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Sample records for monooxygenase gene amoa

  1. Global occurrence of archaeal amoA genes in terrestrial hot springs.

    Science.gov (United States)

    Zhang, Chuanlun L; Ye, Qi; Huang, Zhiyong; Li, Wenjun; Chen, Jinquan; Song, Zhaoqi; Zhao, Weidong; Bagwell, Christopher; Inskeep, William P; Ross, Christian; Gao, Lei; Wiegel, Juergen; Romanek, Christopher S; Shock, Everett L; Hedlund, Brian P

    2008-10-01

    Despite the ubiquity of ammonium in geothermal environments and the thermodynamic favorability of aerobic ammonia oxidation, thermophilic ammonia-oxidizing microorganisms belonging to the crenarchaeota kingdom have only recently been described. In this study, we analyzed microbial mats and surface sediments from 21 hot spring samples (pH 3.4 to 9.0; temperature, 41 to 86 degrees C) from the United States, China, and Russia and obtained 846 putative archaeal ammonia monooxygenase large-subunit (amoA) gene and transcript sequences, representing a total of 41 amoA operational taxonomic units (OTUs) at 2% identity. The amoA gene sequences were highly diverse, yet they clustered within two major clades of archaeal amoA sequences known from water columns, sediments, and soils: clusters A and B. Eighty-four percent (711/846) of the sequences belonged to cluster A, which is typically found in water columns and sediments, whereas 16% (135/846) belonged to cluster B, which is typically found in soils and sediments. Although a few amoA OTUs were present in several geothermal regions, most were specific to a single region. In addition, cluster A amoA genes formed geographic groups, while cluster B sequences did not group geographically. With the exception of only one hot spring, principal-component analysis and UPGMA (unweighted-pair group method using average linkages) based on the UniFrac metric derived from cluster A grouped the springs by location, regardless of temperature or bulk water pH, suggesting that geography may play a role in structuring communities of putative ammonia-oxidizing archaea (AOA). The amoA genes were distinct from those of low-temperature environments; in particular, pair-wise comparisons between hot spring amoA genes and those from sympatric soils showed less than 85% sequence identity, underscoring the distinctness of hot spring archaeal communities from those of the surrounding soil system. Reverse transcription-PCR showed that amoA genes were

  2. Global Occurrence of Archaeal amoA Genes in Terrestrial Hot Springs▿

    Science.gov (United States)

    Zhang, Chuanlun L.; Ye, Qi; Huang, Zhiyong; Li, WenJun; Chen, Jinquan; Song, Zhaoqi; Zhao, Weidong; Bagwell, Christopher; Inskeep, William P.; Ross, Christian; Gao, Lei; Wiegel, Juergen; Romanek, Christopher S.; Shock, Everett L.; Hedlund, Brian P.

    2008-01-01

    Despite the ubiquity of ammonium in geothermal environments and the thermodynamic favorability of aerobic ammonia oxidation, thermophilic ammonia-oxidizing microorganisms belonging to the crenarchaeota kingdom have only recently been described. In this study, we analyzed microbial mats and surface sediments from 21 hot spring samples (pH 3.4 to 9.0; temperature, 41 to 86°C) from the United States, China, and Russia and obtained 846 putative archaeal ammonia monooxygenase large-subunit (amoA) gene and transcript sequences, representing a total of 41 amoA operational taxonomic units (OTUs) at 2% identity. The amoA gene sequences were highly diverse, yet they clustered within two major clades of archaeal amoA sequences known from water columns, sediments, and soils: clusters A and B. Eighty-four percent (711/846) of the sequences belonged to cluster A, which is typically found in water columns and sediments, whereas 16% (135/846) belonged to cluster B, which is typically found in soils and sediments. Although a few amoA OTUs were present in several geothermal regions, most were specific to a single region. In addition, cluster A amoA genes formed geographic groups, while cluster B sequences did not group geographically. With the exception of only one hot spring, principal-component analysis and UPGMA (unweighted-pair group method using average linkages) based on the UniFrac metric derived from cluster A grouped the springs by location, regardless of temperature or bulk water pH, suggesting that geography may play a role in structuring communities of putative ammonia-oxidizing archaea (AOA). The amoA genes were distinct from those of low-temperature environments; in particular, pair-wise comparisons between hot spring amoA genes and those from sympatric soils showed less than 85% sequence identity, underscoring the distinctness of hot spring archaeal communities from those of the surrounding soil system. Reverse transcription-PCR showed that amoA genes were

