina A.; Sanchez-Ponce, Raymundo; Corlew, Melany M.; Rush, Roger; Felts, Andrew S.; Manka, Jason; Bates, Brittney S.; Venable, Daryl F.; Rodriguez, Alice L.; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

    2012-01-01

    Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates. PMID:22711749

  11. An Updated Review of Tyrosinase Inhibitors

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    Te-Sheng Chang

    2009-05-01

    Full Text Available Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase, which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors newly discovered from natural and synthetic sources. The inhibitory strength is compared with that of a standard inhibitor, kojic acid, and their inhibitory mechanisms are discussed.

  12. Red wine contains a potent inhibitor of phenolsulphotransferase.

    Science.gov (United States)

    Littlewood, J T; Glover, V; Sandler, M

    1985-01-01

    Many ethanolic drinks, especially red wine, contain potent inhibitors of phenolsulphotransferase. At a dilution of 1/75 from the original beverage, extracts from six types of red wine inhibited human platelet phenolsulphotransferase P by a mean of 99% and human platelet phenolsulphotransferase M by 12%. Such extracts had no significant effect on rat liver monoamine oxidase A or human platelet monoamine oxidase B. The inhibitors, which have not yet been identified, can be extracted into ethyl acetate at acid or neutral pH. Thus, they are not monoamines. Flavonoid phenols are plausible candidates. As phenolsulphotransferase M and P are involved in the metabolism of many phenols, including drugs, the inhibition of these enzymes could result in the enhancement of pharmacological potency and have important clinical consequences. PMID:3857069

  13. Biophysical and physicochemical methods differentiate highly ligand-efficient human D-amino acid oxidase inhibitors.

    Science.gov (United States)

    Lange, Jos H M; Venhorst, Jennifer; van Dongen, Maria J P; Frankena, Jurjen; Bassissi, Firas; de Bruin, Natasja M W J; den Besten, Cathaline; de Beer, Stephanie B A; Oostenbrink, Chris; Markova, Natalia; Kruse, Chris G

    2011-10-01

    Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target-ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1-6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target-ligand residence times for 5 and 6 as compared to 1-4, based on the observed k(off) values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1-6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  14. Will the Amaranthus tuberculatus Resistance Mechanism to PPO-Inhibiting Herbicides Evolve in Other Amaranthus Species?

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    Chance W. Riggins

    2012-01-01

    Full Text Available Resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO has been slow to evolve and, to date, is confirmed for only four weed species. Two of these species are members of the genus Amaranthus L. Previous research has demonstrated that PPO-inhibitor resistance in A. tuberculatus (Moq. Sauer, the first weed to have evolved this type of resistance, involves a unique codon deletion in the PPX2 gene. Our hypothesis is that A. tuberculatus may have been predisposed to evolving this resistance mechanism due to the presence of a repetitive motif at the mutation site and that lack of this motif in other amaranth species is why PPO-inhibitor resistance has not become more common despite strong herbicide selection pressure. Here we investigate inter- and intraspecific variability of the PPX2 gene—specifically exon 9, which includes the mutation site—in ten amaranth species via sequencing and a PCR-RFLP assay. Few polymorphisms were observed in this region of the gene, and intraspecific variation was observed only in A. quitensis. However, sequencing revealed two distinct repeat patterns encompassing the mutation site. Most notably, A. palmeri S. Watson possesses the same repetitive motif found in A. tuberculatus. We thus predict that A. palmeri will evolve resistance to PPO inhibitors via the same PPX2 codon deletion that evolved in A. tuberculatus.

  15. Genome wide association mapping for the tolerance to the polyamine oxidase inhibitor guazatine in Arabidopsis thaliana

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    Kostadin Evgeniev eAtanasov

    2016-04-01

    Full Text Available Guazatine is a potent inhibitor of polyamine oxidase (PAO activity. In agriculture, guazatine is used as non-systemic contact fungicide efficient in the protection of cereals and citrus fruits against disease. The composition of guazatine is complex, mainly constituted by a mixture of synthetic guanidated polyamines (polyaminoguanidines. Here we have studied the effects from exposure to guazatine in the weed Arabidopsis thaliana. We report that micromolar concentrations of guazatine are sufficient to inhibit growth of Arabidopsis seedlings and induce chlorosis, whereas germination is barely affected. We observed the occurrence of quantitative variation in the response to guazatine between 107 randomly chosen Arabidopsis accessions. This enabled us to undertake genome-wide association (GWA mapping that identified a locus on chromosome one associated with guazatine tolerance. CHLOROPHYLLASE 1 (CLH1 within this locus was studied as candidate gene, together with its paralog (CLH2. The analysis of independent clh1-2, clh1-3, clh2-3, clh2-2 and double clh1-2 clh2-3 mutant alleles indicated that CLH1 and/or CLH2 loss-of-function or expression down-regulation promote guazatine tolerance in Arabidopsis. We report a natural mechanism by which Arabidopsis populations can overcome toxicity by the fungicide guazatine.

  16. Molecular mechanism of the relation of monoamine oxidase B and its inhibitors to Parkinson's disease: possible implications of glial cells.

    Science.gov (United States)

    Nagatsu, T; Sawada, M

    2006-01-01

    Monoamine oxidases A and B (MAO A and MAO B) are the major enzymes that catalyze the oxidative deamination of monoamine neurotaransmitters such as dopamine (DA), noradrenaline, and serotonin in the central and peripheral nervous systems. MAO B is mainly localized in glial cells. MAO B also oxidizes the xenobiotic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to a parkinsonism-producing neurotoxin, 1-methyl-4-phenyl-pyridinium (MPP+). MAO B may be closely related to the pathogenesis of Parkinson's disease (PD), in which neuromelanin-containing DA neurons in the substantia nigra projecting to the striatum in the brain selectively degenerate. MAO B degrades the neurotransmitter DA that is deficient in the nigro-striatal region in PD, and forms H2O2 and toxic aldehyde metabolites of DA. H2O2 produces highly toxic reactive oxygen species (ROS) by Fenton reaction that is catalyzed by iron and neuromelanin. MAO B inhibitors such as L-(-)-deprenyl (selegiline) and rasagiline are effective for the treatment of PD. Concerning the mechanism of the clinical efficacy of MAO B inhibitors in PD, the inhibition of DA degradation (a symptomatic effect) and also the prevention of the formation of neurotoxic DA metabolites, i.e., ROS and dopamine derived aldehydes have been speculated. As another mechanism of clinical efficacy, MAO B inhibitors such as selegiline are speculated to have neuroprotective effects to prevent progress of PD. The possible mechanism of neuroprotection of MAO B inhibitors may be related not only to MAO B inhibition but also to induction and activation of multiple factors for anti-oxidative stress and anti-apoptosis: i.e., catalase, superoxide dismutase 1 and 2, thioredoxin, Bcl-2, the cellular poly(ADP-ribosyl)ation, and binding to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Furthermore, it should be noted that selegiline increases production of neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial

  17. In vitro oxidation of indoleacetic acid by soluble auxin-oxidases and peroxidases from maize roots

    International Nuclear Information System (INIS)

    Beffa, R.; Martin, H.V.; Pilet, P.E.

    1990-01-01

    Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl 2 and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [ 3 H]indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol

  18. Effects of phenylated compounds of methylglyoxal bis(guanylhydrazone) on diamine oxidase activity from rat small intestine.

    Science.gov (United States)

    Balaña-Fouce, R; Pulido, T G; Escudero, D O; Sanz-Sanchez, F

    1986-01-01

    Two phenylated compounds of methylglyoxal bis(guanylhydrazone), potentially inhibitors of diamine oxidase activity, have been synthesized: phenylglyoxal bis(guanylhydrazone) and diphenylglyoxal bis(guanylhydrazone). Their inhibitory capacity was tested: while PGBG was able to reduce the enzyme activity by 50% at 1.3 microM, DPGBG was only able to reduce diamine oxidase activity by less than 2% at a concentration 1000-fold higher. The inhibition of PGBG was non-competitive and the Ki calculated by a Dixon plot was estimated as 1.7 microM.

  19. A Plastid Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast Differentiation1

    Science.gov (United States)

    Josse, Eve-Marie; Simkin, Andrew J.; Gaffé, Joël; Labouré, Anne-Marie; Kuntz, Marcel; Carol, Pierre

    2000-01-01

    The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development. PMID:10938359

  20. Critical role of NADPH oxidase in neuronal oxidative damage and microglia activation following traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Quan-Guang Zhang

    Full Text Available BACKGROUND: Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O(2(-, and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI. METHODOLOGY/PRINCIPAL FINDINGS: The results revealed that NADPH oxidase activity in the cerebral cortex and hippocampal CA1 region increases rapidly following controlled cortical impact in male mice, with an early peak at 1 h, followed by a secondary peak from 24-96 h after TBI. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, revealed that the O(2(- induction occurred in neurons at 1 h after TBI. Pre- or post-treatment with the NADPH oxidase inhibitor, apocynin markedly inhibited microglial activation and oxidative stress damage. Apocynin also attenuated TBI-induction of the Alzheimer's disease proteins β-amyloid and amyloid precursor protein. Finally, both pre- and post-treatment of apocynin was also shown to induce significant neuroprotection against TBI. In addition, a NOX2-specific inhibitor, gp91ds-tat was also shown to exert neuroprotection against TBI. CONCLUSIONS/SIGNIFICANCE: As a whole, the study demonstrates that NADPH oxidase activity and superoxide production exhibit a biphasic elevation in the hippocampus and cortex following TBI, which contributes significantly to the pathology of TBI via mediation of oxidative stress damage, microglial activation, and AD protein induction in the brain following TBI.

  1. NADPH oxidase is involved in regulation of gene expression and ROS overproduction in soybean (Glycine max L. seedlings exposed to cadmium

    Directory of Open Access Journals (Sweden)

    Jagna Chmielowska-Bąk

    2017-06-01

    Full Text Available Cadmium-induced oxidative burst is partially mediated by NADPH oxidase. The aim of the present research was to evaluate the role of NADPH oxidase in soybeans’ response to short-term cadmium stress. The application of an NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI, affected expression of two Cd-inducible genes, encoding DOF1 and MYBZ2 transcription factors. This effect was observed after 3 h of treatment. Interestingly, Cd-dependent increases in NADPH oxidase activity occurred only after a period of time ranging from 6 and 24 h of stress. Stimulation of the enzyme correlated in time with a significant accumulation of reactive oxygen species (ROS. Further analysis revealed that pharmacological inhibition of NADPH oxidase activity during 24 h of Cd stress does not affect Cd uptake, seedling growth, or the level of lipid peroxidation. The role of NADPH oxidase in the response of soybean seedlings to short-term Cd exposure is discussed.

  2. Rasagiline: A second-generation monoamine oxidase type-B inhibitor for the treatment of Parkinson's disease.

    Science.gov (United States)

    Chen, Jack J; Ly, Anh-Vuong

    2006-05-15

    The pharmacology, pharmacokinetics, clinical efficacy, and safety of rasagiline are reviewed. Rasagiline is a novel, investigational propargylamine that irreversibly and selectively inhibits monoamine oxidase type B (MAO-B). Rasagiline demonstrates complete and selective inhibition of MAO-B and is at least five times more potent than selegiline. Unlike selegiline, which is metabolized to amphetamine derivatives, rasagiline is biotransformed to the nonamphetamine compound aminoindan. Clinical studies have revealed that rasagiline is associated with improved outcomes in patients with early Parkinson's disease (PD) and also reduces "off" time in patients with moderate to advanced PD with motor fluctuations. Rasagiline is rapidly absorbed by the gastrointestinal tract and readily crosses the blood-brain barrier. The optimal therapeutic dosage is 0.5-1 mg administered orally once daily. Rasagiline appears to be well tolerated, although elderly patients may be more prone to treatment-emergent adverse cardiovascular and psychiatric effects. At the recommended therapeutic dosage of up to 1 mg once daily, tyramine restriction is unnecessary. In addition to MAO-B inhibition, rasagiline has demonstrated neuroprotective properties in experimental laboratory models. The mechanisms whereby rasagiline exerts neuroprotective effects are multifactorial and include upregulation of cellular antioxidant activity and antiapoptotic factors. Rasagiline is an investigational selective and irreversible inhibitor of MAO-B that has demonstrated efficacy and safety for the treatment of PD. Whether rasagiline is associated with clinically significant neuroprotection is the subject of ongoing clinical trials.

  3. Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

    Science.gov (United States)

    Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

    2012-08-01

    Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (Ppenis. Apocynin treatment of sickle mice reversed (P0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. © 2012 International Society for Sexual Medicine.

  4. Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

    Science.gov (United States)

    Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

    2013-09-01

    Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

  5. Subnanomolar indazole-5-carboxamide inhibitors of monoamine oxidase B (MAO-B) continued: indications of iron binding, experimental evidence for optimised solubility and brain penetration.

    Science.gov (United States)

    Tzvetkov, Nikolay T; Antonov, Liudmil

    2017-12-01

    Pharmacological and physicochemical studies of N-unsubstituted indazole-5-carboxamides (subclass I) and their structurally optimised N1-methylated analogues (subclass II), initially developed as drug and radioligand candidates for the treatment and diagnosis of Parkinson's disease (PD), are presented. The compounds are highly brain permeable, selective, reversible, and competitive monoamine oxidase B (MAO-B) inhibitors with improved water-solubility and subnanomolar potency (pIC 50  >8.8). Using a well-validated, combined X-ray/modelling technology platform, we performed a semi-quantitative analysis of the binding modes of all compounds and investigated the role of the indazole N1 position for their MAO-B inhibitory activity. Moreover, compounds NTZ-1006, 1032, and 1441 were investigated for their ability to bind Fe 2+ and Fe 3+ ions using UV-visible spectroscopy.

  6. Add-on treatment of benzoate for schizophrenia: a randomized, double-blind, placebo-controlled trial of D-amino acid oxidase inhibitor.

    Science.gov (United States)

    Lane, Hsien-Yuan; Lin, Ching-Hua; Green, Michael F; Hellemann, Gerhard; Huang, Chih-Chia; Chen, Po-Wei; Tun, Rene; Chang, Yue-Cung; Tsai, Guochuan E

    2013-12-01

    In addition to dopaminergic hyperactivity, hypofunction of the N-methyl-d-aspartate receptor (NMDAR) has an important role in the pathophysiology of schizophrenia. Enhancing NMDAR-mediated neurotransmission is considered a novel treatment approach. To date, several trials on adjuvant NMDA-enhancing agents have revealed beneficial, but limited, efficacy for positive and negative symptoms and cognition. Another method to enhance NMDA function is to raise the levels of d-amino acids by blocking their metabolism. Sodium benzoate is a d-amino acid oxidase inhibitor. To examine the clinical and cognitive efficacy and safety of add-on treatment of sodium benzoate for schizophrenia. A randomized, double-blind, placebo-controlled trial in 2 major medical centers in Taiwan composed of 52 patients with chronic schizophrenia who had been stabilized with antipsychotic medications for 3 months or longer. Six weeks of add-on treatment of 1 g/d of sodium benzoate or placebo. The primary outcome measure was the Positive and Negative Syndrome Scale (PANSS) total score. Clinical efficacy and adverse effects were assessed biweekly. Cognitive functions were measured before and after the add-on treatment. Benzoate produced a 21% improvement in PANSS total score and large effect sizes (range, 1.16-1.69) in the PANSS total and subscales, Scales for the Assessment of Negative Symptoms-20 items, Global Assessment of Function, Quality of Life Scale and Clinical Global Impression and improvement in the neurocognition subtests as recommended by the National Institute of Mental Health's Measurement and Treatment Research to Improve Cognition in Schizophrenia initiative, including the domains of processing speed and visual learning. Benzoate was well tolerated without significant adverse effects. Benzoate adjunctive therapy significantly improved a variety of symptom domains and neurocognition in patients with chronic schizophrenia. The preliminary results show promise for d-amino acid oxidase

  7. Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.).

    Science.gov (United States)

    Rahman, Andi Nur Faidah; Ohta, Mayumi; Nakatani, Kazuya; Hayashi, Nobuyuki; Fujita, Shuji

    2012-04-11

    Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).

  8. A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin

    Energy Technology Data Exchange (ETDEWEB)

    Budayova-Spano, Monika, E-mail: spano@embl-grenoble.fr [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble (France); Bonneté, Françoise; Ferté, Natalie [Centre de Recherche en Matière Condensée et Nanosciences, Campus de Luminy, Case 913, 13288 Marseille (France); El Hajji, Mohamed [Sanofi-Aventis, 371 Rue du Professeur Blayac, 34184 Montpellier (France); Meilleur, Flora [Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble (France); Blakeley, Matthew Paul [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Castro, Bertrand [Sanofi-Aventis, 371 Rue du Professeur Blayac, 34184 Montpellier (France); European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France)

    2006-03-01

    Neutron diffraction data of hydrogenated recombinant urate oxidase enzyme (Rasburicase), complexed with a purine-type inhibitor 8-azaxanthin, was collected to 2.1 Å resolution from a crystal grown in D{sub 2}O by careful control and optimization of crystallization conditions via knowledge of the phase diagram. Deuterium atoms were clearly seen in the neutron-scattering density map. Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 Å resolution using the LADI instrument from a crystal (grown in D{sub 2}O) with volume 1.8 mm{sup 3}. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 Å) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.

  9. Effect of NADPH oxidase inhibitor-apocynin on the expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 exposed renal ischemia/reperfusion injury in rats

    Directory of Open Access Journals (Sweden)

    Zhiming Li

    2015-01-01

    Full Text Available This study was designed to evaluate whether NADPH oxidase inhibitor (apocynin preconditioning induces expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 to protect against renal ischemia/reperfusion (I/R injury (RI/RI in rats. Rats were pretreated with 50 mg/kg apocynin, then subjected to 45 min ischemia and 24 h reperfusion. The results indicated that apocynin preconditioning improved the recovery of renal function and nitroso-redox balance, reduced oxidative stress injury and inflammation damage, and upregulated expression of SHP-1 as compared to RI/RI group. Therefore our study demonstrated that apocynin preconditioning provided a protection to the kidney against I/R injury in rats partially through inducing expression of SHP-1.

  10. Mitochondrial terminal alternative oxidase and its enhancement by thermal stress in the coral symbiont Symbiodinium

    Science.gov (United States)

    Oakley, Clinton A.; Hopkinson, Brian M.; Schmidt, Gregory W.

    2014-06-01

    A terminal electron acceptor alternative to mitochondrial cytochrome c oxidase (COX), mitochondrial alternative oxidase (AOX), is ubiquitous in higher plants and represented in nearly every algal taxon but is poorly documented in dinoflagellates. AOX competes for electrons with the conventional COX and has been hypothesized to function as a means of reducing oxidative stress in mitochondria, as well as a potential mechanism for ameliorating thermal and other physiological stressors. Here, the presence of an active AOX in cultured Symbiodinium was assayed by the response of oxygen consumption to the AOX inhibitor salicylhydroxamic acid (SHAM) and the COX inhibitor cyanide (CN). CN-insensitive, SHAM-sensitive oxygen consumption was found to account for a large portion (26 %) of Symbiodinium dark respiration and is consistent with high levels of AOX activity. This experimental evidence of the existence of a previously unreported terminal oxidase was further corroborated by analysis of publicly available Symbiodinium transcriptome data. The potential for enhanced AOX expression to play a compensatory role in mediating thermal stress was supported by inhibitor assays of cultured Symbiodinium at low (18 °C), moderate (26 °C), and high (32 °C) temperature conditions. Maximum capacity of the putative AOX pathway as a proportion of total dark oxygen consumption was found to increase from 26 % at 26 °C to 45 % and 53 % at 18 °C and 32 °C, respectively, when cells were acclimated to the treatment temperatures. Cells assayed at 18 and 32 °C without acclimation exhibited either the same or lower AOX capacity as controls, suggesting that the AOX protein is upregulated under temperature stress. The physiological implications for the presence of AOX in the coral/algal symbiosis and its potential role in response to many forms of biotic and abiotic stress, particularly oxidative stress, are discussed.

  11. Paraquat and maneb co-exposure induces noradrenergic locus coeruleus neurodegeneration through NADPH oxidase-mediated microglial activation

    International Nuclear Information System (INIS)

    Hou, Liyan; Zhang, Cong; Wang, Ke; Liu, Xiaofang; Wang, Hongwei; Che, Yuning; Sun, Fuqiang; Zhou, Xueying; Zhao, Xiulan; Wang, Qingshan

    2017-01-01

    Highlights: • Microglial activation induced by paraquat and maneb precedes noradrenergic neurodegeneration in locus coeruleus. • NADPH oxidase activation contributes to microglia-mediated neuroinflammation and related noradrenergic neurodegeneration. • Inhibition of NADPH oxidase by apocynin protects noradrenergic neurons against paraquat and maneb-induced toxicity. - Abstract: Co-exposure to paraquat (PQ) and maneb (Mb) has been shown to increase the risk of Parkinson’s disease (PD) and dopaminergic (DA) neurodegeneration in the substantia nigra pars compacta (SNpc) is observed in PQ and Mb-treated experimental animals. The loss of noradrenergic locus coeruleus (LC/NE) neurons in brainstem is a common feature shared by multiple neurodegenerative diseases, including PD. However, whether PQ and Mb is able to damage LC/NE neurons remains undefined. In this study, mice treated with combined PQ and Mb displayed progressive LC/NE neurodegeneration. Time course studies revealed that the activation of microglia preceded LC/NE neurodegeneration. Mechanistically, the activation of NADPH oxidase contributed to microglial activation and subsequent LC/NE neurodegeneration. We found that PQ and Mb co-exposure induced activation of NADPH oxidase as shown by increased superoxide production and membrane translocation of p47 phox , a cytosolic subunit of NADPH oxidase. Inhibition of NADPH oxidase by apocynin, a widely used NADPH oxidase inhibitor, suppressed microglial activation and gene expressions of proinflammatory factors. Furthermore, reduced activation of nuclear factor-κB (NF-κB) pathway was observed in apocynin-treated mice. More importantly, inhibition of NADPH oxidase by apocynin afforded LC/NE neuroprotection against PQ and Mb-induced neurotoxicity. Thus, our findings revealed the critical role NADPH oxidase-mediated microglial activation in driving LC/NE neurodegeneration induced by PQ and Mb, providing new insights into the pathogenesis of environmental

  12. Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study

    Czech Academy of Sciences Publication Activity Database

    Kopečný, D.; Briozzo, P.; Popelková, H.; Šebela, M.; Končitíková, R.; Spíchal, Lukáš; Nisler, Jaroslav; Madzak, C.; Frébort, Ivo; Laloue, M.; Houba-Herin, N.

    2010-01-01

    Roč. 92, č. 8 (2010), s. 1052-1062 ISSN 0300-9084 R&D Projects: GA ČR GA522/08/0555; GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : benzylurea * crystal structure * cytokinin oxidase/dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 3.787, year: 2010

  13. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

    Science.gov (United States)

    Turrens, J F; Bickar, D; Lehninger, A L

    1986-06-01

    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

  14. The lysyl oxidase inhibitor β-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats

    Directory of Open Access Journals (Sweden)

    María Miana

    2015-06-01

    Full Text Available Extracellular matrix (ECM remodelling of the adipose tissue plays a pivotal role in the pathophysiology of obesity. The lysyl oxidase (LOX family of amine oxidases, including LOX and LOX-like (LOXL isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its involvement in adipose tissue dysfunction in obesity is unclear. In this study, we observed that LOX is the main isoenzyme expressed in human adipose tissue and that its expression is strongly upregulated in samples from obese individuals that had been referred to bariatric surgery. LOX expression was also induced in the adipose tissue from male Wistar rats fed a high-fat diet (HFD. Interestingly, treatment with β-aminopropionitrile (BAPN, a specific and irreversible inhibitor of LOX activity, attenuated the increase in body weight and fat mass that was observed in obese animals and shifted adipocyte size toward smaller adipocytes. BAPN also ameliorated the increase in collagen content that was observed in adipose tissue from obese animals and improved several metabolic parameters – it ameliorated glucose and insulin levels, decreased homeostasis model assessment (HOMA index and reduced plasma triglyceride levels. Furthermore, in white adipose tissue from obese animals, BAPN prevented the downregulation of adiponectin and glucose transporter 4 (GLUT4, as well as the increase in suppressor of cytokine signaling 3 (SOCS3 and dipeptidyl peptidase 4 (DPP4 levels, triggered by the HFD. Likewise, in the TNFα-induced insulin-resistant 3T3-L1 adipocyte model, BAPN prevented the downregulation of adiponectin and GLUT4 and the increase in SOCS3 levels, and consequently normalised insulin-stimulated glucose uptake. Therefore, our data provide evidence that LOX plays a pathologically relevant role in the metabolic dysfunction induced by obesity and emphasise the interest of novel pharmacological interventions that target adipose tissue fibrosis and LOX

  15. Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

    Science.gov (United States)

    Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

    2017-03-11

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Discovery of Monoamine Oxidase A Inhibitors Derived from in silico Docking

    International Nuclear Information System (INIS)

    Jo, Geunhyeong; Sung, Suhyun; Lee, Younggiu; Kim, Bonggyu; Yoon, Junwie; Lee, Hyeok; Ahn, Joonghoon; Lim, Yoongho; Ji, Sangyun; Koh, Dongsoo

    2012-01-01

    MAOA inhibitors (MAOAIs) have been used as antidepressants for over forty years. Iproniazid was introduced in 1957, but it was withdrawn because of hepatotoxicity. Tranylcypromine was developed in the mid-1960s, withdrawn from the market because of problems related to hypertension, then reintroduced for limited usage. Many MAOAIs have been developed and used for treating atypical depression after the failure of other classes of antidepressant drugs such as selective serotonin reuptake inhibitors and tricyclic antidepressants. While iproniazid and tranylcypromine were nonselective MAOAIs, a selective MAOAI, clorgyline, was introduced in the latter half of the 1960s. Recently, more selective and safe MAOAIs, namely moclobemide, toloxatone, and tetrindol, were launched. However, their side effects and activities need further improvement. Therefore, we have made efforts to discover new MAOAIs. In conclusion, even though the number of compounds tested here is not enough for evaluation, the current result demonstrates that phenylpyrazole moiety is not necessary for showing good inhibitory effects. Because benzoflavanones have not previously been reported to act on MAOA as inhibitors, and the inhibitory effect of one of benzo-flavones, 3-(4-methoxyphenyl)-2,3-dihydro-1H-benzo[ f ] chromen-1-one used in this study is comparable to that of clorgyline which is known as MAOA inhibitor,31 our findings are meaningful

  17. Discovery of Monoamine Oxidase A Inhibitors Derived from in silico Docking

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Geunhyeong; Sung, Suhyun; Lee, Younggiu; Kim, Bonggyu; Yoon, Junwie; Lee, Hyeok; Ahn, Joonghoon; Lim, Yoongho [Konkuk Univ., Seoul (Korea, Republic of); Ji, Sangyun [Rural Development Administration, Suwon (Korea, Republic of); Koh, Dongsoo [Dongduk Women' s Univ., Seoul (Korea, Republic of)

    2012-11-15

    MAOA inhibitors (MAOAIs) have been used as antidepressants for over forty years. Iproniazid was introduced in 1957, but it was withdrawn because of hepatotoxicity. Tranylcypromine was developed in the mid-1960s, withdrawn from the market because of problems related to hypertension, then reintroduced for limited usage. Many MAOAIs have been developed and used for treating atypical depression after the failure of other classes of antidepressant drugs such as selective serotonin reuptake inhibitors and tricyclic antidepressants. While iproniazid and tranylcypromine were nonselective MAOAIs, a selective MAOAI, clorgyline, was introduced in the latter half of the 1960s. Recently, more selective and safe MAOAIs, namely moclobemide, toloxatone, and tetrindol, were launched. However, their side effects and activities need further improvement. Therefore, we have made efforts to discover new MAOAIs. In conclusion, even though the number of compounds tested here is not enough for evaluation, the current result demonstrates that phenylpyrazole moiety is not necessary for showing good inhibitory effects. Because benzoflavanones have not previously been reported to act on MAOA as inhibitors, and the inhibitory effect of one of benzo-flavones, 3-(4-methoxyphenyl)-2,3-dihydro-1H-benzo[ f ] chromen-1-one used in this study is comparable to that of clorgyline which is known as MAOA inhibitor,31 our findings are meaningful.

  18. A Small Molecule Polyamine Oxidase Inhibitor Blocks Androgen-Induced Oxidative Stress and Delays Prostate Cancer Progression in the TRAMP Mouse Model

    Science.gov (United States)

    Basu, Hirak S.; Thompson, Todd A.; Church, Dawn R.; Clower, Cynthia C.; Mehraein-Ghomi, Farideh; Amlong, Corey A.; Martin, Christopher T.; Woster, Patrick M.; Lindstrom, Mary J.; Wilding, George

    2009-01-01

    High levels of reactive oxygen species (ROS) present in human prostate epithelia are an important etiological factor in prostate cancer (CaP) occurrence, recurrence and progression. Androgen induces ROS production in the prostate by a yet unknown mechanism. Here, to the best of our knowledge, we report for the first time that androgen induces an overexpression of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in the polyamine oxidation pathway. As prostatic epithelia produce a large excess of polyamines, the androgen-induced polyamine oxidation that produces H2O2 could be a major reason for the high ROS levels in the prostate epithelia. A small molecule polyamine oxidase inhibitor N,N'-butanedienyl butanediamine (MDL 72,527 or CPC-200) effectively blocks androgen-induced ROS production in human CaP cells as well as significantly delays CaP progression and death in animals developing spontaneous CaP. These data demonstrate that polyamine oxidation is not only a major pathway for ROS production in prostate, but inhibiting this pathway also successfully delays prostate cancer progression. PMID:19773450

  19. Molecular insights into human monoamine oxidase (MAO) inhibition by 1,4-naphthoquinone: evidences for menadione (vitamin K3) acting as a competitive and reversible inhibitor of MAO.

    Science.gov (United States)

    Coelho Cerqueira, Eduardo; Netz, Paulo Augusto; Diniz, Cristiane; Petry do Canto, Vanessa; Follmer, Cristian

    2011-12-15

    Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic and exogenous amines and its inhibitors have therapeutic value for several conditions including affective disorders, stroke, neurodegenerative diseases and aging. The discovery of 2,3,6-trimethyl-1,4-naphthoquinone (TMN) as a nonselective and reversible inhibitor of MAO, has suggested 1,4-naphthoquinone (1,4-NQ) as a potential scaffold for designing new MAO inhibitors. Combining molecular modeling tools and biochemical assays we evaluate the kinetic and molecular details of the inhibition of human MAO by 1,4-NQ, comparing it with TMN and menadione. Menadione (2-methyl-1,4-naphthoquinone) is a multitarget drug that acts as a precursor of vitamin K and an inducer of mitochondrial permeability transition. Herein we show that MAO-B was inhibited competitively by 1,4-NQ (K(i)=1.4 μM) whereas MAO-A was inhibited by non-competitive mechanism (K(i)=7.7 μM). Contrasting with TMN and 1,4-NQ, menadione exhibited a 60-fold selectivity for MAO-B (K(i)=0.4 μM) in comparison with MAO-A (K(i)=26 μM), which makes it as selective as rasagiline. Fluorescence and molecular modeling data indicated that these inhibitors interact with the flavin moiety at the active site of the enzyme. Additionally, docking studies suggest the phenyl side groups of Tyr407 and Tyr444 (for MAO-A) or Tyr398 and Tyr435 (for MAO-B) play an important role in the interaction of the enzyme with 1,4-NQ scaffold through forces of dispersion as verified for menadione, TMN and 1,4-NQ. Taken together, our findings reveal the molecular details of MAO inhibition by 1,4-NQ scaffold and show for the first time that menadione acts as a competitive and reversible inhibitor of human MAO. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Inhibitors of polyamine metabolism: review article.

    Science.gov (United States)

    Wallace, H M; Fraser, A V

    2004-07-01

    The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases. The early development of polyamine biosynthetic single enzyme inhibitors such as alpha-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer. The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N1-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.

  1. Glucose oxidase variants with improved properities

    OpenAIRE

    Fischer, Rainer; Ostafe, Raluca; Prodanovic, Radivoje

    2014-01-01

    Source: WO14173822A3 [EN] The technology provided herein relates to novel variants of microbial glucose oxidase with improved properties, more specifically to polypeptides having glucose oxidase activity as their major enzymatic activity; to nucleic acid molecules encoding said glucose oxidases; vectors and host cells containing the nucleic acids and methods for producing the glucose oxidase; compositions comprising said glucose oxidase; methods for the preparation and production of such enzy...

  2. Interactions of Desmethoxyyangonin, a Secondary Metabolite from Renealmia alpinia, with Human Monoamine Oxidase-A and Oxidase-B

    Directory of Open Access Journals (Sweden)

    Narayan D. Chaurasiya

    2017-01-01

    Full Text Available Renealmia alpinia (Zingiberaceae, a medicinal plant of tropical rainforests, is used to treat snakebites and other injuries and also as a febrifuge, analgesic, antiemetic, antiulcer, and anticonvulsant. The dichloromethane extract of R. alpinia leaves showed potent inhibition of human monoamine oxidases- (MAOs- A and B. Phytochemical studies yielded six known compounds, including pinostrobin 1, 4′-methyl ether sakuranetin 2, sakuranetin 3, pinostrobin chalcone 4, yashabushidiol A 5, and desmethoxyyangonin 6. Compound 6 displayed about 30-fold higher affinity for MAO-B than MAO-A, with Ki values of 31 and 922 nM, respectively. Kinetic analysis of inhibition and equilibrium-dialysis dissociation assay of the enzyme-inhibitor complex showed reversible binding of desmethoxyyangonin 6 with MAO-A and MAO-B. The binding interactions of compound 6 in the active site of the MAO-A and MAO-B isoenzymes, investigated through molecular modeling algorithms, confirmed preferential binding of desmethoxyyangonin 6 with MAO-B compared to MAO-A. Selective reversible inhibitors of MAO-B, like desmethoxyyangonin 6, may have important therapeutic significance for the treatment of neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease.

  3. Dietary Phenolic Compounds Interfere with the Fate of Hydrogen Peroxide in Human Adipose Tissue but Do Not Directly Inhibit Primary Amine Oxidase Activity

    Directory of Open Access Journals (Sweden)

    Christian Carpéné

    2016-01-01

    Full Text Available Resveratrol has been reported to inhibit monoamine oxidases (MAO. Many substrates or inhibitors of neuronal MAO interact also with other amine oxidases (AO in peripheral organs, such as semicarbazide-sensitive AO (SSAO, known as primary amine oxidase, absent in neurones, but abundant in adipocytes. We asked whether phenolic compounds (resveratrol, pterostilbene, quercetin, and caffeic acid behave as MAO and SSAO inhibitors. AO activity was determined in human adipose tissue. Computational docking and glucose uptake assays were performed in 3D models of human AO proteins and in adipocytes, respectively. Phenolic compounds fully inhibited the fluorescent detection of H2O2 generated during MAO and SSAO activation by tyramine and benzylamine. They also quenched H2O2-induced fluorescence in absence of biological material and were unable to abolish the oxidation of radiolabelled tyramine and benzylamine. Thus, phenolic compounds hampered H2O2 detection but did not block AO activity. Only resveratrol and quercetin partially impaired MAO-dependent [14C]-tyramine oxidation and behaved as MAO inhibitors. Phenolic compounds counteracted the H2O2-dependent benzylamine-stimulated glucose transport. This indicates that various phenolic compounds block downstream effects of H2O2 produced by biogenic or exogenous amine oxidation without directly inhibiting AO. Phenolic compounds remain of interest regarding their capacity to limit oxidative stress rather than inhibiting AO.

  4. Role of catechins on ET-1 induced stimulation of PLD and NADPH oxidase activities in pulmonary smooth muscle cells: Determination of the probable mechanism by molecular docking studies.

    Science.gov (United States)

    Chakraborti, Sajal; Sarkar, Jaganmay; Bhuyan, Rajabrata; Chakraborti, Tapati

    2017-12-05

    Treatment of human pulmonary artery smooth muscle cells with ET-1 stimulated PLD and NADPH oxidase activities, which were inhibited upon pretreatment with bosentan (ET-1 receptor antagonist), FIPI (PLD inhibitor), apocynin (NADPH oxidase inhibitor) and EGCG & ECG (catechins having galloyl group), but not EGC & EC (catechins devoid of galloyl group). Herein, we determined the probable mechanism by which the galloyl group containing catechins inhibit ET-1 induced stimulation of PLD activity by molecular docking analyses based on our biochemical studies. ET-1 induced stimulation of PLD activity was inhibited by SecinH3 (inhibitor of cytohesin). Arf-6 and cytohesin-1 were associated in the cell membrane, which was not inhibited by the catechins during ET-1 treatment to the cells. However, EGCG and ECG inhibited binding of GTPγS with Arf-6 even in presence of cytohesin-1. The molecular docking analyses revealed that the galloyl group containing catechins (EGCG/ECG) with cytohesin1-Arf6GDP, but not the non-galloyl-containing catechins (EGC and EC), prevents GDP/GTP exchange in Arf-6 which seems to be an important mechanism for inhibition of ET-1 induced activation of PLD and subsequently increase in NADPH oxidase activities.

  5. Comprehensive review of rasagiline, a second-generation monoamine oxidase inhibitor, for the treatment of Parkinson's disease.

    Science.gov (United States)

    Chen, Jack J; Swope, David M; Dashtipour, Khashayar

    2007-09-01

    Inhibitors of monoamine oxidase (MAO) with selectivity and specificity for MAO type B (MAO-B) prolong the duration of action of both endogenously and exogenously derived dopamine. Rasagiline [N-propargyl-l(R)-aminoindan] is a second-generation propargylamine pharmacophore that selectively and irreversibly inhibits brain MAO-B and is specifically designed for the treatment of Parkinson's disease (PD). The aim of this study was to review the pharmacology, tolerability, and clinical efficacy of rasagiline in the treatment of PD. MEDLINE (1966-April 2007), the Cochrane Database of Systematic Reviews, and International Pharmaceutical Abstracts (1970-April 2007) were searched for original research and review articles published in English. The search terms were monoamine oxidase, neuroprotection, Parkinson disease, propargylamine, rasagiline, and selegiline. The reference lists of articles were also consulted, as was information provided by the manufacturer of rasagiline. Data from 63 clinical and laboratory studies were analyzed. Based on the results from those studies, we concluded that rasagiline PO QD, at the therapeutic dosage range of 0.5 to 1 rag/d, is effective and well tolerated and completely, selectively, and specifically inhibited MAO-B. Pharmacologically, rasagiline was found to be Rasagiline was effective both as monotherapy in early PD and as adjunctive treatment in patients with advancing PD and motor fluctuations. As monotherapy, rasagiline provided modest yet clinically meaningful benefit. A randomized, double-blind, placebo-controlled study found that, after 26 weeks of treatment, the adjusted effect size for total Unified Parkinson's Disease Rating Scale score was -4.20 (95% CI, -5.66 to -2.73) for rasagiline 1 mg/d versus placebo (P rasagiline is initiated early (before the need for dopaminergic agents) rather than later. In patients with more advanced disease who received treatment with dopaminergic agents, rasagiline and entacapone were associated

  6. Synergistic effects between catalase inhibitors and modulators of nitric oxide metabolism on tumor cell apoptosis.

    Science.gov (United States)

    Scheit, Katrin; Bauer, Georg

    2014-10-01

    Inhibitors of catalase (such as ascorbate, methyldopa, salicylic acid and neutralizing antibodies) synergize with modulators of nitric oxide (NO) metabolism (such as arginine, arginase inhibitor, NO synthase-inducing interferons and NO dioxygenase inhibitors) in the singlet oxygen-mediated inactivation of tumor cell protective catalase. This is followed by reactive oxygen species (ROS)-dependent apoptosis induction. TGF-beta, NADPH oxidase-1, NO synthase, dual oxidase-1 and caspase-9 are characterized as essential catalysts in this process. The FAS receptor and caspase-8 are required for amplification of ROS signaling triggered by individual compounds, but are dispensable when the synergistic effect is established. Our findings explain the antitumor effects of catalase inhibitors and of compounds that target NO metabolism, as well as their synergy. These data may have an impact on epidemiological studies related to secondary plant compounds and open new perspectives for the establishment of novel antitumor drugs and for the improvement of established chemotherapeutics. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Effects of befloxatone, a reversible selective monoamine oxidase-A inhibitor, on psychomotor function and memory in healthy subjects.

    Science.gov (United States)

    Warot, D; Berlin, I; Patat, A; Durrieu, G; Zieleniuk, I; Puech, A J

    1996-10-01

    Befloxatone is a new reversible and selective monoamine oxidase (MAO-A) inhibitor that has been shown to have antidepressant activity in various animal models. To assess the effects of single oral doses of befloxatone (5, 10, and 20 mg) on psychomotor performance and memory, a randomized, double-blind, five-way, crossover study with both placebo and amitriptyline (50 mg) was carried out in 15 healthy male volunteers. Psychomotor and cognitive functions were evaluated using both objective measures, including Critical Flicker Frequency (CFF), Choice Reaction Time (CRT), Digit Symbol Substitution Test (DSST), and a picture memory test and subjective measures, including Visual Analog Scales (VAS) and Addiction Research Center Inventory (ARCI), before and 2, 4, and 8 hours after administration. Pupil diameter was recorded by videopupillography. Single doses of befloxatone from 5 to 20 mg did not result in any detrimental effects on skilled performance and memory. In contrast, amitriptyline significantly impaired arousal (CFF), speed of reaction (CRT), information processing (DSST) and long-term memory (delayed free recall of pictures) and produced subjective sedation from 2 to 8 hours after administration. At the doses studied amitriptyline induced miosis but befloxatone did not modify pupil diameter. There was no evidence in this study to suggest that befloxatone, at the doses studied, has any sedative or amnesic effects in healthy subjects.