  3. Comparison among amoA Primers Suited for Quantification and Diversity Analyses of Ammonia-Oxidizing Bacteria in Soil

    Science.gov (United States)

    Shimomura, Yumi; Morimoto, Sho; Hoshino, Yuko Takada; Uchida, Yoshitaka; Akiyama, Hiroko; Hayatsu, Masahito

    2012-01-01

    Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment. PMID:22075625

  4. amoA Gene abundances and nitrification potential rates suggest that benthic ammonia-oxidizing bacteria and not Archaea dominate N cycling in the Colne Estuary, United Kingdom.

    Science.gov (United States)

    Li, Jialin; Nedwell, David B; Beddow, Jessica; Dumbrell, Alex J; McKew, Boyd A; Thorpe, Emma L; Whitby, Corinne

    2015-01-01

    Nitrification, mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), is important in global nitrogen cycling. In estuaries where gradients of salinity and ammonia concentrations occur, there may be differential selections for ammonia-oxidizer populations. The aim of this study was to examine the activity, abundance, and diversity of AOA and AOB in surface oxic sediments of a highly nutrified estuary that exhibits gradients of salinity and ammonium. AOB and AOA communities were investigated by measuring ammonia monooxygenase (amoA) gene abundance and nitrification potentials both spatially and temporally. Nitrification potentials differed along the estuary and over time, with the greatest nitrification potentials occurring mid-estuary (8.2 μmol N grams dry weight [gdw](-1) day(-1) in June, increasing to 37.4 μmol N gdw(-1) day(-1) in January). At the estuary head, the nitrification potential was 4.3 μmol N gdw(-1) day(-1) in June, increasing to 11.7 μmol N gdw(-1) day(-1) in January. At the estuary head and mouth, nitrification potentials fluctuated throughout the year. AOB amoA gene abundances were significantly greater (by 100-fold) than those of AOA both spatially and temporally. Nitrosomonas spp. were detected along the estuary by denaturing gradient gel electrophoresis (DGGE) band sequence analysis. In conclusion, AOB dominated over AOA in the estuarine sediments, with the ratio of AOB/AOA amoA gene abundance increasing from the upper (freshwater) to lower (marine) regions of the Colne estuary. These findings suggest that in this nutrified estuary, AOB (possibly Nitrosomonas spp.) were of major significance in nitrification. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. A first insight into the occurrence and expression of functional amoA and accA genes of autotrophic and ammonia-oxidizing bathypelagic Crenarchaeota of Tyrrhenian Sea

    Science.gov (United States)

    Yakimov, Michail M.; Cono, Violetta La; Denaro, Renata

    2009-05-01

    The autotrophic and ammonia-oxidizing crenarchaeal assemblage at offshore site located in the deep Mediterranean (Tyrrhenian Sea, depth 3000 m) water was studied by PCR amplification of the key functional genes involved in energy (ammonia mono-oxygenase alpha subunit, amoA) and central metabolism (acetyl-CoA carboxylase alpha subunit, accA). Using two recently annotated genomes of marine crenarchaeons, an initial set of primers targeting archaeal accA-like genes was designed. Approximately 300 clones were analyzed, of which 100% of amoA library and almost 70% of accA library were unambiguously related to the corresponding genes from marine Crenarchaeota. Even though the acetyl-CoA carboxylase is phylogenetically not well conserved and the remaining clones were affiliated to various bacterial acetyl-CoA/propionyl-CoA carboxylase genes, the pool of archaeal sequences was applied for development of quantitative PCR analysis of accA-like distribution using TaqMan ® methodolgy. The archaeal accA gene fragments, together with alignable gene fragments from the Sargasso Sea and North Pacific Subtropical Gyre (ALOHA Station) metagenome databases, were analyzed by multiple sequence alignment. Two accA-like sequences, found in ALOHA Station at the depth of 4000 m, formed a deeply branched clade with 64% of all archaeal Tyrrhenian clones. No close relatives for residual 36% of clones, except of those recovered from Eastern Mediterranean, was found, suggesting the existence of a specific lineage of the crenarchaeal accA genes in deep Mediterranean water. Alignment of Mediterranean amoA sequences defined four cosmopolitan phylotypes of Crenarchaeota putative ammonia mono-oxygenase subunit A gene occurring in the water sample from the 3000 m depth. Without exception all phylotypes fell into Deep Marine Group I cluster that contain the vast majority of known sequences recovered from global deep-sea environment. Remarkably, three phylotypes accounted for 91% of all Mediterranean