  8. Molecular Modeling of Peroxidase and Polyphenol Oxidase: Substrate Specificity and Active Site Comparison

    Directory of Open Access Journals (Sweden)

    Lalida Shank

    2010-09-01

    Full Text Available Peroxidases (POD and polyphenol oxidase (PPO are enzymes that are well known to be involved in the enzymatic browning reaction of fruits and vegetables with different catalytic mechanisms. Both enzymes have some common substrates, but each also has its specific substrates. In our computational study, the amino acid sequence of grape peroxidase (ABX was used for the construction of models employing homology modeling method based on the X-ray structure of cytosolic ascorbate peroxidase from pea (PDB ID:1APX, whereas the model of grape polyphenol oxidase was obtained directly from the available X-ray structure (PDB ID:2P3X. Molecular docking of common substrates of these two enzymes was subsequently studied. It was found that epicatechin and catechin exhibited high affinity with both enzymes, even though POD and PPO have different binding pockets regarding the size and the key amino acids involved in binding. Predicted binding modes of substrates with both enzymes were also compared. The calculated docking interaction energy of trihydroxybenzoic acid related compounds shows high affinity, suggesting specificity and potential use as common inhibitor to grape ascorbate peroxidase and polyphenol oxidase.

  9. 5-(2-Aminopropyl)indole (5-IT): a psychoactive substance used for recreational purposes is an inhibitor of human monoamine oxidase (MAO).

    Science.gov (United States)

    Herraiz, Tomás; Brandt, Simon D

    2014-01-01

    5-(2-Aminopropyl)indole (5-IT) is a psychoactive compound that has recently been associated with several fatal and non-fatal intoxications in a number of European countries. There are indications that acute effects may include symptoms of monoaminergic (e.g. serotonin) toxicity and one mechanism involved in the increase of serotonin levels includes the inhibition of monoamine oxidase. This study investigated the effect of 5-IT on human MAO-A and -B isozymes using kynuramine as the substrate. Substrate conversion to 4-hydroxyquinoline was monitored by high-performance liquid chromatography coupled to diode array detection. This method was employed to determine the extent of MAO inhibition (IC50 and Ki ) and it was found that 5-IT was a selective, competitive and reversible inhibitor of MAO-A. 5-IT revealed a relatively potent ability to inhibit MAO-A (IC50  =1.6 μM and Ki  =0.25 μM) while MAO-B inhibition was not observed (0-500 μM 5-IT). Under identical experimental conditions, other established inhibitors of MAO-A and antidepressants provided the following IC50 values: clorgyline 16 nM, harmaline 20 nM, toloxatone 6.7 μM and moclobemide >500 μM. These data indicated that 5-IT was less potent than clorgyline and harmaline but more potent than toloxatone and moclobemide under the in-vitro conditions studied. The inhibition of MAO-A suggests that 5-IT by itself or in combination with other substances may be able to potentiate serotonergic/monoaminergic effects and further studies are needed to clarify its relevance to the adverse effects reported for 5-IT. Copyright © 2013 John Wiley & Sons, Ltd.

  10. Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

    Science.gov (United States)

    Majumdar, Arkajo; Kar, Rup Kumar

    2018-07-01

    Plasma membrane (PM) H + -ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O 2 ˙ - , respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H + -ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H + -ATPase inhibitor). Conversely, H + -ATPase activity retarded in response to different ROS scavengers [CuCl 2 , N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl 2 and diphenyleneiodonium (DPI)], while H 2 O 2 promoted PM H + -ATPase activity at lower concentrations. Repressing effects of Ca +2 antagonists (La +3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H + -ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H + -ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

  11. Pro-inflammatory effects of interleukin-17A on vascular smooth muscle cells involve NAD(P)H- oxidase derived reactive oxygen species.

    Science.gov (United States)

    Pietrowski, Eweline; Bender, Bianca; Huppert, Jula; White, Robin; Luhmann, Heiko J; Kuhlmann, Christoph R W

    2011-01-01

    T cells are known for their contribution to the inflammatory element of atherosclerosis. Recently, it has been demonstrated that the Th17 derived cytokine IL-17 is involved in the pro-inflammatory response of vascular smooth muscle cells (VSMC). The aim of the present study was to examine whether reactive oxygen species (ROS) might be involved in this context. The effect of IL-17A on ROS generation was examined using the fluorescent dye 2'7'-dichlorodihydrofluorescein (H(2)DCF) in primary murine VSMC. IL-17A induced an increase in H(2)DCF fluorescence in VSMC, and this effect was blocked by the NAD(P)H-oxidase inhibitor apocynin and siRNA targeting Nox2. The p38-MAPK inhibitors SB203580 and SB202190 dose-dependently reduced the IL-17A induced ROS production. The IL-17A induced release of the pro-inflammatory cytokines IL-6, G-CSF, GM-CSF and MCP-1 from VSMC, as detected by the Luminex technology, was completely abolished by NAD(P)H-oxidase inhibition. Taken together, our data indicate that IL-17A causes the NAD(P)H-oxidase dependent generation of ROS leading to a pro-inflammatory activation of VSMC. Copyright © 2010 S. Karger AG, Basel.

  12. Chlorella induces stomatal closure via NADPH oxidase-dependent ROS production and its effects on instantaneous water use efficiency in Vicia faba.

    Directory of Open Access Journals (Sweden)

    Yan Li

    Full Text Available Reactive oxygen species (ROS have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs. Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2 scavenger, catalase (CAT, significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI, and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM, suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi in Vicia faba via a reduction in leaf transpiration rate (E without a parallel reduction in net photosynthetic rate (Pn assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels.

  13. Development of 2-(Substituted Benzylamino)-4-Methyl-1, 3-Thiazole-5-Carboxylic Acid Derivatives as Xanthine Oxidase Inhibitors and Free Radical Scavengers.

    Science.gov (United States)

    Ali, Md Rahmat; Kumar, Suresh; Afzal, Obaid; Shalmali, Nishtha; Sharma, Manju; Bawa, Sandhya

    2016-04-01

    A series of 2-(substituted benzylamino)-4-methylthiazole-5-carboxylic acid was designed and synthesized as structural analogue of febuxostat. A methylene amine spacer was incorporated between the phenyl ring and thiazole ring in contrast to febuxostat in which the phenyl ring was directly linked with the thiazole moiety. The purpose of incorporating methylene amine was to provide a heteroatom which is expected to favour hydrogen bonding within the active site residues of the enzyme xanthine oxidase. The structure of all the compounds was established by the combined use of FT-IR, NMR and MS spectral data. All the compounds were screened in vitro for their ability to inhibit the enzyme xanthine oxidase as per the reported procedure along with DPPH free radical scavenging assay. Compounds 5j, 5k and 5l demonstrated satisfactory potent xanthine oxidase inhibitory activities with IC50 values, 3.6, 8.1 and 9.9 μm, respectively, whereas compounds 5k, 5n and 5p demonstrated moderate antioxidant activities having IC50 15.3, 17.6 and 19.6 μm, respectively, along with xanthine oxidase inhibitory activity. Compound 5k showed moderate xanthine oxidase inhibitory activity as compared with febuxostat along with antioxidant activity. All the compounds were also studied for their binding affinity in active site of enzyme (PDB ID-1N5X). © 2015 John Wiley & Sons A/S.

  14. The lysyl oxidase inhibitor β-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats.

    Science.gov (United States)

    Miana, María; Galán, María; Martínez-Martínez, Ernesto; Varona, Saray; Jurado-López, Raquel; Bausa-Miranda, Belén; Antequera, Alfonso; Luaces, María; Martínez-González, José; Rodríguez, Cristina; Cachofeiro, Victoria

    2015-06-01

    Extracellular matrix (ECM) remodelling of the adipose tissue plays a pivotal role in the pathophysiology of obesity. The lysyl oxidase (LOX) family of amine oxidases, including LOX and LOX-like (LOXL) isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its involvement in adipose tissue dysfunction in obesity is unclear. In this study, we observed that LOX is the main isoenzyme expressed in human adipose tissue and that its expression is strongly upregulated in samples from obese individuals that had been referred to bariatric surgery. LOX expression was also induced in the adipose tissue from male Wistar rats fed a high-fat diet (HFD). Interestingly, treatment with β-aminopropionitrile (BAPN), a specific and irreversible inhibitor of LOX activity, attenuated the increase in body weight and fat mass that was observed in obese animals and shifted adipocyte size toward smaller adipocytes. BAPN also ameliorated the increase in collagen content that was observed in adipose tissue from obese animals and improved several metabolic parameters - it ameliorated glucose and insulin levels, decreased homeostasis model assessment (HOMA) index and reduced plasma triglyceride levels. Furthermore, in white adipose tissue from obese animals, BAPN prevented the downregulation of adiponectin and glucose transporter 4 (GLUT4), as well as the increase in suppressor of cytokine signaling 3 (SOCS3) and dipeptidyl peptidase 4 (DPP4) levels, triggered by the HFD. Likewise, in the TNFα-induced insulin-resistant 3T3-L1 adipocyte model, BAPN prevented the downregulation of adiponectin and GLUT4 and the increase in SOCS3 levels, and consequently normalised insulin-stimulated glucose uptake. Therefore, our data provide evidence that LOX plays a pathologically relevant role in the metabolic dysfunction induced by obesity and emphasise the interest of novel pharmacological interventions that target adipose tissue fibrosis and LOX activity for

  15. Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

    Science.gov (United States)

    Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

    2015-05-01

    Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Multipotent cholinesterase/monoamine oxidase inhibitors for the treatment of Alzheimer’s disease: design, synthesis, biochemical evaluation, ADMET, molecular modeling, and QSAR analysis of novel donepezil-pyridyl hybrids

    Directory of Open Access Journals (Sweden)

    Bautista-Aguilera OM

    2014-10-01

    Full Text Available Oscar M Bautista-Aguilera,1,* Gerard Esteban,2,* Mourad Chioua,1 Katarina Nikolic,3 Danica Agbaba,3 Ignacio Moraleda,4 Isabel Iriepa,4 Elena Soriano,5 Abdelouahid Samadi,1 Mercedes Unzeta,2 José Marco-Contelles1 1Laboratory of Medicinal Chemistry (Institute of General Organic Chemistry [IQOG], National Research Council [CSIC], Madrid, Spain; 2Department of Biochemistry and Molecular Biology, Institute of Neurosciences, Autonomous Barcelona University, Barcelona, Spain; 3Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia; 4Department of Organic Chemistry, Faculty of Pharmacy, University of Alcalá, Ctra Barcelona, Alcalá de Henares, Spain; 5Synthesis, and Structure of Organic Compounds (SEPCO (IQOG, CSIC, Madrid, Spain *These authors have equally contributed to this work Abstract: The design, synthesis, and biochemical evaluation of donepezil-pyridyl hybrids (DPHs as multipotent cholinesterase (ChE and monoamine oxidase (MAO inhibitors for the potential treatment of Alzheimer’s disease (AD is reported. The 3D-quantitative structure-activity relationship study was used to define 3D-pharmacophores for inhibition of MAO A/B, acetylcholinesterase (AChE, and butyrylcholinesterase (BuChE enzymes and to design DPHs as novel multi-target drug candidates with potential impact in the therapy of AD. DPH14 (­Electrophorus electricus AChE [EeAChE]: half maximal inhibitory concentration [IC50] =1.1±0.3 nM; equine butyrylcholinesterase [eqBuChE]: IC50 =600±80 nM was 318-fold more potent for the inhibition of AChE, and 1.3-fold less potent for the inhibition of BuChE than the reference compound ASS234. DPH14 is a potent human recombinant BuChE (hBuChE inhibitor, in the same range as DPH12 or DPH16, but 13.1-fold less potent than DPH15 for the inhibition of human recombinant AChE (hAChE. Compared with donepezil, DPH14 is almost equipotent for the inhibition of hAChE, and 8.8-fold more potent for h

  17. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum. Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols.

    Science.gov (United States)

    Fraaije, M W; Veeger, C; van Berkel, W J

    1995-11-15

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyphenylglycols and a series of catecholamines. These compounds are converted to the corresponding ketones. Both enantiomers of (nor)epinephrine are substrates for vanillyl-alcohol oxidase, but the R isomer is preferred. Vanillyl-alcohol oxidase is most active with chavicol and eugenol. These 4-allylphenols are converted to coumaryl alcohol and coniferyl alcohol, respectively. Isotopic labeling experiments show that the oxygen atom inserted at the C gamma atom of the side chain is derived from water. The 4-hydroxycinnamyl alcohol products and the substrate analog isoeugenol are competitive inhibitors of vanillyl alcohol oxidation. The binding of isoeugenol to the oxidized enzyme perturbs the optical spectrum of protein-bound FAD. pH-dependent binding studies suggest that vanillyl-alcohol oxidase preferentially binds the phenolate form of isoeugenol (pKa < 6, 25 degrees C). From this and the high pH optimum for turnover, a hydride transfer mechanism involving a p-quinone methide intermediate is proposed for the vanillyl-alcohol-oxidase-catalyzed conversion of 4-allylphenols.

  18. Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells.

    Science.gov (United States)

    Oliveira, Jessica Silva Santos de; Santos, Gabriela da Silva; Moraes, João Alfredo; Saliba, Alessandra Mattos; Barja-Fidalgo, Thereza Christina; Mattos-Guaraldi, Ana Luíza; Nagao, Prescilla Emy

    2018-01-01

    BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

  19. In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase-A and –B Enzymatic Activity

    Science.gov (United States)

    Brooks, Allen F.; Shao, Xia; Quesada, Carole A.; Sherman, Phillip; Scott, Peter J.H.; Kilbourn, Michael R.

    2017-01-01

    The isozymes of monoamine oxidase (MAO-A and MAO-B) are important enzymes involved in the metabolism of numerous biogenic amines, including the neurotransmitters serotonin, dopamine and norepinephrine. Recently, changes in concentrations of MAO-B have been proposed as an in vivo marker of neuroinflammation associated with Alzheimer’s disease. Previous developments of in vivo radiotracers for imaging changes in MAO enzyme expression or activity have utilized the irreversible propargylamine-based suicide inhibitors, or high-affinity reversibly-binding inhibitors. As an alternative approach, we have investigated 1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as metabolic trapping agents for the monoamine oxidases. MAO-mediated oxidation and spontaneous hydrolysis yields 1-[11C]methyl-2,3-dihydro-4-pyridinone as a hydrophilic metabolite that is trapped within brain tissues. Radiotracers with phenyl, biphenyl and 7-coumarinyl ethers were evaluated using microPET imaging in rat and primate brain. No isozyme selectivity for radiotracer trapping was observed in the rat brain for any compound, but in the monkey brain the phenyl ether demonstrated MAO-A selectivity, and the coumarinyl ether showed MAO-B selectivity. These are lead compounds for further development of 1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines with optimized brain pharmacokinetics and isozyme selectivity. PMID:26393369

  20. Oxidase-based biocatalytic processes

    DEFF Research Database (Denmark)

    Ramesh, Hemalata; Woodley, John; Krühne, Ulrich

    interestingbiocatalystsbecause they use a mild oxidant (oxygen) as a substrateas opposed to their chemical counterparts which use strong oxidants such as permanganates. A class of oxidases calledmonoamine oxidases has been used as the central case study for the thesis. The rationale for choosing thissystemis that it has been...

  1. Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.).

    Science.gov (United States)

    Siddiq, M; Dolan, K D

    2017-03-01

    Polyphenol oxidase (PPO) was extracted and characterized from high-bush blueberries. PPO showed an optimum activity at pH 6.1-6.3 and 35°C, with the enzyme showing significant activity over a wide temperature range (25-60°C). Catechol was the most readily oxidized substrate followed by 4-methylcatechol, DL-DOPA, and dopamine. Blueberry PPO showed a K m of 15mM and V max of 2.57 ΔA 420 nm/min×10 -1 , determined with catechol. PPO was completely inactivated in 20min at 85°C, however, after 30minat 75°C it showed about 10% residual activity. Thermal treatment at 55 and 65°C for 30min resulted in the partial inactivation of PPO. Ascorbic acid, sodium diethyldithiocarbamic acid, L-cysteine, and sodium metabisulfite were effective inhibitors of PPO at 1.0mM. Benzoic acid and cinnamic acid series inhibitors showed relatively weak inhibition of PPO (21.8-27.6%), even at as high as 2.0mM concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Isolated sulfite oxidase deficiency.

    Science.gov (United States)

    Rupar, C A; Gillett, J; Gordon, B A; Ramsay, D A; Johnson, J L; Garrett, R M; Rajagopalan, K V; Jung, J H; Bacheyie, G S; Sellers, A R

    1996-12-01

    Isolated sulfite oxidase (SO) deficiency is an autosomal recessively inherited inborn error of sulfur metabolism. In this report of a ninth patient the clinical history, laboratory results, neuropathological findings and a mutation in the sulfite oxidase gene are described. The data from this patient and previously published patients with isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are summarized to characterize this rare disorder. The patient presented neonatally with intractable seizures and did not progress developmentally beyond the neonatal stage. Dislocated lenses were apparent at 2 months. There was increased urine excretion of sulfite and S-sulfocysteine and a decreased concentration of plasma cystine. A lactic acidemia was present for 6 months. Liver sulfite oxidase activity was not detectable but xanthine dehydrogenase activity was normal. The boy died of respiratory failure at 32 months. Neuropathological findings of cortical necrosis and extensive cavitating leukoencephalopathy were reminiscent of those seen in severe perinatal asphyxia suggesting an etiology of energy deficiency. A point mutation that resulted in a truncated protein missing the molybdenum-binding site has been identified.

  3. Synthesis and evaluation of 2-benzylidene-1-tetralone derivatives for monoamine oxidase inhibitory activity.

    Science.gov (United States)

    Amakali, Klaudia T; Legoabe, Lesetja Jan; Petzer, Anel; Petzer, Jacobus P

    2018-05-01

    Chalcone has been identified as a promising lead for the design of monoamine oxidase (MAO) inhibitors. This study attempted to discover potent and selective chalcone-derived MAO inhibitors by synthesising a series consisting of various cyclic chalcone derivatives. The cyclic chalcones were selected based on the possibility that their restricted structures would confer a higher degree of MAO isoform selectivity, and included the following chemical classes: 1-indanone, 1-tetralone, 1-benzosuberone, chromone, thiochromone, 4-chromanone and 4-thiochromanone. The results showed that the cyclic chalcones are in general good potency, and in most instances specific inhibitors of the human MAO-B isoform. Among these compounds, the 4-chromanone derivative was the most potent MAO-B inhibitor with an IC50 value of 0.156 µM. To further investigate the MAO inhibition of cyclic chalcones, a series of twenty-three 2-benzylidene-1-tetralone derivatives were synthesised and evaluated as MAO inhibitors. Most 2-benzylidene-1-tetralones possess good inhibitory activity and specificity for MAO-B with the most potent inhibitor displaying an IC50 value of 0.0064 µM, while the most potent MAO-A inhibitor possessed an IC50 value of 0.754 µM. This study thus shows that certain cyclic chalcones are human MAO-B inhibitors, compounds that could be suitable for the treatment of neurodegenerative disorders such as Parkinson's disease. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. Inducible bilirubin oxidase: A novel function for the mouse cytochrome P450 2A5

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Bakar, A' edah, E-mail: a.abubakar@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Arthur, Dionne Maioha [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide (Australia); Aganovic, Simona [Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Centre, Box 578, S-751 23 Uppsala (Sweden); Ng, Jack C. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide (Australia); Lang, Matti A. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Centre, Box 578, S-751 23 Uppsala (Sweden)

    2011-11-15

    We have previously shown that bilirubin (BR), a breakdown product of haem, is a strong inhibitor and a high affinity substrate of the mouse cytochrome P450 2A5 (CYP2A5). The antioxidant BR, which is cytotoxic at high concentrations, is potentially useful in cellular protection against oxygen radicals if its intracellular levels can be strictly controlled. The mechanisms that regulate cellular BR levels are still obscure. In this paper we provide preliminary evidence for a novel function of CYP2A5 as hepatic 'BR oxidase'. A high-performance liquid chromatography/electrospray ionisation mass spectrometry screening showed that recombinant yeast microsomes expressing the CYP2A5 oxidise BR to biliverdin, as the main metabolite, and to three other smaller products with m/z values of 301, 315 and 333. The metabolic profile is significantly different from that of chemical oxidation of BR. In chemical oxidation the smaller products were the main metabolites. This suggests that the enzymatic reaction is selective, towards biliverdin production. Bilirubin treatment of primary hepatocytes increased the CYP2A5 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A5 compared to cells treated only with CHX. Collectively, the observations suggest that the CYP2A5 is potentially an inducible 'BR oxidase' where BR may accelerate its own metabolism through stabilization of the CYP2A5 protein. It is possible that this metabolic pathway is potentially part of the machinery controlling intracellular BR levels in transient oxidative stress situations, in which high amounts of BR are produced. -- Highlights: Black-Right-Pointing-Pointer CYP2A5 metabolizes bilirubin to biliverdin and dipyrroles. Black-Right-Pointing-Pointer Bilirubin increased the hepatic CYP2A5 protein and activity levels. Black-Right-Pointing-Pointer Bilirubin does not

  5. The properties of B-form monoamine oxidase in mitochondria from monkey platelet.

    Science.gov (United States)

    Obata, Toshio; Aomine, Masahiro

    The present study was examined the effect of the properties of monkey platelet monoamine oxidase (MAO) based on inhibitor sensitivity. Monkey platelet showed a high MAO activity with beta-phenylethylamine (beta-PEA) as substrate and a very low A-form MAO activity with 5 hydroxytryptamine (5-HT) as substrate. Moreover, monkey platelet MAO was sensitive to the drugs deprenyl as B-form MAO inhibitor and less sensitive to clorgyline and harmaline as A form MAO inhibitor with beta-PEA as the B-form MAO substrate. B-form MAO from monkey platelet was more stable against heat treatment at 55 degrees C than B-form MAO in brain. After digestion with trypsin at 37 degrees C for 4 hrs, it was found that MAO from platelet was inhibited about 70% with beta-PEA as substrate with brain. The tricyclic antidepressant imipramine and nortriptyline inhibited B-form MAO activity more potency than B-form MAO in brain. However, when the noncyclic antidepressant nomifensine was used, monkey platelet B-form MAO activities were less potently inhibited. All these reagents were noncompetitive inhibitors of B form MAO in monkey platelet. The present studies demonstrated that monkey platelet MAO is a single of B-form MAO and sensitive to tricyclic antidepressants.

  6. Development of radioiodinated ligands for exploration of brain monoamine oxidase by tomo-scintigraphy

    International Nuclear Information System (INIS)

    Rafii, H.

    1996-01-01

    Monoamine oxidases, MAO, are important in the regulation of monoaminergic neuro-transmissions. The fluctuations in MAO activities has been observed in some psychiatric and neuro-degenerative diseases. Thus, quantification of cerebral MAO activity would be useful for diagnosis and the therapeutic follow-up of these disorders. With the object of doing an in vivo scintigraphic exploration of cerebral MAO by SPECT, we have undertaken to synthesize some radioiodinated MAO inhibitors. In the first part of this work, we have discussed the general properties of the monoamine oxidases and their inhibitors. In the second part we have described the scintigraphic methods. the ligands to be used for MAO exploration, and the radioiodination methods. At last in the third part, the development of three radioiodinated ligands has been presented: - [ 125 I]3-iodopargyline. In vivo results showed that, this radioligand blocked the cerebral MAO-B with moderate selectivity. However, complementary in vivo studies would be needed to define precisely its activity.- [ 125 I]Ro 16-6491. The cerebral fixation of this radioligand was in accordance with the MAO-B sites in the rat brains, but its fixation was too low for scintigraphic exploration in vivo with iodine-123. - [ 125 I]Ro 11-9900. In vivo studies of rat brains showed that the MAO-A sites were bound preferentially by this radioligand. The cerebral biodistribution of this ligand labelled with iodine-123 is considered for use in a model animal nearest to human pathology. (author)

  7. H2O2 and NADPH oxidases involve in regulation of 2-(2-phenylethyl)chromones accumulation during salt stress in Aquilaria sinensis calli.

    Science.gov (United States)

    Wang, Xiaohui; Dong, Xianjuan; Feng, Yingying; Liu, Xiao; Wang, Jinling; Zhang, Zhongxiu; Li, Jun; Zhao, Yunfang; Shi, Shepo; Tu, Pengfei

    2018-04-01

    2-(2-Phenylethyl)chromones are the main compounds responsible for the quality of agarwood, which is widely used in traditional medicines, incenses and perfumes. H 2 O 2 and NADPH oxidases (also known as respiratory burst oxidase homologs, Rbohs) mediate diverse physiological and biochemical processes in environmental stress responses. However, little is known about the function of H 2 O 2 and NADPH oxidases in 2-(2-phenylethyl)chromones accumulation. In this study, we found that salt stress induced a transient increase in content of H 2 O 2 and 2-(2-phenylethyl)chromones accumulation in Aquilaria sinensis calli. Exogenous H 2 O 2 remarkably decreased the production of 2-(2-phenylethyl)chromones, while dimethylthiourea (DMTU), a scavenger of H 2 O 2 , significantly increased 2-(2-phenylethyl)chromones accumulation in salt treated calli. Three new H 2 O 2 -generating genes, named AsRbohA-C, were isolated and characterized from A. sinensis. Salt stress also induced a transient increase in AsRbohA-C expression and NADPH oxidase activity. Furthermore, exogenous H 2 O 2 increased AsRbohA-C expression and NADPH oxidase activity, while DMTU inhibited AsRbohA-C expression and NADPH oxidase activity under salt stress. Moreover, diphenylene iodonium (DPI), the inhibitor of NADPH oxidases, reduced AsRbohA-C expression and NADPH oxidase activity, but significantly induced 2-(2-phenylethyl)chromones accumulation during salt stress. These results clearly demonstrated the central role of H 2 O 2 and NADPH oxidases in regulation of salt-induced 2-(2-phenylethyl)chromones accumulation in A. sinensis calli. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Selective inhibition of monoamine oxidase A by purpurin, an anthraquinone.

    Science.gov (United States)

    Lee, Hyun Woo; Ryu, Hyung Won; Kang, Myung-Gyun; Park, Daeui; Oh, Sei-Ryang; Kim, Hoon

    2017-03-01

    Monoamine oxidase (MAO) catalyzes the oxidation of monoamines that act as neurotransmitters. During a target-based screening of natural products using two isoforms of recombinant human MAO-A and MAO-B, purpurin (an anthraquinone derivative) was found to potently and selectively inhibit MAO-A, with an IC 50 value of 2.50μM, and not to inhibit MAO-B. Alizarin (also an anthraquinone) inhibited MAO-A less potently with an IC 50 value of 30.1μM. Furthermore, purpurin was a reversible and competitive inhibitor of MAO-A with a K i value of 0.422μM. A comparison of their chemical structures suggested the 4-hydroxy group of purpurin might play an important role in its inhibition of MAO-A. Molecular docking simulation showed that the binding affinity of purpurin for MAO-A (-40.0kcal/mol) was higher than its affinity for MAO-B (-33.9kcal/mol), and that Ile 207 and Gly 443 of MAO-A were key residues for hydrogen bonding with purpurin. The findings of this study suggest purpurin is a potent, selective, reversible inhibitor of MAO-A, and that it be considered a new potential lead compound for development of novel reversible inhibitors of MAO-A (RIMAs). Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. In vitro and in vivo evidences that antioxidant action contributes to the neuroprotective effects of the neuronal nitric oxide synthase and monoamine oxidase-B inhibitor, 7-nitroindazole.

    Science.gov (United States)

    Thomas, Bobby; Saravanan, Karuppagounder S; Mohanakumar, Kochupurackal P

    2008-05-01

    The neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI) is neuroprotective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Monoamine oxidase (MAO)-B inhibitory action partially contributes to this effect. We tested the hypothesis that 7-NI could be a powerful hydroxyl radical (OH) scavenger, and interferes with oxidative stress caused by MPTP. We measured OH, reduced glutathione (GSH), as well as superoxide dismutase (SOD) and catalase activities in the nucleus caudatus putamen and substantia nigra of Balb/c mice following MPTP and/or 7-NI administration. The nNOS inhibitor caused dose-dependent inhibition in the production of OH in (i) Fenton-like reaction employing ferrous citrate in a cell-free system in test tubes, (ii) in isolated mitochondrial preparation in presence of MPP+, and (iii) in the striatum of mice systemically treated with MPTP. An MPTP-induced depletion of GSH in both the nuclei was blocked by 7-NI, which was dose-dependent (10-50mg/kg), but independent of MAO-B inhibition. The nNOS-mediated recovery of GSH paralleled attenuation of MPTP-induced depletion of striatal dopamine. MPTP-induced increase in the activities of striatal or nigral SOD and catalase were significantly attenuated by 7-NI treatment. These results suggest potent antioxidant action of 7-NI in its neuroprotective effects against MPTP-induced neurotoxicity.

  10. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

    Science.gov (United States)

    Morre, D. James

    2002-01-01

    The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

  11. Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

    International Nuclear Information System (INIS)

    Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2009-01-01

    Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O 2 ·- generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 phox knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47 phox , p67 phox and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67 phox siRNA. Exposure of MPMVEC obtained from gp91 phox knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to

  12. An ultrafiltration assay for lysyl oxidase

    International Nuclear Information System (INIS)

    Shackleton, D.R.; Hulmes, D.J.

    1990-01-01

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

  13. Exploring flavin-containing carbohydrate oxidases

    NARCIS (Netherlands)

    Ferrari, Alessandro Renato

    2017-01-01

    Oxidases are enzymes capable of removing one or more electrons from their substrate and transfer them to molecular oxygen, forming hydrogen peroxide. Due to their high regio- and enantioselectivity, their use is preferred over traditional organic chemistry methods. Among the oxidases, flavoprotein

  14. Purification and characterization of amine oxidase from soybean seedlings.

    Science.gov (United States)

    Vianello, F; Di Paolo, M L; Stevanato, R; Gasparini, R; Rigo, A

    1993-11-15

    A simple and rapid procedure for purification of soybean seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation was purified by ion-exchange chromatography on a cellulose phosphate column and batch affinity chromatography on 6-aminohexyl-Sepharose. Cyclohexylamine, a competitive inhibitor, was utilized to elute the enzyme. A homogeneous enzyme was obtained with a yield higher than 25%, the content of minor components being lauryl sulfate-polyacrylamide gel electrophoresis. The enzyme is a dimer and contains two Cu2+ ion per molecule. Its EPR spectrum is typical of Cu2+ in a tetragonal symmetry. The enzyme oxidizes cadaverine at high rate, the specific activity being 4.3 mukat/mg. Molecular, spectroscopic, and kinetic properties of this enzyme are reported.

  15. A small molecule polyamine oxidase inhibitor blocks androgen-induced oxidative stress and delays prostate cancer progression in the transgenic adenocarcinoma of the mouse prostate model.

    Science.gov (United States)

    Basu, Hirak S; Thompson, Todd A; Church, Dawn R; Clower, Cynthia C; Mehraein-Ghomi, Farideh; Amlong, Corey A; Martin, Christopher T; Woster, Patrick M; Lindstrom, Mary J; Wilding, George

    2009-10-01

    High levels of reactive oxygen species (ROS) present in human prostate epithelia are an important etiologic factor in prostate cancer (CaP) occurrence, recurrence, and progression. Androgen induces ROS production in the prostate by a yet unknown mechanism. Here, to the best of our knowledge, we report for the first time that androgen induces an overexpression of spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in the polyamine oxidation pathway. As prostatic epithelia produce a large excess of polyamines, the androgen-induced polyamine oxidation that produces H2O2 could be a major reason for the high ROS levels in the prostate epithelia. A small molecule polyamine oxidase inhibitor N,N'-butanedienyl butanediamine (MDL 72,527 or CPC-200) effectively blocks androgen-induced ROS production in human CaP cells, as well as significantly delays CaP progression and death in animals developing spontaneous CaP. These data show that polyamine oxidation is not only a major pathway for ROS production in prostate, but inhibiting this pathway also successfully delays CaP progression.

  16. Spermine oxidase promotes bile canalicular lumen formation through acrolein production.

    Science.gov (United States)

    Uemura, Takeshi; Takasaka, Tomokazu; Igarashi, Kazuei; Ikegaya, Hiroshi

    2017-11-01

    Spermine oxidase (SMOX) catalyzes oxidation of spermine to generate spermidine, hydrogen peroxide (H 2 O 2 ) and 3-aminopropanal, which is spontaneously converted to acrolein. SMOX is induced by a variety of stimuli including bacterial infection, polyamine analogues and acetaldehyde exposure. However, the physiological functions of SMOX are not yet fully understood. We investigated the physiological role of SMOX in liver cells using human hepatocellular carcinoma cell line HepG2. SMOX localized to the bile canalicular lumen, as determined by F-actin staining. Knockdown of SMOX reduced the formation of bile canalicular lumen. We also found that phospho-Akt (phosphorylated protein kinase B) was localized to canalicular lumen. Treatment with Akt inhibitor significantly reduced the formation of bile canalicular lumen. Acrolein scavenger also inhibited the formation of bile canalicular lumen. PTEN, phosphatase and tensin homolog and an inhibitor of Akt, was alkylated in a SMOX-dependent manner. Our results suggest that SMOX plays a central role in the formation of bile canalicular lumen in liver cells by activating Akt pathway through acrolein production.

  17. Brain monoamine oxidase B and A in human parkinsonian dopamine deficiency disorders.

    Science.gov (United States)

    Tong, Junchao; Rathitharan, Gausiha; Meyer, Jeffrey H; Furukawa, Yoshiaki; Ang, Lee-Cyn; Boileau, Isabelle; Guttman, Mark; Hornykiewicz, Oleh; Kish, Stephen J

    2017-09-01

    See Jellinger (doi:10.1093/awx190) for a scientific commentary on this article. The enzyme monoamine oxidases (B and A subtypes, encoded by MAOB and MAOA, respectively) are drug targets in the treatment of Parkinson's disease. Inhibitors of MAOB are used clinically in Parkinson's disease for symptomatic purposes whereas the potential disease-modifying effect of monoamine oxidase inhibitors is debated. As astroglial cells express high levels of MAOB, the enzyme has been proposed as a brain imaging marker of astrogliosis, a cellular process possibly involved in Parkinson's disease pathogenesis as elevation of MAOB in astrocytes might be harmful. Since brain monoamine oxidase status in Parkinson's disease is uncertain, our objective was to measure, by quantitative immunoblotting in autopsied brain homogenates, protein levels of both monoamine oxidases in three different degenerative parkinsonian disorders: Parkinson's disease (n = 11), multiple system atrophy (n = 11), and progressive supranuclear palsy (n = 16) and in matched controls (n = 16). We hypothesized that if MAOB is 'substantially' localized to astroglial cells, MAOB levels should be generally associated with standard astroglial protein measures (e.g. glial fibrillary acidic protein). MAOB levels were increased in degenerating putamen (+83%) and substantia nigra (+10%, non-significant) in multiple system atrophy; in caudate (+26%), putamen (+27%), frontal cortex (+31%) and substantia nigra (+23%) of progressive supranuclear palsy; and in frontal cortex (+33%), but not in substantia nigra of Parkinson's disease, a region we previously reported no increase in astrocyte protein markers. Although the magnitude of MAOB increase was less than those of standard astrocytic markers, significant positive correlations were observed amongst the astrocyte proteins and MAOB. Despite suggestions that MAOA (versus MAOB) is primarily responsible for metabolism of dopamine in dopamine neurons, there was no loss of the

  18. A role for NADPH oxidase in antigen presentation

    Directory of Open Access Journals (Sweden)

    Gail J Gardiner

    2013-09-01

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

  19. Cardiovascular activity of rasagiline, a selective and potent inhibitor of mitochondrial monoamine oxidase B: comparison with selegiline.

    Science.gov (United States)

    Abassi, Zaid A; Binah, Ofer; Youdim, Moussa B H

    2004-10-01

    Selegiline is used for treating Parkinson's disease. Despite its efficacy, the clinical use of selegiline in combination with l-dihydroxphenylalanine in Parkinsonian patients is hampered by cardiovascular complications, such as hypotension. This study was designed to compare in rats the cardiovascular effects of selegiline and rasagiline, their metabolites l-methamphetamine and aminoindan (TVP-136), respectively, and the second rasagiline metabolite non-monoamine oxidase (MAO) inhibitor TVP-1022 (N-propargyl-1S(-)aminoindan). Intravenous (i.v.) administration of selegiline and rasagiline (1 mg kg(-1)) to anaesthetized rats (thiobutabarbital, 100 mg kg(-1), i.p.) did not affect mean arterial pressure (MAP), carotid blood flow (CBF) or carotid vascular resistance (CVR). Selegiline (10 mg kg(-1), i.v.) decreased MAP, CBF and increased CVR. In contrast, rasagiline (10 mg kg(-1), i.v.) caused a small transient decrease in MAP, while CBF and CVR were unchanged. l-methamphetamine (1 mg kg(-1), i.v.) administration provoked a dramatic and long-lasting depressor response, decreased CBF and increased CVR. In contrast, injection of aminoindan or TVP-1022 at a similar dose produced gradual nonsignificant decreases in MAP and CBF. Chronic oral treatment (21 days) of awake rats with selegiline at 10 mg kg(-1) decreased systolic blood pressure (SBP), diastolic blood pressure (DBP), and MAP, whereas heart rate was unaffected. Since the effective MAO-B inhibitory and clinical dose of rasagiline is about one-tenth that of selegiline, administration of 1 mg kg(-1) day(-1) rasagiline resulted in moderate decreases in SBP, DBP, and MAP, which were significantly lower than those caused by the 10 mg kg(-1) day(-1) dose of selegiline. These findings indicate that rasagiline, when given at doses equivalent to selegiline, is less likely to be hypotensive.

  20. Cognitive aspects of congenital learned helplessness and its reversal by the monoamine oxidase (MAO)-B inhibitor deprenyl.

    Science.gov (United States)

    Schulz, Daniela; Mirrione, Martine M; Henn, Fritz A

    2010-02-01

    Cognitive processes are assumed to change with learned helplessness, an animal model of depression, but little is known about such deficits. Here we investigated the role of cognitive and related functions in selectively bred helpless (cLH, n=10), non-helpless (cNLH, n=12) and wild type (WT, n=8) Sprague Dawley rats. The animals were exposed to an open field for 10min on each of two test days. On the third day, an object exploration paradigm was carried out. The animals were later tested for helplessness. Both cLH and cNLH rats were more active than WTs on the first day in the open field. Over trials, cNLH and WT rats lowered their activity less than cLH rats. This resistance-to-habituation co-varied with a resistance to develop helplessness. In cLH rats, higher 'anxiety' or less time spent in the center of the open field co-varied with severe helplessness. In WTs, a greater reactivity to novel objects and to a spatially relocated object predicted lower levels of helplessness. In cLH rats (n=4-5 per group), chronic treatment with a high dose of the monoamine oxidase (MAO)-B inhibitor deprenyl (10mg/kg; i.p.), an anti-Parkinson, nootropic and antidepressant drug, attenuated helplessness. Remarkably, helplessness reversal required the experience of repeated test trials, reminiscent of a learning process. Chronic deprenyl (10mg/kg; i.p.) did not alter locomotion/exploration or 'anxiety' in the open field. In conclusion, helplessness may be related to altered mechanisms of reinforcement learning and working memory, and to abnormalities in MAO-A and/or MAO-B functioning. Copyright 2009 Elsevier Inc. All rights reserved.

  1. NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs.

    Science.gov (United States)

    Saez, Fara; Hong, Nancy J; Garvin, Jeffrey L

    2018-05-01

    Luminal flow augments Na + reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na + , playing a key role in its homeostasis. Whether flow elevations enhance Na + -K + -2Cl - cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.

  2. Lysyl Oxidase Plays a Critical Role in Endothelial Cell Stimulation to Drive Tumor Angiogenesis

    DEFF Research Database (Denmark)

    Baker, Ann-Marie; Bird, Demelza; Welti, Jonathan C

    2013-01-01

    Identification of key molecules that drive angiogenesis is critical for the development of new modalities for the prevention of solid tumor progression. Using multiple models of colorectal cancer, we show that activity of the extracellular matrix-modifying enzyme lysyl oxidase (LOX) is essential...... for stimulating endothelial cells in vitro and angiogenesis in vivo. We show that LOX activates Akt through platelet-derived growth factor receptor ß (PDGFRß) stimulation, resulting in increased VEGF expression. LOX-driven angiogenesis can be abrogated through targeting LOX directly or using inhibitors of PDGFRß...

  3. Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

    Science.gov (United States)

    Kana, Bavesh D.; Weinstein, Edward A.; Avarbock, David; Dawes, Stephanie S.; Rubin, Harvey; Mizrahi, Valerie

    2001-01-01

    The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 102 Pa of pO2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 102 Pa of pO2 or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that

  4. DL-3-n-butylphthalide-Edaravone hybrids as novel dual inhibitors of amyloid-β aggregation and monoamine oxidases with high antioxidant potency for Alzheimer's therapy.

    Science.gov (United States)

    Qiang, Xiaoming; Li, Yan; Yang, Xia; Luo, Li; Xu, Rui; Zheng, Yunxiaozhu; Cao, Zhongcheng; Tan, Zhenghuai; Deng, Yong

    2017-02-15

    Considering the complex etiology of Alzheimer's disease (AD), multifunctional agents may be beneficial for the treatment of this disease. A series of DL-3-n-butylphthalide-Edaravone hybrids were designed, synthesized and evaluated as novel dual inhibitors of amyloid-β aggregation and monoamine oxidases. Among them, compounds 9a-d exhibited good inhibition of self-induced Aβ 1-42 aggregation with inhibition ratio 57.7-71.5%. For MAO, these new hybrids exhibited good balance of inhibition for MAO-A and MAO-B. In addition, all target compounds retained the antioxidant activity of edaravone, showed equal or better antioxidant activity than edaravone. The results of the parallel artificial membrane permeability assay for blood-brain barrier indicated that compounds 9a-d would be able to cross the blood-brain barrier and reach their biological targets in the central nervous system. The promising results in all assays demonstrated that the strategy behind the designing of compounds was rational and favourable. Taken together, these preliminary findings suggested that the compounds with the strongest bioactivity deserves further investigated for pharmacological development in AD therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Understanding the Molecular Determinant of Reversible Human Monoamine Oxidase B Inhibitors Containing 2H-Chromen-2-One Core: Structure-Based and Ligand-Based Derived Three-Dimensional Quantitative Structure-Activity Relationships Predictive Models.

    Science.gov (United States)

    Mladenović, Milan; Patsilinakos, Alexandros; Pirolli, Adele; Sabatino, Manuela; Ragno, Rino

    2017-04-24

    Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of aryalkylamines neurotransmitters with concomitant reduction of oxygen to hydrogen peroxide. Consequently, the enzyme's malfunction can induce oxidative damage to mitochondrial DNA and mediates development of Parkinson's disease. Thus, MAO B emerges as a promising target for developing pharmaceuticals potentially useful to treat this vicious neurodegenerative condition. Aiming to contribute to the development of drugs with the reversible mechanism of MAO B inhibition only, herein, an extended in silico-in vitro procedure for the selection of novel MAO B inhibitors is demonstrated, including the following: (1) definition of optimized and validated structure-based three-dimensional (3-D) quantitative structure-activity relationships (QSAR) models derived from available cocrystallized inhibitor-MAO B complexes; (2) elaboration of SAR features for either irreversible or reversible MAO B inhibitors to characterize and improve coumarin-based inhibitor activity (Protein Data Bank ID: 2V61 ) as the most potent reversible lead compound; (3) definition of structure-based (SB) and ligand-based (LB) alignment rule assessments by which virtually any untested potential MAO B inhibitor might be evaluated; (4) predictive ability validation of the best 3-D QSAR model through SB/LB modeling of four coumarin-based external test sets (267 compounds); (5) design and SB/LB alignment of novel coumarin-based scaffolds experimentally validated through synthesis and biological evaluation in vitro. Due to the wide range of molecular diversity within the 3-D QSAR training set and derived features, the selected N probe-derived 3-D QSAR model proves to be a valuable tool for virtual screening (VS) of novel MAO B inhibitors and a platform for design, synthesis and evaluation of novel active structures. Accordingly, six highly active and selective MAO B inhibitors (picomolar to low nanomolar range of activity) were disclosed as a

  6. Resveratrol prevents high glucose-induced epithelial-mesenchymal transition in renal tubular epithelial cells by inhibiting NADPH oxidase/ROS/ERK pathway.

    Science.gov (United States)

    He, Ting; Guan, Xu; Wang, Song; Xiao, Tangli; Yang, Ke; Xu, Xinli; Wang, Junping; Zhao, Jinghong

    2015-02-15

    Resveratrol (RSV) is reported to have renoprotective activity against diabetic nephropathy, while the mechanisms underlying its function have not been fully elucidated. In this study, we investigate the effect and related mechanism of RSV against high glucose-induced epithelial to mesenchymal transition (EMT) in human tubular epithelial cells (HK-2). A typical EMT is induced by high glucose in HK-2 cells, accompanied by increased levels of reactive oxygen species (ROS). RSV exhibits a strong ability to inhibit high glucose-induced EMT by decreasing intracellular ROS levels via down-regulation of NADPH oxidase subunits NOX1 and NOX4. The activation of extracellular signal-regulated kinase (ERK1/2) is found to be involved in high glucose-induced EMT in HK-2 cells. RSV, like NADPH oxidase inhibitor diphenyleneiodonium, can block ERK1/2 activation induced by high glucose. Our results demonstrate that RSV is a potent agent against high glucose-induced EMT in renal tubular cells via inhibition of NADPH oxidase/ROS/ERK1/2 pathway. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Design, synthesis, pharmacological evaluation, QSAR analysis, molecular modeling and ADMET of novel donepezil-indolyl hybrids as multipotent cholinesterase/monoamine oxidase inhibitors for the potential treatment of Alzheimer's disease.

    Science.gov (United States)

    Bautista-Aguilera, Oscar M; Esteban, Gerard; Bolea, Irene; Nikolic, Katarina; Agbaba, Danica; Moraleda, Ignacio; Iriepa, Isabel; Samadi, Abdelouahid; Soriano, Elena; Unzeta, Mercedes; Marco-Contelles, José

    2014-03-21

    The design, synthesis, and pharmacological evaluation of donepezil-indolyl based amines 7-10, amides 12-16, and carboxylic acid derivatives 5 and 11, as multipotent ASS234 analogs, able to inhibit simultaneously cholinesterase (ChE) and monoamine oxidase (MAO) enzymes for the potential treatment of Alzheimer's disease (AD), is reported. Theoretical studies using 3D-Quantitative Structure-Activity Relationship (3D-QSAR) was used to define 3D-pharmacophores for inhibition of MAO A/B, AChE, and BuChE enzymes. We found that, in general, and for the same substituent, amines are more potent ChE inhibitors (see compounds 12, 13 versus 7 and 8) or equipotent (see compounds 14, 15 versus 9 and 10) than the corresponding amides, showing a clear EeAChE inhibition selectivity. For the MAO inhibition, amides were not active, and among the amines, compound 14 was totally MAO A selective, while amines 15 and 16 were quite MAO A selective. Carboxylic acid derivatives 5 and 11 showed a multipotent moderate selective profile as EeACE and MAO A inhibitors. Propargylamine 15 [N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)prop-2-yn-1-amine] resulted in the most potent hMAO A (IC50 = 5.5 ± 1.4 nM) and moderately potent hMAO B (IC50 = 150 ± 31 nM), EeAChE (IC50 = 190 ± 10 nM), and eqBuChE (IC50 = 830 ± 160 nM) inhibitor. However, the analogous N-allyl and the N-morpholine derivatives 16 and 14 deserve also attention as they show an attractive multipotent profile. To sum up, donepezil-indolyl hybrid 15 is a promising drug for further development for the potential prevention and treatment of AD. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  8. Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter

    Directory of Open Access Journals (Sweden)

    C. Massart

    2014-01-01

    Full Text Available NADPH oxidases (NOXes and dual oxidases (DUOXes generate O2.− and H2O2. Diphenyleneiodonium (DPI inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog 99mTcO4− nor of the K+ cation mimic 86Rb+ in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

  9. Design and synthesis of novel chalcones as potent selective monoamine oxidase-B inhibitors.

    Science.gov (United States)

    Hammuda, Arwa; Shalaby, Raed; Rovida, Stefano; Edmondson, Dale E; Binda, Claudia; Khalil, Ashraf

    2016-05-23

    A novel series of substituted chalcones were designed and synthesized to be evaluated as selective human MAO-B inhibitors. A combination of either methylsulfonyl or trifluoromethyl substituents on the aromatic ketone moiety with a benzodioxol ring on the other end of the chalcone scaffold was investigated. The compounds were tested for their inhibitory activities on both human MAO-A and B. All compounds appeared to be selective MAO-B inhibitors with Ki values in the micromolar to submicromolar range. Molecular modeling studies have been performed to get insight into the binding mode of the synthesized compounds to human MAO-B active site. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  10. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.L.; Rajagopalan, K.V. (Duke Univ. Medical Center, Durham, NC (USA)); London, R.E. (National Institute of Environmental Health Science, Research Triangle Park, NC (USA))

    1989-09-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and {sup 31}P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.

  11. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    International Nuclear Information System (INIS)

    Johnson, J.L.; Rajagopalan, K.V.; London, R.E.

    1989-01-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and 31 P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well

  12. Polyphenol Oxidase Enzyme and Inactivation Methods

    Directory of Open Access Journals (Sweden)

    Leman Yılmaz

    2018-03-01

    Full Text Available Polyphenol oxidase enzyme is found in vegetables and fruits, as well as in some animal organs and microorganisms. Polyphenol oxidase enzyme responsible for enzymatic browning is a group of copper proteins that catalyses the oxidation of phenolic compounds to quinones, which produce brown pigments, commonly found in fruits and vegetables. During the industrial preparation of fruits and vegetables, results of catalytic effect of polyphenol oxidase causes enzymatic browning. Enzymatic browning impairs the appearance of products containing phenolic compounds along with undesirable colour, odor and taste formation and significant loss of nutritional value of the products. This affects the acceptability of the products by the consumers and causes economic losses. In this review, some characteristics of polyphenol oxidase enzyme in different fruits and vegetables have been reviewed and information about chemical antibrowning agents, thermal applications, irradiation applications and alternative methods such as high pressure processing, pulse electric field, supercritical carbon dioxide and ultrasound applications to inactivate this enzyme has been presented.

  13. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    María A. Hidalgo

    2015-01-01

    Full Text Available N-Formyl-methionyl-leucyl-phenylalanine (fMLP and platelet-activating factor (PAF induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8 release and nicotinamide adenine dinucleotide phosphate reduced (NADPH oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP, diphenyleneiodonium (DPI, and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

  14. Testicular Sperm Count and Oxidative Stress Profile in gamma-Irradiated Rats and Response to Treatment with Xanthine Oxidase Inhibitor ''Allopurinol''

    International Nuclear Information System (INIS)

    Tawfik, S.S.; Zahran, A.M.; Azab, Kh.Sh.

    2006-01-01

    Allopurinol is an inhibitor of xanthine oxidase (X O) enzyme and could be useful radiomodifer. X O is present in almost all tissues of mammals. It is considered being one of the major sources of free radicals in the biological systems. The testis is known to be one of the most radiosensitive organs in mammals. Thus, protection for reproductive potential and heredity in the germ cells of these mammals against radiation damage is recommended. Role of allopurinol in ameliorating radiation damage on sperm count and oxidative response in testis of irradiated rats. Male rats were randomly divided into 4 groups (n=12). Group (I) served as controls. Group (II) received i.p., technical allopurinol (30 mg/kg body wt/day suspended in 0.5 ml of sterilized saline for 22 successive days). Group (III) submitted to whole body gamma-radiation as fractionated doses at 1 Gy installment till 8 Gy on 1st, 4th, 7th, 10th, 13th, 16th, 19th and 22nd days of treatment. Group (VI) administrated i.p., technical allopurinol, during which animals were exposed to gamma-irradiation protocol. Experimental observations were performed on the 1st and 15th days after last irradiation fraction or end of treatment. Irradiation resulted in decreases in the levels of total proteins, GSH, NO, DNA and RNA and activities of SOD and Catalase (CAT) in the testicular homogenate. On the contrary, irradiation produced increases in levels of LPX (TBARS) and H 2 O 2 and activities of X O and LDH

  15. The terminal oxidases of Paracoccus denitrificans

    NARCIS (Netherlands)

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

    Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to

  16. Quantitation of immunoadsorbed flavoprotein oxidases by luminol-mediated chemiluminescence.

    Science.gov (United States)

    Hinkkanen, A; Maly, F E; Decker, K

    1983-04-01

    The detection of the flavoenzymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase at the sub-femtomol level was achieved by coupling the reaction of the immunoadsorbed proteins to the peroxidase-catalysed oxidation of luminol. The H2O2-producing oxidases retained their full activity when bound to the respective immobilized antibodies. This fact allowed the concentration of the enzymes from very dilute solutions and the quantitative assay of their activities in the microU range. Due to strict stereoselectivity and the absence of immunological cross-reactivity, the two flavoproteins could be determined in the same solution. This method was used to measure the 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase activities in Escherichia coli RR1 and different Arthrobacter strains cultured under non-inducing conditions. The same activity ratio of 6-hydroxy-L-nicotine oxidase/6-hydroxy-D-nicotine oxidase as in D L-nicotine-induced cells of A. oxidans was observed in non-induced wild type and in riboflavin-requiring (rf-) mutant cells of this aerob.

  17. The monoamine oxidase inhibition properties of selected structural analogues of methylene blue

    International Nuclear Information System (INIS)

    Delport, Anzelle; Harvey, Brian H.; Petzer, Anél; Petzer, Jacobus P.

    2017-01-01

    The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, the present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC 50 = 0.0037 μM), Nile blue (IC 50 = 0.0077 μM) and 1,9-dimethyl methylene blue (IC 50 = 0.018 μM) exhibiting higher potency inhibition compared to MB (IC 50 = 0.07 μM). Nile blue also represents a potent MAO-B inhibitor with an IC 50 value of 0.012 μM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST. - Highlights: • MB analogues, cresyl violet and Nile blue, are high potency MAO-A inhibitors. • Nile blue also represents a potent MAO-B inhibitor. • Potent MAO-A inhibition should alert to potential serotonin toxicity.

  18. The monoamine oxidase inhibition properties of selected structural analogues of methylene blue

    Energy Technology Data Exchange (ETDEWEB)

    Delport, Anzelle [Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520 (South Africa); Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520 (South Africa); Harvey, Brian H. [Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520 (South Africa); Pharmacology, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520 (South Africa); Petzer, Anél [Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520 (South Africa); Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520 (South Africa); Petzer, Jacobus P., E-mail: jacques.petzer@nwu.ac.za [Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520 (South Africa); Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520 (South Africa)

    2017-06-15

    The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, the present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC{sub 50} = 0.0037 μM), Nile blue (IC{sub 50} = 0.0077 μM) and 1,9-dimethyl methylene blue (IC{sub 50} = 0.018 μM) exhibiting higher potency inhibition compared to MB (IC{sub 50} = 0.07 μM). Nile blue also represents a potent MAO-B inhibitor with an IC{sub 50} value of 0.012 μM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST. - Highlights: • MB analogues, cresyl violet and Nile blue, are high potency MAO-A inhibitors. • Nile blue also represents a potent MAO-B inhibitor. • Potent MAO-A inhibition should alert to potential serotonin toxicity.

  19. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72 isoforms

    Directory of Open Access Journals (Sweden)

    Sehgal SA

    2015-07-01

    Full Text Available Sheikh Arslan Sehgal,1,2 Shazia Mannan,2,* Sumaira Kanwal,2,* Ishrat Naveed,1 Asif Mir1 1Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan; 2Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal, Pakistan *These authors contributed equally to this work Abstract: Schizophrenia (SZ, a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with d-amino acid oxidase activator (DAOA, G72. Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor–ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ. Keywords: schizophrenia, bioinformatics, modeling, docking, DAOA, G72, DAO, computer-aided drug designing, phylogenetic analysis, d-amino acid oxidase

  20. Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

    Science.gov (United States)

    Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

    2010-07-01

    Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

  1. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    OpenAIRE

    Jänne, J; Morris, D R

    1984-01-01

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylm...

  2. Confirmation of a blocked amino terminus of sulfhydryl oxidase

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  3. Immobilization of oxidases and their analytical applications

    International Nuclear Information System (INIS)

    Yasinzai, M.

    2007-01-01

    Immobilized enzymes are replacing their soluble counter-parts in nearly every field of application. These enzyme modifications have evolved from a research curiosity into an entire branch of Biotechnology. An immobilization method for flavin containing oxidases and their use in flow injection system is described. An electrochemical detector for H/sub 2/O/sub 2/ is assembled which is used effectively for the determination of glucose using more common glucose oxidase and the simultaneous determination of sugars. The combination of oxidases with hydrolases have been used for the determination of maltose and starch. (author)

  4. NADPH Oxidases: Progress and Opportunities

    OpenAIRE

    San Martin, Alejandra; Griendling, Kathy K.

    2014-01-01

    From the initial discovery in 1999 that NADPH oxidases comprise a family of enzymes to our current focus on drug development to treat multiple pathologies related to this enzyme family, progress has been swift and impressive. We have expanded our understanding of the extent of the family, the basic enzymatic biochemistry, the multiple cellular functions controlled by NADPH oxidases, and their varied roles in physiology and diseases. We have developed numerous cell culture tools, animal models...

  5. Gravity Responsive NADH Oxidase of the Plasma Membrane

    Science.gov (United States)

    Morre, D. James (Inventor)

    2002-01-01

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

  6. Molecular mechanisms of the microsomal mixed function oxidases and biological and pathological implications

    Directory of Open Access Journals (Sweden)

    Arthur I. Cederbaum

    2015-04-01

    Full Text Available The cytochrome P450 mixed function oxidase enzymes play a major role in the metabolism of important endogenous substrates as well as in the biotransformation of xenobiotics. The liver P450 system is the most active in metabolism of exogenous substrates. This review briefly describes the liver P450 (CYP mixed function oxidase system with respect to its enzymatic components and functions. Electron transfer by the NADPH-P450 oxidoreductase is required for reduction of the heme of P450, necessary for binding of molecular oxygen. Binding of substrates to P450 produce substrate binding spectra. The P450 catalytic cycle is complex and rate-limiting steps are not clear. Many types of chemical reactions can be catalyzed by P450 enzymes, making this family among the most diverse catalysts known. There are multiple forms of P450s arranged into families based on structural homology. The major drug metabolizing CYPs are discussed with respect to typical substrates, inducers and inhibitors and their polymorphic forms. The composition of CYPs in humans varies considerably among individuals because of sex and age differences, the influence of diet, liver disease, presence of potential inducers and/or inhibitors. Because of such factors and CYP polymorphisms, and overlapping drug specificity, there is a large variability in the content and composition of P450 enzymes among individuals. This can result in large variations in drug metabolism by humans and often can contribute to drug–drug interactions and adverse drug reactions. Because of many of the above factors, especially CYP polymorphisms, there has been much interest in personalized medicine especially with respect to which CYPs and which of their polymorphic forms are present in order to attempt to determine what drug therapy and what dosage would reflect the best therapeutic strategy in treating individual patients.

  7. A broad distribution of the alternative oxidase in microsporidian parasites.

    Directory of Open Access Journals (Sweden)

    Bryony A P Williams

    2010-02-01

    Full Text Available Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX, a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1 as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.

  8. Analysis of monoamine oxidase (MAO) enzymatic activity by high-performance liquid chromatography-diode array detection combined with an assay of oxidation with a peroxidase and its application to MAO inhibitors from foods and plants.

    Science.gov (United States)

    Herraiz, Tomás; Flores, Andrea; Fernández, Lidia

    2018-01-15

    Monoamine oxidase (MAO) enzymes catalyze the oxidative deamination of biogenic amines and neurotransmitters and produce ammonia, aldehydes, and hydrogen peroxide which is involved in oxidative processes. Inhibitors of MAO-A and -B isozymes are useful as antidepressants and neuroprotectants. The assays of MAO usually measure amine oxidation products or hydrogen peroxide by spectrophotometric techniques. Those assays are often compromised by interfering compounds resulting in poor results. This research describes a new method that combines in the same assay the oxidative deamination of kynuramine to 4-hydroxyquinoline analyzed by HPLC-DAD with the oxidation of tetramethylbenzidine (TMB) (or Amplex Rex) by horseradish peroxidase (HRP) in presence of hydrogen peroxide. The new method was applied to study the inhibition of human MAO-A and -B by bioactive compounds including β-carboline alkaloids and flavonoids occurring in foods and plants. As determined by HPLC-DAD, β-carbolines, methylene blue, kaempferol and clorgyline inhibited MAO-A and methylene blue, 5-nitroindazole, norharman and deprenyl inhibited MAO-B, and all of them inhibited the oxidation of TMB in the same extent. The flavonoids catechin and cyanidin were not inhibitors of MAO by HPLC-DAD but highly inhibited the oxidation of TMB (or Amplex Red) by peroxidase whereas quercetin and resveratrol were moderate inhibitors of MAO-A by HPLC-DAD, but inhibited the peroxidase assay in a higher level. For some phenolic compounds, using the peroxidase-coupled assay to measure MAO activity led to mistaken results. The new method permits to discern between true inhibitors of MAO from those that are antioxidants and which interfere with peroxidase assays but do not inhibit MAO. For true inhibitors of MAO, inhibition as determined by HPLC-DAD correlated well with inhibition of the oxidation of TMB and this approach can be used to assess the in vitro antioxidant activity (less hydrogen peroxide production) resulting

  9. The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress.

    Science.gov (United States)

    Jakubowska, Dagmara; Janicka, Małgorzata

    2017-11-01

    The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H + -ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H + -ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H + -ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H + -ATPase and NADPH oxidase are key factors. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Calcium transport in vesicles energized by cytochrome oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Rosier, Randy N. [Univ. of Rochester, NY (United States)

    1979-01-01

    Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K+ selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K+ flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interaction with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.

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

    Directory of Open Access Journals (Sweden)

    Efthimios A. Andronis

    2014-04-01

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

  12. RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

    Science.gov (United States)

    Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

    2014-10-01

    Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM fMLF and 1μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Lysyl oxidase in colorectal cancer

    DEFF Research Database (Denmark)

    Cox, Thomas R; Erler, Janine T

    2013-01-01

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

  14. Bisulfite compounds as metabolic inhibitors: nonspecific effects on membranes

    Energy Technology Data Exchange (ETDEWEB)

    Luettge, U; Osmond, C B; Ball, E; Brinckmann, E; Kinze, G

    1972-01-01

    Bisulfite compounds are shown to be nonspecific inhibitors of photosynthetic processes and of ion transport in green tissues. CO/sub 2/ fixation and light-dependent transient changes in external pH are inhibited about 50% by 5 x 10/sup -4/M glyoxal-Na-bisulfite. Chloride uptake in the light and in the dark is inhibited to the same extent at this concentration. At 5 x 10/sup -3/M the inhibitor reduces ATP levels in the light and in the dark, and the effects on glycolate oxidase and PEP carboxylase are observed. The extent of inhibition is dependent on time of treatment with glyoxal-Na-bisulfite and freshly prepared NaHSO/sub 3/ is equally as effective as the addition compound. Possible explanations of the bisulfite effects and the relationships to SO/sub 2/ effects on photosynthesis are discussed.

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

    Science.gov (United States)

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

    2010-05-01

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

  16. Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

    Science.gov (United States)

    Shi, Xueyan; Dick, Ryan A.; Ford, Kevin A.; Casida, John E.

    2009-01-01

    Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19 and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19 and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than that of TMX. Imidacloprid (IMI), CLO and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI and 4-hydroxy-thiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid and CLO metabolism in vivo in mice, elevating the brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites. PMID:19391582

  17. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    Science.gov (United States)

    Jänne, J; Morris, D R

    1984-03-15

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.

  18. Protection by deferoxamine from endothelial injury: A possible link with inhibition of intracellular xanthine oxidase

    International Nuclear Information System (INIS)

    Rinaldo, J.E.; Gorry, M.

    1990-01-01

    Hydroxyl radical scavengers and xanthine oxidase inhibitors protect cultured bovine pulmonary endothelial cells (BPAEC) from lytic injury by the endotoxin lipopolysaccharide (LPS). We hypothesized that exposure of BPAEC to cytotoxic concentrations of LPS activated intracellular xanthine oxidase, and that intracellular iron-dependent hydroxyl radical formation (a Fenton reaction) ensued, resulting in cell lysis. To test this, the protective effects of deferoxamine against H2O2 and LPS-induced cytotoxicity to BPAEC was assessed by 51Cr release. Preincubation with 0.4 mM deferoxamine conferred 67 +/- 15% (mean +/- SE) protection from LPS-induced cytotoxicity but 48 h of preincubation were required to induce significant protection. Significant protection form a classical Fenton reaction model, injury by 50 microM H2O2, could be induced by a 1-h preincubation with a 0.4 mM deferoxamine. The dissociated time course suggested that deferoxamine might work by different mechanisms in these models. The effects of LPS and deferoxamine on BPAEC-associated xanthine oxidase (XO) and xanthine dehydrogenase (XD) activity were assessed using a spectrofluorophotometric measurement of the conversion of pterin to isoxanthopterin. BPAEC had 106 +/- 7 microU/mg XD+XO activity; XO activity constituted 48 +/- 1% of total XO+XD activity. LPS at a cytotoxic concentration did not alter XO, XD, or percent XO. Deferoxamine had striking proportional inhibitory effects on XO and XD in intact cells. XO+XD activity fell to 6 +/- 1% of control levels during a 48-h exposure of BPAEC to deferoxamine. Deferoxamine did not inhibit XO+XD ex vivo

  19. Production of rabbit antibodies against purified Glucose oxidase

    Directory of Open Access Journals (Sweden)

    Muhammad Anjum Zia

    2012-02-01

    Full Text Available Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex G-200 column for gel filtration chromatography. It was observed that enzyme achieved 59.37 U mg-1of specific activity with 27.08 fold purity and 64.36% recovery. Purified glucose oxidase was injected into rabbits through intravenous route, to raise the glucose oxidase antibodies. After 30 days incubation period, the rabbits were slaughtered and serum was separated from blood. The antibodies were isolated by ammonium sulfate precipitation and confirmed by agar gel precipitation test. This could be a convenient and low cost alternate assay for the estimation of glucose oxidase in biological fluids. Moreover, such antibodies against the said enzyme could be used in various therapeutic and diagnostic applications.

  20. Chemoenzymatic combination of glucose oxidase with titanium silicalite -1

    DEFF Research Database (Denmark)

    Vennestrøm, Peter Nicolai Ravnborg; Taarning, Esben; Christensen, Claus H.

    2010-01-01

    Zeozymes: A proof-of-concept is presented for the chemoenzymatic combination of titanium silicalite-1 zeolite with glucose oxidase. In this combination, glucose is oxidized to gluconic acid and the H2O2 byproduct formed in situ is used for the simultaneous oxidation of chemical substrates. Both...... a soluble glucose oxidase and a truly integrated heterogeneous combination whereby the oxidase enzyme is anchored onto the zeolite surface are reported....

  1. ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptor-dependent STAT3 activation.

    Directory of Open Access Journals (Sweden)

    Shin-Ei Cheng

    Full Text Available BACKGROUND: Up-regulation of cyclooxygenase (COX-2 and its metabolite prostaglandin E(2 (PGE(2 are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE(2 release remain unclear. PRINCIPAL FINDINGS: Here, we showed that ATPγS induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin, PKC (Gö6983, Gö6976, Ro318220, and Rottlerin, ROS (Edaravone, NADPH oxidase [diphenyleneiodonium chloride (DPI and apocynin], Jak2 (AG490, and STAT3 [cucurbitacin E (CBE] and transfection with siRNAs of PKCα, PKCι, PKCμ, p47(phox, Jak2, STAT3, and cPLA(2 markedly reduced ATPγS-induced COX-2 expression and PGE(2 production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPγS. Moreover, ATPγS-induced ROS generation and p47(phox translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPγS stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, Gö6983, Gö6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells. SIGNIFICANCE: Taken together, these results showed that ATPγS induced COX-2 expression and PGE(2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA(2 signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.

  2. A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.

    Science.gov (United States)

    Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

    2009-09-01

    Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.

  3. Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis.

    Science.gov (United States)

    Young, Luke; May, Benjamin; Pendlebury-Watt, Alice; Shearman, Julia; Elliott, Catherine; Albury, Mary S; Shiba, Tomoo; Inaoka, Daniel Ken; Harada, Shigeharu; Kita, Kiyoshi; Moore, Anthony L

    2014-07-01

    In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues (R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent Km (O2). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOX's catalytic cycle is controlled and since maximum activity is decreased we predict that such mutations result in an increase in the steady state level of at least one O2-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two

  4. Amine oxidases as important agents of pathological processes of rhabdomyolysis in rats.

    Science.gov (United States)

    Gudkova, O O; Latyshko, N V; Shandrenko, S G

    2016-01-01

    In this study we have tested an idea on the important role of amine oxidases (semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase) as an additional source of oxidative/carbonyl stress under glycerol-induced rhabdomyolysis, since the enhanced formation of reactive oxygen species and reactive carbonyl species in a variety of tissues is linked to various diseases. In our experiments we used the sensitive fluorescent method devised for estimation of amine oxidases activity in the rat kidney and thymus as targeted organs under rhabdomyolysis. We have found in vivo the multiple rises in activity of semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase (2-4.5 times) in the corresponding cell fractions, whole cells or their lysates at the 3-6th day after glycerol injection. Aberrant antioxidant activities depended on rhabdomyolysis stage and had organ specificity. Additional treatment of animals with metal chelator ‘Unithiol’ adjusted only the activity of antioxidant enzymes but not amine oxidases in both organs. Furthermore the in vitro experiment showed that Fenton reaction (hydrogen peroxide in the presence of iron) products alone had no effect on semicarbazide-sensitive amine oxidase activity in rat liver cell fraction whereas supplementation with methylglyoxal resulted in its significant 2.5-fold enhancement. Combined action of the both agents had additive effect on semicarbazide-sensitive amine oxidase activity. We can assume that biogenic amine and polyamine catabolism by amine oxidases is upregulated by oxidative and carbonyl stress factors directly under rhabdomyolysis progression, and the increase in catabolic products concentration contributes to tissue damage in glycerol-induced acute renal failure and apoptosis stimulation in thymus.

  5. Estrogen inhibits lysyl oxidase and decreases mechanical function in engineered ligaments.

    Science.gov (United States)

    Lee, Cassandra A; Lee-Barthel, Ann; Marquino, Louise; Sandoval, Natalie; Marcotte, George R; Baar, Keith

    2015-05-15

    Women are more likely to suffer an anterior cruciate ligament (ACL) rupture than men, and the incidence of ACL rupture in women rises with increasing estrogen levels. We used an engineered ligament model to determine how an acute rise in estrogen decreases the mechanical properties of ligaments. Using fibroblasts isolated from human ACLs from male or female donors, we engineered ligaments and determined that ligaments made from female ACL cells had more collagen and were equal in strength to those made from male ACL cells. We then treated engineered ligaments for 14 days with low (5 pg/ml), medium (50 pg/ml), or high (500 pg/ml) estrogen, corresponding to the range of in vivo serum estrogen concentrations and found that collagen within the grafts increased without a commensurate increase in mechanical strength. Mimicking the menstrual cycle, with 12 days of low estrogen followed by 2 days of physiologically high estrogen, resulted in a decrease in engineered ligament mechanical function with no change in the amount of collagen in the graft. The decrease in mechanical stiffness corresponded with a 61.7 and 76.9% decrease in the activity of collagen cross-linker lysyl oxidase with 24 and 48 h of high estrogen, respectively. Similarly, grafts treated with the lysyl oxidase inhibitor β-aminoproprionitrile (BAPN) for 24 h showed a significant decrease in ligament mechanical strength [control (CON) = 1.58 ± 0.06 N; BAPN = 1.06 ± 0.13 N] and stiffness (CON = 7.7 ± 0.46 MPa; BAPN = 6.1 ± 0.71 MPa) without changing overall collagen levels (CON = 396 ± 11.5 μg; BAPN = 382 ± 11.6 μg). Together, these data suggest that the rise in estrogen during the follicular phase decreases lysyl oxidase activity in our engineered ligament model and if this occurs in vivo may decrease the stiffness of ligaments and contribute to the elevated rate of ACL rupture in women. Copyright © 2015 the American Physiological Society.

  6. Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin

    NARCIS (Netherlands)

    Heuvel, van den R.H.H.; Berg, van den W.A.M.; Rovida, S.; Berkel, van W.J.H.

    2004-01-01

    The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol

  7. Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte thellungiella: role of the plastid terminal oxidase as an alternative electron sink.

    Science.gov (United States)

    Stepien, Piotr; Johnson, Giles N

    2009-02-01

    The effects of short-term salt stress on gas exchange and the regulation of photosynthetic electron transport were examined in Arabidopsis (Arabidopsis thaliana) and its salt-tolerant close relative Thellungiella (Thellungiella halophila). Plants cultivated on soil were challenged for 2 weeks with NaCl. Arabidopsis showed a much higher sensitivity to salt than Thellungiella; while Arabidopsis plants were unable to survive exposure to greater than 150 mM salt, Thellugiella could tolerate concentrations as high as 500 mM with only minimal effects on gas exchange. Exposure of Arabidopsis to sublethal salt concentrations resulted in stomatal closure and inhibition of CO2 fixation. This lead to an inhibition of electron transport though photosystem II (PSII), an increase in cyclic electron flow involving only PSI, and increased nonphotochemical quenching of chlorophyll fluorescence. In contrast, in Thellungiella, although gas exchange was marginally inhibited by high salt and PSI was unaffected, there was a large increase in electron flow involving PSII. This additional electron transport activity is oxygen dependent and sensitive to the alternative oxidase inhibitor n-propyl gallate. PSII electron transport in Thellungiella showed a reduced sensitivity to 2'-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenylether, an inhibitor of the cytochrome b6f complex. At the same time, we observed a substantial up-regulation of a protein reacting with antibodies raised against the plastid terminal oxidase. No such up-regulation was seen in Arabidopsis. We conclude that in salt-stressed Thellungiella, plastid terminal oxidase acts as an alternative electron sink, accounting for up to 30% of total PSII electron flow.

  8. JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

    Directory of Open Access Journals (Sweden)

    Ling Yan

    2013-01-01

    Full Text Available Excessive fluoride may cause central nervous system (CNS dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS, and NADPH oxidase (NOX is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells.

  9. JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

    Science.gov (United States)

    Yan, Ling; Liu, Shengnan; Wang, Chen; Wang, Fei; Song, Yingli; Yan, Nan; Xi, Shuhua; Liu, Ziyou; Sun, Guifan

    2013-01-01

    Excessive fluoride may cause central nervous system (CNS) dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS), and NADPH oxidase (NOX) is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular O2 ·− and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL) resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells. PMID:24072958

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

    Science.gov (United States)

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

    2016-07-09

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

  11. Correlation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity with blood-brain barrier monoamine oxidase activity

    International Nuclear Information System (INIS)

    Kalaria, R.N.; Mitchell, M.J.; Harik, S.I.

    1987-01-01

    Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes parkinsonism in humans and subhuman primates, but not in rats and many other laboratory animals; mice are intermediate in their susceptibility. Since MPTP causes selective dopaminergic neurotoxicity when infused directly into rat substantia nigra, the authors hypothesized that systemic MPTP may be metabolized by monoamine oxidase and/or other enzymes in rat brain capillaries and possibly other peripheral organs and thus prevented from reaching its neuronal sites of toxicity. They tested this hypothesis by assessing monoamine oxidase in isolated cerebral microvessels of humans, rats, and mice by measuring the specific binding of [ 3 H]pargyline, an irreversible monoamine oxidase inhibitor, and by estimating the rates of MPTP and benzylamine oxidation. [ 3 H]Pargyline binding to rat cerebral microvessels was about 10-fold higher than to human or mouse microvessels. Also, MPTP oxidation by rat brain microvessels was about 30-fold greater than by human microvessels; mouse microvessels yielded intermediate values. These results may explain, at least in part, the marked species differences in susceptibility to systemic MPTP. They also suggest the potential importance of enzyme barriers at the blood-brain interface that can metabolize toxins not excluded by structural barriers, and may provide biological bases for developing therapeutic strategies for the prevention of MPTP-induced neurotoxicity and other neurotoxic conditions including, possibly, Parkinson's disease

  12. Iododerivative of pargyline: A potential tracer for the exploration of monoamine oxidase sites by SPECT

    International Nuclear Information System (INIS)

    Lena, Isabelle; Ombetta, Jean-Edouard; Chalon, Sylvie; Dognon, Anne-Marie; Baulieu, Jean-Louis; Frangin, Yves; Garreau, Lucette; Besnard, Jean-Claude; Guilloteau, Denis

    1995-01-01

    Monoamine oxidases are important in the regulation of monoaminergic neurotransmission. An increase in monoamine oxidase B (MAO B) has been observed in some neurodegenerative diseases, and therefore quantification of cerebral MAO B activity by SPECT would be useful for the diagnosis and therapeutic follow-up of these disorders. We have developed an iodinated derivative of pargyline, a selective inhibitor of MAO B, in order to explore this enzyme by SPECT. Stable bromo and iodo derivatives of pargyline were synthesized and chemically characterized. The radioiodinated ligand [ 125 I]-2-iodopargyline was obtained with high specific activity from the bromo precursor by nucleophilic exchange. Affinity and selectivity of 2-iodopargyline were tested in vitro. Biodistribution study of [ 125 I]-2-iodopargyline was performed in rats. Radioiodinated ligand were obtained in a no-carrier-added form. 2-iodopargyline has a higher in vitro affinity for MAO B than pargyline. However, the in vitro selectivity for MAO B was better for pargyline than for 2-iodopargyline. Ex vivo autoradiographic studies and in vivo saturation studies with selective inhibitors of MAO showed that the cerebral biodistribution of [ 125 I]-2-iodopargyline in the rat is consistent with high level binding to MAO B sites in the pineal gland and in the thalamus. In conclusion, 2-iodopargyline preferentially binds in vivo to MAO B sites with high affinity. However, its selectivity for MAO B in rats is not very high, whereas this ligand binds to a lesser extent to MAO A. It will be then of great value to evaluate the specificity of 2-iodopargyline in humans. This new ligand labeled with 123 I should therefore be a suitable tool for SPECT exploration of MAO B in the human brain

  13. EPA:DHA 6:1 prevents angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase- and COX-derived oxidative stress.

    Science.gov (United States)

    Niazi, Zahid Rasul; Silva, Grazielle C; Ribeiro, Thais Porto; León-González, Antonio J; Kassem, Mohamad; Mirajkar, Abdur; Alvi, Azhar; Abbas, Malak; Zgheel, Faraj; Schini-Kerth, Valérie B; Auger, Cyril

    2017-12-01

    Eicosapentaenoic acid:docosahexaenoic acid (EPA:DHA) 6:1, an omega-3 polyunsaturated fatty acid formulation, has been shown to induce a sustained formation of endothelial nitric oxide (NO) synthase-derived NO, a major vasoprotective factor. This study examined whether chronic intake of EPA:DHA 6:1 prevents hypertension and endothelial dysfunction induced by angiotensin II (Ang II) in rats. Male Wister rats received orally corn oil or EPA:DHA 6:1 (500 mg kg -1 per day) before chronic infusion of Ang II (0.4 mg kg -1 per day). Systolic blood pressure was determined by tail cuff sphingomanometry, vascular reactivity using a myograph, oxidative stress using dihydroethidium and protein expression by immunofluorescence and western blot analysis. Ang II-induced hypertension was associated with reduced acetylcholine-induced relaxations of secondary branch mesenteric artery rings affecting the endothelium-dependent hyperpolarization (EDH)- and the NO-mediated relaxations, both of which were improved by the NADPH oxidase inhibitor VAS-2870. The Ang II treatment induced also endothelium-dependent contractile responses (EDCFs), which were abolished by the cyclooxygenase (COX) inhibitor indomethacin. An increased level of vascular oxidative stress and expression of NADPH oxidase subunits (p47 phox and p22 phox ), COX-1 and COX-2, endothelial NO synthase and Ang II type 1 receptors were observed in the Ang II group, whereas SK Ca and connexin 37 were downregulated. Intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction by improving both the NO- and EDH-mediated relaxations, and by reducing EDCFs and the expression of target proteins. The present findings indicate that chronic intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction in rats, most likely by preventing NADPH oxidase- and COX-derived oxidative stress.

  14. Optimization of glucose oxidase production by Aspergillus niger

    African Journals Online (AJOL)

    user

    2011-02-28

    Feb 28, 2011 ... manganese, cobalt, thioglycolic acid, and gluconic acid according to (Liu et al., .... In this experiment duplicate media of glucose 10% were adjusted at different ... Glucose oxidase as a pharmaceutical anti oxidant Drug. Devt. ... Plush KS, Hellmuth K, Rinas U (1996). kinetics of glucose oxidase excretion by ...

  15. Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase

    NARCIS (Netherlands)

    Muntyan, M.S.; Cherepanov, D.A.; Malinen, A.M.; Bloch, D.A.; Sorokin, D.Y.; Severina, I.I.; Ivashina, T.V.; Lahti, R.; Muyzer, G.; Skulachev, V.P.