  6. [Abundances of ammonia-oxidizing archaeal accA and amoA genes in response to NO2 - and NO3 - of hot springs in Yunnan province].

    Science.gov (United States)

    Song, Zhaoqi; Wang, Li; Zhou, Enmin; Wang, Fengping; Xiao, Xiang; Zhang, Chuanlun; Li, Wenjun

    2014-12-04

    Yunnan hot springs have highly diverseammonia-oxidizing archaea (AOA), which are autotrophic and can fix CO2 using the 3-hydroxypropionate/ 4-hydroxybutyrate (HP/HD) pathway. In this study, we investigated the abundances of prokaryotic 16S rRNA gene and archaeal accA and amoA genes in the sediments of hot springs of Yunnan Province, and analysed the correlations between the above gene abundances and environmental factors. We selected the sediments of twenty representative hot springs, and detected the gene abundances by quantitative polymerase chain reaction (qPCR). The principal component analysis (PCA) and the Mantel test in the R software package were performed for the correlations of gene abundance and environmental variables. The bacterial and archaeal 16S rRNA gene abundances were from 6.6 x 10(7) to 4.19 x 10(11) and from 1.27 x 10(6) to 1.51 x 10(11) copies/g sediment, respectively; Archaeal accA and amoA genes were from 8.89 x 10(3) to 6.49 x 10(5) and from 7.64 x 10(3) to 4.36 x 10(5) copies/g sediment, respectively. The results of mantel test showed that accA gene was significantly (R = 0.98, P < 0.001) correlated with amoA gene; Both of them also were correlated significantly with NO2- and NO3 -, but not with pH. The abundances of bacterial and archaeal 16S rRNA genes and the ratio between them varied significantly among Yunnan hot springs. The archaealaccA and amoA genes showed significant correlation with each other, validating our previous finding that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.

  7. Abundance and activity of 16S rRNA, amoA and nifH bacterial genes during assisted phytostabilization of mine tailings

    Science.gov (United States)

    Nelson, Karis N.; Neilson, Julia W.; Root, Robert A.; Chorover, Jon; Maier, Raina M.

    2014-01-01

    Mine tailings in semiarid regions are highly susceptible to erosion and are sources of dust pollution and potential avenues of human exposure to toxic metals. One constraint to revegetation of tailings by phytostabilization is the absence of microbial communities critical for biogeochemical cycling of plant nutrients. The objective of this study was to evaluate specific genes as in situ indicators of biological soil response during phytoremediation. The abundance and activity of 16S rRNA, nifH, and amoA were monitored during a nine month phytostabilization study using buffalo grass and quailbush grown in compost-amended, metalliferous tailings. The compost amendment provided a greater than 5-log increase in bacterial abundance, and survival of this compost-inoculum was more stable in planted treatments. Despite increased abundance, the activity of the introduced community was low, and significant increases were not detected until six and nine months in quailbush, and unplanted compost and buffalo grass treatments, respectively. In addition, increased abundances of nitrogen-fixation (nifH) and ammonia-oxidizing (amoA) genes were observed in rhizospheres of buffalo grass and quailbush, respectively. Thus, plant establishment facilitated the short term stabilization of introduced bacterial biomass and supported the growth of two key nitrogen-cycling populations in compost-amended tailings. PMID:25495940

  8. Abundance and Activity of 16S rRNA, AmoA and NifH Bacterial Genes During Assisted Phytostabilization of Mine Tailings.