    2015-01-01

    Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which

  16. Serum diamine oxidase activity in patients with histamine intolerance.

    Science.gov (United States)

    Manzotti, G; Breda, D; Di Gioacchino, M; Burastero, S E

    2016-03-01

    Intolerance to various foods, excluding bona fide coeliac disease and lactose intolerance, represents a growing cause of patient visits to allergy clinics.Histamine intolerance is a long-known, multifaceted clinical condition triggered by histamine-rich foods and alcohol and/or by drugs that liberate histamine or block diamine oxidase (DAO), the main enzyme involved in the metabolism of ingested histamine. Histamine limitation diets impose complex, non-standardized restrictions that may severely impact the quality of life of patients. We retrospectively evaluated 14 patients who visited allergy outpatient facilities in northern Italy with a negative diagnosis for IgE-mediated food hypersensitivity, coeliac disease, conditions related to gastric hypersecretion, and systemic nickel hypersensitivity, and who previously underwent a histamine limitation diet with benefits for their main symptoms. Serum diamine oxidase levels and the clinical response to diamine oxidase supplementation were investigated. We found that 10 out of 14 patients had serum DAO activityintolerance. Moreover, 13 out of 14 patients subjectively reported a benefit in at least one of the disturbances related to food intolerances following diamine oxidase supplementation. The mean value (±SD) of diamine oxidase activity in the cohort of patients with histamine intolerance symptoms was 7.04±6.90 U/mL compared to 39.50±18.16 U/mL in 34 healthy controls (P=0.0031). In patients with symptoms triggered by histamine-rich food, measuring the serum diamine oxidase activity can help identify subjects who can benefit from a histamine limitation diet and/or diamine oxidase supplementation.Properly designed, controlled studies investigating histamine intolerance that include histamine provocation are indispensable for providing insights into the area of food intolerances, which are currently primarily managed with non-scientific approaches in Italy. © The Author(s) 2015.

  17. Structure and activity of Aspergillus nidulans copper amine oxidase

    DEFF Research Database (Denmark)

    McGrath, Aaron P; Mithieux, Suzanne M; Collyer, Charles A

    2011-01-01

    Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related...... enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in α-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 Å. The enzyme is a dimer...... with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity....

  18. Rac1-NADPH oxidase signaling promotes CD36 activation under glucotoxic conditions in pancreatic beta cells.

    Science.gov (United States)

    Elumalai, Suma; Karunakaran, Udayakumar; Lee, In Kyu; Moon, Jun Sung; Won, Kyu Chang

    2017-04-01

    We recently reported that cluster determinant 36 (CD36), a fatty acid transporter, plays a pivotal role in glucotoxicity-induced β-cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression. Emerging evidence suggests that the small GTPase Rac1 is involved in the pathogenesis of beta cell dysfunction in type 2 diabetes (T2D). The primary objective of the current study was to determine the role of Rac1 in CD36 activation and its impact on β-cell dysfunction in diabetes mellitus. To address this question, we subjected INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) in the presence or absence of Rac1 inhibition either by NSC23766 (Rac1 GTPase inhibitor) or small interfering RNA. High glucose exposure in INS-1 and human beta cells (1.1b4) resulted in the activation of Rac1 and induced cell apoptosis. Rac1 activation mediates NADPH oxidase (NOX) activation leading to elevated ROS production in both cells. Activation of the Rac1-NOX complex by high glucose levels enhanced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. The inhibition of Rac1 by NSC23766 inhibited NADPH oxidase activity and ROS generation induced by high glucose concentrations in INS-1 & human 1.1b4 beta cells. Inhibition of Rac1-NOX complex activation by NSC23766 significantly reduced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. In addition, Rac1 inhibition by NSC23766 significantly reduced high glucose-induced mitochondrial dysfunction. Furthermore, NADPH oxidase inhibition by VAS2870 also attenuated high glucose-induced ROS generation and cell apoptosis. These results suggest that Rac1-NADPH oxidase dependent CD36 expression contributes to high glucose-induced beta cell dysfunction and cell death. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Radiosynthesis of [11C]brofaromine, a potential tracer for imaging monoamine oxidase A

    International Nuclear Information System (INIS)

    Ametamey, S.M.; Beer, H.-F.; Guenther, I.; Antonini, A.; Leenders, K.L.; Waldmeier, P.C.; Schubiger, P.A.

    1996-01-01

    Brofaromine(4-5(-methoxy-7-bromobenzofuranyl)-2-piperidine-HCl) is a potent and selective inhibitor of monoamine oxidase (MAO) A. Two methods for its synthesis and a preliminary positron emission tomography (PET) evaluation in monkey brain are described. The first method, at low carrier concentration of CO 2 , consisted of direct O-methylation of (4-(5-hydroxy-7-bromobenzofuranyl)-2-piperidine). The total radiochemical yield achieved ranged from 30 to 50% (from end of bombardment [EOB] and decay corrected) with an overall synthesis time of 45 min. The second approach, with high carrier amounts of CO 2 arising from inherent target problems, was accomplished in a three-step route involving protection of secondary amino functionality, O-methylation and deprotection. The total radiochemical yield was 10% (from EOB and decay corrected) with a total synthesis time of 70 min. For both methods methylation was achieved using the classical methylating agent [ 11 C]CH 3 I, and radiochemical purity was higher than 98%. PET evaluation of the radioligand in a Rhesus monkey showed a high uptake of radioactivity in the brain. Using the irreversible MAO-A inhibitor clorgyline and reversible MAO-A inhibitors moclobemide and brofaromine, three blockade experiments were designed to determine the extent of specific binding of [ 11 C]brofaromine to MAO-A. No apparent decrease in accumulation of radioactivity in the monkey brain was observed when compared to a baseline scan

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

    Science.gov (United States)

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

    2001-01-01

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

  1. Monoamine oxidase-dependent endoplasmic reticulum-mitochondria dysfunction and mast cell degranulation lead to adverse cardiac remodeling in diabetes.

    Science.gov (United States)

    Deshwal, Soni; Forkink, Marleen; Hu, Chou-Hui; Buonincontri, Guido; Antonucci, Salvatore; Di Sante, Moises; Murphy, Michael P; Paolocci, Nazareno; Mochly-Rosen, Daria; Krieg, Thomas; Di Lisa, Fabio; Kaludercic, Nina

    2018-02-19

    Monoamine oxidase (MAO) inhibitors ameliorate contractile function in diabetic animals, but the mechanisms remain unknown. Equally elusive is the interplay between the cardiomyocyte alterations induced by hyperglycemia and the accompanying inflammation. Here we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in reactive oxygen species that causes permeability transition pore opening and mitochondrial dysfunction. These events occur upstream of endoplasmic reticulum (ER) stress and are abolished by the MAO inhibitor pargyline, highlighting the role of these flavoenzymes in the ER/mitochondria cross-talk. In vivo, streptozotocin administration to mice induced oxidative changes and ER stress in the heart, events that were abolished by pargyline. Moreover, MAO inhibition prevented both mast cell degranulation and altered collagen deposition, thereby normalizing diastolic function. Taken together, these results elucidate the mechanisms underlying MAO-induced damage in diabetic cardiomyopathy and provide novel evidence for the role of MAOs in inflammation and inter-organelle communication. MAO inhibitors may be considered as a therapeutic option for diabetic complications as well as for other disorders in which mast cell degranulation is a dominant phenomenon.

  2. Plasma diamine oxidase levels in pregnancy complicated by threatened abortion.

    OpenAIRE

    Legge, M; Duff, G B

    1981-01-01

    Plasma diamine oxidase levels were assayed in 66 patients who presented with pregnancy complicated by threatened abortion. Levels within the normal range were associated with continuing pregnancies, whereas levels below the normal range were associated with subsequent abortion. Among those patients in whom gestation was greater than eight weeks, 66.6% of diamine oxidase levels correctly predicted the pregnancy outcome. Assay of the diamine oxidase levels at eight weeks of gestation or less ga...

  3. Metavanadate causes cellular accumulation of copper and decreased lysyl oxidase activity

    International Nuclear Information System (INIS)

    Cui, Changtai T.; Uriu-Adams, Janet Y.; Tchaparian, Eskouhie H.; Keen, Carl L.; Rucker, Robert B.

    2004-01-01

    Selected indices of copper metabolism in weanling rats and fibroblast cultures were progressively altered in response to increased levels of sodium metavanadate. In diets, vanadium was added in amounts ranging from 0 to 80 μg V/g of diet, that is, 0-1.6 μmol V/g of diet. In fibroblast cultures, vanadium ranged from 0 to 400 nmol V/ml. The inhibition of P-ATPase-7A activity by metavanadate, important to copper egress from cells, was a primary focus. In skin, and tendon, the copper concentration was increased in response to increased dietary levels of metavanadate, whereas lysyl oxidase activity, a secreted cuproprotein, was reduced. The reduction in lysyl oxidase activity was also accompanied by reduced redox cycling potential of isolated fractions of lysyl oxidase, presumably due to reduced lysyltyrosyl quinone (LTQ) formation at the active site of lysyl oxidase. In contrast, liver copper concentrations and plasma ceruloplasmin activity were not affected by metavanadate exposure. However, semicarbazide-sensitive benzylamine oxidase (SCBO) activity, which was taken as an indirect measure of vascular adhesive protein-1 (VAP-1), was increased. In cultured fibroblasts, cellular copper was also increased and lysyl oxidase decreased in response to metavanadate. Moreover, the steady-state levels of atp7a and lysyl oxidase mRNAs were not affected by addition of metavanadate to culture medium up to 200 nmol/ml. Taken together, these data suggest that pathways involving copper egress and lysyl oxidase activation are particularly sensitive to metavanadate exposure through processes that are predominately posttranslational

  4. Oxidases as Breast Cancer Oncogens

    National Research Council Canada - National Science Library

    Yeldandi, Anjana

    2000-01-01

    ...) in a non-tumorigenic human mammary epithelial cell line to ascertain whether oxidase overexpressing cells undergo transformation when exposed to substrate xanthine for XOX and uric acid for UOX...

  5. Plasma diamine oxidase levels in pregnancy complicated by threatened abortion.

    Science.gov (United States)

    Legge, M; Duff, G B

    1981-02-01

    Plasma diamine oxidase levels were assayed in 66 patients who presented with pregnancy complicated by threatened abortion. Levels within the normal range were associated with continuing pregnancies, whereas levels below the normal range were associated with subsequent abortion. Among those patients in whom gestation was greater than eight weeks, 66.6% of diamine oxidase levels correctly predicted the pregnancy outcome. Assay of the diamine oxidase levels at eight weeks of gestation or less gave little useful information.

  6. Effects of Greek legume plant extracts on xanthine oxidase, catalase and superoxide dismutase activities.

    Science.gov (United States)

    Spanou, Chrysoula I; Veskoukis, Aristidis S; Stagos, Dimitrios; Liadaki, Kalliopi; Aligiannis, Nectarios; Angelis, Apostolos; Skaltsounis, Alexios-Leandros; Anastasiadi, Maria; Haroutounian, Serkos A; Kouretas, Dimitrios

    2012-03-01

    Legumes are considered to have beneficial health implications, which have been attributed to their phytochemical content. Polyphenols are considered the most important phytochemical compounds extensively studied for their antioxidant properties. The aim of the present study was to examine the effects of potent antioxidant legume plant extracts on xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. XO exerts a dual role, as it is the major contributor of free radicals during exercise while it generates uric acid, the most potent antioxidant molecule in plasma. CAT and SOD are two of the main enzymes of the antioxidant defence of tissues. We demonstrate that the majority of the extracts inhibited XO activity, but they had no effect on CAT inhibition and SOD induction when used at low concentrations. These results imply that the tested extracts may be considered as possible source of novel XO inhibitors. However, we have shown that allopurinol administration, a known XO inhibitor, before exercise reduces performance and induces oxidative stress in rats. Considering the fact that the extracts examined had an inhibitory effect on XO activity, possibly posing a restriction in their characterization as antioxidants, phytochemical antioxidant administration before exercise should probably be reconsidered.

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

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Oxidase screening test for gonorrhea. 866.2420... screening test for gonorrhea. (a) Identification. An oxidase screening test for gonorrhea is an in vitro... of gonorrhea. (b) Classification. Class III (premarket approval) (transitional device). (c) Date PMA...

  8. Distinguishing the Roles of Thylakoid Respiratory Terminal Oxidases in the Cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Ermakova, Maria; Huokko, Tuomas; Richaud, Pierre; Bersanini, Luca; Howe, Christopher J; Lea-Smith, David J; Peltier, Gilles; Allahverdiyeva, Yagut

    2016-06-01

    Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and the membrane-localized respiratory terminal oxidases (RTOs), cytochrome c oxidase (Cox) and cytochrome bd quinol oxidase (Cyd), are present in the photosynthetic electron transfer chain of Synechocystis sp. PCC 6803. However, the role of individual RTOs and their relative importance compared with other electron sinks are poorly understood, particularly under light. Via membrane inlet mass spectrometry gas exchange, chlorophyll a fluorescence, P700 analysis, and inhibitor treatment of the wild type and various mutants deficient in RTOs, Flv1/3, and photosystem I, we investigated the contribution of these complexes to the alleviation of excess electrons in the photosynthetic chain. To our knowledge, for the first time, we demonstrated the activity of Cyd in oxygen uptake under light, although it was detected only upon inhibition of electron transfer at the cytochrome b6f site and in ∆flv1/3 under fluctuating light conditions, where linear electron transfer was drastically inhibited due to impaired photosystem I activity. Cox is mostly responsible for dark respiration and competes with P700 for electrons under high light. Only the ∆cox/cyd double mutant, but not single mutants, demonstrated a highly reduced plastoquinone pool in darkness and impaired gross oxygen evolution under light, indicating that thylakoid-based RTOs are able to compensate partially for each other. Thus, both electron sinks contribute to the alleviation of excess electrons under illumination: RTOs continue to function under light, operating on slower time ranges and on a limited scale, whereas Flv1/3 responds rapidly as a light-induced component and has greater capacity. © 2016 American Society of Plant Biologists. All Rights Reserved.

  9. MONOAMINE OXIDASE: RADIOTRACER DEVELOPMENT AND HUMAN STUDIES.

    Energy Technology Data Exchange (ETDEWEB)

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

    2000-09-28

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

  10. Catalytic aspects of a copper(II) complex: biological oxidase to ...

    Indian Academy of Sciences (India)

    BISWAJIT CHOWDHURY

    2017-10-03

    Oct 3, 2017 ... made with a Jasco model V-730 UV-Vis spectrophotometer. ..... Ligand-induced coordination changes ... Fet3 protein from yeast, a multinuclear copper oxidase ... of mutants of the multicopper oxidase Fet3p Biochem-.

  11. Platelet monoamine oxidase: specific activity and turnover number in headache

    International Nuclear Information System (INIS)

    Summers, K.M.; Brown, G.K.; Craig, I.W.; Peatfield, R.; Rose, F.C.

    1982-01-01

    Monoamine oxidase turnover numbers (molecules of substrate converted to product per minute per active site) have been calculated for the human platelet enzyme using [ 3 H]pargyline. Headache patients with high and low monoamine oxidase specific activities relative to controls were found to have turnover numbers very close to those for controls. This finding suggests that their specific activities vary because of differences in the concentration of active monoamine oxidase molecules, rather than differences in the ability of those enzyme molecules to catalyse the deamination reaction. (Auth.)

  12. Immobilization of xanthine oxidase on a polyaniline silicone support.

    Science.gov (United States)

    Nadruz, W; Marques, E T; Azevedo, W M; Lima-Filho, J L; Carvalho, L B

    1996-03-01

    A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

  13. Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.

    Science.gov (United States)

    Brueggeman, Andrew J; Kuehler, Daniel; Weeks, Donald P

    2014-09-01

    Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides. © 2014 Society for Experimental Biology, Association of Applied Biologists and

  14. High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

    Directory of Open Access Journals (Sweden)

    Jessica K Leet

    Full Text Available Using transgenic zebrafish (fli1:egfp that stably express enhanced green fluorescent protein (eGFP within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1 screening chemicals for cardiovascular toxicity and (2 prioritizing chemicals for future hypothesis

  15. Xanthine oxidase in human skeletal muscle following eccentric exercise

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik; Orthenblad, N.

    1997-01-01

    the increase in xanthine oxidase in the muscle there were no detectable changes in the levels of muscle malondialdehyde or in plasma antioxidant capacity up to 4 days post-exercise. 5. It is concluded that eccentric exercise leads to an increased level of xanthine oxidase in human muscle and that the increase...

  16. Construction of mutant glucose oxidases with increased dye-mediated dehydrogenase activity.

    Science.gov (United States)

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

    2012-11-02

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

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

    Science.gov (United States)

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

    2012-01-01

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

  18. Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

    NARCIS (Netherlands)

    Tamayo Ramos, J.A.; Berkel, van W.J.H.; Graaff, de L.H.

    2012-01-01

    BACKGROUND: Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated. RESULTS: The

  19. Design, synthesis and inhibitory activities of 8-(substituted styrol-formamido)phenyl-xanthine derivatives on monoamine oxidase B.

    Science.gov (United States)

    Hu, Suwen; Nian, Siyun; Qin, Kuiyou; Xiao, Tong; Li, Lingna; Qi, Xiaolu; Ye, Faqing; Liang, Guang; Hu, Guoxin; He, Jincai; Yu, Yinfei; Song, Bo

    2012-01-01

    The design and synthesis of two series of 8-(substituted styrol-formamido)phenyl-xanthine derivatives are described. Their in vitro monoamine oxidase B (MAO-B) inhibition were tested and the effect of substituents on the N-7, phenyl and the substituted positions are discussed. It was observed that compound 9b displayed significant MAO-B inhibition activity and selectivity, fluorine substitution plays a key role in the selectivity of MAO-B inhibition, and the styrol-formamido group at position-3' may enhance the activity and selectivity of 8-phenyl-xanthine analogues. These results suggest that such compounds may be utilized for the development of new candidate MAO-B inhibitors for treatment of Parkinson's disease.

  20. Cytochemical Localization of Glucose Oxidase in Peroxisomes of Aspergillus niger

    NARCIS (Netherlands)

    Veenhuis, Marten; Dijken, Johannes Pieter van

    1980-01-01

    The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both

  1. Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A2

    International Nuclear Information System (INIS)

    Gamache, D.A.; Kornberg, L.J.; Bartolf, M.; Franson, R.C.

    1986-01-01

    Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1- 14 C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A 2 (PLA 2 ) activity (64.3-545.6 nmols/min/mg). The PLA 2 was maximally active in the neutral-alkaline pH range, was Ca 2+ -dependent, and was unaffected by the addition of xanthine. PLA 2 activity was totally inhibited by 1mM EDTA whereas radical production by optimal concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA 2 activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca 2+ -dependent PLA 2 measured in various tissue homogenates (≤ 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA 2 may have influenced previously published reports, and such studies should be interpreted cautiously

  2. Physcio chemical analysis of browning inhibitors treated solanum turberosum powder

    International Nuclear Information System (INIS)

    Alizai, M.N.K.; Abid, H.

    2008-01-01

    White potatoes (Solanum turberosum) were procured from agriculture Research Institute Tarnab Farm Peshawar to use for the preparation of potato powder. The process involves sorting. Washing, peeling slicing, blanching, treating with poly phenol oxidase inhibitors, dehydration, grinding and packing. All these parameters used in process were standardized. Chemical analysis of fresh potato and potato powder were carried out. Microbiological examination, functional properties and storage life studies of the potato powder were also performed. The product prepared by drying in cabinet dryer at 55 C for 7 hours was off white colour potatoes chips which was grinded to make off white potato powder. The potato powder possessed taste and texture. (author)

  3. Identification of the NADPH Oxidase 4 Inhibiting Principle of Lycopus europaeus

    Directory of Open Access Journals (Sweden)

    Silvia Revoltella

    2018-03-01

    Full Text Available NADPH oxidase 4 (Nox4 has recently been implicated as driving force in cellular senescence. Thus, there is growing interest to develop Nox4 inhibitors, which might be valuable agents for cosmeceutical applications. Alpine plants represent a valuable source for the identification of novel bioactive natural products with anti-ageing effects, especially substances that protect plants against UV radiation, which is also known to contribute to the ageing of human skin. Therefore, the aim of this study was to identify novel Nox4 inhibitors from alpine plants. Within an initial screening of extracts of alpine plants on their ability to inhibit Nox4 activity in HEK cells, the methanolic extract of the subaerial parts of Lycopus europaeus showed a strong inhibition of Nox4 (81% chemiluminescence quenching and a simultaneously high cell viability (91% vitality. Rosmarinic acid was isolated and identified as the major compound in this bioactive extract. It showed a dose dependent inhibitory activity on Nox4 with an IC50 of 1 µM. Moreover, it also showed a significant inhibitory activity on Nox2 in the low micromolar range, whereas no inhibition of Nox5 was detected. Further investigations confirmed that the observed effects of rosmarinic acid on Nox2 and Nox4 are real inhibitory activities, and not due to ROS scavenging effects. Therefore, L. europaeus, which we demonstrated to be a good source of rosmarinic acid, has great potential for usage in cosmeceutical products with anti-ageing activity.

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

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2012-11-01

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

  5. Protein structural development of threadfin bream ( Nemipterus spp.) surimi gels induced by glucose oxidase.

    Science.gov (United States)

    Wang, Lei; Fan, Daming; Fu, Lulu; Jiao, Xidong; Huang, Jianlian; Zhao, Jianxin; Yan, Bowen; Zhou, Wenguo; Zhang, Wenhai; Ye, Weijian; Zhang, Hao

    2018-01-01

    This study investigated the effect of glucose oxidase on the gel properties of threadfin bream surimi. The gel strength of surimi increased with the addition of 0.5‰ glucose oxidase after two-step heating. Based on the results of the chemical interactions, the hydrophobic interaction and disulfide bond of glucose oxidase-treated surimi samples increased compared with the control samples at the gelation temperature and gel modori temperature. The surface hydrophobicity of samples with glucose oxidase and glucose increased significantly ( p glucose oxidase induced more α-helixes to turn into a more elongated random and flocculent structure. Glucose oxidase changes the secondary structure of the surimi protein, making more proteins depolarize and stretch and causing actomyosin to accumulate to each other, resulting in the formation of surimi gel.

  6. Functional expression of amine oxidase from Aspergillus niger (AO-I) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kolaríková, Katerina; Galuszka, Petr; Sedlárová, Iva; Sebela, Marek; Frébort, Ivo

    2009-01-01

    The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.

  7. Production of rabbit antibodies against purified Glucose oxidase

    OpenAIRE

    Zia,Muhammad Anjum; Ain,Qurat-ul; Iftikhar,Tehreema; Abbas,Rao Zahid; Rahman,Khalil-ur

    2012-01-01

    Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose) resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex ...

  8. The use of galactose oxidase in lipid labeling

    International Nuclear Information System (INIS)

    Radin, N.S.; Evangelatos, G.P.

    1981-01-01

    Galactose oxidase can be used to oxidize the terminal carbon atom of lipids containing galactose or N-acetylgalactosamine, and the resultant aldehyde group can be reduced back to the original carbinol with radioactive borohydride. The efficiency of the first reaction has been investigated systematically by using [6- 3 H]galactosyl ceramide as substrate and measuring the amount of radioactive water formed. This enabled us to establish that the addition of catalase and peroxidase greatly speeded the oxidation, that phosphate and PIPES buffers were the best among those tested, that the reaction continued for 24 hr without a second addition of galactose oxidase, and that the optimum concentration of organic solvent (tetrahydrofuran) was 50%. The suggestion if made that a similar set of variables be studied for each lipid or nonlipid by the same basic technique: labeling by the oxidase/borohydride method and use of the resultant compound as substrate

  9. Synergism between demethylation inhibitor fungicides or gibberellin inhibitor plant growth regulators and bifenthrin in a pyrethroid-resistant population of Listronotus maculicollis (Coleoptera: Curculionidae).

    Science.gov (United States)

    Ramoutar, D; Cowles, R S; Requintina, E; Alm, S R

    2010-10-01

    In 2007-2008, the "annual bluegrass weevil," Listronotus maculicollis Kirby (Coleoptera: Curculionidae), a serious pest of Poa annua L. (Poales: Poaceae) on U.S. golf courses, was shown to be resistant to two pyrethroids, bifenthrin and lambda-cyhalothrin. In 2008, we showed that bifenthrin resistance was principally mediated by oxidase detoxification (cytochrome P450 [P450]). P450s can be inhibited by demethylation inhibitor fungicides and gibberellin inhibitor plant growth regulators, both of which are commonly used on golf courses. We tested these compounds for synergistic activity with bifenthin against a pyrethroid-resistant population of L. maculicollis. The LD50 value for bifenthrin was significantly reduced from 87 ng per insect (without synergists) to 9.6-40 ng per insect after exposure to the fungicides fenarimol, fenpropimorph, prochloraz, propiconazole, and pyrifenox and the plant growth regulators flurprimidol, paclobutrazol, and trinexapac-ethyl. Simulated field exposure with formulated products registered for use on turf revealed enhanced mortality when adult weevils were exposed to bifenthrin (25% mortality, presented alone) combined with field dosages of propiconizole, fenarimol, flurprimidol, or trinexapac-ethyl (range, 49-70% mortality).

  10. Molecular Basis for Converting (2S-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

    Directory of Open Access Journals (Sweden)

    Simon Burgener

    2017-12-01

    Full Text Available Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

  11. Amine oxidase from lentil seedlings: energetic domains and effect of temperature on activity.

    Science.gov (United States)

    Moosavi-Nejad, S Z; Rezaei-Tavirani, M; Padiglia, A; Floris, G; Moosavi-Movahedi, A A

    2001-07-01

    Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).

  12. Chemical mechanism of D-amino acid oxidase from Rhodotorula gracilis: pH dependence of kinetic parameters.

    Science.gov (United States)

    Ramón, F; Castillón, M; De La Mata, I; Acebal, C

    1998-01-01

    The variation of kinetic parameters of d-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. d-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the beta-carboxylic group of the inhibitor. PMID:9461524

  13. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms.

    Science.gov (United States)

    Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

    2015-01-01

    Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ.

  14. Lysyl oxidase in cancer research

    DEFF Research Database (Denmark)

    Perryman, Lara; Erler, Janine Terra

    2014-01-01

    Metastasis is the main reason for cancer-associated deaths and therapies are desperately needed to target the progression of cancer. Lysyl oxidase (LOX) plays a pivotal role in cancer progression, including metastasis, and is therefore is an attractive therapeutic target. In this review we...

  15. High-level expression of the native barley alpha-amylase/subtilisin inhibitor in Pichia pastoris

    DEFF Research Database (Denmark)

    Micheelsen, Pernille Ollendorff; Ostergaard, Peter Rahbek; Lange, Lene

    2008-01-01

    An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed...... and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants...

  16. Systemic Manifestations in Pyridox(am)ine 5'-Phosphate Oxidase Deficiency.

    Science.gov (United States)

    Guerriero, Réjean M; Patel, Archana A; Walsh, Brian; Baumer, Fiona M; Shah, Ankoor S; Peters, Jurriaan M; Rodan, Lance H; Agrawal, Pankaj B; Pearl, Phillip L; Takeoka, Masanori

    2017-11-01

    Pyridoxine is converted to its biologically active form pyridoxal-5-phosphate (P5P) by the enzyme pyridox(am)ine 5'-phosphate oxidase and serves as a cofactor in nearly 200 reactions in the central nervous system. Pyridox(am)ine 5'-phosphate oxidase deficiency leads to P5P dependent epilepsy, typically a neonatal- or infantile-onset epileptic encephalopathy treatable with P5P or in some cases, pyridoxine. Following identification of retinopathy in a patient with pyridox(am)ine 5'-phosphate oxidase deficiency that was reversible with P5P therapy, we describe the systemic manifestations of pyridox(am)ine 5'-phosphate oxidase deficiency. A series of six patients with homozygous mutations of PNPO, the gene coding pyridox(am)ine 5'-phosphate oxidase, were evaluated in our center over the course of two years for phenotyping of neurological and systemic manifestations. Five of six were born prematurely, three had anemia and failure to thrive, and two had elevated alkaline phosphatase. A movement disorder was observed in two children, and a reversible retinopathy was observed in the most severely affected infant. All patients had neonatal-onset epilepsy and were on a continuum of developmental delay to profound encephalopathy. Electroencephalographic features included background slowing and disorganization, absent sleep features, and multifocal and generalized epileptiform discharges. All the affected probands carried a homozygous PNPO mutation (c.674 G>T, c.686 G>A and c.352G>A). In addition to the well-described epileptic encephalopathy, pyridox(am)ine 5'-phosphate oxidase deficiency causes a range of neurological and systemic manifestations. A movement disorder, developmental delay, and encephalopathy, as well as retinopathy, anemia, and failure to thrive add to the broadening clinical spectrum of P5P dependent epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.).

    Science.gov (United States)

    Dogan, Serap; Turan, Yusuf; Ertürk, Hatibe; Arslan, Oktay

    2005-02-09

    In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH(4))(2)SO(4) precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V(max)/K(m) values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic

  18. Preclinical profile of befloxatone, a new reversible MAO-A inhibitor.

    Science.gov (United States)

    Curet, O; Damoiseau-Ovens, G; Sauvage, C; Sontag, N; Avenet, P; Depoortere, H; Caille, D; Bergis, O; Scatton, B

    1998-12-01

    Befloxatone, a novel oxazolidinone derivative, is a potent, selective and reversible monoamine oxidase A (MAO-A) inhibitor in vitro (K1A = 1.9-3.6 nM) and ex vivo (ED50 MAO-A = 0.02 mg/kg, p.o.). It does not interact with a large number of receptors, monoamine transporters or other amine oxidases. Binding studies with [3H]-befloxatone in rat brain sections show that it labels with high affinity (Kd = 1.3 nM) a single population of sites with the pharmacological characteristics and regional distribution of MAO-A. In the rat brain, befloxatone (0.75 mg/kg, i.p.) increases tissue levels of monoamines and decreases levels of their deaminated metabolites. Acute administration of befloxatone (0.75 mg/kg, i.p.) induces an increase in extracellular striatal dopamine and cortical norepinephrine but not cortical serotonin levels in the rat. Befloxatone (1 mg/kg, i.p.) potently inhibits the firing rate of serotonergic neurons, partially decreases the firing of noradrenergic neurons and has no effect on the firing of dopaminergic neurons (a mirror image of its effects on monoamine release in terminal regions), suggesting that the relative effects of befloxatone on monoamine release may be governed by autoreceptor-mediated control of monoaminergic neurons at the cell body level. Befloxatone (0.03-0.3 mg/kg, p.o.) exhibits potent activity in behavioural models predictive of antidepressant activity. Befloxatone (up to 1.5 mg/kg, p.o.) does not potentiate the pressor effects of orally administered tyramine at centrally active doses and duodenal [3H]-befloxatone binding is displaced by increasing doses of orally administered tyramine (0.1-40 mg/kg, i.p.). These results suggest that befloxatone is a potent reversible MAO-A inhibitor with antidepressant potential and a wide safety margin with regard to the potentiation of the pressor effect of tyramine.

  19. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dokyoung; Jun, Yong Woong; Ahn, Kyo Han [POSTECH, Pohang (Korea, Republic of)

    2014-05-15

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

  20. Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit.

    Science.gov (United States)

    Bravo, Karent; Osorio, Edison

    2016-04-15

    Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. [Experimental rationale for the parameters of a rapid method for oxidase activity determination].

    Science.gov (United States)

    Butorina, N N

    2010-01-01

    Experimental rationale is provided for the parameters of a rapid (1-2-min) test to concurrently determine the oxidase activity of all bacteria grown on the membrane filter after water filtration. Oxidase reagents that are the aqueous solutions of tetramethyl-p-phenylenediamine dihydrochloride and demethyl-p-phenylenediamine dihydrochloride have been first ascertained to exert no effect on the viability and enzymatic activity of bacteria after one-hour contact. An algorithm has been improved for the rapid oxidase activity test: the allowable time for bacteria to contact oxidase reagents and procedures for minimizing the effect on bacterial biochemical activity following the contact. An accelerated method based on lactose medium with tergitol 7 and Endo agar has been devised to determine coliform bacteria, by applying the rapid oxidase test: the time of a final response is 18-24 hours. The method has been included into GOST 52426-2005.

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

    Science.gov (United States)

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

    1985-01-01

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

  3. Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila

    Directory of Open Access Journals (Sweden)

    Alessandro R. Ferrari

    2018-01-01

    Full Text Available The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615 and XP_003665713 (MtVAO713, were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

  4. Antiglycation, radical scavenging, and semicarbazide-sensitive amine oxidase inhibitory activities of acetohydroxamic acid in vitro

    Directory of Open Access Journals (Sweden)

    Liu YH

    2017-07-01

    Full Text Available Yuh-Hwa Liu,1,2,* Yeh-Lin Lu,3,* Der-Zen Liu,4 Wen-Chi Hou5 1Division of Gastroenterology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; 2Department of General Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 3Department of Pharmacy, Taipei Medical University, Taipei, Taiwan; 4Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan; 5Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan *These authors contributed equally to this work Abstract: Advanced glycation endproducts (AGEs can promote intracellular reactive oxygen species production, and the levels of AGEs are highly correlated with cardiovascular disease and diabetes complications. Acetohydroxamic acid (acetH is a bacterial urease inhibitor drug used to treat kidney stones and infections in the urinary tract, and hydroxyurea (HU is a drug used for antineoplasm and sickle cell diseases. Both acetH and HU are hydroxamic acid derivatives. It was found that acetH and HU at 2.5 or 5 mM showed anti-AGE formation by lowering the AGEs’ fluorescent intensities and Nε-(carboxymethyllysine formation in bovine serum albumin/galactose models, and both showed better and significant differences (P<0.05 compared to the positive control of aminoguanidine. Regarding radical scavenging activities, the half-inhibition concentrations (IC50 of acetH against α,α-diphenyl-β-picrylhydrazyl radical and hydroxyl radical were 34.86 and 104.42 µM, respectively. The IC50 of acetH against semicarbazide-sensitive amine oxidase was 10.56 µM, and acetH showed noncompetitive inhibition respective to the substrates (benzylamine. The antiglycation, antioxidant, and semicarbazide-sensitive amine oxidase inhibitory activities of acetH prove that it has the potential for treating cardiovascular disease and diabetes complications and it needs further investigation in animal models. Keywords: acetH, AGEs, Nε

  5. Purification and Characterization of Polyphenol Oxidase, Peroxidase and Lipoxygenase from Freshly Cut Lettuce (L. sativa

    Directory of Open Access Journals (Sweden)

    Vural Gökmen

    2011-01-01

    Full Text Available Enzymatic reactions taking place in minimally processed vegetables are considered as a major problem, because they adversely affect sensorial and nutritional quality. Polyphenol oxidase (PPO, peroxidase (POD and lipoxygenase (LOX from lettuce were purified on a column packed with positively charged diethylaminoethyl (DEAE cellulose by applying pH gradient elution from pH=4.0 to 9.0. The main purified fractions (PPO1 and PPO4, POD1 and POD2, LOX1 and LOX2 were characterized for enzyme concentration-reaction rate relationship, thermal stability, pH activity and kinetic parameters. Kinetic properties of each isoform were considerably different. Cysteine was found as the most effective inhibitor of both fractions of PPO. Kinetic parameters of lettuce POD were presented using guaiacol at various H2O2 concentrations. β-carotene directly influences lettuce LOX in the reaction medium available for the catalytic conversion of linoleic acid into hydroperoxides. Ascorbic and oxalic acids appear as effective PPO inhibitors, protecting phenolic compounds against oxidation in lettuce. Understanding the characteristics of deteriorative enzymes becomes important to maintain suitable conditions for fresh-like quality of lettuce. The results can be useful to keep the nutritional quality of minimally processed lettuce during shelf-life.

  6. From caffeine to fish waste: amine compounds present in food and drugs and their interactions with primary amine oxidase.

    LENUS (Irish Health Repository)

    Olivieri, Aldo

    2011-07-01

    Tissue bound primary amine oxidase (PrAO) and its circulating plasma-soluble form are involved, through their catalytic activity, in important cellular roles, including the adhesion of lymphocytes to endothelial cells during various inflammatory conditions, the regulation of cell growth and maturation, extracellular matrix deposition and maturation and glucose transport. PrAO catalyses the oxidative deamination of several xenobiotics and has been linked to vascular toxicity, due to the generation of cytotoxic aldehydes. In this study, a series of amines and aldehydes contained in food and drugs were tested via a high-throughput assay as potential substrates or inhibitors of bovine plasma PrAO. Although none of the compounds analyzed were found to be substrates for the enzyme, a series of molecules, including caffeine, the antidiabetics phenformin and tolbutamide and the antimicrobial pentamidine, were identified as PrAO inhibitors. Although the inhibition observed was in the millimolar and micromolar range, these data show that further work will be necessary to elucidate whether the interaction of ingested biogenic or xenobiotic amines with PrAO might adversely affect its biological roles.

  7. Vanillyl-alcohol oxidase, a tasteful biocatalyst

    NARCIS (Netherlands)

    Heuvel, van den R.H.H.; Fraaije, M.W.; Mattevi, A.; Laane, C.; Berkel, van W.J.H.

    2001-01-01

    The covalent flavoenzyme vanillyl-alcohol oxidase (VAO) is a versatile biocatalyst. It converts a wide range of phenolic compounds by catalysing oxidation, deamination, demethylation, dehydrogenation and hydroxylation reactions. The production of natural vanillin, 4-hydroxybenzaldehyde, coniferyl

  8. Dgroup: DG01277 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available DG01277 Chemical ... DGroup Pirlindole ... D08392 ... Pirlindole (INN) D08393 ... Pirlindole hydrochloride Neuropsychi...atric agent ... DG01568 ... MAO inhibitor ... DG01558 ... Monoamine oxidase A inhibitor ... Reversible monoamine oxidase A (MAO-A) inhibitor MAOA [HSA:4128] [KO:K00274] ...

  9. Oral administration of D-alanine in monkeys robustly increases plasma and cerebrospinal fluid levels but experimental D-amino acid oxidase inhibitors had minimal effect.

    Science.gov (United States)

    Rojas, Camilo; Alt, Jesse; Ator, Nancy A; Wilmoth, Heather; Rais, Rana; Hin, Niyada; DeVivo, Michael; Popiolek, Michael; Tsukamoto, Takashi; Slusher, Barbara S

    2016-09-01

    Hypofunction of the N-methyl-d-aspartate (NMDA) receptor is thought to exacerbate psychosis in patients diagnosed with schizophrenia. Consistent with this hypothesis, D-alanine, a co-agonist at the glycine site of the NMDA receptor, was shown to improve positive and cognitive symptoms when used as add-on therapy for schizophrenia treatment. However, D-alanine had to be administered at high doses (~7 g) to observe clinical effects. One possible reason for the high dose is that D-alanine could be undergoing oxidation by D-amino acid oxidase (DAAO) before it reaches the brain. If this is the case, the dose could be reduced by co-administration of D-alanine with a DAAO inhibitor (DAAOi). Early studies with rodents showed that co-administration of D-alanine with 5-chloro-benzo[d]isoxazol-3-ol (CBIO), a prototype DAAOi, significantly enhanced the levels of extracellular D-alanine in the frontal cortex compared with D-alanine alone. Further, the use of CBIO reduced the dose of D-alanine needed to attenuate prepulse inhibition deficits induced by dizocilpine. The objective of the work reported herein was to confirm the hypothesis that DAAO inhibition can enhance D-alanine exposure in a species closer to humans: non-human primates. We report that while oral D-alanine administration to baboons (10 mg/kg) enhanced D-alanine plasma and CSF levels over 20-fold versus endogenous levels, addition of experimental DAAOi to the regimen exhibited a 2.2-fold enhancement in plasma and no measurable effect on CSF levels. The results provide caution regarding the utility of DAAO inhibition to increase D-amino acid levels as treatment for patients with schizophrenia. © The Author(s) 2016.

  10. Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes

    NARCIS (Netherlands)

    Veenhuis, M.; Wendelaar Bonga, S.E.

    1979-01-01

    The distribution of catalase, amino acid oxidase, α-hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based

  11. Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity.

    Directory of Open Access Journals (Sweden)

    Laura Jeacock

    2017-03-01

    Full Text Available Aquaglyceroporins (AQPs transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM, octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited.

  12. Lysyl Oxidase and the Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Tong-Hong Wang

    2016-12-01

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

  13. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

    International Nuclear Information System (INIS)

    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-01-01

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47 phox , p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF-α induces

  14. Dual functional cholinesterase and MAO inhibitors for the treatment of Alzheimer's disease: synthesis, pharmacological analysis and molecular modeling of homoisoflavonoid derivatives.

    Science.gov (United States)

    Wang, Yali; Sun, Yang; Guo, Yueyan; Wang, Zechen; Huang, Ling; Li, Xingshu

    2016-01-01

    Because of the complexity of Alzheimer's disease (AD), the multi-target-directed ligand (MTDL) strategy is expected to provide superior effects for the treatment of AD, instead of the classic one-drug-one-target strategy. In this context, we focused on the design, synthesis and evaluation of homoisoflavonoid derivatives as dual acetyl cholinesterase (AChE) and monoamine oxidase (MAO-B) inhibitors. Among all the synthesized compounds, compound 10 provided a desired balance of AChE and hMAO-B inhibition activities, with IC50 value of 3.94 and 3.44 μM, respectively. Further studies revealed that compound 10 was a mixed-type inhibitor of AChE and an irreversible inhibitor of hMAO-B, which was also confirmed by molecular modeling studies. Taken together, the data indicated that 10 was a promising dual functional agent for the treatment of AD.

  15. Plasma amine oxidase activities in Norrie disease patients with an X-chromosomal deletion affecting monoamine oxidase.

    Science.gov (United States)

    Murphy, D L; Sims, K B; Karoum, F; Garrick, N A; de la Chapelle, A; Sankila, E M; Norio, R; Breakefield, X O

    1991-01-01

    Two individuals with an X-chromosomal deletion were recently found to lack the genes encoding monoamine oxidase type A (MAO-A) and MAO-B. This abnormality was associated with almost total (90%) reductions in the oxidatively deaminated urinary metabolites of the MAO-A substrate, norepinephrine, and with marked (100-fold) increases in an MAO-B substrate, phenylethylamine, confirming systemic functional consequences of the genetic enzyme deficiency. However, urinary concentrations of the deaminated metabolites of dopamine and serotonin (5-HT) were essentially normal. To investigate other deaminating systems besides MAO-A and MAO-B that might produce these metabolites of dopamine and 5-HT, we examined plasma amine oxidase (AO) activity in these two patients and two additional patients with the same X-chromosomal deletion. Normal plasma AO activity was found in all four Norrie disease-deletion patients, in four patients with classic Norrie disease without a chromosomal deletion, and in family members of patients from both groups. Marked plasma amine metabolite abnormalities and essentially absent platelet MAO-B activity were found in all four Norrie disease-deletion patients, but in none of the other subjects in the two comparison groups. These results indicate that plasma AO is encoded by gene(s) independent of those for MAO-A and MAO-B, and raise the possibility that plasma AO, and perhaps the closely related tissue AO, benzylamine oxidase, as well as other atypical AOs or MAOs encoded independently from MAO-A and MAO-B may contribute to the oxidative deamination of dopamine and 5-HT in humans.

  16. Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.

    Science.gov (United States)

    Barlow, J N; Zhang, Z; John, P; Baldwin, J E; Schofield, C J

    1997-03-25

    1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.

  17. Genetics Home Reference: isolated sulfite oxidase deficiency

    Science.gov (United States)

    ... and Management Resources (1 link) GeneReview: Isolated Sulfite Oxidase Deficiency General Information from MedlinePlus (5 links) Diagnostic Tests Drug Therapy Genetic Counseling Palliative Care Surgery and ...

  18. Up-Streaming Process for Glucose Oxidase by Thermophilic Penicillium sp. in Shake Flask

    OpenAIRE

    Muhammad Mohsin JAVED; Aroosh SHABIR; Sana ZAHOOR; Ikram UL-HAQ

    2012-01-01

    The present study is concerned with the production of glucose oxidase (GOD) from thermophilic Penicillium sp. in 250 mL shake flask. Fourteen different strains of thermophilic Penicillium sp. were isolated from the soil and were screened for glucose oxidase production. IIBP-13 strain gave maximum extra-cellular glucose oxidase production as compared to other isolates. Effect of submerged fermentation in shaking and static conditions, different carbon sources and incubation period on the produ...

  19. Two X-linked chronic granulomatous disease patients with unusual NADPH oxidase properties

    NARCIS (Netherlands)

    Wolach, Baruch; Broides, Arnon; Zeeli, Tal; Gavrieli, Ronit; de Boer, Martin; van Leeuwen, Karin; Levy, Jacov; Roos, Dirk

    2011-01-01

    Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two

  20. Structural studies of alternative oxidase (AOX) from moniliophthora perniciosa, the causal agent of witches' broom disease in cacao: a membrane-associated protein

    International Nuclear Information System (INIS)

    Oliveira, J.F.; Prado, P.F.V.; Tiezzi, H.O.; Dias, S.M.G.; Ambrosio, A.L.B.; Thomazella, D.P.T.; Teixeira, P.J.P.L.; Pereira, G.A.G.

    2012-01-01

    Full text: Alternative oxidase (AOX) is a protein attached to the inner mitochondrial membrane that receives electrons directly from reduced ubiquinone and catalyzes the reduction of oxygen to water. AOX is a non-proton motive terminal quinol oxidase that enables cell respiration to continue even in the presence of inhibitors targeting the complexes of the respiratory chain. This protein is present in higher plants, pathogenic fungi and some parasites. The structural characterization of AOX becomes interesting due to its potential as a fungicide target. AOX is predicted to be a monotopic interfacial membrane protein interacting with a single leaflet of the lipid bilayer, rather than transmembrane. Amino acid sequence analysis reveals the presence of two conserved glutamate-histidine motifs, identifying it as a member of the diiron carboxylate protein family. The AOX model is defined by two pairs of helices forming a four helix bundle and an additional hydrophobic connecting sequence between the two helical pairs is proposed to act as the membrane anchoring region. In this work we aim at production, purification and crystallization of the AOX protein from M. perniciosa for further structural studies of this membrane-associated protein, by X-ray protein crystallography (author)

  1. Development of radioiodinated ligands for exploration of brain monoamine oxidase by tomo-scintigraphy; Developpement de ligands radioactifs pour l'exploration des monoamines oxydases cerebrales en tomoscintigraphie

    Energy Technology Data Exchange (ETDEWEB)

    Rafii, H

    1996-07-01

    Monoamine oxidases, MAO, are important in the regulation of monoaminergic neuro-transmissions. The fluctuations in MAO activities has been observed in some psychiatric and neuro-degenerative diseases. Thus, quantification of cerebral MAO activity would be useful for diagnosis and the therapeutic follow-up of these disorders. With the object of doing an in vivo scintigraphic exploration of cerebral MAO by SPECT, we have undertaken to synthesize some radioiodinated MAO inhibitors. In the first part of this work, we have discussed the general properties of the monoamine oxidases and their inhibitors. In the second part we have described the scintigraphic methods. the ligands to be used for MAO exploration, and the radioiodination methods. At last in the third part, the development of three radioiodinated ligands has been presented: - [{sup 125}I]3-iodopargyline. In vivo results showed that, this radioligand blocked the cerebral MAO-B with moderate selectivity. However, complementary in vivo studies would be needed to define precisely its activity.- [{sup 125}I]Ro 16-6491. The cerebral fixation of this radioligand was in accordance with the MAO-B sites in the rat brains, but its fixation was too low for scintigraphic exploration in vivo with iodine-123. - [{sup 125}I]Ro 11-9900. In vivo studies of rat brains showed that the MAO-A sites were bound preferentially by this radioligand. The cerebral biodistribution of this ligand labelled with iodine-123 is considered for use in a model animal nearest to human pathology. (author)

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

    Science.gov (United States)

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

  3. Blockade of TGF-β 1 Signalling Inhibits Cardiac NADPH Oxidase Overactivity in Hypertensive Rats

    Directory of Open Access Journals (Sweden)

    José Luis Miguel-Carrasco

    2012-01-01

    Full Text Available NADPH oxidases constitute a major source of superoxide anion (⋅O2 - in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-β 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-β 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-β 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-β 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-β 1.

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

    Science.gov (United States)

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

    1977-06-17

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

  5. Assessment of Enzyme Inhibition: A Review with Examples from the Development of Monoamine Oxidase and Cholinesterase Inhibitory Drugs.

    Science.gov (United States)

    Ramsay, Rona R; Tipton, Keith F

    2017-07-15

    The actions of many drugs involve enzyme inhibition. This is exemplified by the inhibitors of monoamine oxidases (MAO) and the cholinsterases (ChE) that have been used for several pharmacological purposes. This review describes key principles and approaches for the reliable determination of enzyme activities and inhibition as well as some of the methods that are in current use for such studies with these two enzymes. Their applicability and potential pitfalls arising from their inappropriate use are discussed. Since inhibitor potency is frequently assessed in terms of the quantity necessary to give 50% inhibition (the IC 50 value), the relationships between this and the mode of inhibition is also considered, in terms of the misleading information that it may provide. Incorporation of more than one functionality into the same molecule to give a multi-target-directed ligands (MTDLs) requires careful assessment to ensure that the specific target effects are not significantly altered and that the kinetic behavior remains as favourable with the MTDL as it does with the individual components. Such factors will be considered in terms of recently developed MTDLs that combine MAO and ChE inhibitory functions.

  6. Evaluation of the Expression of Amine Oxidase Proteins in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Woo Young Sun

    2017-12-01

    Full Text Available We aimed to evaluate the expression of amine oxidase proteins in breast cancer and their clinical implications. We performed immunohistochemical staining of amine oxidase proteins (LOX, lysyl oxidase, AOC3, amine oxidase, MAOA, monoamine oxidase A, MAOB, monoamine oxidase B. Based on their hormone receptors, such as estrogen receptor (ER and progesterone receptor (PR, human epidermal growth factor receptor 2 (HER-2, and Ki-67 immunohistochemical staining, breast cancer was divided into four molecular subtypes: luminal A, luminal B, HER-2 type, and triple-negative breast cancer (TNBC. Luminal A was observed in 380 cases (49.4%, luminal B in 224 (29.1%, HER-2 type in 68 (8.8%, and TNBC in 98 (12.7%. Stromal AOC3, MAO-A, and MAO-B expression varied according to molecular subtypes. Stromal AOC3 expression was high in luminal B and HER-2 type and MAO-A expression was high in luminal A and luminal B (p < 0.001. MAO-B expression was higher in TNBC than in other subtypes (p = 0.020. LOX positivity was associated with high histological grade (p < 0.001 and high Ki-67 labeling index (LI (p = 0.009, and stromal AOC3 positivity was associated with high histological grade (p = 0.001, high Ki-67 LI (p < 0.001, and HER-2 positivity (p = 0.002. MAO-A positivity was related to low histological grade (p < 0.001, ER positivity, PR positivity (p < 0.001, and low Ki-67 LI (p < 0.001. In univariate analysis, MAO-A positivity was related to short disease-free survival in HER-2 type (p = 0.013, AOC3 negativity was related to short disease-free survival and overall survival in ER-positive breast cancer, PR-positive breast cancer, HER-2-negative breast cancer, and lymph node metastasis. In conclusion, the expression of amine oxidase proteins varies depending on the molecular subtype of breast cancer. Stromal AOC3 expression was high in luminal B and HER-2 type, and MAO-A expression was high in luminal A and luminal B.

  7. Behavioral effects of α,α,β,β-tetradeutero-5-MeO-DMT in rats: comparison with 5-MeO-DMT administered in combination with a monoamine oxidase inhibitor

    Science.gov (United States)

    Halberstadt, Adam L.; Nichols, David E.; Geyer, Mark A.

    2012-01-01

    RATIONALE Ayahuasca is a psychoactive tea prepared from a combination of plants that contain a hallucinogenic tryptamine and monoamine oxidase inhibitors (MAOIs). Behavioral Pattern Monitor (BPM) experiments demonstrated that the combination of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and a behaviorally inactive dose of an MAOA inhibitor such as harmaline or clorgyline induces biphasic effects on locomotor activity in rats, initially reducing locomotion and then increasing activity as time progresses. OBJECTIVES The present study investigated whether the biphasic locomotor profile induced by the combination of 5-MeO-DMT and an MAOI is a consequence of a reduction in the rate of 5-MeO-DMT metabolism. This hypothesis was tested using a deuterated derivative of 5-MeO-DMT (α,α,β,β-tetradeutero-5-MeO-DMT) that is resistant to metabolism by MAO. RESULTS Confirming our previous findings, 1.0 mg/kg 5-MeO-DMT (s.c.) had biphasic effects on locomotor activity in rats pretreated with a behaviorally inactive dose of the nonselective MAOI pargyline (10 mg/kg). Administration of 5-MeO-DMT alone, even at doses greater than 1.0 mg/kg, produced only reductions in locomotor activity. Although low doses of α,α,β,β-tetradeutero-5-MeO-DMT (0.3 and 1.0 mg/kg, s.c.) produced only hypoactivity in the BPM, a dose of 3.0 mg/kg induced a biphasic locomotor profile similar to that produced by the combination of 5-MeO-DMT and an MAOI. Receptor binding studies demonstrated that deuterium substitution had little effect on the affinity of 5-MeO-DMT for a wide variety of neurotransmitter binding sites. CONCLUSIONS The finding with α,α,β,β-tetradeutero-5-MeO-DMT indicates that the hyperactivity induced by 5-MeO-DMT after MAO inhibition is a consequence of reduced metabolism of 5-MeO-DMT, leading to prolonged occupation of central serotonin receptors. These results demonstrate that deuterated tryptamines may be useful in behavioral and pharmacological studies to mimic the effects of

  8. Tea polyphenols alleviate high fat and high glucose-induced endothelial hyperpermeability by attenuating ROS production via NADPH oxidase pathway.

    Science.gov (United States)

    Zuo, Xuezhi; Tian, Chong; Zhao, Nana; Ren, Weiye; Meng, Yi; Jin, Xin; Zhang, Ying; Ding, Shibin; Ying, Chenjiang; Ye, Xiaolei

    2014-03-02

    Hyperglycemia-induced endothelial hyperpermeability is crucial to cardiovascular disorders and macro-vascular complications in diabetes mellitus. The objective of this study is to investigate the effects of green tea polyphenols (GTPs) on endothelial hyperpermeability and the role of nicotinamide adenine dinucleotide phosphate (NADPH) pathway. Male Wistar rats fed on a high fat diet (HF) were treated with GTPs (0, 0.8, 1.6, 3.2 g/L in drinking water) for 26 weeks. Bovine aortic endothelial cells (BAECs) were treated with high glucose (HG, 33 mmol/L) and GTPs (0.0, 0.4, or 4 μg/mL) for 24 hours in vitro. The endothelial permeabilities in rat aorta and monolayer BAECs were measured by Evans blue injection method and efflux of fluorescein isothiocyanate (FITC)-dextran, respectively. The reactive oxygen species (ROS) levels in rat aorta and monolayer BAECs were measured by dihydroethidium (DHE) and 2', 7'-dichloro-fluorescein diacetate (DCFH-DA) fluorescent probe, respectively. Protein levels of NADPH oxidase subunits were determined by Western-blot. HF diet-fed increased the endothelial permeability and ROS levels in rat aorta while HG treatments increased the endothelial permeability and ROS levels in cultured BAECs. Co-treatment with GTPs alleviated those changes both in vivo and in vitro. In in vitro studies, GTPs treatments protected against the HG-induced over-expressions of p22phox and p67phox. Diphenylene iodonium chloride (DPI), an inhibitor of NADPH oxidase, alleviated the hyperpermeability induced by HG. GTPs could alleviate endothelial hyperpermeabilities in HF diet-fed rat aorta and in HG treated BAECs. The decrease of ROS production resulting from down-regulation of NADPH oxidase contributed to the alleviation of endothelial hyperpermeability.

  9. Polyphenol oxidase activity and antioxidant properties of Yomra apple (Malus communis L.) from Turkey.

    Science.gov (United States)

    Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

    2014-12-01

    In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  11. Multi-Copper Oxidases and Human Iron Metabolism

    Science.gov (United States)

    Vashchenko, Ganna; MacGillivray, Ross T. A.

    2013-01-01

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

  12. Metabonomic strategy for the investigation of the mode of action of the phytotoxin (5S,8R,13S,16R)-(-)-pyrenophorol using 1H nuclear magnetic resonance fingerprinting.

    Science.gov (United States)

    Aliferis, Konstantinos A; Chrysayi-Tokousbalides, Maria

    2006-03-08

    The biochemical mode of action of (5S,8R,13S,16R)-(-)-pyrenophorol isolated from a Drechslera avenae pathotype was investigated by using metabolic fingerprinting. (1)H NMR spectra of crude leaf extracts from untreated Avena sterilis seedlings and A. sterilis seedlings treated with pyrenophorol were compared with those obtained from treatments with the herbicides diuron, glyphosate, mesotrione, norflurazon, oxadiazon, and paraquat. Multivariate analysis was carried out to group treatments according to the mode of action of the phytotoxic substances applied. Analysis results revealed that none of the herbicide treatments fitted the pyrenophorol model and indicate that the effect of the phytotoxin on A. sterilis differs than those caused by glyphosate, mesotrione, norflurazon, oxadiazon, paraquat, and diuron, which inhibit 5-enolpyruvylshikimate-3-phosphate synthase, 4-hydroxyphenyl-pyruvate-dioxygenase, phytoene desaturase, protoporphyrinogen oxidase, photosystem I, and photosystem II, respectively. The method applied, combined with appropriate data preprocessing and analysis, was found to be rapid for the screening of phytotoxic substances for metabolic effects.

  13. Homozygous variegate porphyria presenting with developmental and language delay in childhood.

    Science.gov (United States)

    Pinder, V A E; Holden, S T; Deshpande, C; Siddiqui, A; Mellerio, J E; Wraige, E; Powell, A M

    2013-10-01

    Variegate porphyria is an autosomal dominant disorder that usually presents with photosensitivity and acute neurological crises in adulthood. It is caused by heterozygous mutations in the protoporphyrinogen oxidase gene (PPOX). A rarer variant, homozygous variegate porphyria (HVP), presents in childhood with recurrent skin blisters and scarring. More variable features of HVP are short stature, brachydactyly, nystagmus, epilepsy, developmental delay and mental retardation. We describe a child who presented with nystagmus, developmental delay and ataxia, combined with a photosensitive eruption. Analysis of porphyrins in plasma, urine and stool supported a clinical diagnosis of HVP. DNA from the patient showed that he is compound heterozygous for two novel missense mutations in the PPOX coding region: c.169G>C (p.Gly57Arg) and c.1259C>G (Pro420Arg). Interestingly, cranial magnetic resonance imaging showed an absence of myelin, a feature not previously reported in HVP, which expands the differential diagnosis of childhood hypomyelinating leucoencephalopathies. © 2013 British Association of Dermatologists.

  14. Synthesis and biological evaluations of a series of thaxtomin analogues.

    Science.gov (United States)

    Zhang, Hongbo; Wang, Qingpeng; Ning, Xin; Hang, Hang; Ma, Jing; Yang, Xiande; Lu, Xiaolin; Zhang, Jiabao; Li, Yonghong; Niu, Congwei; Song, Haoran; Wang, Xin; Wang, Peng George

    2015-04-15

    Thaxtomins are a unique family of phytotoxins with unique 4-nitroindole and diketopiperazine fragments possessing potential herbicidal activities. This work presents the total synthesis of natural product thaxtomin C and its analogues. The extensive structure-activity relationship study screens four effective compounds, including thaxtomin A and thaxtomin C. It is indicated that 4-nitro indole fragment is essential for phytotoxicity, while benzyl and m-hydroxybenzyl substituents on the diketopiperazine ring are favorable for the efficacy. The N-methylations on indole and diketopiperazine show weak influence on the herbicidal activities. The four selected compounds show effective herbicidal activities against Brassica campestris, Amaranthus retroflexus, and Abutilon theophrasti, which are comparable or better than dichlobenil, even at a dosage of 187.5 g ha(-1). Moreover, these four compounds show good crop-selective properties to different crops and exhibit moderate protoporphyrinogen oxidase (PPO) enzyme inhibition. The antifungal results indicate that thaxtomin C displays inhibition to a wide range of fungi.

  15. Engineering glucose oxidase to minimize the influence of oxygen on sensor response

    International Nuclear Information System (INIS)

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

    2014-01-01

    Glucose oxidase (GOx) is an important industrial enzyme and is recognized as the gold standard for monitoring blood glucose. However, due to its inherent oxidase property, the presence of oxygen affects electrochemical measurements of venous blood glucose employing artificial electron mediators. We therefore attempted to engineer Penicillium amagasakiense-derived GOx into a dehydrogenase by focusing on the amino acid residues predicted to interact with oxygen. Our rational amino acid substitution approach resulted in the construction of the Ser114Ala/Phe355Leu mutant, which has an 11-fold decrease in oxidase activity and 2.8-fold increase in dehydrogenase activity compared with wild-type GOx. As a result, the dehydrogenase/oxidase activity ratio of the engineered enzyme was 32-fold greater than that of the wild-type enzyme. The enzyme sensor constructed with Ser114Ala/Phe355Leu was considerably less affected by oxygen than the wild-type GOx-based sensor at lower glucose concentrations

  16. The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily.

    Science.gov (United States)

    Wongnate, Thanyaporn; Chaiyen, Pimchai

    2013-07-01

    Enzymes in the glucose-methanol-choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared with that of other enzymes in the GMC family for which structural and mechanistic information is available, including glucose oxidase, choline oxidase, cholesterol oxidase, cellobiose dehydrogenase, aryl-alcohol oxidase, and pyridoxine 4-oxidase. Other enzymes in the family that have been newly discovered or for which less information is available are also discussed. A large primary kinetic isotope effect was observed for the flavin reduction when 2-d-D-glucose was used as a substrate, but no solvent kinetic isotope effect was detected for the flavin reduction step. The reaction of P2O is consistent with a hydride transfer mechanism in which there is stepwise formation of d-glucose alkoxide prior to the hydride transfer. Site-directed mutagenesis of P2O and pH-dependence studies indicated that His548 is a catalytic base that facilitates the deprotonation of C2-OH in D-glucose. This finding agrees with the current mechanistic model for aryl-alcohol oxidase, glucose oxidase, cellobiose dehydrogenase, methanol oxidase, and pyridoxine 4-oxidase, but is different from that of cholesterol oxidase and choline oxidase. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation. © 2013 The Authors Journal compilation © 2013 FEBS.

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

    Science.gov (United States)

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

    2016-11-21

    Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

  18. Size-selective QD@MOF core-shell nanocomposites for the highly sensitive monitoring of oxidase activities.

    Science.gov (United States)

    Wang, Ke; Li, Nan; Zhang, Jing; Zhang, Zhiqi; Dang, Fuquan

    2017-01-15

    In this work, we proposed a novel and facile method to monitor oxidase activities based on size-selective fluorescent quantum dot (QD)@metal-organic framework (MOF) core-shell nanocomposites (CSNCPs). The CSNCPs were synthesized from ZIF-8 and CdTe QDs in aqueous solution in 40min at room temperature with stirring. The prepared CdTe@ZIF-8 CSNCPs , which have excellent water dispersibility and stability, displays distinct fluorescence responses to hole scavengers of different molecular sizes (e.g., H 2 O 2 , substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H 2 O 2 can efficiently quench the fluorescence of CdTe@ZIF-8 CSNCPs over a linearity range of 1-100nM with a detection limit of 0.29nM, whereas large molecules such as substrate and oxidase have very little effect on its fluorescence. Therefore, the highly sensitive detection of oxidase activities was achieved by monitoring the fluorescence quenching of CdTe@ZIF-8 CSNCPs by H 2 O 2 produced in the presence of substrate and oxidase, which is proportional to the oxidase activities. The linearity ranges of the uricase and glucose oxidase activity are 0.1-50U/L and 1-100U/L, respectively, and their detection limits are 0.024U/L and 0.26U/L, respectively. Therefore, the current QD@MOF CSNCPs based sensing system is a promising, widely applicable means of monitoring oxidase activities in biochemical research. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. The antioxidant properties, cytotoxicity and monoamine oxidase ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... Department of Pharmaceutical Chemistry, North-West University, Private Bag X6001, Potchefstroom 2520, ..... on the inhibition of the catabolism of serotonin, .... Structure of human monoamine oxidase B, a drug target for.

  20. Investigation of antihemolytic, xanthine oxidase inhibition ...

    African Journals Online (AJOL)

    Abbreviations: SVEs: Salvia Verbenaca L. aerial part Extracts; CrE: Crud Extract; ChE: Chloroform Extract ; EAE: Ethyl Acetate Extract; AqE : Aqueous Extract ; ROS: Reactive Oxygen Spices; AAPH : 2,2, -Azobis (2-AmidinoPropane) Dihydrochloride ; DPPH: DiPhenyl- Picryl-Hydrazyl; XO: Xanthine Oxidase; Gen: Gentamicin ...

  1. Structural studies of alternative oxidase (AOX) from moniliophthora perniciosa, the causal agent of witches' broom disease in cacao: a membrane-associated protein

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, J.F.; Prado, P.F.V.; Tiezzi, H.O.; Dias, S.M.G.; Ambrosio, A.L.B. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Thomazella, D.P.T.; Teixeira, P.J.P.L.; Pereira, G.A.G. [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

    2012-07-01

    Full text: Alternative oxidase (AOX) is a protein attached to the inner mitochondrial membrane that receives electrons directly from reduced ubiquinone and catalyzes the reduction of oxygen to water. AOX is a non-proton motive terminal quinol oxidase that enables cell respiration to continue even in the presence of inhibitors targeting the complexes of the respiratory chain. This protein is present in higher plants, pathogenic fungi and some parasites. The structural characterization of AOX becomes interesting due to its potential as a fungicide target. AOX is predicted to be a monotopic interfacial membrane protein interacting with a single leaflet of the lipid bilayer, rather than transmembrane. Amino acid sequence analysis reveals the presence of two conserved glutamate-histidine motifs, identifying it as a member of the diiron carboxylate protein family. The AOX model is defined by two pairs of helices forming a four helix bundle and an additional hydrophobic connecting sequence between the two helical pairs is proposed to act as the membrane anchoring region. In this work we aim at production, purification and crystallization of the AOX protein from M. perniciosa for further structural studies of this membrane-associated protein, by X-ray protein crystallography (author)

  2. Biphenyl Modulates the Expression and Function of Respiratory Oxidases in the Polychlorinated-Biphenyls Degrader Pseudomonas pseudoalcaligenes KF707

    Directory of Open Access Journals (Sweden)

    Federica Sandri

    2017-06-01

    Full Text Available Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(caa3 type oxidases (Caa3 and Ccaa3, two cbb3 type oxidases (Cbb31 and Cbb32, and one bd type cyanide-insensitive quinol oxidase (CIO. While the activity and expression of both Cbb31 and Cbb32 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb32 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb31 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb32 and C(caa3 deletion mutants. These results, together, reveal that both function and expression of cbb3 and caa3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.

  3. Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

    Science.gov (United States)

    Thrower, J S; Blalock, R; Klinman, J P

    2001-08-14

    1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

  4. Expression Studies of Gibberellin Oxidases in Developing Pumpkin Seeds1

    Science.gov (United States)

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

    2003-01-01

    Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA12-aldehyde, GA12, GA15, GA24, GA25, and GA9 to GA14-aldehyde, GA14, GA37, GA36, GA13, and GA4, respectively. Recombinant 2-ox protein oxidized GA9, GA4, and GA1 to GA51, GA34, and GA8, respectively. Previously cloned GA 7-oxidase revealed additional 3β-hydroxylation activity of GA12. Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2β,3β-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed. PMID:12644672

  5. Discovery, characterization, and kinetic analysis of an alditol oxidase from streptomyces coelicolor

    NARCIS (Netherlands)

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

    2007-01-01

    A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each

  6. S-adenosylmethionine decarboxylase inhibitors: new aryl and heteroaryl analogues of methylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Stanek, J; Caravatti, G; Capraro, H G; Furet, P; Mett, H; Schneider, P; Regenass, U

    1993-01-08

    A series of 3-acylbenzamidine (amidino)hydrazones 7a-h, the corresponding (hetero)aromatic congeners 7i-p, and 3,3'-bis-amidino-biaryls 25a-e were synthesized. The hydrazones 7a-p were prepared by conversion of the corresponding acyl nitriles 1a,c-d,i,n-p to the imido esters 3a,c-d,i and the amidines 5a,c-d,h-i, followed by a reaction with aminoguanidine, or vice versa. Similarly, the biaryl 3,3'-dinitriles 23a-e were converted, via the imino esters 24a-c or the imino thioesters 27d-e, to the diamidines 25a-e. These new products are conformationally constrained analogues of methylglyoxal bis(guanylhydrazone) (MGBG). They are up to 100 times more potent as inhibitors of rat liver S-adenosylmethionine decarboxylase (SMDC) and generally less potent inhibitors of rat small intestine diamine oxidase (DAO) than MGBG. Some of these SAMDC inhibitors, e.g., compounds 7a, 7e, 7i, 25a, and 25d, have shown antiproliferative effects against T24 human bladder carcinoma cells. These products, whose structure-activity relationships are discussed, are of interest as potential anticancer agents and drugs for the treatment of protozoal and Pneumocystis carinii infections.

  7. Increased xanthine oxidase during labour--implications for oxidative stress.

    Science.gov (United States)

    Many, A; Roberts, J M

    1997-11-01

    Xanthine dehydrogenase/oxidase (XDH/XO) produces uric acid. When in the oxidase form, this production is coupled with the generation of free radicals. Hypoxia-reperfusion enhances conversion of XDH to XO. Since the placenta is exposed to short periods of hypoxia reperfusion during labour, 17 placentae of pregnancy terminated by elective caesarean section and five placentae of pregnancies terminated by caesarean section during labour were examined for XDH/XO activity. It was found that XO activity was higher in the placentae of labouring women (P = 0.003), which suggests that labour enhances conversion of XDH to XO, facilitating free radical production.

  8. Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

    Science.gov (United States)

    Gabler, M; Fischer, L

    1999-08-01

    The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1. 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers. The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).

  9. Monochloramine produces reactive oxygen species in liver by converting xanthine dehydrogenase into xanthine oxidase.

    Science.gov (United States)

    Sakuma, Satoru; Miyoshi, Emi; Sadatoku, Namiko; Fujita, Junko; Negoro, Miki; Arakawa, Yukio; Fujimoto, Yohko

    2009-09-15

    In the present study, we assessed the influence of monochloramine (NH(2)Cl) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, NH(2)Cl (2.5-20 microM) dose-dependently enhanced XO activity concomitant with a decrease in XD activity, implying that NH(2)Cl can convert XD into the reactive oxygen species (ROS) producing form XO. The NH(2)Cl (5 microM)-induced XD/XO interconversion in the rat liver cytosol was completely inhibited when added in combination with an inhibitor of NH(2)Cl methionine (25 microM). A sulfhydryl reducing agent, dithiothreitol at concentrations of 0.1, 1 and 5 mM also dose-dependently reversed the NH(2)Cl (5 microM)-induced XD/XO interconversion. These imply that NH(2)Cl itself acts on the XD/XO interconversion, and that this conversion occurs at the cysteine residues in XD. Furthermore, using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, it was found that NH(2)Cl could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an XO inhibitor, allopurinol. These results suggest that NH(2)Cl has the potential to convert XD into XO in the liver, which in turn may induce the ROS generation in this region.

  10. Mediation of glycosylated and partially-deglycosylated glucose oxidase of Aspergillus niger by a ferrocene-derivatised detergent.

    Science.gov (United States)

    Fraser, D M; Zakeeruddin, S M; Grätzel, M

    1992-01-30

    A ferrocene-derivatised detergent, (11-ferrocenylundecyl) trimethylammonium bromide (FTMAB), when oxidised to the corresponding ferricinium ion, was found by electrochemical studies to be an effective electron acceptor for reduced glucose oxidase of Aspergillus niger (EC 1.13.4) and thus acts as a electron-transfer mediator between glucose oxidase and a working electrode held at a potential sufficiently positive to reoxidise reduced FTMAB. An increase in mediating activity was produced when FTMAB was present in concentrations above its critical micelle concentration. An 'enzyme electrode' was formed by adsorption of glucose oxidase and FTMAB surfactant on a graphite rod. The electrode functioned as an amperometric biosensor for glucose in phosphate-buffered saline solution. A mixed micelle of glucose oxidase and FTMAB, probably adsorbed on the electrode surface, appears to be advantageous for the amperometric determination of glucose. Additionally, glucose oxidase was treated with alpha-mannosidase. When this partially-deglycosylated glucose oxidase was incorporated in an enzyme electrode, a 100-fold increase in the second-order rate constant (k) for electron transfer between the enzyme and FTMAB was observed, together with increased current densities, with respect to the equivalent values for FTMAB and commercial glucose oxidase. The use of deglycosylated enzymes in biosensors is suggested.

  11. Intramolecular electron transfer in ascorbate oxidase is enhanced in the presence of oxygen

    DEFF Research Database (Denmark)

    Farver, O; Wherland, S; Pecht, I

    1994-01-01

    Intramolecular electron transfer from the type 1 copper center to the type 3 copper(II) pair is induced in the multi-copper enzyme, ascorbate oxidase, following pulse radiolytic reduction of the type 1 Cu(II) ion. In the presence of a slight excess of dioxygen over ascorbate oxidase, interaction...... between the trinuclear copper center and O2 is observed even with singly reduced ascorbate oxidase molecules. Under these conditions, the rate constant for intramolecular electron transfer from type 1 Cu(I) to type 3 Cu(II) increases 5-fold to 1100 +/- 300 s-1 (20 degrees C, pH 5.8) as compared...

  12. Polyacrylic acid-coated cerium oxide nanoparticles: An oxidase mimic applied for colorimetric assay to organophosphorus pesticides.

    Science.gov (United States)

    Zhang, Shi-Xiang; Xue, Shi-Fan; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-11-15

    It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Xanthine oxidase activity regulates human embryonic brain cells growth

    Directory of Open Access Journals (Sweden)

    Kevorkian G. A.

    2011-10-01

    Full Text Available Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22 in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90. XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p < 0.05. On day 12th the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96 and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05. In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.

  14. Age influences the effects of nicotine and monoamine oxidase inhibition on mood-related behaviors in rats.

    Science.gov (United States)

    Villégier, Anne-Sophie; Gallager, Brittney; Heston, Jon; Belluzzi, James D; Leslie, Frances M

    2010-03-01

    Epidemiological studies have demonstrated a comorbidity of smoking with depression and anxiety, particularly during adolescence. However, few animal studies have considered possible synergistic interactions between nicotine and other tobacco smoke constituents, such as monoamine oxidase (MAO) inhibitors, in the regulation of mood. The aim of the study was to test the hypothesis that nicotine combined with the irreversible MAO inhibitor, tranylcypromine, will differentially affect depression- and anxiety-related behaviors in adolescent and adult rats. Nicotine (0, 0.05, 0.2 mg/kg, s.c.) and tranylcypromine (3 mg/kg, i.p.) were tested separately, or together, on male rats aged postnatal days 30 and 68, in three mood-related behavioral tests: forced swim test (FST), elevated plus maze (EPM), and open field. Nicotine (0.2 mg/kg) in adults significantly decreased floating time in the FST and increased time spent in the open arm of the EPM, with no change in locomotor activity. Tranylcypromine pretreatment combined with nicotine (0.2 mg/kg) significantly increased locomotor activity and time spent in the center of the open field. Whereas nicotine alone had no significant effect on adolescents, it significantly increased locomotor activity and decreased floating time in the FST when combined with tranylcypromine pretreatment. There is an age-dependent effect of nicotine, alone and in combination with MAO inhibition, on mood-related behaviors. Whereas nicotine alone induces mood improvement in adults, it has no effect on adolescents. Nicotine combined with tranylcypromine has unique, age-dependent effects. Thus, experimental studies of smoking should consider both age and other tobacco constituents, such as MAO inhibitors, as critical factors.

  15. Ratiometric glucose sensing based on fluorescent oxygen films and glucose oxidase

    Directory of Open Access Journals (Sweden)

    Fengyu Su

    2017-06-01

    Full Text Available A new two-layer sensor film was constructed for sensing glucose based on glucose oxidase and oxygen sensing material. The first layer of film containing the oxygen sensor and intra-reference material was polymerized, then the second layer of glucose oxidase and glutaraldehyde was formed on the oxygen sensor layer. The two-layer sensor film has a resolution up to 0.05 mM and a detection range from 0 to 5 mM to glucose. The effects of pH and temperature on the sensing performance were systematically investigated. The selective detection of glucose among other monosaccharides, such as fructose, mannose and galactose indicated that the sensing film has excellent selectivity. The prepared sensor was successfully applied for glucose sample detection of glucose concentration in artificial tears. Keywords: Glucose sensor, Glucose oxidase, Fluorescence, Oxygen film, Diabetes

  16. Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Tamayo-Ramos Juan Antonio

    2012-12-01

    Full Text Available Abstract Background Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated. Results The laccase-like multicopper oxidases McoA, McoB and McoG from the commonly used cell factory Aspergillus niger were homologously expressed, purified and analyzed for their biocatalytic potential. All three recombinant enzymes were monomers with apparent molecular masses ranging from 80 to 110 kDa. McoA and McoG resulted to be blue, whereas McoB was yellow. The newly obtained oxidases displayed strongly different activities towards aromatic compounds and synthetic dyes. McoB exhibited high catalytic efficiency with N,N-dimethyl-p-phenylenediamine (DMPPDA and 2,2-azino-di(3-ethylbenzthiazoline sulfonic acid (ABTS, and appeared to be a promising biocatalyst. Besides oxidizing a variety of phenolic compounds, McoB catalyzed successfully the decolorization and detoxification of the widely used textile dye malachite green. Conclusions The A. niger McoA, McoB, and McoG enzymes showed clearly different catalytic properties. Yellow McoB showed broad substrate specificity, catalyzing the oxidation of several phenolic compounds commonly present in different industrial effluents. It also harbored high decolorization and detoxification activity with the synthetic dye malachite green, showing to have an interesting potential as a new industrial biocatalyst.

  17. Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose

    International Nuclear Information System (INIS)

    Xia, Xiaodong; Long, Yunfei; Wang, Jianxiu

    2013-01-01

    Highlights: ► A glucose oxidase/gold nanocluster conjugates formed by etching chemistry. ► Integration of the bioactivities and fluorescence properties within a single unit. ► These conjugates serve as novel fluorescent probe for glucose. -- Abstract: Creation and application of noble metal nanoclusters have received continuous attention. By integrating enzyme activity and fluorescence for potential applications, enzyme-capped metal clusters are more desirable. This work demonstrated a glucose oxidase (an enzyme for glucose)-functionalized gold cluster as probe for glucose. Under physiological conditions, such bioconjugate was successfully prepared by an etching reaction, where tetrakis (hydroxylmethyl) phosphonium-protected gold nanoparticle and thioctic acid-modified glucose oxidase were used as precursor and etchant, respectively. These bioconjugates showed unique fluorescence spectra (λ em max = 650 nm, λ ex max = 507 nm) with an acceptable quantum yield (ca. 7%). Moreover, the conjugated glucose oxidase remained active and catalyzed reaction of glucose and dissolved O 2 to produce H 2 O 2 , which quenched quantitatively the fluorescence of gold clusters and laid a foundation of glucose detection. A linear range of 2.0 × 10 −6 –140 × 10 −6 M and a detection limit of 0.7 × 10 −6 M (S/N = 3) were obtained. Also, another horseradish peroxidase/gold cluster bioconjugate was produced by such general synthesis method. Such enzyme/metal cluster bioconjugates represented a promising class of biosensors for biologically important targets in organelles or cells

  18. Influence of iodinated contrast media on the activities of histamine inactivating enzymes diamine oxidase and histamine N-methyltransferase in vitro.

    Science.gov (United States)

    Kuefner, M A; Feurle, J; Petersen, J; Uder, M; Schwelberger, H G

    2014-01-01

    Iodinated contrast media can cause pseudoallergic reactions associated with histamine release in significant numbers of patients. To clarify whether these adverse reactions may be aggravated by a compromised histamine catabolism we asked if radiographic contrast agents in vitro inhibit the histamine inactivating enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HMT). Nine iodinated contrast agents were tested in vitro. Following pre-incubation of purified porcine kidney DAO and recombinant human HMT with 0.1-10mM of the respective contrast medium (H2O and specific inhibitors of DAO and HMT as controls) enzyme activities were determined by using radiometric micro assays. None of the contrast media irrespective of their structure showed significant inhibition of the activities of DAO and HMT. Pre-incubation of the enzymes with specific inhibitors led to complete inhibition of the respective enzymatic activity. The iodinated contrast media tested in vitro did not exhibit inhibition of histamine converting enzymes at physiologically relevant concentrations. However due to the in vitro character of this study these results do not directly reflect the in vivo situation. Copyright © 2012 SEICAP. Published by Elsevier Espana. All rights reserved.

  19. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin [Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, 100191 (China); Wei, Ling [Beijing Center for Physical & Chemical Analysis, Beijing 100089 (China); Liu, Yutong [School of Life Science, Beijing Normal University, Beijing 100875 (China); Liao, Jieying [Department of Translational Medicine, Xiamen Institute of Rare Earth Materials, Chinese Academy of Sciences, Xiamen 361024 (China); Gao, Hui-Ming [Model Animal Research Center of Nanjing University, Nanjing 211800 (China); Zhou, Hui, E-mail: hardhui@gmail.com [Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, 100191 (China)

    2017-05-01

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to

  20. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

    International Nuclear Information System (INIS)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin; Wei, Ling; Liu, Yutong; Liao, Jieying; Gao, Hui-Ming; Zhou, Hui

    2017-01-01

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm 2 induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm 2 ) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91 phox , p47 phox and p40 phox ); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47 phox and p67 phox translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our

  1. Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

    Science.gov (United States)

    Abduvali Valiev; Zumrut B. Ogel; Dier D. Klepzig

    2009-01-01

    In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase....