    Science.gov (United States)

    Nelson, Karis N; Neilson, Julia W; Root, Robert A; Chorover, Jon; Maier, Raina M

    2015-01-01

    Mine tailings in semiarid regions are highly susceptible to erosion and are sources of dust pollution and potential avenues of human exposure to toxic metals. One constraint to revegetation of tailings by phytostabilization is the absence of microbial communities critical for biogeochemical cycling of plant nutrients. The objective of this study was to evaluate specific genes as in situ indicators of biological soil response during phytoremediation. The abundance and activity of 16S rRNA, nifH, and amoA were monitored during a nine month phytostabilization study using buffalo grass and quailbush grown in compost-amended, metalliferous tailings. The compost amendment provided a greater than 5-log increase in bacterial abundance, and survival of this compost-inoculum was more stable in planted treatments. Despite increased abundance, the activity of the introduced community was low, and significant increases were not detected until six and nine months in quailbush, and unplanted compost and buffalo grass treatments, respectively. In addition, increased abundances of nitrogen-fixation (nifH) and ammonia-oxidizing (amoA) genes were observed in rhizospheres of buffalo grass and quailbush, respectively. Thus, plant establishment facilitated the short term stabilization of introduced bacterial biomass and supported the growth of two key nitrogen-cycling populations in compost-amended tailings.

  9. [Association of kynurenine-3-monooxygenase gene with schizophrenia].

    Science.gov (United States)

    Golimbet, V E; Lezheiko, T V; Alfimova, M V; Abramova, L I; Kondrat'ev, N V

    2014-06-01

    Neurotoxic products produced during tryptophan metabolism via the kynurenine pathway could be involved in schizophrenia pathogenesis. It has been shown that kynurenine-3-monooxygenase (KMO) is indirectly involved in these products' formation. KMO polymorphic loci rs2275163 (C/T) and rs1053230 (A/G) were examined in 187 schizophrenia patients and 229 healthy subjects. A genetic combination of allele T and genotype GG was observed more often in a patient group compared with healthy controls (p = 0.003, OR 2.0 (95% CI 1.2-2.9). In the latter group, this combination was associated with schizophrenia endophenotype (p = 0.04), which manifested in a higher expression of schizotypal personality traits assessed using the MMPI test.

  10. Diversity of Archaea and detection of crenarchaeotal amoA genes in the rivers Rhine and Têt

    OpenAIRE

    Herfort, L.; Kim, J.H.; Coolen, M.J.L.; Abbas, B.; Schouten, S.; Herndl, G.J.; Sinninghe Damste, J.S.

    2009-01-01

    Pelagic archaeal phylogenetic diversity and the potential for crenarchaeotal nitrification of Group 1.1a were determined in the rivers Rhine and Têt by 16S rRNA sequencing, catalyzed reported deposition-fluorescence in situ hybridization (CARD–FISH) and quantification of 16S rRNA and functional genes. Euryarchaeota were, for the first time, detected in temperate river water even though a net predominance of crenarchaeotal phylotypes was found. Differences in phylogenic distribution were obser...

  11. The abundance of functional genes, cbbL, nifH, amoA and apsA, and bacterial community structure of intertidal soil from Arabian Sea.

    Science.gov (United States)

    Keshri, Jitendra; Yousuf, Basit; Mishra, Avinash; Jha, Bhavanath

    2015-06-01

    The Gulf of Cambay is a trumpet-shaped inlet of the Arabian Sea, located along the west coast of India and confronts a high tidal range with strong water currents. The region belongs to a semi-arid zone and saline alkaline intertidal soils are considered biologically extreme. The selected four soil types (S1-S4) were affected by salinity, alkalinity and sodicity. Soil salinity ranged from 20 to 126 dS/m, soil pH 8.6-10.0 with high sodium adsorption ratio (SAR) and exchangeable sodium percentage (ESP). Abundance of the key functional genes like cbbL, nifH, amoA and apsA involved in biogeochemical cycling were targeted using qPCR, which varied from (2.36 ± 0.03) × 10(4) to (2.87 ± 0.26) × 10(8), (1.18 ± 0.28) × 10(6) to (1.01 ± 0.26) × 10(9), (1.41 ± 0.21) × 10(6) to (1.29 ± 0.05) × 10(8) and (8.47 ± 0.23) × 10(4) to (1.73 ± 0.01) × 10(6) per gram dry weight, respectively. The microbial community structure revealed that soils S1 and S3 were dominated by phylum Firmicutes whereas S4 and S2 showed an abundance of Proteobacterial clones. These soils also represented Bacteroidetes, Chloroflexi, Actinobacteria, Planctomycetes and Acidobacteria clones. Molecular phylogeny showed a significant variation in the bacterial community distribution among the intertidal soil types. A high number of novel taxonomic units were observed which makes the intertidal zone a unique reservoir of unidentified bacterial taxa that may be explored further. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. Influence of kynurenine 3-monooxygenase (KMO) gene polymorphism on cognitive function in schizophrenia✰,✰✰

    Science.gov (United States)

    Wonodi, Ikwunga; McMahon, Robert P.; Krishna, Nithin; Mitchell, Braxton D.; Liu, Judy; Glassman, Matthew; Hong, L. Elliot; Gold, James M.

    2015-01-01

    Background Cognitive deficits compromise quality of life and productivity for individuals with schizophrenia and have no effective treatments. Preclinical data point to the kynurenine pathway of tryptophan metabolism as a potential target for pro-cognitive drug development. We have previously demonstrated association of a kynurenine 3-monooxygenase (KMO) gene variant with reduced KMO gene expression in postmortem schizophrenia cortex, and neurocognitive endophenotypic deficits in a clinical sample. KMO encodes kynurenine 3-monooxygenase (KMO), the rate-limiting microglial enzyme of cortical kynurenine metabolism. Aberration of the KMO gene might be the proximal cause of impaired cortical kynurenine metabolism observed in schizophrenia. However, the relationship between KMO variation and cognitive function in schizophrenia is unknown. This study examined the effects of the KMO rs2275163C>T C (risk) allele on cognitive function in schizophrenia. Methods We examined the association of KMO polymorphisms with general neuropsychological performance and P50 gating in a sample of 150 schizophrenia and 95 healthy controls. Results Consistent with our original report, the KMO rs2275163C>T C (risk) allele was associated with deficits in general neuropsychological performance, and this effect was more marked in schizophrenia compared with controls. Additionally, the C (Arg452) allele of the missense rs1053230C>T variant (KMO Arg452Cys) showed a trend effect on cognitive function. Neither variant affected P50 gating. Conclusions These data suggest that KMO variation influences a range of cognitive domains known to predict functional outcome. Extensive molecular characterization of this gene would elucidate its role in cognitive function with implications for vertical integration with basic discovery. PMID:25464917

  13. Influence of kynurenine 3-monooxygenase (KMO) gene polymorphism on cognitive function in schizophrenia.

    Science.gov (United States)

    Wonodi, Ikwunga; McMahon, Robert P; Krishna, Nithin; Mitchell, Braxton D; Liu, Judy; Glassman, Matthew; Hong, L Elliot; Gold, James M

    2014-12-01

    Cognitive deficits compromise quality of life and productivity for individuals with schizophrenia and have no effective treatments. Preclinical data point to the kynurenine pathway of tryptophan metabolism as a potential target for pro-cognitive drug development. We have previously demonstrated association of a kynurenine 3-monooxygenase (KMO) gene variant with reduced KMO gene expression in postmortem schizophrenia cortex, and neurocognitive endophenotypic deficits in a clinical sample. KMO encodes kynurenine 3-monooxygenase (KMO), the rate-limiting microglial enzyme of cortical kynurenine metabolism. Aberration of the KMO gene might be the proximal cause of impaired cortical kynurenine metabolism observed in schizophrenia. However, the relationship between KMO variation and cognitive function in schizophrenia is unknown. This study examined the effects of the KMO rs2275163C>T C (risk) allele on cognitive function in schizophrenia. We examined the association of KMO polymorphisms with general neuropsychological performance and P50 gating in a sample of 150 schizophrenia and 95 healthy controls. Consistent with our original report, the KMO rs2275163C>T C (risk) allele was associated with deficits in general neuropsychological performance, and this effect was more marked in schizophrenia compared with controls. Additionally, the C (Arg452) allele of the missense rs1053230C>T variant (KMO Arg452Cys) showed a trend effect on cognitive function. Neither variant affected P50 gating. These data suggest that KMO variation influences a range of cognitive domains known to predict functional outcome. Extensive molecular characterization of this gene would elucidate its role in cognitive function with implications for vertical integration with basic discovery. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Isoeugenol monooxygenase and its putative regulatory gene are located in the eugenol metabolic gene cluster in Pseudomonas nitroreducens Jin1.