  2. Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

    International Nuclear Information System (INIS)

    Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

    2011-01-01

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O 3 ) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O 3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O 3 fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O 3 , determined from the mRNA levels of the major allergens. We conclude that O 3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: → O 3 reduces the viability of ragweed pollen. → ROS and allergens of ragweed pollen were not affected by O 3 exposure. → O 3 enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. → O 3 increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

  3. Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

    Energy Technology Data Exchange (ETDEWEB)

    Pasqualini, Stefania, E-mail: spas@unipg.it [Department of Applied Biology, University of Perugia, Perugia (Italy); Tedeschini, Emma; Frenguelli, Giuseppe [Department of Applied Biology, University of Perugia, Perugia (Italy); Wopfner, Nicole; Ferreira, Fatima [Department of Molecular Biology, CD Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg (Austria); D' Amato, Gennaro [Division of Respiratory and Allergic Diseases, ' A. Cardarelli' High Speciality Hospital, Naples (Italy); Ederli, Luisa [Department of Applied Biology, University of Perugia, Perugia (Italy)

    2011-10-15

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O{sub 3}) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O{sub 3} fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O{sub 3} fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O{sub 3}, determined from the mRNA levels of the major allergens. We conclude that O{sub 3} can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: > O{sub 3} reduces the viability of ragweed pollen. > ROS and allergens of ragweed pollen were not affected by O{sub 3} exposure. > O{sub 3} enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. > O{sub 3} increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

  4. The effect of orexin-A on cardiac dysfunction mediated by NADPH oxidase-derived superoxide anion in ventrolateral medulla.

    Directory of Open Access Journals (Sweden)

    Jun Chen

    Full Text Available Hypocretin/orexin-producing neurons, located in the perifornical region of the lateral hypothalamus area (LHA and projecting to the brain sites of rostral ventrolateral medulla (RVLM, involve in the increase of sympathetic activity, thereby regulating cardiovascular function. The current study was designed to test the hypothesis that the central orexin-A (OXA could be involved in the cardiovascular dysfunction of acute myocardial infarction (AMI by releasing NAD(PH oxidase-derived superoxide anion (O2 (- generation in RVLM, AMI rat model established by ligating the left anterior descending (LAD coronary artery to induce manifestation of cardiac dysfunction, monitored by the indicators as heart rate (HR, heart rate variability (HRV, mean arterial pressure (MAP and left intraventricular pressure. The results showed that the expressions of OXA in LHA and orexin 1 receptor (OX1R increased in RVLM of AMI rats. The double immunofluorescent staining indicated that OX1R positive cells and NAD(PH oxidative subunit gp91phox or p47phox-immunoreactive (IR cells were co-localized in RVLM. Microinjection of OXA into the cerebral ventricle significantly increased O2 (- production and mRNA expression of NAD(PH oxidase subunits when compared with aCSF-treated ones. Exogenous OXA administration in RVLM produced pressor and tachycardiac effects. Furthermore, the antagonist of OX1R and OX2R (SB-408124 and TCS OX2 29, respectively or apocynin (APO, an inhibitor of NAD(PH oxidase, partly abolished those cardiovascular responses of OXA. HRV power spectral analysis showed that exogenous OXA led to decreased HF component of HRV and increased LF/HF ratio in comparison with aCSF, which suggested that OXA might be related to sympathovagal imbalance. As indicated by the results, OXA might participate in the central regulation of cardiovascular activities by disturbing the sympathovagal balance in AMI, which could be explained by the possibility that OXR and NAD(PH-derived O

  5. Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

    Science.gov (United States)

    Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

    2017-01-01

    Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

  6. Xanthine oxidase activity and free radical generation in patients with sepsis syndrome

    DEFF Research Database (Denmark)

    Galley, H F; Davies, Michael Jonathan; Webster, N R

    1996-01-01

    OBJECTIVE: To determine xanthine oxidase activity, free radical concentrations, and lipid peroxidation in patients with sepsis syndrome compared with noninfected critically ill patients. DESIGN: A prospective observational study. SETTING: A nine-bed intensive care unit in a university teaching......). CONCLUSIONS: Patients with sepsis have xanthine oxidase activation, high free-radical concentrations, and evidence of free radical damage. The finding that xanthine oxidase activity was lower in those patients who died, coupled with increased lactate concentrations implies more severe ischemia with incomplete...... to the Acute Physiology and Chronic Health Evaluation (APACHE) II score or to the presence of organ dysfunction. The mean ascorbyl radical concentration (arbitrary units) determined by electron paramagnetic resonance following spin trapping was increased in patients compared with healthy subjects (p

  7. Synthesis and characterization of [{sup 125}I]N-(2-aminoethyl)-4-iodobenzamide as a selective monoamine oxidase B inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Rafii, Hamid; Chalon, Sylvie; Ombetta, Jean-Edouard; Frangin, Yves; Garreau, Lucette; Dognon, Anne-Marie; Lena, Isabelle; Bodard, Sylvie; Vilar, Marie-Paule; Besnard, Jean-Claude; Guilloteau, Denis

    1995-07-01

    We described the radiosynthesis of an analog of Ro 16-6491, [{sup 125}I]N-(2-aminoethyl)-4-iodobenzamide, for SPECT exploration of the monoamine oxidase B (MAO-B) in human brain. The radiolabelling was carried out by nucleophilic exchange of the brominated precursor at solid-state phase in presence of ammonium sulphate. The radiochemical purity of radioiodinated product was higher than 95%. In comparison with Ro 16-6491, the in vitro studies showed a good selectivity of stable N-(2-aminoethyl)-4-iodobenzamide for MAO-B but a slightly lower affinity. Biodistribution studies in the rat showed a high and selective uptake of this compound in the pineal gland 1 h after i.v. injection. The cerebral uptake was low, but the coupling of [{sup 125}I]N-(2-aminoethyl)-4-iodobenzamide with a lipophilic radical to enhance the passage through the blood-brain barrier can be envisaged.

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

    Science.gov (United States)

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

    2014-04-01

    The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-D-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. © 2014 The Protein Society.

  9. Age-related ultrastructural and monoamine oxidase changes in the rat optic nerve.

    Science.gov (United States)

    Taurone, S; Ripandelli, G; Minni, A; Lattanzi, R; Miglietta, S; Pepe, N; Fumagalli, L; Micera, A; Pastore, F S; Artico, M

    2016-01-01

    The aim of this paper is to study the morphology and the distribution of the monoamine oxidase enzymatic system in the optic nerve of 4 month-old Wistar (young) and 28 month-old Wistar (old) rats. The optic nerve was harvested from 20 young and old rats. The segment of optic nerve was divided longitudinally into two pieces, each 0.1 mm in length. The first piece was used for transmission electron microscopy. The second piece was stained with histochemical reaction for monoamine oxidase. The agerelated changes in the optic nerve of rats include micro-anatomical details, ultrastructure and monoamine oxidase histochemical staining. A strong decrease of the thin nerve fibers and a swelling of the thick ones can be observed in optic nerve fibers of old rats. Increased monoamine oxidase histochemical staining of the optic nerve of aged rats is well demonstrated. The increase of meningeal shealth and the decrease of thin nerve fibers of the optic nerve in old rats are well documented. Morphological, ultrastructural and histochemical changes observed in optic nerve fibers of the old rats show a close relation with aging.

  10. The antioxidant properties, cytotoxicity and monoamine oxidase ...

    African Journals Online (AJOL)

    Tarchonanthus camphoratus (camphor bush) has been widely used for numerous medicinal purposes. The aim of the present study was to evaluate the antioxidant properties, cytotoxicity and monoamine oxidase inhibition activities of the crude dichloromethane leaf extract of T. camphoratus. The antioxidant activities were ...

  11. Update on the pharmacology of selective inhibitors of MAO-A and MAO-B: focus on modulation of CNS monoamine neurotransmitter release.

    Science.gov (United States)

    Finberg, John P M

    2014-08-01

    Inhibitors of monoamine oxidase (MAO) were initially used in medicine following the discovery of their antidepressant action. Subsequently their ability to potentiate the effects of an indirectly-acting sympathomimetic amine such as tyramine was discovered, leading to their limitation in clinical use, except for cases of treatment-resistant depression. More recently, the understanding that: a) potentiation of indirectly-acting sympathomimetic amines is caused by inhibitors of MAO-A but not by inhibitors of MAO-B, and b) that reversible inhibitors of MAO-A cause minimal tyramine potentiation, has led to their re-introduction to clinical use for treatment of depression (reversible MAO-A inhibitors and new dose form MAO-B inhibitor) and treatment of Parkinson's disease (MAO-B inhibitors). The profound neuroprotective properties of propargyl-based inhibitors of MAO-B in preclinical experiments have drawn attention to the possibility of employing these drugs for their neuroprotective effect in neurodegenerative diseases, and have raised the question of the involvement of the MAO-mediated reaction as a source of reactive free radicals. Despite the long-standing history of MAO inhibitors in medicine, the way in which they affect neuronal release of monoamine neurotransmitters is still poorly understood. In recent years, the detailed chemical structure of MAO-B and MAO-A has become available, providing new possibilities for synthesis of mechanism-based inhibitors. This review describes the latest advances in understanding the way in which MAO inhibitors affect the release of the monoamine neurotransmitters dopamine, noradrenaline and serotonin (5-HT) in the CNS, with an accent on the importance of these effects for the clinical actions of the drugs. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

    Science.gov (United States)

    Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

    2013-04-10

    Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, Gloeophyllum trabeum

    Science.gov (United States)

    Diane Dietrich; Casey Crooks

    2009-01-01

    A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5’UTR...

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

    NARCIS (Netherlands)

    Hackenberg, C.; Kern, R.; Hüge, J; Stal, L.J.; Tsuji, Y.; Kopka, J.; Shiraiwa, Y.; Bauwe, H.; Hagemann, M.

    2011-01-01

    Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to

  15. Cyanobacterial lactate oxidases serve as essential partners of N2-fixation and evolved to photorespiratory glycolate oxidases in plants

    NARCIS (Netherlands)

    Hackenberg, C.; Kern, R.; Hüge, J.; Stal, L.J.; Tsuji, Y.; Kopka, J.; Shiraiwa, Y.; Bauwe, H.; Hagemann, M.

    2011-01-01

    Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to

  16. Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species*

    Science.gov (United States)

    Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

    2012-01-01

    The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species. PMID:22287551

  17. Quantum chemical modeling of the inhibition mechanism of monoamine oxidase by oxazolidinone and analogous heterocyclic compounds.

    Science.gov (United States)

    Erdem, Safiye Sağ; Özpınar, Gül Altınbaş; Boz, Ümüt

    2014-02-01

    Monoamine oxidase (MAO, EC 1.4.3.4) is responsible from the oxidation of a variety of amine neurotransmitters. MAO inhibitors are used for the treatment of depression or Parkinson's disease. They also inhibit the catabolism of dietary amines. According to one hypothesis, inactivation results from the formation of a covalent adduct to a cysteine residue in the enzyme. If the adduct is stable enough, the enzyme is inhibited for a long time. After a while, enzyme can turn to its active form as a result of adduct breakdown by β-elimination. In this study, the proposed inactivation mechanism was modeled and tested by quantum chemical calculations. Eight heterocyclic methylthioamine derivatives were selected to represent the proposed covalent adducts. Activation energies related to their β-elimination reactions were calculated using ab initio and density functional theory methods. Calculated activation energies were in good agreement with the relative stabilities of the hypothetical adducts predicted in the literature by enzyme inactivation measurements.

  18. Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases

    Directory of Open Access Journals (Sweden)

    Gábor Sirokmány

    2018-06-01

    Full Text Available Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels. Keywords: Peroxidasin, NADPH oxidase, Hydrogen peroxide, Collagen IV, Sulfilimine

  19. A Biocatalytic One-Pot Approach for the Preparation of Lignin Oligomers Using an Oxidase/Peroxidase Cascade Enzyme System

    NARCIS (Netherlands)

    Habib, Mohamed H. M.; Deuss, Peter J.; Loncar, Nikola; Trajkovic, Milos; Fraaije, Marco W.

    2017-01-01

    Synthetic lignin was prepared biocatalytically in a one-pot, two-step reaction using an oxidase/peroxidase cascade enzyme system. Using eugenol in combination with eugenol oxidase and a peroxidase, lignin-like material was produced. The cascade reaction takes advantage of the ability of the oxidase

  20. Plasma diamine oxidase activity in asthmatic children

    Directory of Open Access Journals (Sweden)

    Kyoichiro Toyoshima

    1996-01-01

    Full Text Available Histamine plays an important role in the development of asthmatic symptoms. Diamine oxidase (DAO histaminase, which inactivates histamine, is located in the intestine and kidney and is released into plasma. Plasma DAO activity in asthmatic children was measured by a recently developed high performance liquid chromatographic method using histamine as the DAO substrate. Diamine oxidase activity was higher in severely asthmatic children than in those with mild asthma. A time course study during the acute exacerbation phase revealed that DAO activity rose during acute asthmatic attacks and then decreased gradually over several days. Although the mechanisms of plasma DAO activity increase during acute asthmatic attacks could not be explained, data showed that plasma DAO activity is an important index of histamine metabolism in asthmatics and may relate to some mechanisms of acute exacerbation of airway inflammation. Consequently, fluctuations in plasma DAO can be used as one of various indices of instability in management of asthma.

  1. Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Xiaodong [College of Chemistry and Chemical Engineering, Central South University, Changsha 410083 (China); School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201 (China); Long, Yunfei, E-mail: l_yunfei927@163.com [School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201 (China); Wang, Jianxiu, E-mail: jxiuwang@csu.edu.cn [College of Chemistry and Chemical Engineering, Central South University, Changsha 410083 (China)

    2013-04-15

    Highlights: ► A glucose oxidase/gold nanocluster conjugates formed by etching chemistry. ► Integration of the bioactivities and fluorescence properties within a single unit. ► These conjugates serve as novel fluorescent probe for glucose. -- Abstract: Creation and application of noble metal nanoclusters have received continuous attention. By integrating enzyme activity and fluorescence for potential applications, enzyme-capped metal clusters are more desirable. This work demonstrated a glucose oxidase (an enzyme for glucose)-functionalized gold cluster as probe for glucose. Under physiological conditions, such bioconjugate was successfully prepared by an etching reaction, where tetrakis (hydroxylmethyl) phosphonium-protected gold nanoparticle and thioctic acid-modified glucose oxidase were used as precursor and etchant, respectively. These bioconjugates showed unique fluorescence spectra (λ{sub em} {sub max} = 650 nm, λ{sub ex} {sub max} = 507 nm) with an acceptable quantum yield (ca. 7%). Moreover, the conjugated glucose oxidase remained active and catalyzed reaction of glucose and dissolved O{sub 2} to produce H{sub 2}O{sub 2}, which quenched quantitatively the fluorescence of gold clusters and laid a foundation of glucose detection. A linear range of 2.0 × 10{sup −6}–140 × 10{sup −6} M and a detection limit of 0.7 × 10{sup −6} M (S/N = 3) were obtained. Also, another horseradish peroxidase/gold cluster bioconjugate was produced by such general synthesis method. Such enzyme/metal cluster bioconjugates represented a promising class of biosensors for biologically important targets in organelles or cells.

  2. Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae*

    Science.gov (United States)

    Nguyen, Don Trinh; Göpfert, Jens Christian; Ikezawa, Nobuhiro; MacNevin, Gillian; Kathiresan, Meena; Conrad, Jürgen; Spring, Otmar; Ro, Dae-Kyun

    2010-01-01

    Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature. PMID:20351109

  3. Significance of membrane bioreactor design on the biocatalytic performance of glucose oxidase and catalase: Free vs. immobilized enzyme systems

    DEFF Research Database (Denmark)

    Morthensen, Sofie Thage; Meyer, Anne S.; Jørgensen, Henning

    2017-01-01

    Membrane separation of xylose and glucose can be accomplished via oxidation of glucose to gluconic acid by enzymatic glucose oxidase catalysis. Oxygen for this reaction can be supplied via decomposition of hydrogen peroxide by enzymatic catalase catalysis. In order to maximize the biocatalytic...... productivity of glucose oxidase and catalase (gluconic acid yield per total amount of enzyme) the following system set-ups were compared: immobilization of glucose oxidase alone; co-immobilization of glucose oxidase and catalase; glucose oxidase and catalase free in the membrane bioreactor. Fouling......-induced enzyme immobilization in the porous support of an ultrafiltration membrane was used as strategy for entrapment of glucose oxidase and catalase. The biocatalytic productivity of the membrane reactor was found to be highly related to the oxygen availability, which in turn depended on the reactor...

  4. Conformational lock and dissociative thermal inactivation of lentil seedling amine oxidase.

    Science.gov (United States)

    Moosavi-Nejad, S Zahra; Moosavi-Movahedi, Ali-Akbar; Rezaei-Tavirani, Mostafa; Floris, Giovanni; Medda, Rosaria

    2003-03-31

    The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 degrees C. The thermal inactivation curves were not linear at 52 and 57 degrees C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 degrees C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.

  5. The glucose oxidase-peroxidase assay for glucose

    Science.gov (United States)

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

  6. [The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation].

    Science.gov (United States)

    Stasia, Marie-José

    2007-05-01

    Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.

  7. In Vitro Effects of Cognitives and Nootropics on Mitochondrial Respiration and Monoamine Oxidase Activity.

    Science.gov (United States)

    Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

    2017-10-01

    Impairment of mitochondrial metabolism, particularly the electron transport chain (ETC), as well as increased oxidative stress might play a significant role in pathogenesis of Alzheimer's disease (AD). Some effects of drugs used for symptomatic AD treatment may be related to their direct action on mitochondrial function. In vitro effects of pharmacologically different cognitives (galantamine, donepezil, rivastigmine, 7-MEOTA, memantine) and nootropic drugs (latrepirdine, piracetam) were investigated on selected mitochondrial parameters: activities of ETC complexes I, II + III, and IV, citrate synthase, monoamine oxidase (MAO), oxygen consumption rate, and hydrogen peroxide production of pig brain mitochondria. Complex I activity was decreased by galantamine, donepezil, and memantine; complex II + III activity was increased by galantamine. None of the tested drugs caused significant changes in the rate of mitochondrial oxygen consumption, even at high concentrations. Except galantamine, all tested drugs were selective MAO-A inhibitors. Latrepirdine, donepezil, and 7-MEOTA were found to be the most potent MAO-A inhibitors. Succinate-induced mitochondrial hydrogen peroxide production was not significantly affected by the drugs tested. The direct effect of cognitives and nootropics used in the treatment of AD on mitochondrial respiration is relatively small. The safest drugs in terms of disturbing mitochondrial function appear to be piracetam and rivastigmine. The MAO-A inhibition by cognitives and nootropics may also participate in mitochondrial neuroprotection. The results support the future research aimed at measuring the effects of currently used drugs or newly synthesized drugs on mitochondrial functioning in order to understand their mechanism of action.

  8. Interrupted reperfusion reduces the activation of NADPH oxidase after cerebral I/R injury.

    Science.gov (United States)

    Shen, Jia; Bai, Xiao-Yin; Qin, Yuan; Jin, Wei-Wei; Zhou, Jing-Yin; Zhou, Ji-Ping; Yan, Ying-Gang; Wang, Qiong; Bruce, Iain C; Chen, Jiang-Hua; Xia, Qiang

    2011-06-15

    Interrupted reperfusion reduces ischemia/reperfusion (I/R) injury. This study was designed to determine whether NADPH oxidase participates in the neural protection against global I/R injury after interrupted reperfusion. Mice were randomly divided into five groups: sham (sham-operated), I/R (20-min global I/R), RR (I/R+interrupted reperfusion), Apo (I/R+apocynin administration), and RR+Apo. Behavioral tests (pole test, beam walking, and Morris water maze) and Nissl staining were undertaken in all five groups; superoxide levels, expression of gp91(phox) and p47(phox), p47(phox) translocation, and Rac1 activation were measured in the sham, I/R, and RR groups. The motor coordination, bradykinesia, and spatial learning and memory, as well as the neuron survival rates, were better in the RR, Apo, and RR+Apo groups than in the I/R group. The NADPH oxidase-dependent superoxide levels, p47(phox) and gp91(phox) expression, p47(phox) translocation, and Rac1 activation were lower in the RR group than in the I/R group. In conclusion, the neural protective effect of interrupted reperfusion is at least partly mediated by decreasing the expression and assembly of NADPH oxidase and the levels of NADPH oxidase-derived superoxide. The most striking reduction Rac1-GTP in the RR group suggests that interrupted reperfusion also acts on the activation of assembled NADPH oxidase by reducing the availability of Rac1-GTP. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Molla Gianluca

    2010-04-01

    Full Text Available Abstract Background Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO. Results Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies. Conclusions Comparison of our results with those published on non-covalent (type I COs expressed in recombinant form (either in E. coli or Streptomyces spp., shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.

  10. Activation of PAR-1/NADPH Oxidase/ROS Signaling Pathways is Crucial for the Thrombin-Induced sFlt-1 Production in Extravillous Trophoblasts: Possible Involvement in the Pathogenesis of Preeclampsia

    Directory of Open Access Journals (Sweden)

    Qi-tao Huang

    2015-03-01

    Full Text Available Backgrounds/Aims: Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1 expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT. Methods: An EVT cell line (HRT-8/SVneo was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS production were determined by DCFH-DA. Results: Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Conclusions: Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future.

  11. Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia.

    Science.gov (United States)

    Huang, Qi-Tao; Chen, Jian-Hong; Hang, Li-Lin; Liu, Shi-San; Zhong, Mei

    2015-01-01

    Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT). An EVT cell line (HRT-8/SVneo) was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production were determined by DCFH-DA. Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA) directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase) could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future. © 2015 S. Karger AG, Basel.

  12. Wound-induced ethylene synthesis and expression and formation of 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, phenylalanine ammonia-lyase, and peroxidase in wounded mesocarp tissue of Cucurbita maxima.

    Science.gov (United States)

    Kato, M; Hayakawa, Y; Hyodo, H; Ikoma, Y; Yano, M

    2000-04-01

    1-Aminocyclopropane-1-carboxylate (ACC) synthase was rapidly induced in mesocarp tissue of Cucurbita maxima after wounding in the cut surface layer in 1 mm thickness (ca. 9 cells) (first layer) in both the enzyme activity and the levels of transcript. This led to a rapid accumulation of ACC and hence ethylene production. In the inside tissue (1-2 mm) (second layer), no significant induction of ACC synthase was observed, which resulted in a low level of ACC, although ethylene was evolved at a much lower rate than the first one. In contrast to ACC synthase, ACC oxidase was induced markedly in both the first and second layers and the development of its activity and the levels of mRNA remained high until later stages. It was considered that wound ethylene was closely associated with the development of ACC oxidase, since 2,5-norbornadiene (NBD), an inhibitor of ethylene action, substantially suppressed it. Phenylalanine ammonia-lyase (PAL) greatly increased in activity after wounding similarly to that of ACC synthase, in which increase in PAL activity occurred predominantly in the first layer. Induction of peroxidase activity after wounding had a close correlation in profile with that of ACC oxidase in that marked increases in the activity were observed in both the first and second layers and were strongly suppressed by NBD application. Four peroxidase isozymes were found by PAGE, among which a fraction was newly detected after wounding.

  13. Surface reconstitution of glucose oxidase onto a norbornylogous bridge self-assembled monolayer

    International Nuclear Information System (INIS)

    Liu Jingquan; Paddon-Row, Michael N.; Gooding, J. Justin

    2006-01-01

    An electrode construct was fabricated in which a self-assembled monolayer containing a novel norbornylogous bridge was covalently attached to flavin adenine dinucleotide (FAD), the redox active centre of several oxidase enzymes. The electrochemistry of the construct was investigated before and after the reconstitution of glucose oxidase around the surface bound FAD. Rapid rates of electron transfer were observed both before and after the reconstitution of biocatalytically active enzyme. However, no biocatalytic activity was observed under anaerobic conditions suggesting the a lack of enzyme turnover through direct electron transfer. It is proposed that a decrease in the electronic coupling between the redox active FAD and the electrode following reconstitution of the glucose oxidase - a probable consequence of the FAD being immersed in a protein environment - was responsible for the inability of the enzyme to be turned over under anaerobic conditions

  14. Genetic defects of cytochrome c oxidase assembly

    Czech Academy of Sciences Publication Activity Database

    Pecina, Petr; Houšťková, H.; Hansíková, H.; Zeman, J.; Houštěk, Josef

    2004-01-01

    Roč. 53, Suppl. 1 (2004), s. S213-S223 ISSN 0862-8408 R&D Projects: GA ČR GA303/03/0749 Institutional research plan: CEZ:AV0Z5011922 Keywords : cytochrome c oxidase * mitochondrial disorders Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.140, year: 2004

  15. Preferential inhibition of xanthine oxidase by 2-amino-6-hydroxy-8-mercaptopurine and 2-amino-6-purine thiol

    Directory of Open Access Journals (Sweden)

    Mukhopadhyay Anup

    2007-05-01

    Full Text Available Abstract Background The anticancer drug, 6-mercaptopurine (6MP is subjected to metabolic clearance through xanthine oxidase (XOD mediated hydroxylation, producing 6-thiouric acid (6TUA, which is excreted in urine. This reduces the effective amount of drug available for therapeutic efficacy. Co-administration of allopurinol, a suicide inhibitor of XOD, which blocks the hydroxylation of 6MP inadvertently enhances the 6MP blood level, counters this reduction. However, allopurinol also blocks the hydroxylation of hypoxanthine, xanthine (released from dead cancer cells leading to their accumulation in the body causing biochemical complications such as xanthine nephropathy. This necessitates the use of a preferential XOD inhibitor that selectively inhibits 6MP transformation, but leaves xanthine metabolism unaffected. Results Here, we have characterized two such unique inhibitors namely, 2-amino-6-hydroxy-8-mercaptopurine (AHMP and 2-amino-6-purinethiol (APT on the basis of IC50 values, residual activity in bi-substrate simulative reaction and the kinetic parameters like Km, Ki, kcat. The IC50 values of AHMP for xanthine and 6MP as substrate are 17.71 ± 0.29 μM and 0.54 ± 0.01 μM, respectively and the IC50 values of APT for xanthine and 6MP as substrates are 16.38 ± 0.21 μM and 2.57 ± 0.08 μM, respectively. The Ki values of XOD using AHMP as inhibitor with xanthine and 6MP as substrate are 5.78 ± 0.48 μM and 0.96 ± 0.01 μM, respectively. The Ki values of XOD using APT as inhibitor with xanthine and 6MP as substrate are 6.61 ± 0.28 μM and 1.30 ± 0.09 μM. The corresponding Km values of XOD using xanthine and 6MP as substrate are 2.65 ± 0.02 μM and 6.01 ± 0.03 μM, respectively. The results suggest that the efficiency of substrate binding to XOD and its subsequent catalytic hydroxylation is much superior for xanthine in comparison to 6MP. In addition, the efficiency of the inhibitor binding to XOD is much more superior when 6MP is the

  16. Diethylglyoxal bis(guanylhydrazone): a novel highly potent inhibitor of S-adenosylmethionine decarboxylase with promising properties for potential chemotherapeutic use.

    Science.gov (United States)

    Elo, H; Mutikainen, I; Alhonen-Hongisto, L; Laine, R; Jänne, J

    1988-07-01

    Diethylglyoxal bis(guanylhydrazone) (DEGBG), a novel analog of the antileukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) was synthesized. It was found to be the most powerful inhibitor of yeast S-adenosylmethionine decarboxylase (AdoMetDC) so far studied (Ki approx. 9 nM). This property, together with the finding that the compound is a weaker inhibitor of intestinal diamine oxidase than are MGBG and its glyoxal, ethylglyoxal and ethylmethylglyoxal analogs, makes the compound a promising candidate as a polyamine antimetabolite for chemotherapy studies. DEGBG was also found to potentiate the antiproliferative effect of the ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine against mouse L1210 leukemia cells in vitro. DEGBG increased several-fold the intracellular putrescine concentration of cultured L1210 cells, just as MGBG and its ethylglyoxal analog are known to do. The results strongly suggest that DEGBG is worth further studies. Combined with previous studies, they also made possible the construction of some empirical rules concerning the structure-activity relationships of bis(guanylhydrazone) type inhibitors of AdoMetDC. The identity of DEGBG was confirmed by a single-crystal X-ray analysis and by 1H- and 13C-NMR spectroscopy. It consisted of the same isomer as MGBG and several of its analogs are known to consist of.

  17. Salicylic Acid Regulation of Respiration in Higher Plants: Alternative Oxidase Expression.

    Science.gov (United States)

    Rhoads, DM; McIntosh, L

    1992-01-01

    Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (4 days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid (SA) in the appendix is very low early on D-1, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in SA by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of SA to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to SA was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of aox1. PMID:12297672

  18. Combined effect of loss of the caa3 oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments.

    Science.gov (United States)

    Zhou, Guangqi; Yin, Jianhua; Chen, Haijiang; Hua, Yijie; Sun, Linlin; Gao, Haichun

    2013-09-01

    Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (EA). Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa3- and cbb3-type) and a bd-type quinol oxidase. As aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain vastly unexplored. In this work, we discovered that the cbb3-type oxidase is the predominant system for respiration of oxygen (O2), especially when O2 is abundant. Under microaerobic conditions, the bd-type quinol oxidase has a significant role in addition to the cbb3-type oxidase. In contrast, multiple lines of evidence suggest that under test conditions the caa3-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression. In addition, expression of both cbb3- and bd-type oxidases is under direct control of Crp (cAMP receptor protein) but not the well-established redox regulator Fnr (fumarate nitrate regulator) of canonical systems typified in Escherichia coli. These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa3-type oxidase and switch of the regulatory system for respiration.

  19. NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

    International Nuclear Information System (INIS)

    Newaz, Mohammad; Blanton, Ahmad; Fidelis, Paul; Oyekan, Adebayo

    2005-01-01

    Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). L-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p 2+ -dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53-7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and

  20. Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase-producing Streptomyces cavourensis strain NEAE-42

    OpenAIRE

    El-Naggar, Noura El-Ahmady; El-Shweihy, Nancy M.; El-Ewasy, Sara M.

    2016-01-01

    Background Due to broad range of clinical and industrial applications of cholesterol oxidase, isolation and screening of bacterial strains producing extracellular form of cholesterol oxidase is of great importance. Results One hundred and thirty actinomycete isolates were screened for their cholesterol oxidase activity. Among them, a potential culture, strain NEAE-42 is displayed the highest extracellular cholesterol oxidase activity. It was selected and identified as Streptomyces cavourensis...

  1. Crystallization of carbohydrate oxidase from Microdochium nivale

    Czech Academy of Sciences Publication Activity Database

    Dušková, Jarmila; Dohnálek, Jan; Skálová, Tereza; Ostergaard, L. H.; Fuglsang, C. C.; Kolenko, Petr; Štěpánková, Andrea; Hašek, Jindřich

    2009-01-01

    Roč. 65, č. 6 (2009), s. 638-640 ISSN 1744-3091 R&D Projects: GA AV ČR IAA500500701; GA ČR GA305/07/1073 Institutional research plan: CEZ:AV0Z40500505 Keywords : carbohydrate oxidase * crystallization * data processing Subject RIV: CD - Macromolecular Chemistry Impact factor: 0.551, year: 2009

  2. Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees.

    Science.gov (United States)

    Haruta, M; Murata, M; Kadokura, H; Homma, S

    1999-03-01

    An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica). Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized. The deduced protein sequences showed identities of 85.3 to 97.5%. Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6. These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar.

  3. Ratiometric glucose sensing based on fluorescent oxygen films and glucose oxidase

    OpenAIRE

    Fengyu Su; Liqiang Zhang; Xiangxing Kong; Fred Lee; Yanqing Tian; Deirdre R. Meldrum

    2017-01-01

    A new two-layer sensor film was constructed for sensing glucose based on glucose oxidase and oxygen sensing material. The first layer of film containing the oxygen sensor and intra-reference material was polymerized, then the second layer of glucose oxidase and glutaraldehyde was formed on the oxygen sensor layer. The two-layer sensor film has a resolution up to 0.05 mM and a detection range from 0 to 5 mM to glucose. The effects of pH and temperature on the sensing performance were systemati...

  4. Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits.

    Science.gov (United States)

    Smith, Susan M E; Min, Jaeki; Ganesh, Thota; Diebold, Becky; Kawahara, Tsukasa; Zhu, Yerun; McCoy, James; Sun, Aiming; Snyder, James P; Fu, Haian; Du, Yuhong; Lewis, Iestyn; Lambeth, J David

    2012-06-22

    NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Kinetic Resolution of sec-Thiols via Enantioselective Oxidation with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase

    NARCIS (Netherlands)

    Pickl, Mathias; Swoboda, Alexander; Romero, Elvira; Winkler, Christoph; Binda, Claudia; Mattevi, Andrea; Faber, Kurt; Fraaije, Marco

    2018-01-01

    Various flavoprotein oxidases were recently shown to oxidize prim-thiols. Here we extend this reactivity towards sec-thiols via structure-guided engineering of 5-(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of rac-sec-thiols

  6. Polyphenol oxidase activity from three sicilian artichoke [ Cynara cardunculus L. Var. scolymus L. (Fiori)] cultivars: studies and technological application on minimally processed production.

    Science.gov (United States)

    Todaro, Aldo; Peluso, Orazio; Catalano, Anna Eghle; Mauromicale, Giovanni; Spagna, Giovanni

    2010-02-10

    Several papers helped with the development of more methods to control browning, or study thermal polyphenol oxidase (PPO) inactivation, but did not provide any solutions to technological process problems and food process improvement. Artichokes [ Cynara cardunculus L. var. scolymus L. (Fiori)] are susceptible to browning; this alteration could affect and reduce the suitability for its use, fresh or processed. Within this study, the catecholase and cresolase activities of PPO from three different Sicilian artichokes cultivar were characterized with regard to substrate specificity and enzyme kinetics, optimum pH and temperature, temperature and pH stability, and inhibitor test; all of the results were used for technological purposes, particularly to optimize minimally processed productions (ready-to-eat and cook-chilled artichokes).

  7. Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

    Directory of Open Access Journals (Sweden)

    Sluse F.E.

    1998-01-01

    Full Text Available Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression or short-term (post-translational modification, allosteric activation regulated. Electron distribution (partitioning between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach. Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon and with harmful reactive oxygen species formation.

  8. Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

    Science.gov (United States)

    Vanlerberghe, G C; McIntosh, L

    1992-09-01

    Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

  9. Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells

    Directory of Open Access Journals (Sweden)

    Lee I-Ta

    2012-11-01

    Full Text Available Abstract Background In bacteria-induced glomerulonephritis, Toll-like receptor 4 (TLR4 activation by lipopolysaccharide (LPS, a key component of the outer membranes of Gram-negative bacteria can increase oxidative stress and the expression of vascular cell adhesion molecule-1 (VCAM-1, which recruits leukocytes to the glomerular mesangium. However, the mechanisms underlying VCAM-1 expression induced by LPS are still unclear in human renal mesangial cells (HRMCs. Results We demonstrated that LPS induced VCAM-1 mRNA and protein levels associated with an increase in the promoter activity of VCAM-1, determined by Western blot, RT-PCR, and promoter assay. LPS-induced responses were inhibited by transfection with siRNAs of TLR4, myeloid differentiation factor 88 (MyD88, Nox2, Nox4, p47phox, c-Src, p38 MAPK, activating transcription factor 2 (ATF2, and p300 or pretreatment with the inhibitors of reactive oxygen species (ROS, edaravone, NADPH oxidase [apocynin (APO or diphenyleneiodonium chloride (DPI], c-Src (PP1, p38 MAPK (SB202190, and p300 (GR343. LPS induced NADPH oxidase activation, ROS production, and p47phox translocation from the cytosol to the membrane, which were reduced by PP1 or c-Src siRNA. We observed that LPS induced TLR4, MyD88, c-Src, and p47phox complex formation determined by co-immunoprecipitation and Western blot. We further demonstrated that LPS stimulated ATF2 and p300 phosphorylation and complex formation via a c-Src/NADPH oxidase/ROS/p38 MAPK pathway. Up-regulation of VCAM-1 led to enhancing monocyte adhesion to HRMCs challenged with LPS, which was inhibited by siRNAs of c-Src, p47phox, p38 MAPK, ATF2, and p300 or pretreatment with an anti-VCAM-1 neutralizing antibody. Conclusions In HRMCs, LPS-induced VCAM-1 expression was, at least in part, mediated through a TLR4/MyD88/ c-Src/NADPH oxidase/ROS/p38 MAPK-dependent p300 and ATF2 pathway associated with recruitment of monocyte adhesion to kidney. Blockade of these pathways may

  10. Layer-by-Layer Assembly of Glucose Oxidase on Carbon Nanotube Modified Electrodes.

    Science.gov (United States)

    Suroviec, Alice H

    2017-01-01

    The use of enzymatically modified electrodes for the detection of glucose or other non-electrochemically active analytes is becoming increasingly common. Direct heterogeneous electron transfer to glucose oxidase has been shown to be kinetically difficult, which is why electron transfer mediators or indirect detection is usually used for monitoring glucose with electrochemical sensors. It has been found, however, that electrodes modified with single or multi-walled carbon nanotubes (CNTs) demonstrate fast heterogeneous electron transfer kinetics as compared to that found for traditional electrodes. Incorporating CNTs into the assembly of electrochemical glucose sensors, therefore, affords the possibility of facile electron transfer to glucose oxidase, and a more direct determination of glucose. This chapter describes the methods used to use CNTs in a layer-by-layer structure along with glucose oxidase to produce an enzymatically modified electrode with high turnover rates, increased stability and shelf-life.

  11. Optimization of cholesterol oxidase production by Brevibacterium sp ...

    African Journals Online (AJOL)

    An ultrasound-assisted emulsification as a pretreatment for cholesterol oxidase production by submerge fermentation using Brevibacterium sp. in a batch system was studied. Medium improvement for the production employing response surface methodology (RSM) was optimized in this paper. The concentration of ...

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

    International Nuclear Information System (INIS)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-01-01

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na 2 SeO 3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant (K m ) values and maximal reaction velocity (V max ) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

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

    Science.gov (United States)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Julia Leclerc

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

  15. Association between plasma activities of semicarbazide-sensitive amine oxidase and angiotensin-converting enzyme in patients with type 1 diabetes mellitus

    DEFF Research Database (Denmark)

    Boomsma, F; Pedersen-Bjergaard, U; Agerholm-Larsen, Birgit

    2005-01-01

    AIMS/HYPOTHESIS: Plasma semicarbazide-sensitive amine oxidase (SSAO) is elevated in patients with type 1 and type 2 diabetes and has been implicated in the pathophysiology of diabetic late complications. The regulation of SSAO production remains unknown. We studied correlations between plasma SSAO...... activity and parameters associated with diabetic late complications. METHODS: Plasma SSAO was measured in a well-characterised group of 287 patients with type 1 diabetes. Standard statistical methods were used to investigate correlations with clinical parameters and components of the renin.......001, r=0.27) was found between plasma SSAO and serum ACE activity in patients untreated with ACE inhibitors or angiotensin II receptor antagonists (n=221), but plasma SSAO did not differ by ACE I/D genotype. Plasma SSAO correlated positively with duration of diabetes, HbA(1)c and plasma renin...

  16. Antioxidant Activity of Some Plant Extracts Towards Xanthine Oxidase, Lipoxygenase and Tyrosinase

    Directory of Open Access Journals (Sweden)

    Pi-Yu Chen

    2009-08-01

    Full Text Available Natural products have the potential to be developed into new drugs for the treatment of various diseases. The aim of the present study was to screen the antioxidant activities of some common edible fruits, garden plants and medicinal plants indigenous to Taiwan. This was performed by assessing the activities of lipoxygenase, xanthine oxidase and tyrosinase following incubation with extracts from these plants. A further aim was to use HPLC-DAD and tyrosinase to chromatographically identify the antioxidative constituents obtained from an extract exhibiting strong antioxidative properties. The acetone extracts of 27 cultivated plant species from Taiwan were tested for antioxidant activities towards xanthine oxidase, tyrosinase and lipoxygenase using spectrophotometric assays. Koelreuteria henryi, Prunus campanulata, and Rhodiola rosea showed the highest xanthine oxidase inhibitory activities. Camellia sinensis, Rhodiola rosea, and Koelreuteria henryi exhibited good tyrosinase inhibitory activities and potent anti-lipoxygenase activities. As Koelreuteria henryi had notable significant inhibitory activities towards xanthine oxidase, tyrosinase, and lipoxygenase, it was further tested with tyrosinase and HPLC-DAD. The results from this part of the study revealed that the more powerful the antioxidant capability of the extracted component, the greater the decrease in peak height obtained after reacting with tyrosinase. Additional studies are warranted to further characterize the compounds responsible for the antioxidant properties of the examined extracts.

  17. Crystallization and preliminary X-ray analysis of Aspergillus oryzae catechol oxidase

    International Nuclear Information System (INIS)

    Kaljunen, Heidi; Gasparetti, Chiara; Kruus, Kristiina; Rouvinen, Juha; Hakulinen, Nina

    2011-01-01

    Catechol oxidase from A. oryzae was crystallized by the hanging-drop vapour-diffusion method. Catechol oxidase is an enzyme that catalyzes the oxidation of o-diphenols to the corresponding o-quinones. It is a copper-containing enzyme with a binuclear copper active site. Here, the crystallization and multiple-wavelength anomalous dispersion data collection of catechol oxidase from the mould fungus Aspergillus oryzae are described. During the purification, three forms of the enzyme (39.3, 40.5 and 44.3 kDa) were obtained. A mixture of these three forms was initially crystallized and gave crystals that diffracted to 2.5 Å resolution and belonged to space group P3 2 21, with unit-cell parameters a = b = 118.9, c = 84.5 Å, α = β = 90, γ = 120°. A preparation containing only the shorter form (39.3 kDa) produced crystals that diffracted to 2.9 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 51.8, b = 95.3, c = 139.5 Å, α = β = γ = 90°

  18. Inhibitory effects of drugs on the metabolic activity of mouse and human aldehyde oxidases and influence on drug-drug interactions.