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    Ryu, Ji-Young; Seo, Jiyoung; Unno, Tatsuya; Ahn, Joong-Hoon; Yan, Tao; Sadowsky, Michael J; Hur, Hor-Gil

    2010-03-01

    The plant-derived phenylpropanoids eugenol and isoeugenol have been proposed as useful precursors for the production of natural vanillin. Genes involved in the metabolism of eugenol and isoeugenol were clustered in region of about a 30 kb of Pseudomonas nitroreducens Jin1. Two of the 23 ORFs in this region, ORFs 26 (iemR) and 27 (iem), were predicted to be involved in the conversion of isoeugenol to vanillin. The deduced amino acid sequence of isoeugenol monooxygenase (Iem) of strain Jin1 had 81.4% identity to isoeugenol monooxygenase from Pseudomonas putida IE27, which also transforms isoeugenol to vanillin. Iem was expressed in E. coli BL21(DE3) and was found to lead to isoeugenol to vanillin transformation. Deletion and cloning analyses indicated that the gene iemR, located upstream of iem, is required for expression of iem in the presence of isoeugenol, suggesting it to be the iem regulatory gene. Reverse transcription, real-time PCR analyses indicated that the genes involved in the metabolism of eugenol and isoeugenol were differently induced by isoeugenol, eugenol, and vanillin.

  15. Phylogenetic diversity and spatial distribution of the microbial community associated with the Caribbean deep-water sponge Polymastia cf. corticata by 16S rRNA, aprA, and amoA gene analysis.

    Science.gov (United States)

    Meyer, Birte; Kuever, Jan

    2008-08-01

    Denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA, aprA, and amoA genes demonstrated that a phylogenetically diverse and complex microbial community was associated with the Caribbean deep-water sponge Polymastia cf. corticata Ridley and Dendy, 1887. From the 38 archaeal and bacterial 16S rRNA phylotypes identified, 53% branched into the sponge-specific, monophyletic sequence clusters determined by previous studies (considering predominantly shallow-water sponge species), whereas 26% appeared to be P. cf. corticata specifically associated microorganisms ("specialists"); 21% of the phylotypes were confirmed to represent seawater- and sediment-derived proteobacterial species ("contaminants") acquired by filtration processes from the host environment. Consistently, the aprA and amoA gene-based analyses indicated the presence of environmentally derived sulfur- and ammonia-oxidizers besides putative sponge-specific sulfur-oxidizing Gammaproteobacteria and Alphaproteobacteria and a sulfate-reducing archaeon. A sponge-specific, endosymbiotic sulfur cycle as described for marine oligochaetes is proposed to be also present in P. cf. corticata. Overall, the results of this work support the recent studies that demonstrated the sponge species specificity of the associated microbial community while the biogeography of the host collection site has only a minor influence on the composition. In P. cf. corticata, the specificity of the sponge-microbe associations is even extended to the spatial distribution of the microorganisms within the sponge body; distinct bacterial populations were associated with the different tissue sections, papillae, outer and inner cortex, and choanosome. The local distribution of a phylotype within P. cf. corticata correlated with its (1) phylogenetic affiliation, (2) classification as sponge-specific or nonspecifically associated microorganism, and (3) potential ecological role in the host sponge.

  16. Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection

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    Walker M Andrew

    2010-07-01

    Full Text Available Abstract Background Plant cytochrome P450 monooxygenases (CYP mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines. Results Cloning of genomic DNA and cDNA revealed that the CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS in the upstream region and three candidate polyadenylation (PolyA sites in the downstream region of CYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression. Conclusions This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression

  17. In planta functions of cytochrome P450 monooxygenase genes in the phytocassane biosynthetic gene cluster on rice chromosome 2.

    Science.gov (United States)

    Ye, Zhongfeng; Yamazaki, Kohei; Minoda, Hiromi; Miyamoto, Koji; Miyazaki, Sho; Kawaide, Hiroshi; Yajima, Arata; Nojiri, Hideaki; Yamane, Hisakazu; Okada, Kazunori

    2018-06-01

    In response to environmental stressors such as blast fungal infections, rice produces phytoalexins, an antimicrobial diterpenoid compound. Together with momilactones, phytocassanes are among the major diterpenoid phytoalexins. The biosynthetic genes of diterpenoid phytoalexin are organized on the chromosome in functional gene clusters, comprising diterpene cyclase, dehydrogenase, and cytochrome P450 monooxygenase genes. Their functions have been studied extensively using in vitro enzyme assay systems. Specifically, P450 genes (CYP71Z6, Z7; CYP76M5, M6, M7, M8) on rice chromosome 2 have multifunctional activities associated with ent-copalyl diphosphate-related diterpene hydrocarbons, but the in planta contribution of these genes to diterpenoid phytoalexin production remains unknown. Here, we characterized cyp71z7 T-DNA mutant and CYP76M7/M8 RNAi lines to find that potential phytoalexin intermediates accumulated in these P450-suppressed rice plants. The results suggested that in planta, CYP71Z7 is responsible for C2-hydroxylation of phytocassanes and that CYP76M7/M8 is involved in C11α-hydroxylation of 3-hydroxy-cassadiene. Based on these results, we proposed potential routes of phytocassane biosynthesis in planta.

  18. Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph

    OpenAIRE

    Angela V. Smirnova; Peter F. Dunfield

    2018-01-01

    Methanotrophs are a specialized group of bacteria that can utilize methane (CH4) as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO), of either a soluble, cytoplasmic type (sMMO), or a particulate, membrane-bound type (pMMO). Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silv...

  19. Abundance and activity of 16S rRNA, amoA and nifH bacterial genes during assisted phytostabilization of mine tailings

    OpenAIRE

    Nelson, Karis N.; Neilson, Julia W.; Root, Robert A.; Chorover, Jon; Maier, Raina M.

    2015-01-01

    Mine tailings in semiarid regions are highly susceptible to erosion and are sources of dust pollution and potential avenues of human exposure to toxic metals. One constraint to revegetation of tailings by phytostabilization is the absence of microbial communities critical for biogeochemical cycling of plant nutrients. The objective of this study was to evaluate specific genes as in situ indicators of biological soil response during phytoremediation. The abundance and activity of 16S rRNA, nif...

  20. Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

    Science.gov (United States)

    Lao, Shu-Hua; Huang, Xiao-Hui; Huang, Hai-Jian; Liu, Cheng-Wen; Zhang, Chuan-Xi; Bao, Yan-Yuan

    2015-11-01

    The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Methanobactin from Methylocystis sp. strain SB2 affects gene expression and methane monooxygenase activity in Methylosinus trichosporium OB3b.

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    Farhan Ul-Haque, Muhammad; Kalidass, Bhagyalakshmi; Vorobev, Alexey; Baral, Bipin S; DiSpirito, Alan A; Semrau, Jeremy D

    2015-04-01

    Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl2, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl2, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

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

    2015-02-01

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

  3. Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph.

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    Smirnova, Angela V; Dunfield, Peter F

    2018-03-06

    Methanotrophs are a specialized group of bacteria that can utilize methane (CH₄) as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO), of either a soluble, cytoplasmic type (sMMO), or a particulate, membrane-bound type (pMMO). Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silvestris is a facultative methanotroph able to grow on several multicarbon substrates in addition to methane. We constructed transcriptional fusions of the mmo promoters of Methyloferula stellata and Methylocella silvestris to a promoterless gfp in order to compare their transcriptional regulation in response to different growth substrates, in the genetic background of both organisms. The following patterns were observed: (1) The mmo promoter of the facultative methanotroph Methylocella silvestris was either transcriptionally downregulated or repressed by any growth substrate other than methane in the genetic background of Methylocella silvetris ; (2) Growth on methane alone upregulated the mmo promoter of Methylocella silvetris in its native background but not in the obligate methanotroph Methyloferula stellata ; (3) The mmo promoter of Methyloferula stellata was constitutive in both organisms regardless of the growth substrate, but with much lower promoter activity than the mmo promoter of Methylocella silvetris . These results support a conclusion that a different mode of transcriptional regulation of sMMO contributes to the facultative lifestyle of Methylocella silvetris compared to the obligate methanotroph Methyloferula stellata .