    Science.gov (United States)

    Takaoka, Naoki; Sanoh, Seigo; Okuda, Katsuhiro; Kotake, Yaichiro; Sugahara, Go; Yanagi, Ami; Ishida, Yuji; Tateno, Chise; Tayama, Yoshitaka; Sugihara, Kazumi; Kitamura, Shigeyuki; Kurosaki, Mami; Terao, Mineko; Garattini, Enrico; Ohta, Shigeru

    2018-04-17

    As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug-drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC 50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O 6 -benzylguanine as substrates. 17β-Estradiol, menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC 50 values. We also evaluated the potential DDI between an AOX substrate (O 6 -benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (C max ) and the area under the plasma concentration-time curve (AUC 0-24 ) of O 6 -benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Effects of glucose oxidase on the growth performance, serum ...

    African Journals Online (AJOL)

    Martina

    2016-02-06

    Feb 6, 2016 ... The experiment was conducted to investigate the effects of diets supplemented with ... Keywords: Glucose oxidase, intestinal health, performance, swine ..... This work was supported by the National High-Tech Research and ...

  20. Determination of the rate constant for neuronal and extra-neuronal monoamine oxidase

    International Nuclear Information System (INIS)

    Cassis, L.; Ludwig, J.; Trendelenburg, U.

    1986-01-01

    In the rat vas deferens, neuronal deamination of 3 H-(-) noradrenaline ( 3 H-NA) to 3 H-dihydroxyphenethylglycol ( 3 HDOPEG) cannot be inhibited by pretreatment with a monoamine oxidase (MAO) inhibitor. However, in the extraneuronal compartment of the rat heart, inhibition of MAO abolishes the formation of 3 HDOPEG. To clarify this discrepancy, the authors determined the rate constant for MAO (/sup k/mao/) neuronally (rat vas deferens) and extraneuronally (rat heart). For neuronal /sup k/mao, vasa deferentia were incubated with 3 HNA for 300 minutes, and the cumulative formation of 3 HDOPEG measured. The delay in time before 3 HDOPEG achieves steady state (/sup tau/system), is inversely proportional to /sup k/mao. Because /sup tau/system is very short for neuronal MAO, an appreciable delay was only achieved after partial inhibition of MAO with various parglyline concentrations. To relate to the uninhibited enzyme, the percentage inhibition by pargyline was then determined in homogenate preparations. For extraneuronal MAO, a similar procedure was performed in perfused rat hearts. Results show a significantly greater /sup k/mao of neuronal origin, (/sup k/mao = .57min - 1) which when related to the fractional size of the neuronal compartment suggests a very high activity of neuronal MAO

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

    Directory of Open Access Journals (Sweden)

    Coppée Jean-Yves

    2010-02-01

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

  2. Identification of the Mitochondrial Heme Metabolism Complex.

    Science.gov (United States)

    Medlock, Amy E; Shiferaw, Mesafint T; Marcero, Jason R; Vashisht, Ajay A; Wohlschlegel, James A; Phillips, John D; Dailey, Harry A

    2015-01-01

    Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.

  3. Comparative study between dimethyl sulfoxide (dmso), allopurinol and urate oxidase administration in nephrotoxic rats induced with

    International Nuclear Information System (INIS)

    Heibashy, M.I.A.; El-Nahla, A.M.; Ibrahim, A.I.; Saleh, Sh.Y.A.

    2010-01-01

    This study was conducted to show whether DMSO, allopurinol and urate oxidase could offer ameliorating effects against abnormal alterations in kidney function tests in gentamicin (GM) induced nephrotoxic rats . Two experiments were carried out, the first one showed that daily injection of 80 mg GM/kg b. wt interapertonealy (I.P) for two weeks induced acute renal failure indicated by significant elevation in serum urea, creatinine, uric acid, potassium, inorganic phosphorus, TBARS and PTH and a significant decline in serum sodium, total and ionized calcium when compared with their corresponding values in saline injected rats. In the second experiment, comparisons were made between GM induced nephrotoxic rats and other nephrotoxic groups received daily pf I.P injection of DMSO (4 ml/kg b.wt), allopurinol (1.5 mg/100 g b.wt) and urate oxidase (10 mg/100 g b.wt) for 30 days after the incidence of nephrotoxicity. At all intervals, 10,20 and 30 days; serum urea, creatinine, uric acid, potassium, inorganic phosphorus, TBARS and PTH in DMSO, allopurinol and urate oxidase treated groups exhibited significant reduction than nephrotoxic untreated rats. During the same intervals, the levels of serum total and ionized calcium showed an opposite trend. serum sodium level did not show any significant difference between all treated groups except after 20 days , it was increased significantly in urate oxidase treated group and after 30 days in both allopurinol and urate oxidase treated groups. in term time intervals, a significant correction was recorded on the level of most measured parameters. in nephritic rats, the administration of DMSO, allopurinol or urate oxidase led to a significant amelioration effects in the kidney function tests and urate oxidase was the best protective.

  4. A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases

    Directory of Open Access Journals (Sweden)

    Tom A. Ewing

    2018-01-01

    Full Text Available Vanillyl alcohol oxidase (VAO and eugenol oxidase (EUGO are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol and a EUGO variant (V436I with increased activity towards chavicol (4-allylphenol and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

  5. A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

    Science.gov (United States)

    Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

    2018-01-14

    Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

  6. Cytochrome oxidase as an indicator of ice storage and frozen storage

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2001-01-01

    in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used...... as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degreesC....

  7. On the Possibility of Uphill Intramolecular Electron Transfer in Multicopper Oxidases: Electrochemical and Quantum Chemical Study of Bilirubin Oxidase

    Czech Academy of Sciences Publication Activity Database

    Shleev, S.; Andoralov, V.; Falk, M.; Reimann, C. T.; Ruzgas, T.; Srnec, Martin; Ryde, U.; Rulíšek, Lubomír

    2012-01-01

    Roč. 24, č. 7 (2012), s. 1524-1540 ISSN 1040-0397 Grant - others:7th Framework Program(XE) NMP4-SL-2009-229255 Institutional research plan: CEZ:AV0Z40550506 Keywords : bilirubin oxidase * intramolecular electron transfer * rate-limiting catalytic step * reorganization energy * QM/MM calculations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.817, year: 2012

  8. Gene cloning and characterization of NADH oxidase from ...

    African Journals Online (AJOL)

    The genome search of Thermococcus kodakarensis revealed three open reading frames, Tk0304, Tk1299 and Tk1392 annotated as nicotinamide adenine dinucleotide (NADH) oxidases. This study deals with cloning, and characterization of Tk0304. The gene, composed of 1320 nucleotides, encodes a protein of 439 ...

  9. Mapping the primary structure of copper/topaquinone-containing methylamine oxidase from Aspergillus niger.

    Science.gov (United States)

    Lenobel, R; Sebela, M; Frébort, I

    2005-01-01

    The amino acid sequence of methylamine oxidase (MeAO) from the fungus Aspergillus niger was analyzed using mass spectrometry (MS). First, MeAO was characterized by an accurate molar mass of 72.4 kDa of the monomer measured using MALDI-TOF-MS and by a pI value of 5.8 determined by isoelectric focusing. MALDI-TOF-MS revealed a clear peptide mass fingerprint after tryptic digestion, which did not provide any relevant hit when searched against a nonredundant protein database and was different from that of A. niger amine oxidase AO-I. Tandem mass spectrometry with electrospray ionization coupled to liquid chromatography allowed unambiguous reading of six peptide sequences (11-19 amino acids) and seven sequence tags (4-15 amino acids), which were used for MS BLAST homology searching. MeAO was found to be largely homologous to a hypothetical protein AN7641.2 (EMBL/GenBank protein-accession code EAA61827) from Aspergillus nidulans FGSC A4 with a theoretical molar mass of 76.46 kDa and pI 6.14, which belongs to the superfamily of copper amine oxidases. The protein AN7641.2 is only little homologous to the amine oxidase AO-I (32% identity, 49 % similarity).

  10. Oxidase catalysis via aerobically generated hypervalent iodine intermediates

    Science.gov (United States)

    Maity, Asim; Hyun, Sung-Min; Powers, David C.

    2018-02-01

    The development of sustainable oxidation chemistry demands strategies to harness O2 as a terminal oxidant. Oxidase catalysis, in which O2 serves as a chemical oxidant without necessitating incorporation of oxygen into reaction products, would allow diverse substrate functionalization chemistry to be coupled to O2 reduction. Direct O2 utilization suffers from intrinsic challenges imposed by the triplet ground state of O2 and the disparate electron inventories of four-electron O2 reduction and two-electron substrate oxidation. Here, we generate hypervalent iodine reagents—a broadly useful class of selective two-electron oxidants—from O2. This is achieved by intercepting reactive intermediates of aldehyde autoxidation to aerobically generate hypervalent iodine reagents for a broad array of substrate oxidation reactions. The use of aryl iodides as mediators of aerobic oxidation underpins an oxidase catalysis platform that couples substrate oxidation directly to O2 reduction. We anticipate that aerobically generated hypervalent iodine reagents will expand the scope of aerobic oxidation chemistry in chemical synthesis.

  11. Biodegradation of phenolic compounds with oxidases from sorghum and non-defined mixed bacterium media

    International Nuclear Information System (INIS)

    Obame, C. E. L.; Savadogo, P. W.; Mamoudou, D. H.; Dembele, R. H.; Traore, A. S.

    2009-01-01

    The biodegradation of the phenolic compounds is performed using oxidative enzymes, e. g. polyphenol oxidases (PPOs) and peroxidases (POXs). These oxidases displaying a wide spectrum for the oxidation of phenolic compounds were isolated either from sorghum or mixed bacteria. Spectrophotometric methods were used to assess the monophenolase and diphenolase activities of PPOs as well as the hydrogen-dependant oxidation of POXs. (Author)

  12. Biodegradation of phenolic compounds with oxidases from sorghum and non-defined mixed bacterium media

    Energy Technology Data Exchange (ETDEWEB)

    Obame, C. E. L.; Savadogo, P. W.; Mamoudou, D. H.; Dembele, R. H.; Traore, A. S.

    2009-07-01

    The biodegradation of the phenolic compounds is performed using oxidative enzymes, e. g. polyphenol oxidases (PPOs) and peroxidases (POXs). These oxidases displaying a wide spectrum for the oxidation of phenolic compounds were isolated either from sorghum or mixed bacteria. Spectrophotometric methods were used to assess the monophenolase and diphenolase activities of PPOs as well as the hydrogen-dependant oxidation of POXs. (Author)

  13. Study on the preparation of immobilized glucose oxidase membrane and its application in clinic analysis

    International Nuclear Information System (INIS)

    Yu Ye; Cao Jin; Su Zongxian; Chen Zixiong

    1990-01-01

    The paper deals with the preparation of immobilized glucose oxidase membrane by using two steps irradiation (irradiation gratfting, irradiation entrapping). Some properties of membrane were discussed. The immobilized glucose oxidase membrane with oxygen electrode and oxygen analyser can be satisfied with the clinic analysis for the determination of serum glucose

  14. D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects.

    Science.gov (United States)

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

    2012-10-01

    Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells.

  15. Inhibition of monoamine oxidase B (MAO-B) by Chinese herbal medicines.

    Science.gov (United States)

    Lin, R D; Hou, W C; Yen, K Y; Lee, M H

    2003-11-01

    Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic amines accompaned by the release of H2O2. Two subtypes, MAO-A and MAO-B, exist on the basis of their specificities to substrates and inhibitors. The regulation of MAO-B activity is important in the treatment of neurodegenerative diseases. Twenty-seven species of plants used in traditional Chinese medicines, selected from an enthnobotanical survey, were used in an investigation of their inhibitory effect on MAO-B in rat brain homogenates. The 50% aqueous methanol extracts of four active extracts, Arisaema amurense, Lilium brownii var. colchesteri, Lycium chinense, and Uncaria rhynchophylla, exhibited the best activity and selectivity towards MAO-B with IC50 values of 0.44, 0.29, 0.40, and 0.03 mg/ml, respectively. A kinetic study of MAO-B inhibition by the four extracts using the Lineweaver-Burk plot for each active extract revealed the IC50 concentrations, and results show that: Ki = 0.59 mg/ml for A. amurense for the mixed-type mode, Ki = 0.58 mg/ml for L. brownii var. colchesteri for the mixed-type mode, Ki = 5.01 mg/ml for L. chinense for the uncompetitive mode, and Ki = 0.02 mg/ml for U. rhynchophylla for the uncompetitive mode. These may therefore be candidates for use in delaying the progressive degeneration caused by neurological diseases.

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

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei, E-mail: hmliu2004@126.com [Huazhong University of Science and Technology, School of Chemistry and Chemical Engineering (China)

    2016-03-15

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na{sub 2}SeO{sub 3} as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant (K{sub m}) values and maximal reaction velocity (V{sub max}) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  17. Random mutagenesis of aspergillus niger and process optimization for enhanced production of glucose oxidase

    International Nuclear Information System (INIS)

    Haq, I.; Nawaz, A.; Mukhtar, A.N.H.; Mansoor, H.M.Z.; Ameer, S.M.

    2014-01-01

    The study deals with the improvement of wild strain Aspergillus niger IIB-31 through random mutagenesis using chemical mutagens. The main aim of the work was to enhance the glucose oxidase (GOX) yield of wild strain (24.57+-0.01 U/g of cell mass) through random mutagenesis and process optimization. The wild strain of Aspergillus niger IIB-31 was treated with chemical mutagens such as Ethyl methane sulphonate (EMS) and nitrous acid for this purpose. Mutagen treated 98 variants indicating the positive results were picked and screened for the glucose oxidase production using submerged fermentation. EMS treated E45 mutant strain gave the highest glucose oxidase production (69.47 + 0.01 U/g of cell mass), which was approximately 3-folds greater than the wild strain IIB-31. The preliminary cultural conditions for the production of glucose oxidase using submerged fermentation from strain E45 were also optimized. The highest yield of GOD was obtained using 8% glucose as carbon and 0.3% peptone as nitrogen source at a medium pH of 7.0 after an incubation period of 72 hrs at 30 degree. (author)

  18. Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

    International Nuclear Information System (INIS)

    Cervelli, Manuela; Grillo, Rosalba; Woster, Patrick M; Casero, Robert A Jr; Mariottini, Paolo; Bellavia, Gabriella; Fratini, Emiliano; Amendola, Roberto; Polticelli, Fabio; Barba, Marco; Federico, Rodolfo; Signore, Fabrizio; Gucciardo, Giacomo

    2010-01-01

    Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials

  19. Spermine oxidase (SMO activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

    Directory of Open Access Journals (Sweden)

    Gucciardo Giacomo

    2010-10-01

    Full Text Available Abstract Background Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC. This study investigates the expression of spermine oxidase (SMO and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. Methods BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. Results Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. Conclusions This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

  20. Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; Gorren, A. C.; Dekker, H. L.; Nieboer, P.; van Gelder, B. F.; Muijsers, A. O.

    1992-01-01

    Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of

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

    Directory of Open Access Journals (Sweden)

    Polticelli Fabio

    2012-06-01

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

  2. Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco 1

    Science.gov (United States)

    Vanlerberghe, Greg C.; McIntosh, Lee

    1992-01-01

    Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein. Images Figure 3 Figure 4 PMID:16652932

  3. Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

    NARCIS (Netherlands)

    Winter, Remko T.; Heuts, Dominic P. H. M.; Rijpkema, Egon M. A.; van Bloois, Edwin; Wijma, Hein J.; Fraaije, Marco W.

    We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene

  4. Interferon gamma/NADPH oxidase defence system in immunity and cancer

    Czech Academy of Sciences Publication Activity Database

    Hodný, Zdeněk; Reiniš, Milan; Hubáčková, Soňa; Vašicová, Pavla; Bartek, Jiří

    -, 01 Sep (2015) ISSN 2162-4011 Institutional support: RVO:68378050 ; RVO:61388971 Keywords : IFNγ * NADPH oxidase * immunity * cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.266, year: 2014

  5. Identification of 5-(1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophene-2-Carboxamides as Novel and Selective Monoamine Oxidase B Inhibitors Used to Improve Memory and Cognition.

    Science.gov (United States)

    Kaplan, Alan P; Keenan, Terence; Scott, Roderick; Zhou, Xianbo; Bourchouladze, Rusiko; McRiner, Andrew J; Wilson, Mark E; Romashko, Darlene; Miller, Regina; Bletsch, Matthew; Anderson, Gary; Stanley, Jennifer; Zhang, Adia; Lee, Dong; Nikpur, John

    2017-12-20

    Initial work in Drosophila and mice demonstrated that the transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) is a master control gene for memory formation. The relationship between CREB and memory has also been found to be true in other species, including aplysia and rats. It is thus well-established that CREB activation plays a central role in memory enhancement and that CREB is activated during memory formation. On the basis of these findings, a phenotypic high-throughput screening campaign utilizing a CRE-luciferase (CRE-Luci) SK-N-MC cell line was performed to identify compounds that enhance transcriptional activation of the CRE promoter with a suboptimal dose of forskolin. A number of small-molecule hits of unknown mechanisms of action were identified in the screening campaign, including HT-0411. Follow-up studies suggested that the CREB activation by HT-0411 is attributed to its specific and selective inhibition of monoamine oxidase B (MAO-B). Further, HT-0411 was shown to improve 24 h memory in rodents in a contextual fear conditioning model. This report describes the lead optimization of a series of 5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl) thiophene-2-carboxamides that were identified as novel, potent, and selective inhibitors of MAO-B. Extensive SAR studies and in vivo behavioral evaluations of this and other related analogue series identified a number of potential clinical development candidates; ultimately, compound 8f was identified as a candidate molecule with high selectivity toward MAO-B (29-56 nM) over MAO-A (19% inhibition at a screening concentration of 50 μM), an excellent profile against a panel of other enzymes and receptors, good pharmacokinetic properties in rodents and dogs, and efficacy in multiple rodent memory models.

  6. RXR agonists inhibit high glucose-induced upregulation of inflammation by suppressing activation of the NADPH oxidase-nuclear factor-κB pathway in human endothelial cells.

    Science.gov (United States)

    Ning, R B; Zhu, J; Chai, D J; Xu, C S; Xie, H; Lin, X Y; Zeng, J Z; Lin, J X

    2013-12-13

    An inflammatory response induced by high glucose is a cause of endothelial dysfunction in diabetes and is an important contributing link to atherosclerosis. Diabetes is an independent risk factor of atherosclerosis and activation of retinoid X receptor (RXR) has been shown to exert anti-atherogenic effects. In the present study, we examined the effects of the RXR ligands 9-cis-retinoic acid (9-cis-RA) and SR11237 on high glucose-induced inflammation in human umbilical endothelial vein endothelial cells (HUVECs) and explored the potential mechanism. Our results showed that the inflammation induced by high-glucose in HUVECs was mainly mediated by the activation of nuclear factor-B (NF- κB). High glucose-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were in comparison, significantly decreased by treatment with RXR. The effect of RXR agonists was mainly due to the inhibition of NF-κB activation. Using pharmacological inhibitors and siRNA, we confirmed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was an upstream activator of NF-κB. Furthermore, RXR agonists significantly inhibited high glucose-induced activation of NADPH oxidase and significantly decreased the production of reactive oxygen species (ROS). To explore whether the rapid inhibitory effects of RXR agonists were in fact mediated by RXR, we examined the effect of RXR downregulation by RXR siRNA. Our results showed that RXR siRNA largely abrogated the effects of RXR agonists, suggesting the requirement of RXR expression. Therefore, we have shown that RXR is involved in the regulation of NADPH oxidase- NF-κB signal pathway, as the RXR ligands antagonized the inflammatory response in HUVECs induced by high glucose.

  7. In vitro metabolism of a linear furanocoumarin (8-methoxypsoralen, xanthotoxin) by mixed-function oxidases of larvae of black swallowtail butterfly and fall armyworm

    International Nuclear Information System (INIS)

    Bull, D.L.

    1986-01-01

    Studies were made of the comparative in vitro metabolism of ( 14 C)xanthotoxin and( 14 C)aldrin by homogenate preparations of midguts and bodies (carcass minus digestive tract and head) of last-stage larvae of the black swallowtail butterfly (Papilio polyxenes Fabr.) and the fall armyworm (Spodoptera frugiperda (J. E. Smith)). The two substrates were metabolized by 10,000g supernatant microsomal preparations from both species. Evidence gained through the use of a specific inhibitor and cofactor indicated that mixed-function microsomal oxidases were major factors in the metabolism and that the specific activity of this enzyme system was considerably higher in midgut preparations from P. polyxenes than in similar preparations from S. frugiperda. Aldrin was metabolized 3-4 times faster by P. polyxenes, and xanthotoxin 6-6.5 times faster

  8. Isolation and properties of polyphenol oxidase from basidiocarps of Lactarius pergamenus Fr. (Fr. fungi

    Directory of Open Access Journals (Sweden)

    M. V. Tsivinska

    2015-04-01

    Full Text Available Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr. fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6. Three isoforms of polyphenol oxidase (PPO were discovered and two isoforms (1-1 and 1-2 were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6. Specific activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule of the enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 ºC, and at +70 ºC the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content of the milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m– and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

  9. Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase

    DEFF Research Database (Denmark)

    Wang, Wenzhong; Winther, Jakob R; Thorpe, Colin

    2007-01-01

    centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter...... and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative......The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox...

  10. Crystallization of carbohydrate oxidase from Microdochium nivale

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  11. Zinc Oxide Nanoparticle Induces Microglial Death by NADPH-Oxidase-Independent Reactive Oxygen Species as well as Energy Depletion.

    Science.gov (United States)

    Sharma, Anuj Kumar; Singh, Vikas; Gera, Ruchi; Purohit, Mahaveer Prasad; Ghosh, Debabrata

    2017-10-01

    Zinc oxide nanoparticle (ZnO-NP) is one of the most widely used engineered nanoparticles. Upon exposure, nanoparticle can eventually reach the brain through various routes, interact with different brain cells, and alter their activity. Microglia is the fastest glial cell to respond to any toxic insult. Nanoparticle exposure can activate microglia and induce neuroinflammation. Simultaneous to activation, microglial death can exacerbate the scenario. Therefore, we focused on studying the effect of ZnO-NP on microglia and finding out the pathway involved in the microglial death. The present study showed that the 24 h inhibitory concentration 50 (IC 50 ) of ZnO-NP for microglia is 6.6 μg/ml. Early events following ZnO-NP exposure involved increase in intracellular calcium level as well as reactive oxygen species (ROS). Neither of NADPH oxidase inhibitors, apocynin, (APO) and diphenyleneiodonium chloride (DPIC) were able to reduce the ROS level and rescue microglia from ZnO-NP toxicity. In contrary, N-acetyl cysteine (NAC) showed opposite effect. Exogenous supplementation of superoxide dismutase (SOD) reduced ROS significantly even beyond control level but partially rescued microglial viability. Interestingly, pyruvate supplementation rescued microglia near to control level. Following 10 h of ZnO-NP exposure, intracellular ATP level was measured to be almost 50 % to the control. ZnO-NP-induced ROS as well as ATP depletion both disturbed mitochondrial membrane potential and subsequently triggered the apoptotic pathway. The level of apoptosis-inducing proteins was measured by western blot analysis and found to be upregulated. Taken together, we have deciphered that ZnO-NP induced microglial apoptosis by NADPH oxidase-independent ROS as well as ATP depletion.

  12. Activation of JNK and c-Jun is involved in glucose oxidase-mediated cell death of human lymphoma cells.

    Science.gov (United States)

    Son, Young-Ok; Jang, Yong-Suk; Shi, Xianglin; Lee, Jeong-Chae

    2009-12-31

    Mitogen-activated protein kinases (MAPK) affect the activation of activator protein-1 (AP-1), which plays an important role in regulating a range of cellular processes. However, the roles of these signaling factors on hydrogen peroxide (H(2)O(2))-induced cell death are unclear. This study examined the effects of H(2)O(2) on the activation of MAPK and AP-1 by exposing the cells to H(2)O(2) generated by either glucose oxidase or a bolus addition. Exposing BJAB or Jurkat cells to H(2)O(2) affected the activities of MAPK differently according to the method of H(2)O(2) exposure. H(2)O(2) increased the AP-1-DNA binding activity in these cells, where continuously generated H(2)O(2) led to an increase in mainly the c-Fos, FosB and c-Jun proteins. The c-Jun-NH(2)-terminal kinase (JNK)-mediated activation of c-Jun was shown to be related to the H(2)O(2)-induced cell death. However, the suppression of H(2)O(2)-induced oxidative stress by either JNK inhibitor or c-Jun specific antisense transfection was temporary in the cells exposed to glucose oxidase but not to a bolus H(2)O(2). This was associated with the disruption of death signaling according to the severe and prolonged depletion of reduced glutathione. Overall, these results suggest that H(2)O(2) may decide differently the mode of cell death by affecting the intracellular redox state of thiol-containing antioxidants, and this depends more closely on the duration exposed to H(2)O(2) than the concentration of this agent.

  13. A study on the reactions of plant copper amine oxidase with C3 and C4 aliphatic diamines.

    Science.gov (United States)

    Sebela, M; Frébort, I; Lemr, K; Brauner, F; Pec, P

    2000-12-01

    The paper reports a study on the reactions of grass pea (Lathyrus sativus) amine oxidase (GPAO) with several aliphatic diamines. The influence of the chain length and of unsaturations in the molecules was examined. Kinetic measurements confirmed that trans-, i.e., (E)-2-butene-1,4-diamine (TDABE) and cis-, i.e., (Z)-2-butene-1,4-diamine (CDABE) could be classified as good substrates. Propane-1,3-diamine (DAP) and propene-1,3-diamine (DAPE) were only weakly oxidized, whereas 1,3-diamino-2-propanol (DAPL) was not utilized as a substrate. Contrary to the inactivator 2-butyne-1,4-diamine (DABI), DAPE was shown to be only a competitive inhibitor. DAP itself did not inhibit the catalytic activity. Irreversible inhibition of the activity occurred only after the incubation of GPAO with DABI; other diamines were without this effect. Differential pulse polarography and chromatofocusing confirmed that the aminoaldehyde product of DABI oxidation binds to the enzyme. Activity assay of pea aminoaldehyde dehydrogenase enabled us to detect the products of the oxidation of TDABE, CDABE, and DAP by GPAO. As the product of DAP oxidation, 3-amino-propanal (APAL) was detected by mass spectrometry and confirmed to be a potent noncompetitive inhibitor of GPAO. The absorption changes that occurred in the course of the reaction of GPAO with the diamines were investigated using rapid-scanning spectrophotometry. DABI, TDABE, CDABE, DAP, and DAPE reacted with GPAO providing characteristic maxima of the Cu(I)-semiquinolamine species that is formed in the catalytic cycle. The results presented here confirm that with the exception of DAPL, all the studied diamines could be classified as GPAO substrates, but only DABI can be considered as a mechanism-based inhibitor.

  14. Carbon Nanotube Modified Screen Printed Electrodes: Pyranose Oxidase Immobilization Platform for Amperometric Enzyme Sensors

    Directory of Open Access Journals (Sweden)

    Dilek ODACI DEMIRKOL

    2017-03-01

    Full Text Available Here, a novel enzymatic biosensor was developed using multiwalled carbon nanotube including screen printed electrodes (MWCNT-SPE. Pyranose oxidase (PyOx was immobilized on the electrode surface by way of gelatin membrane and then cross-linked using glutaraldehyde. Glucose was detected at -0.7 V (vs. Ag/AgCl by watching consumed oxygen in enzymatic reaction after addition substrate. After optimization of pH and enzyme loading, the linearity was found in the range of 0.1–1.0 mM of glucose. After that, the effect of MCNT on the current was tested. Also the enzymatic biosensor including glucose oxidase instead of pyranose oxidase was prepared and the biosensor response followed for glucose. Furthermore, this system was tested for glucose analysis in soft drinks.

  15. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells

    International Nuclear Information System (INIS)

    Eum, Sung Yong; Andras, Ibolya; Hennig, Bernhard; Toborek, Michal

    2009-01-01

    Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.

  16. Crosstalk between HDAC6 and Nox2-based NADPH oxidase mediates HIV-1 Tat-induced pro-inflammatory responses in astrocytes

    Directory of Open Access Journals (Sweden)

    Gi Soo Youn

    2017-08-01

    Full Text Available Histone deacetylase 6 (HDAC6 likely is important in inflammatory diseases. However, how HDAC6 exerts its effect on inflammatory processes remains unclear. HIV-1 transactivator of transcription (Tat activates NADPH oxidase resulting in generation of reactive oxygen species (ROS, leading to extensive neuro-inflammation in the central nervous system. We investigated the correlation of HDAC6 and NADPH oxidase in HIV-1 Tat-stimulated astrocytes. HDAC6 knockdown attenuated HIV-1 Tat-induced ROS generation and NADPH oxidase activation. HDAC6 knockdown suppressed HIV-1 Tat-induced expression of NADPH oxidase subunits, such as Nox2, p47phox, and p22phox. Specific inhibition of HDAC6 using tubastatin A suppressed HIV-1 Tat-induced ROS generation and activation of NADPH oxidase. N-acetyl cysteine, diphenyl iodonium, and apocynin suppressed HIV-1 Tat-induced expression of HDAC6 and the pro-inflammatory chemokines CCL2, CXCL8, and CXCL10. Nox2 knockdown attenuated HIV-1 Tat-induced HDAC6 expression and subsequent expression of chemokines. The collective results point to the potential crosstalk between HDAC6 and NADPH oxidase, which could be a combined therapeutic target for relief of HIV-1 Tat-mediated neuro-inflammation. Keywords: HIV-1 Tat, HDAC6, NADPH oxidase, ROS, Inflammation, Astrocytes

  17. Enhanced production of glucose oxidase from UVmutant of ...

    African Journals Online (AJOL)

    UV rays were used as mutagen in wild type strain of Aspergillus niger for enhanced production of glucose oxidase. After mutangenization and selection, mutant A. niger strains, resistant to 2-deoxy-Dglucose were obtained. The mutants showed 1.57 and 1.98 fold increase in activities of extra and intra cellular glucose ...

  18. in Escherichia coli with native cholesterol oxidase expressed

    African Journals Online (AJOL)

    The structure and bio-activity of an endogenous cholesterol oxidase from Brevibacterium sp. was compared to the same enzyme exogenously expressed in Escherichia coli BL21 (DE3) with and without N- or C-terminal his-tags. The different proteins were purified with affinity and subtractive protocols. The specific activity of ...

  19. Switching an O2 sensitive glucose oxidase bioelectrode into an almost insensitive one by cofactor redesign.

    Science.gov (United States)

    Tremey, Emilie; Suraniti, Emmanuel; Courjean, Olivier; Gounel, Sébastien; Stines-Chaumeil, Claire; Louerat, Frédéric; Mano, Nicolas

    2014-06-04

    In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.

  20. Nanoporous gold assembly of glucose oxidase for electrochemical biosensing

    DEFF Research Database (Denmark)

    Xiao, Xinxin; Ulstrup, Jens; Li, Hui

    2014-01-01

    Nanoporous gold (NPG) is composed of three-dimensional (3D) bicontinuous nanostructures with large surface area. Nano-channels inside NPG provide an ideal local environment for immobilization of enzyme molecules with expected stabilization of the protein molecules. In this work, glucose oxidase (...

  1. Sphingosine 1-Phosphate-Induced ICAM-1 Expression via NADPH Oxidase/ROS-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells

    Science.gov (United States)

    Lin, Chih-Chung; Yang, Chien-Chung; Cho, Rou-Ling; Wang, Chen-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-01-01

    The intercellular adhesion molecule-1 (ICAM-1) expression is frequently correlated with the lung inflammation. In lung injury, sphingosine-1-phosphate (S1P, bioactive sphingolipid metabolite), participate gene regulation of adhesion molecule in inflammation progression and aggravate tissue damage. To investigate the transduction mechanisms of the S1P in pulmonary epithelium, we demonstrated that exposure of HPAEpiCs (human pulmonary alveolar epithelial cells) to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCδ), PF431396 (PYK2), diphenyleneiodonium chloride (DPI), apocynin (NADPH oxidase), Edaravone (ROS), and Bay11-7082 (NF-κB). Consistently, knockdown with siRNA transfection of PKCδ, PYK2, p47phox, and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A) and Gi/o-coupled receptor antagonist (GPA2) also blocked the upregulation of ICAM-1 protein and mRNA induced by S1P. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCδ-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-κB p65 phosphorylation and nuclear translocation in HPAEpiCs. Activated NF-κB was blocked by Rottlerin, PF431396, APO, DPI, or Edaravone. Besides, the results of monocyte adhesion assay indicated that S1P-induced ICAM-1 expression on HPAEpiCs can enhance the monocyte attachments. In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway. We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with

  2. Variations in epidermal cytochrome oxidase activity after local irradiation

    International Nuclear Information System (INIS)

    Itoiz, M.E.; Rey, B.M. de; Cabrini, R.L.

    1982-01-01

    Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16krad. The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied. At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles. (author)

  3. Development of Galactose Biosensor Based on Functionalized ZnO Nanorods with Galactose Oxidase

    Directory of Open Access Journals (Sweden)

    K. Khun

    2012-01-01

    Full Text Available The fabrication of galactose biosensor based on functionalised ZnO nanorods is described. The galactose biosensor was developed by immobilizing galactose oxidase on ZnO nanorods in conjunction with glutaraldehyde as a cross-linker molecule. The IRAS study provided evidence for the interaction of galactose oxidase with the surface of ZnO nanorods. The electromotive force (EMF response of the galactose biosensor was measured by potentiometric method. We observed that the proposed biosensor has a linear detection range over a concentration range from 10 mM to 200 mM with good sensitivity of 89.10±1.23 mV/decade. In addition, the proposed biosensor has shown fast time response of less than 10 s and a good selectivity towards galactose in the presence of common interferents such as ascorbic acid, uric acid, glucose, and magnesium ions. The galactose biosensor based on galactose oxidase immobilized ZnO nanorods has a shelf life more than four weeks.

  4. Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

    Science.gov (United States)

    Kadota, Yasuhiro; Shirasu, Ken; Zipfel, Cyril

    2015-08-01

    Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 Å resolution using a twinned crystal

    International Nuclear Information System (INIS)

    Mizutani, Kimihiko; Toyoda, Mayuko; Sagara, Kenta; Takahashi, Nobuyuki; Sato, Atsuko; Kamitaka, Yuji; Tsujimura, Seiya; Nakanishi, Yuji; Sugiura, Toshiyuki; Yamaguchi, Shotaro; Kano, Kenji; Mikami, Bunzo

    2010-01-01

    The crystal structure of bilirubin oxidase (BOD) from M. verrucaria has been determined at 2.3 Å resolution using a merohedrally twinned crystal. BOD has four copper-coordination sites that are almost identical to those of other multicopper oxidases and is also very similar to them in overall structure. Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6 3 . Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33–2.3 Å. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD

  6. Acupuncture elicits neuroprotective effect by inhibiting NAPDH oxidase-mediated reactive oxygen species production in cerebral ischaemia.

    Science.gov (United States)

    Shi, Guang-Xia; Wang, Xue-Rui; Yan, Chao-Qun; He, Tian; Yang, Jing-Wen; Zeng, Xiang-Hong; Xu, Qian; Zhu, Wen; Du, Si-Qi; Liu, Cun-Zhi

    2015-12-10

    In the current study, we aimed to investigate whether NADPH oxidase, a major ROS-producing enzyme, was involved in the antioxidant effect of acupuncture on cognitive impairment after cerebral ischaemia. The cognitive function, infract size, neuron cell loss, level of superoxide anion and expression of NADPH oxidase subunit in hippocampus of two-vessel occlusion (2VO) rats were determined after 2-week acupuncture. Furthermore, the cognitive function and production of O2(-) were determined in the presence and absence of NADPH oxidase agonist (TBCA) and antagonist (Apocynin). The effect of acupuncture on cognitive function after cerebral ischaemia in gp91phox-KO mice was evaluated by Morris water maze. Acupuncture reduced infarct size, attenuated overproduction of O2(-), and reversed consequential cognitive impairment and neuron cell loss in 2VO rats. The elevations of gp91phox and p47phox after 2VO were significantly decreased after acupuncture treatment. However, no differences of gp91phox mRNA were found among any experimental groups. Furthermore, these beneficial effects were reversed by TBCA, whereas apocynin mimicked the effect of acupuncture by improving cognitive function and decreasing O2(-) generation. Acupuncture failed to improve the memory impairment in gp91phox KO mice. Full function of the NADPH oxidase enzyme plays an important role in neuroprotective effects against cognitive impairment via inhibition of NAPDH oxidase-mediated oxidative stress.

  7. The cytochemical demonstration of catalase and D-amino acid oxidase in the microbodies of teleost kidney cells

    NARCIS (Netherlands)

    Veenhuis, M.; Wendelaar Bonga, S.D.

    1977-01-01

    The distribution of catalase and D-amino acid oxidase, marker enzymes for peroxisomes, was determined cytochemically in the kidney tubules of an euryhaline teleost, the three-spined stickleback. Catalase activity was localized with the diaminobenzidine technique. The presence of D-amino acid oxidase

  8. Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis).

    Science.gov (United States)

    Cankar, Katarina; van Houwelingen, Adèle; Goedbloed, Miriam; Renirie, Rokus; de Jong, René M; Bouwmeester, Harro; Bosch, Dirk; Sonke, Theo; Beekwilder, Jules

    2014-03-18

    (+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144±10μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

    Science.gov (United States)

    Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

    2013-01-01

    Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems. Copyright © 2011 John Wiley & Sons, Ltd.

  10. Methodology to assay CYP2E1 mixed function oxidase catalytic activity and its induction

    Directory of Open Access Journals (Sweden)

    Arthur I. Cederbaum

    2014-01-01

    Full Text Available The cytochrome P450 mixed function oxidase enzymes are the major catalysts involved in drug metabolism. There are many forms of P450. CYP2E1 metabolizes many toxicologically important compounds including ethanol and is active in generating reactive oxygen species. Since several of the contributions in the common theme series “Role of CYP2E1 and Oxidative/Nitrosative Stress in the Hepatotoxic Actions of Alcohol” discuss CYP2E1, this methodology review describes assays on how CYP2E1 catalytic activity and its induction by ethanol and other inducers can be measured using substrate probes such as the oxidation of para-nitrophenol to para-nitrocatechol and the oxidation of ethanol to acetaldehyde. Approaches to validate that a particular reaction e.g. oxidation of a drug or toxin is catalyzed by CYP2E1 or that induction of that reaction is due to induction of CYP2E1 are important and specific examples using inhibitors of CYP2E1, anti-CYP2E1 IgG or CYP2E1 knockout and knockin mice will be discussed.

  11. Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

    Directory of Open Access Journals (Sweden)

    Faccio Greta

    2010-08-01

    Full Text Available Abstract Background Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. Results In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Conclusions Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

  12. Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

    Science.gov (United States)

    Faccio, Greta; Kruus, Kristiina; Buchert, Johanna; Saloheimo, Markku

    2010-08-20

    Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

  13. Immobilization of glucose oxidase on sepharose by UV-initiated graft copolymerization

    International Nuclear Information System (INIS)

    D'Angiuro, L.; Cremonesi, P.

    1982-01-01

    The performance of a new method of enzyme immobilization based on photochemically initiated direct graft copolymerization was recently investigated. The immobilization reaction can be carried out in a simple way and by carefully selecting the reaction conditions, the enzyme-graft copolymer can be obtained as the main reaction product. Coupling efficiency of glucose oxidase has been found to depend only on the amount of photocatalyst (FeCl 3 ) fixed on Sepharose used as polysaccharide support. Small quantities of glycidylmethacrylate (GMA) (0.25 g/g dry Sepharose) are sufficient but necessary to achieve the best enzyme coupling efficiency (20-40%). Enzyme immobilization occurs very rapidly and the entire reaction occurs within 60 min. Reaction patterns and physicochemical characteristics of the obtained enzyme-graft copolymers exclude the glucose oxidase entrapment: therefore a covalent attachment mechanism may be proposed. The kinetic parameters of immobilized glucose oxidase (K/sub m/' = 2.0 x 10 -2 M) are quite similar to those of free enzyme (K/sub m/ = 1.93 x 10 -2 M), and no diffusion limitation phenomena are evidenced in samples having different enzyme or polymer content. Lyophilization, thermostability, and long-term continuous operation also have been investigated. The advantages of this method over that using vinylenzyme copolymerization are discussed

  14. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jian-Ching; Rebrin, Igor [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States); Klichko, Vladimir; Orr, William C. [Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275 (United States); Sohal, Rajindar S., E-mail: sohal@usc.edu [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States)

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  15. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir; Orr, William C.; Sohal, Rajindar S.

    2010-01-01

    Research highlights: → Cytochrome c oxidase loses catalytic activity during the aging process. → Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. → Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H 2 O 2 generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  16. Biocatalytic properties and structural analysis of eugenol oxidase from Rhodococcus jostii RHA1 : a versatile oxidative biocatalyst

    NARCIS (Netherlands)

    Nguyen, Quoc-Thai; De Gonzalo, Gonzalo; Binda, Claudia; Rioz, Ana; Mattevi, Andrea; Fraaije, Marco

    2016-01-01

    Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 was previously shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase resulting in a broadened substrate scope and a deeper insight into its structural

  17. Process technology for the application of d-amino acid oxidases in pharmaceutical intermediate manufacturing

    DEFF Research Database (Denmark)

    Tindal, Stuart; Carr, Reuben; Archer, Ian V. J.

    2011-01-01

    Recent advances in biocatalysis have seen increased interest in the use of D-amino acid oxidase to synthesize optically pure amino acids. However, the creation of a genuine oxidase based platform technology will require suitable process technology as well as an understanding of the challenges...... and opportunities of a wider portfolio of synthetic targets. In this article we address some of the recent progress in process technology to enable the future development of a generic platform technology....

  18. Extracellular oxidases and the transformation of solubilised low-rank coal by wood-rot fungi

    Energy Technology Data Exchange (ETDEWEB)

    Ralph, J.P. [Flinders Univ. of South Australia, Bedford Park (Australia). School of Biological Sciences; Graham, L.A. [Flinders Univ. of South Australia, Bedford Park (Australia). School of Biological Sciences; Catcheside, D.E.A. [Flinders Univ. of South Australia, Bedford Park (Australia). School of Biological Sciences

    1996-12-31

    The involvement of extracellular oxidases in biotransformation of low-rank coal was assessed by correlating the ability of nine white-rot and brown-rot fungi to alter macromolecular material in alkali-solubilised brown coal with the spectrum of oxidases they produce when grown on low-nitrogen medium. The coal fraction used was that soluble at 3.0{<=}pH{<=}6.0 (SWC6 coal). In 15-ml cultures, Gloeophyllum trabeum, Lentinus lepideus and Trametes versicolor produced little or no lignin peroxidase, manganese (Mn) peroxidase or laccase activity and caused no change to SWC6 coal. Ganoderma applanatum and Pycnoporus cinnabarinus also produced no detectable lignin or Mn peroxidases or laccase yet increased the absorbance at 400 nm of SWC6 coal. G. applanatum, which produced veratryl alcohol oxidase, also increased the modal apparent molecular mass. SWC6 coal exposed to Merulius tremellosus and Perenniporia tephropora, which secreted Mn peroxidases and laccase and Phanerochaete chrysosporium, which produced Mn and lignin peroxidases was polymerised but had unchanged or decreased absorbance. In the case of both P. chrysosporium and M. tremellosus, polymerisation of SWC6 coal was most extensive, leading to the formation of a complex insoluble in 100 mM NaOH. Rigidoporus ulmarius, which produced only laccase, both polymerised and reduced the A{sub 400} of SWC6 coal. P. chrysosporium, M. tremellosus and P. tephropora grown in 10-ml cultures produced a spectrum of oxidases similar to that in 15-ml cultures but, in each case, caused more extensive loss of A{sub 400}, and P. chrysosporium depolymerised SWC6 coal. It is concluded that the extracellular oxidases of white-rot fungi can transform low-rank coal macromolecules and that increased oxygen availability in the shallower 10-ml cultures favours catabolism over polymerisation. (orig.)

  19. A multicopper oxidase-related protein is essential for insect viability, longevity and ovary development.

    Science.gov (United States)

    Peng, Zeyu; Green, Peter G; Arakane, Yasuyuki; Kanost, Michael R; Gorman, Maureen J

    2014-01-01

    Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.

  20. Pseudo-bi-enzyme glucose sensor: ZnS hollow spheres and glucose oxidase concerted catalysis glucose.

    Science.gov (United States)

    Shuai, Ying; Liu, Changhua; Wang, Jia; Cui, Xiaoyan; Nie, Ling

    2013-06-07

    This work creatively uses peroxidase-like ZnS hollow spheres (ZnS HSs) to cooperate with glucose oxidase (GOx) for glucose determinations. This approach is that the ZnS HSs electrocatalytically oxidate the enzymatically generated H2O2 to O2, and then the O2 circularly participates in the previous glucose oxidation by glucose oxidase. Au nanoparticles (AuNPs) and carbon nanotubes (CNTs) are used as electron transfer and enzyme immobilization matrices, respectively. The biosensor of glucose oxidase-carbon nanotubes-Au nanoparticles-ZnS hollow spheres-gold electrode (GOx-CNT-AuNPs-ZnS HSs-GE) exhibits a rapid response, a low detection limit (10 μM), a wide linear range (20 μM to 7 mM) as well as good anti-interference, long-term longevity and reproducibility.

  1. Abscinazole-F1, a conformationally restricted analogue of the plant growth retardant uniconazole and an inhibitor of ABA 8'-hydroxylase CYP707A with no growth-retardant effect.

    Science.gov (United States)

    Todoroki, Yasushi; Kobayashi, Kyotaro; Shirakura, Minaho; Aoyama, Hikaru; Takatori, Kokichi; Nimitkeatkai, Hataitip; Jin, Mei-Hong; Hiramatsu, Saori; Ueno, Kotomi; Kondo, Satoru; Mizutani, Masaharu; Hirai, Nobuhiro

    2009-09-15

    To develop a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, seed dormancy, and other various physiological events, we designed and synthesized conformationally restricted analogues of uniconazole (UNI), a well-known plant growth retardant, which inhibits a biosynthetic enzyme (ent-kaurene oxidase) of gibberellin as well as ABA 8'-hydroxylase. Although most of these analogues were less effective than UNI in inhibition of ABA 8'-hydroxylase and rice seedling growth, we found that a lactol-bridged analogue with an imidazole is a potent inhibitor of ABA 8'-hydroxylase but not of plant growth. This compound, abscinazole-F1, induced drought tolerance in apple seedlings upon spray treatment with a 10 microM solution.

  2. Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

    Science.gov (United States)

    Khadivi Derakshan, Fatemeh; Darvishi, Farshad; Dezfulian, Mehrouz; Madzak, Catherine

    2017-08-01

    Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

  3. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    Science.gov (United States)

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. NADPH Oxidase, NOX1, Mediates Vascular Injury in Ischemic Retinopathy

    Science.gov (United States)

    Deliyanti, Devy; Rana, Indrajeetsinh; Miller, Antonia G.; Agrotis, Alex; Armani, Roksana; Szyndralewiez, Cédric; Wingler, Kirstin; Touyz, Rhian M.; Cooper, Mark E.; Jandeleit-Dahm, Karin A.; Schmidt, Harald H.H.W.

    2014-01-01

    Abstract Aims: Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. Results: Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. Innovation: Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. Conclusions: Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies. Antioxid. Redox Signal. 20, 2726–2740. PMID:24053718

  5. Selectivity Profiling and Biological Activity of Novel β-Carbolines as Potent and Selective DYRK1 Kinase Inhibitors.

    Directory of Open Access Journals (Sweden)

    Katharina Rüben

    Full Text Available DYRK1A is a pleiotropic protein kinase with diverse functions in cellular regulation, including cell cycle control, neuronal differentiation, and synaptic transmission. Enhanced activity and overexpression of DYRK1A have been linked to altered brain development and function in Down syndrome and neurodegenerative diseases such as Alzheimer's disease. The β-carboline alkaloid harmine is a high affinity inhibitor of DYRK1A but suffers from the drawback of inhibiting monoamine oxidase A (MAO-A with even higher potency. Here we characterized a series of novel harmine analogs with minimal or absent MAO-A inhibitory activity. We identified several inhibitors with submicromolar potencies for DYRK1A and selectivity for DYRK1A and DYRK1B over the related kinases DYRK2 and HIPK2. An optimized inhibitor, AnnH75, inhibited CLK1, CLK4, and haspin/GSG2 as the only off-targets in a panel of 300 protein kinases. In cellular assays, AnnH75 dose-dependently reduced the phosphorylation of three known DYRK1A substrates (SF3B1, SEPT4, and tau without negative effects on cell viability. AnnH75 inhibited the cotranslational tyrosine autophosphorylation of DYRK1A and threonine phosphorylation of an exogenous substrate protein with similar potency. In conclusion, we have characterized an optimized β-carboline inhibitor as a highly selective chemical probe that complies with desirable properties of drug-like molecules and is suitable to interrogate the function of DYRK1A in biological studies.

  6. NADPH oxidase: an enzyme for multicellularity?

    Science.gov (United States)

    Lalucque, Hervé; Silar, Philippe

    2003-01-01

    Multicellularity has evolved several times during the evolution of eukaryotes. One evolutionary pressure that permits multicellularity relates to the division of work, where one group of cells functions as nutrient providers and the other in specialized roles such as defence or reproduction. This requires signalling systems to ensure harmonious development of multicellular structures. Here, we show that NADPH oxidases are specifically present in organisms that differentiate multicellular structures during their life cycle and are absent from unicellular life forms. The biochemical properties of these enzymes make them ideal candidates for a role in intercellular signalling.

  7. Ag-doped CdO nanocatalysts: Preparation, characterization and catechol oxidase activity

    Science.gov (United States)

    El-Kemary, Maged; El-Mehasseb, Ibrahim; El-Shamy, Hany

    2018-06-01

    Silver doped cadmium oxide (Ag/CdO) nanoparticles with an average size of 41 nm have been successfully synthesized via thermal decomposition and liquid impregnation technique. The structural characterization has been performed by using several spectroscopic techniques, e.g., X-ray diffraction (XRD), scanning electron microscopy (SEM) and fourier-transform infrared (FT-IR). The catechol oxidase has been studied by UV-visible absorption spectroscopy and fourier-transform infrared as well as the mechanism has been assured by cyclic voltammetry and fluorescence spectroscopy. The results indicate that the oxidation does not occur in the presence of unsupported cadmium oxide particles by silver and in the same time, the catechol oxidase activity of silver doped CdO nanoparticles were improved by about three orders of magnitude than silver ions.

  8. ERK1/2 pathway is involved in renal gluconeogenesis inhibition under conditions of lowered NADPH oxidase activity.

    Science.gov (United States)

    Winiarska, Katarzyna; Jarzyna, Robert; Dzik, Jolanta M; Jagielski, Adam K; Grabowski, Michal; Nowosielska, Agata; Focht, Dorota; Sierakowski, Bartosz

    2015-04-01

    The aim of this study was to elucidate the mechanisms involved in the inhibition of renal gluconeogenesis occurring under conditions of lowered activity of NADPH oxidase (Nox), the enzyme considered to be one of the main sources of reactive oxygen species in kidneys. The in vitro experiments were performed on primary cultures of rat renal proximal tubules, with the use of apocynin, a selective Nox inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a potent superoxide radical scavenger. In the in vivo experiments, Zucker diabetic fatty (ZDF) rats, a well established model of diabetes type 2, were treated with apocynin solution in drinking water. The main in vitro findings are the following: (1) both apocynin and TEMPOL attenuate the rate of gluconeogenesis, inhibiting the step catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the process; (2) in the presence of the above-noted compounds the expression of PEPCK and the phosphorylation of transcription factor CREB and ERK1/2 kinases are lowered; (3) both U0126 (MEK inhibitor) and 3-(2-aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione (ERK inhibitor) diminish the rate of glucose synthesis via mechanisms similar to those of apocynin and TEMPOL. The observed apocynin in vivo effects include: (1) slight attenuation of hyperglycemia; (2) inhibition of renal gluconeogenesis; (3) a decrease in renal PEPCK activity and content. In view of the results summarized above, it can be concluded that: (1) the lowered activity of the ERK1/2 pathway is of importance for the inhibition of renal gluconeogenesis found under conditions of lowered superoxide radical production by Nox; (2) the mechanism of this phenomenon includes decreased PEPCK expression, resulting from diminished activity of transcription factor CREB; (3) apocynin-evoked inhibition of renal gluconeogenesis contributes to the hypoglycemic action of this compound observed in diabetic animals. Thus, the study has

  9. Season-controlled changes in biochemical constituents and oxidase enzyme activities in tomato (Lycopersicon esculentum Mill.).

    Science.gov (United States)

    Sen, Supatra; Mukherji, S

    2009-07-01

    Season-controlled changes in biochemical constituents viz. carotenoids (carotene and xanthophyll) and pectic substances along with IAA-oxidase and polyphenol oxidase (PPO) enzyme activities were estimated/assayed in leaves of Lycopersicon esculentum Mill. (tomato) in two developmental stages--pre-flowering (35 days after sowing) and post-flowering (75 days after sowing) in three different seasons--summer rainy and winter Carotenoid content along with pectic substances were highest in winter and declined significantly in summer followed by rainy i.e. winter > summer > rainy. Carotenoid content was significantly higher in the pre-flowering as compared to post-flowering in all three seasons while pectic substances increased in the post-flowering as compared to pre-flowering throughout the annual cycle. IAA oxidase and PPO enzyme activities were enhanced in rainy and decreased sharply in summer and winter i.e. rainy > summer > winter. Both the enzymes exhibited higher activity in the post-flowering stage as compared to pre-flowering in all three seasons. These results indicate winter to be the most favourable season for tomato plants while rainy season environmental conditions prove to be unfavourable (stressful) with diminished content of carotenoid and pectic substances and low activities of IAA oxidase and PPO, ultimately leading to poor growth and productivity.

  10. Electrochemical L-Lactic Acid Sensor Based on Immobilized ZnO Nanorods with Lactate Oxidase

    Directory of Open Access Journals (Sweden)

    Kimleang Khun

    2012-02-01

    Full Text Available In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF response of L-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10−4–1 × 100 mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards L-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks.

  11. Electrochemical L-lactic acid sensor based on immobilized ZnO nanorods with lactate oxidase.

    Science.gov (United States)

    Ibupoto, Zafar Hussain; Shah, Syed Muhammad Usman Ali; Khun, Kimleang; Willander, Magnus

    2012-01-01

    In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF) response of l-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10(-4)-1 × 10(0) mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards l-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks.

  12. Thermal Characterization of Purified Glucose Oxidase from A Newly Isolated Aspergillus Niger UAF-1

    Science.gov (United States)

    Anjum Zia, Muhammad; Khalil-ur-Rahman; K. Saeed, Muhammad; Andaleeb, Fozia; I. Rajoka, Muhammad; A. Sheikh, Munir; A. Khan, Iftikhar; I. Khan, Azeem

    2007-01-01

    An intracellular glucose oxidase was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger UAF-1. The enzyme was purified to a yield of 28.43% and specific activity of 135 U mg−1 through ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The enzyme showed high affinity for D-glucose with a Km value of 2.56 mM. The enzyme exhibited optimum catalytic activity at pH 5.5. Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40°C. The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol−1 and free energy of denaturation 103.63 kJ mol−1. These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays. PMID:18193107

  13. Serum xanthine oxidase profile in stressed Marwari sheep from arid tracts in India

    Directory of Open Access Journals (Sweden)

    Maan R.

    2012-08-01

    Full Text Available The present investigation was aimed to determine serum xanthine oxidase profile in stressed Marwari breed of sheep belonging to arid tracts in Rajasthan, India. Extreme hot and cold ambiences were considered as stress conditions to the animals. Blood samples were collected to obtain sera during moderate, extreme hot and cold ambiences. The mean value of serum xanthine oxidase during moderate ambience was 93.33±1.11 mU L-1.The mean value of serum xanthine oxidase was significantly (p≤0.05higher during hot and significantly (p≤0.05 lower during cold ambiences as compared to moderate mean value serving as control. The sex and age effects were significant (p≤0.05 in all ambiences. The mean values were significantly (p≤0.05 higher in males than females. In each ambience the age effect showed a significant (p≤0.05 increase in the mean values being highest in the animals of 2.5-4.5 years of age. The effects of extreme ambiences were observed on the male and female animals of all age groups as revealed by various interactions studied viz. ambience X age; ambience X sex and age X sex (p≤0.01. Further sex effect was present in the animals of each age group. It can be concluded that serum xanthine oxidase can be used as an effective marker to assess oxidative stress in these animals. Mean values obtained from large number of animals during moderate ambience will help in providing physiological reference values for future research and clinical interpretations.

  14. Isolation and purification of pyranose 2-oxidase from Phanerochaete chrysosporium and characterization of gene structure and regulation

    Science.gov (United States)

    Theodorus H. de Koker; Michael D. Mozuch; Daniel Cullen; Jill Gaskell; Philip J. Kersten

    2004-01-01

    Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified. 13C-nuclear magnetic resonance confirmed production of D- arabino -hexos-2-ulose (glucosone) from D-glucose with the oxidase. Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of...

  15. PAM-OBG: A monoamine oxidase B specific prodrug that inhibits MGMT and generates DNA interstrand crosslinks, potentiating temozolomide and chemoradiation therapy in intracranial glioblastoma

    Science.gov (United States)

    Sharpe, Martyn A.; Raghavan, Sudhir; Baskin, David S.

    2018-01-01

    Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of glioblastoma with respect to patient survival. In addition to elevation of O6-methylguanine DNA methyltransferase (MGMT), down-regulation of three DNA repair pathways; canonical mismatch repair (MMR), Non-Homologous End-Joining (NHEJ), and Homologous Recombination (HR) are correlated with poor patient outcome. We have designed and tested both in vitro and in vivo, a monoamine oxidase B (MAOB) specific prodrug, PAM-OBG, that is converted by glioma MAOB into the MGMT inhibitor O6-benzylguanine (O6BG) and the DNA crosslinking agent acrolein. In cultured glioma cells, we show that PAM-OBG is converted to O6BG, inhibiting MGMT and sensitizing cells to DNA alkylating agents such as BCNU, CCNU, and Temozolomide (TMZ). In addition, we demonstrate that the acrolein generated is highly toxic in glioma treated with an inhibitor of Nucleotide Excision Repair (NER). In mouse intracranial models of primary human glioma, we show that PAM-OBG increases survival of mice treated with either BCNU or CCNU by a factor of six and that in a chemoradiation model utilizing six rounds of TMZ/2Gy radiation, pre-treatment with PAM-OBG more than doubled survival time. PMID:29844863

  16. Inducible bilirubin oxidase: A novel function for the mouse cytochrome P450 2A5

    International Nuclear Information System (INIS)

    Abu-Bakar, A'edah; Arthur, Dionne Maioha; Aganovic, Simona; Ng, Jack C.; Lang, Matti A.

    2011-01-01

    We have previously shown that bilirubin (BR), a breakdown product of haem, is a strong inhibitor and a high affinity substrate of the mouse cytochrome P450 2A5 (CYP2A5). The antioxidant BR, which is cytotoxic at high concentrations, is potentially useful in cellular protection against oxygen radicals if its intracellular levels can be strictly controlled. The mechanisms that regulate cellular BR levels are still obscure. In this paper we provide preliminary evidence for a novel function of CYP2A5 as hepatic “BR oxidase”. A high-performance liquid chromatography/electrospray ionisation mass spectrometry screening showed that recombinant yeast microsomes expressing the CYP2A5 oxidise BR to biliverdin, as the main metabolite, and to three other smaller products with m/z values of 301, 315 and 333. The metabolic profile is significantly different from that of chemical oxidation of BR. In chemical oxidation the smaller products were the main metabolites. This suggests that the enzymatic reaction is selective, towards biliverdin production. Bilirubin treatment of primary hepatocytes increased the CYP2A5 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A5 compared to cells treated only with CHX. Collectively, the observations suggest that the CYP2A5 is potentially an inducible “BR oxidase” where BR may accelerate its own metabolism through stabilization of the CYP2A5 protein. It is possible that this metabolic pathway is potentially part of the machinery controlling intracellular BR levels in transient oxidative stress situations, in which high amounts of BR are produced. -- Highlights: ► CYP2A5 metabolizes bilirubin to biliverdin and dipyrroles. ► Bilirubin increased the hepatic CYP2A5 protein and activity levels. ► Bilirubin does not change the hepatic CYP2A5 mRNA levels. ► Co-treatment with a protein synthesis inhibitor

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purifi...

  18. Lysyl oxidases regulate fibrillar collagen remodelling in idiopathic pulmonary fibrosis

    NARCIS (Netherlands)

    Tjin, Gavin; White, Eric S; Faiz, Alen; Sicard, Delphine; Tschumperlin, Daniel J; Mahar, Annabelle; Kable, Eleanor P W; Burgess, Janette K

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with feweffective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linkingmediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4)] might contribute to disease

  19. DNA damage and decrease of cellular oxidase activity in piglet ...

    African Journals Online (AJOL)

    DNA damage and decrease of cellular oxidase activity in piglet sertoli cells exposed to gossypol. Ming Zhang, Hui Yuan, Zuping He, Liyun Yuan, Jine Yi, Sijun Deng, Li Zhu, Chengzhi Guo, Yin Lu, Jing Wu, Lixin Wen, Qiang Wei, Liqun Xue ...

  20. Lactate Biosensor Based on Cellulose Acetate Membrane Bound Lactate Oxidase

    Directory of Open Access Journals (Sweden)

    Suman

    2007-05-01

    Full Text Available Lactate biosensor was fabricated by immobilizing lactate oxidase in cellulose acetate membrane and by mounting over the sensing part of Pt electrode (working and connected to Ag/AgCl electrode (reference along with auxillary electrode through potentiostat. The enzyme electrode was anodically polarized at +400 mV to generate electrons from H2O2, which was formed from oxidation of serum lactate by immobilized lactate oxidase. The minimum detection limit of the electrode was 0.1mmoles/L and sensitivity of the sensor was 0.008 mA/mM/L lactate. Assay coefficients of variation were < 2% .A good correlation (r=0.99 was found between lactate values obtained by colorimetric method and lactate biosensor. The self-life of the biosensor was 18 days at 4ºC and enzyme electrode can be re-used 150 times without any significant loss in enzyme activity.

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

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

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

  2. Boosting the oxidase mimicking activity of nanoceria by fluoride capping: rivaling protein enzymes and ultrasensitive F- detection

    Science.gov (United States)

    Liu, Biwu; Huang, Zhicheng; Liu, Juewen

    2016-07-01

    Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement.Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement. Electronic supplementary information (ESI) available: Methods, TMB oxidation kinetics and control experiments. See DOI: 10.1039/c6nr02730j

  3. NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

    International Nuclear Information System (INIS)

    Liu Qing; He Xiaoqing; Liu Yongsheng; Du Bingbing; Wang Xiaoyan; Zhang Weisheng; Jia Pengfei; Dong Jingmei; Ma Jianxiu; Wang Xiaohu; Li Sha; Zhang Hong

    2008-01-01

    Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91 phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47 phox was translocated to the cell membrane and localized with p22 phox and gp91 phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

  4. The microglial NADPH oxidase complex as a source of oxidative stress in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Landreth Gary E

    2006-11-01

    Full Text Available Abstract Alzheimer's disease is the most common cause of dementia in the elderly, and manifests as progressive cognitive decline and profound neuronal loss. The principal neuropathological hallmarks of Alzheimer's disease are the senile plaques and the neurofibrillary tangles. The senile plaques are surrounded by activated microglia, which are largely responsible for the proinflammatory environment within the diseased brain. Microglia are the resident innate immune cells in the brain. In response to contact with fibrillar beta-amyloid, microglia secrete a diverse array of proinflammatory molecules. Evidence suggests that oxidative stress emanating from activated microglia contribute to the neuronal loss characteristic of this disease. The source of fibrillar beta-amyloid induced reactive oxygen species is primarily the microglial nicotinamide adenine dinucleotide phosphate (NADPH oxidase. The NADPH oxidase is a multicomponent enzyme complex that, upon activation, produces the highly reactive free radical superoxide. The cascade of intracellular signaling events leading to NADPH oxidase assembly and the subsequent release of superoxide in fibrillar beta-amyloid stimulated microglia has recently been elucidated. The induction of reactive oxygen species, as well as nitric oxide, from activated microglia can enhance the production of more potent free radicals such as peroxynitrite. The formation of peroxynitrite causes protein oxidation, lipid peroxidation and DNA damage, which ultimately lead to neuronal cell death. The elimination of beta-amyloid-induced oxidative damage through the inhibition of the NADPH oxidase represents an attractive therapeutic target for the treatment of Alzheimer's disease.

  5. Effect of heat treatment on polyphenol oxidase and peroxidase ...

    African Journals Online (AJOL)

    Effect of heat treatment (55°C/20 min) on polyphenol oxidase (PPO) and peroxidase (POD) activities and total phenolic compounds was investigated in Algerian dates (Deglet Nour variety) at Tamar (fully ripe) stage and in dates stored for 5 months at ambient temperature and in cold storage (10°C). Results obtained ...

  6. Identification of the stereochemical requirements in the 4-aryl-2-cycloalkylidenhydrazinylthiazole scaffold for the design of selective human monoamine oxidase B inhibitors.

    Science.gov (United States)

    D'Ascenzio, Melissa; Carradori, Simone; Secci, Daniela; Mannina, Luisa; Sobolev, Anatoly P; De Monte, Celeste; Cirilli, Roberto; Yáñez, Matilde; Alcaro, Stefano; Ortuso, Francesco

    2014-05-15

    Exploring the effect that substituents on the cycloaliphatic ring had on the inhibitory activity against human monoamine oxidase B of a series of 4-aryl-2-cycloalkylidenhydrazinylthiazoles led to the synthesis of a new series of 2-methylcyclopentyl and 3-methylcyclopentyl derivatives which were tested in vitro as mixtures of diastereoisomers. In fact, due to the presence of a chiral center on the cycloaliphatic ring and a trisubstituted CN bond, they exist as four diastereoisomers ((E)-(R), (E)-(S), (Z)-(R), (Z)-(S)). 4-(2,4-Difluorophenyl)-2-(2-(3-methylcyclopentylidene)hydrazinyl)thiazole was chosen as a model to investigate the influence of stereochemical requirements on the inhibitory activity against hMAO-B of these derivatives after a stereoconservative synthesis and semi-preparative HPLC diastereoseparation. (R)-(Z) isomer of this compound was endowed with a potent and selective hMAO-B inhibition higher than that of reference drugs as also corroborated by molecular modeling studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Structure of choline oxidase in complex with the reaction product glycine betaine.

    Science.gov (United States)

    Salvi, Francesca; Wang, Yuan-Fang; Weber, Irene T; Gadda, Giovanni

    2014-02-01

    Choline oxidase from Arthrobacter globiformis, which is involved in the biosynthesis of glycine betaine from choline, has been extensively characterized in its mechanistic and structural properties. Despite the knowledge gained on the enzyme, the details of substrate access to the active site are not fully understood. The `loop-and-lid' mechanism described for the glucose-methanol-choline enzyme superfamily has not been confirmed for choline oxidase. Instead, a hydrophobic cluster on the solvent-accessible surface of the enzyme has been proposed by molecular dynamics to control substrate access to the active site. Here, the crystal structure of the enzyme was solved in complex with glycine betaine at pH 6.0 at 1.95 Å resolution, allowing a structural description of the ligand-enzyme interactions in the active site. This structure is the first of choline oxidase in complex with a physiologically relevant ligand. The protein structures with and without ligand are virtually identical, with the exception of a loop at the dimer interface, which assumes two distinct conformations. The different conformations of loop 250-255 define different accessibilities of the proposed active-site entrance delimited by the hydrophobic cluster on the other subunit of the dimer, suggesting a role in regulating substrate access to the active site.

  8. Liquid-Phase Packaging of a Glucose Oxidase Solution with Parylene Direct Encapsulation and an Ultraviolet Curing Adhesive Cover for Glucose Sensors

    Directory of Open Access Journals (Sweden)

    Seiichi Takamatsu

    2010-06-01

    Full Text Available We have developed a package for disposable glucose sensor chips using Parylene encapsulation of a glucose oxidase solution in the liquid phase and a cover structure made of an ultraviolet (UV curable adhesive. Parylene was directly deposited onto a small volume (1 μL of glucose oxidase solution through chemical vapor deposition. The cover and reaction chamber were constructed on Parylene film using a UV-curable adhesive and photolithography. The package was processed at room temperature to avoid denaturation of the glucose oxidase. The glucose oxidase solution was encapsulated and unsealed. Glucose sensing was demonstrated using standard amperometric detection at glucose concentrations between 0.1 and 100 mM, which covers the glucose concentration range of diabetic patients. Our proposed Parylene encapsulation and UV-adhesive cover form a liquid phase glucose-oxidase package that has the advantages of room temperature processing and direct liquid encapsulation of a small volume solution without use of conventional solidifying chemicals.

  9. Hofmeister effects on the glucose oxidase hydrogel-modified electrode

    International Nuclear Information System (INIS)

    Suzuki, Aimi; Tsujimura, Seiya

    2016-01-01

    We describe the consistent effect of salts in the electrolyte solution on glucose oxidation current production in the redox hydrogel-modified electrode containing glucose oxidase as an electrocatalyst and Os complex mediator. The ions affect not only on the electron transfer between the enzyme and the Os complex, but also on the hydrogel structure. This study found that the degree of the effect can be characterized by Hofmeister series. The relative decrease in oxidization current is the lowest in the middle of the Hofmeister series, and increases monotonically on either side. An increase of ionic strength inhibits the electron transfer from the active site of glucose oxidase to Os complex. In addition to this, the kosmotropic anions, which are strongly hydrated, caused hydrogel deswelling (shrinking). The more chaotropic an ion is, the more it adsorbs to uncharged parts of polymer/enzyme with dispersion force, and the swelling of the hydrogel decreases the catalytic current. This study impacts the design of hydrogel electrode and selection of electrolyte ions for bioelectronic applications.

  10. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum.Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols

    OpenAIRE

    Fraaije, Marco W.; Veeger, Cees; Berkel, Willem J.H. van

    1995-01-01

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyph...

  11. Effect of retinal impulse blockage on cytochrome oxidase-poor interpuffs in the macaque striate cortex: quantitative EM analysis of neurons.

    Science.gov (United States)

    Wong-Riley, M T; Trusk, T C; Kaboord, W; Huang, Z

    1994-09-01

    One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase-rich puffs in its supragranular layers. Neurons in puffs have been classified as type A, B, and C in ascending order of cytochrome oxidase content, with type C cells being the most vulnerable to retinal impulse blockade. The present study aimed at analysing cytochrome oxidase-poor interpuffs with reference to their metabolic cell types and the effect of intraretinal tetrodotoxin treatment. The same three metabolic types were found in interpuffs, except that type B and C neurons were smaller and less cytochrome oxidase-reactive in interpuffs than in puffs. Type A neurons had small perikarya, low levels of cytochrome oxidase, and received exclusively symmetric axosomatic synapses. The largest neurons were pyramidal, type B cells with moderate cytochrome oxidase activity and were also contacted exclusively by symmetric axosomatic synapses. Type C cells medium-sized with a rich supply of large, darkly reactive mitochondria and possessed all the characteristics of GABAergic neurons. They were the only cell type that received both symmetric and asymmetric axosomatic synapses. Two weeks of monocular tetrodotoxin blockade in adult monkeys caused all three major cell types in deprived interpuffs to suffer a significant downward shift in the size and cytochrome oxidase reactivity of their mitochondria, but the effects were more severe in type B and C neurons. In nondeprived interpuffs, all three cell types gained both in size and absolute number of mitochondria, and type A cells also had an elevated level of cytochrome oxidase, indicating that they might be functioning at a competitive advantage over cells in deprived columns. However, type B and C neurons showed a net loss of darkly reactive mitochondria, indicating that these cells became less active. Thus, mature interpuff neurons remained vulnerable to retinal impulse blockade and the metabolic capacity of these cells remains tightly

  12. New coumarin derivatives: design, synthesis and use as inhibitors of hMAO.

    Science.gov (United States)

    He, Xu; Chen, Yan-Yan; Shi, Jing-Bo; Tang, Wen-Jiang; Pan, Zhi-Xiang; Dong, Zhi-Qiang; Song, Bao-An; Li, Jun; Liu, Xin-Hua

    2014-07-15

    A series new 2H-chromene-3-carboxamides (4a-4i) and S-2H-chromene-3-carbothioates (5j-5t) were synthesized and evaluated as monoamine oxidase A and B inhibitors. Among them, compound 5k (IC50=0.21μM, IC50 iproniazid=7.65μM) showed the most activity and higher MAO-B selectivity (189.2-fold vs 1-fold) with respect to the MAO-A isoform. The need to clarify at a 3D level some important molecular aspects of discussed SAR, we undertaked a number of docking simulations to better assess. The steric effect was analyzed interms of both posing and scoring by investigating the nature of the binding interactions. The docking results of active compound 5k with hMAO-B complex indicated that conserved residue ILE 199 was important for ligand binding via Sigma-Pi interaction. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Nox1 oxidase suppresses influenza a virus-induced lung inflammation and oxidative stress.

    Directory of Open Access Journals (Sweden)

    Stavros Selemidis

    Full Text Available Influenza A virus infection is an ongoing clinical problem and thus, there is an urgent need to understand the mechanisms that regulate the lung inflammation in order to unravel novel generic pharmacological strategies. Evidence indicates that the Nox2-containing NADPH oxidase enzyme promotes influenza A virus-induced lung oxidative stress, inflammation and dysfunction via ROS generation. In addition, lung epithelial and endothelial cells express the Nox1 isoform of NADPH oxidase, placing this enzyme at key sites to regulate influenza A virus-induced lung inflammation. The aim of this study was to investigate whether Nox1 oxidase regulates the inflammatory response and the oxidative stress to influenza infection in vivo in mice. Male WT and Nox1-deficient (Nox1(-/y mice were infected with the moderately pathogenic HkX-31 (H3N2, 1×10(4 PFU influenza A virus for analysis of bodyweight, airways inflammation, oxidative stress, viral titre, lung histopathology, and cytokine/chemokine expression at 3 and 7 days post infection. HkX-31 virus infection of Nox1(-/y mice resulted in significantly greater: loss of bodyweight (Day 3; BALF neutrophilia, peri-bronchial, peri-vascular and alveolar inflammation; Nox2-dependent inflammatory cell ROS production and peri-bronchial, epithelial and endothelial oxidative stress. The expression of pro-inflammatory cytokines including CCL2, CCL3, CXCL2, IL-1β, IL-6, GM-CSF and TNF-α was higher in Nox1(-/y lungs compared to WT mice at Day 3, however, the expression of CCL2, CCL3, CXCL2, IFN-γ and the anti-inflammatory cytokine IL-10 were lower in lungs of Nox1(-/y mice vs. WT mice at Day 7. Lung viral titre, and airways infiltration of active CD8(+ and CD4(+ T lymphocytes, and of Tregs were similar between WT and Nox1(-/y mice. In conclusion, Nox1 oxidase suppresses influenza A virus induced lung inflammation and oxidative stress in mice particularly at the early phases of the infection. Nox1 and Nox2 oxidases appear

  14. Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

    Science.gov (United States)

    Zhan, Tao; Zhang, Kai; Chen, Yangyan; Lin, Yongjun; Wu, Gaobing; Zhang, Lili; Yao, Pei; Shao, Zongze; Liu, Ziduo

    2013-01-01

    Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops. PMID:24223901

  15. Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution.

    Directory of Open Access Journals (Sweden)

    Tao Zhan

    Full Text Available Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO, we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

  16. Thermal and pH stabilities of partially purified polyphenol oxidase ...

    African Journals Online (AJOL)

    Dr. Paul Chidoka Chikezie

    2012-07-30

    Jul 30, 2012 ... indices of the two PPO extracts is summarized in Table. 1. At the end of the .... activity near neutral pH values (Jime´nez-Atie´nzar et al.,. 2004; Dogan and Dogan, 2004). ..... oxidase from white cherry fruit (Starks gold). GIDA.

  17. Synthesis of Amide and Ester Derivatives of Cinnamic Acid and Its Analogs: Evaluation of Their Free Radical Scavenging and Monoamine Oxidase and Cholinesterase Inhibitory Activities.

    Science.gov (United States)

    Takao, Koichi; Toda, Kazuhiro; Saito, Takayuki; Sugita, Yoshiaki

    2017-01-01

    A series of cinnamic acid derivatives, amides (1-12) and esters (13-22), were synthesized, and structure-activity relationships for antioxidant activity, and monoamine oxidases (MAO) A and B, acetylcholinesterase, and butyrylcholinesterase (BChE) inhibitory activities were analyzed. Among the synthesized compounds, compounds 1-10, 12-18, and rosmarinic acid (23), which contained catechol, o-methoxyphenol or 5-hydroxyindole moieties, showed potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Compounds 9-11, 15, 17-22 showed potent and selective MAO-B inhibitory activity. Compound 20 was the most potent inhibitor of MAO-B. Compounds 18 and 21 showed moderate BChE inhibitory activity. In addition, compound 18 showed potent antioxidant activity and MAO-B inhibitory activity. In a comparison of the cinnamic acid amides and esters, the amides exhibited more potent DPPH free radical scavenging activity, while the esters showed stronger inhibitory activities against MAO-B and BChE. These results suggested that cinnamic acid derivatives such as compound 18, p-coumaric acid 3,4-dihydroxyphenethyl ester, and compound 20, p-coumaric acid phenethyl ester, may serve as lead compounds for the development of novel MAO-B inhibitors and candidate lead compounds for the prevention or treatment of Alzheimer's disease.

  18. Radioenzymatic and immunhistochemical demonstration of mono-amine oxidase in different mammals with regard to degenerative disorders of the central nervous system

    International Nuclear Information System (INIS)

    Konradi, C.

    1987-05-01

    Monoamine oxidase (MAO), an enzyme of the outer mitochondrial membrane, is involved in the degradation of biogenic amines. Its role in the metabolism of neurotransmitters in the brain like catecholamines and serotonin is of special importance. Pharmacological interests in neurological and psychiatric disorders require detailed investigations, especially through the discovery of two MAO-subtypes (MAO-A and MAO-B). Thus MAO-inhibitors offer the possibility of specific medical therapies. Activity of MAO-subtypes in several animal species and different tissues including human brain was determined biochemically via a radioenzymatic method. Examination was carried out for mode of action of both subtypes and response to several substrates and inhibitors. Aim was a survey about distinctive characteristics of MAO-A and MAO-B in one species as well as to others. Furthermore investigations about neuronal and glial distribution took place by histochemical and immuncyto-chemical methods. The histochemical method, which proofs the advantage to clear off pharmacological questions was carried out in the locus coeruleus of Meriones unguiculatus. Monoclonal antibodies against both MAO-subtypes were applied in the human brainstem and compared to polyclonal antibodies against tyrosine hydroxylase (TH). The most striking outcome was a lack of MAO in the neurons of substantia nigra, although TH-antibodies gave positive results. Hence questions remain open to explain the beneficial effect MAO-B-inhibitor l-deprenyl in dopamine-neuron degenerative disorders affecting substantia nigra. In particular the results require rethinking of the roles of MAO-A and MAO-B in human brain and the mode and site of action of drugs affecting their efficacy. Furthermore biochemical MAO-models in animals and their transferability to pharmacology in humans should be applied with limitations. This work is a further development of techniques applicable for human post mortem brain analysis. 152 refs., 21 figs

  19. Cytokinin oxidase from Phaseolus vulgaris callus tissues. Enhanced in vitro activity of the enzyme in the presence of copper-imidazole complexes

    International Nuclear Information System (INIS)

    Chatfield, J.M.; Armstrong, D.J.

    1987-01-01

    The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N 6 -(Δ 2 -isopentenyl)-adenine-2,8- 3 H (i 6 Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N 6 -side chain of i 6 Ade

  20. Evaluation of different disinfectants on the performance of an on-meter dosed amperometric glucose-oxidase-based glucose meter.

    Science.gov (United States)

    Sarmaga, Don; Dubois, Jeffrey A; Lyon, Martha E

    2011-11-01

    Off-meter dosed photometric glucose-oxidase-based glucose meters have been reported to be susceptible to interference by hydrogen-peroxide-based disinfecting agents. The objective of this study was to determine if a single application of hydrogen-peroxide-containing Accel® wipe to disinfect an on-meter dosed amperometric glucose-oxidase-based glucose meter will influence its performance. The performance of five on-meter dosed amperometric glucose-oxidase-based glucose meters was determined before and after disinfecting the devices with a single application of either CaviWipes® (14.3% isopropanol and 0.23% diisobutyl-phenoxy-ethoxyethyl dimethyl benzyl ammonium chloride) or Accel (0.5% hydrogen peroxide) wipes. Replicate glucose measurements were conducted before disinfecting the devices, immediately after disinfecting, and then 1 and 2 min postdisinfecting, with measurements in triplicate. Analysis was sequentially completed for five different meters. Results were analyzed by a two-way analysis of variance (Analyze-it software). No clinical ( .05) in glucose concentration were detected when the on-meter dosed amperometric glucose-oxidase-based glucose meters were disinfected with either CaviWipes or Accel wipes and measured immediately or 1 or 2 min postdisinfecting. No clinically significant difference in glucose concentration was detected between meters (glucose oxidase amperometric-based glucose meters are not analytically susceptible to interference by a single application of hydrogen-peroxide-containing Accel disinfectant wipes. © 2011 Diabetes Technology Society.