  4. Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph

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    Angela V. Smirnova

    2018-03-01

    Full Text Available Methanotrophs are a specialized group of bacteria that can utilize methane (CH4 as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO, of either a soluble, cytoplasmic type (sMMO, or a particulate, membrane-bound type (pMMO. Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silvestris is a facultative methanotroph able to grow on several multicarbon substrates in addition to methane. We constructed transcriptional fusions of the mmo promoters of Methyloferula stellata and Methylocella silvestris to a promoterless gfp in order to compare their transcriptional regulation in response to different growth substrates, in the genetic background of both organisms. The following patterns were observed: (1 The mmo promoter of the facultative methanotroph Methylocella silvestris was either transcriptionally downregulated or repressed by any growth substrate other than methane in the genetic background of Methylocella silvetris; (2 Growth on methane alone upregulated the mmo promoter of Methylocella silvetris in its native background but not in the obligate methanotroph Methyloferula stellata; (3 The mmo promoter of Methyloferula stellata was constitutive in both organisms regardless of the growth substrate, but with much lower promoter activity than the mmo promoter of Methylocella silvetris. These results support a conclusion that a different mode of transcriptional regulation of sMMO contributes to the facultative lifestyle of Methylocella silvetris compared to the obligate methanotroph Methyloferula stellata.

  5. The Kynurenine 3-Monooxygenase Encoding Gene, BcKMO, Is Involved in the Growth, Development, and Pathogenicity of Botrytis cinerea

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    Kang Zhang

    2018-05-01

    Full Text Available A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea. A novel pathogenicity-related gene BcKMO, which encodes kynurenine 3-monooxygenase (KMO, was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO-complementing mutant (BCG183/BcKMO were similar to those of the wild-type (WT strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/BcKMO. Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H2O2, and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP and mitogen-activated protein kinase (MAPK signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1, Pka2, PkaR, Bcg2, Bcg3, bmp1, and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea. Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea.

  6. The Kynurenine 3-Monooxygenase Encoding Gene, BcKMO, Is Involved in the Growth, Development, and Pathogenicity of Botrytis cinerea.

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    Zhang, Kang; Yuan, Xuemei; Zang, Jinping; Wang, Min; Zhao, Fuxin; Li, Peifen; Cao, Hongzhe; Han, Jianmin; Xing, Jihong; Dong, Jingao

    2018-01-01

    A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea . A novel pathogenicity-related gene BcKMO , which encodes kynurenine 3-monooxygenase (KMO), was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO -complementing mutant (BCG183/ BcKMO ) were similar to those of the wild-type (WT) strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/ BcKMO . Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H 2 O 2 , and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1 , Pka2 , PkaR , Bcg2 , Bcg3 , bmp1 , and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea . Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea .

  7. Reverse-transcriptional gene expression of anammox and ammonia-oxidizing archaea and bacteria in soybean and rice paddy soils of Northeast China.

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    Wang, Jing; Dong, Hailiang; Wang, Weidong; Gu, Ji-Dong

    2014-03-01

    The relative gene expression of hydrazine oxidoreductase encoding gene (hzo) for anaerobic ammonium oxidizing bacteria (anammox) and ammonia monooxygenase encoding gene (amoA) for both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in Sanjiang Plain soybean and rice paddy soils of Northeast China was investigated by using real-time reverse-transcriptional quantitative PCR. Metabolically active populations of anammox, AOA, and AOB in rice paddy soils were evident by the presence and successful quantification of hzo mRNA and amoA mRNA genes. The expression ratio of amoA gene for both AOA and AOB varied between soybean soils and different rice paddy soils while the expression of hzo gene for anammox was detectable only in rice paddy soils by showing a diverse relative expression ratio in each soil sample. Gene expression of both archaeal and bacterial amoA genes in rice paddy soils differed among the three sampling depths, but that of hzo was not. Both archaeal and bacterial amoA genes showed an increase trend of expression level with continuation of rice paddy cultivation, but the low expression ratio of hzo gene indicated a relatively small contribution of anammox in overall removal of inorganic nitrogen through N2 even under anoxic and high nitrogen input in agriculture. Bacterial amoA gene from two soybean fields and three rice paddy fields were also analyzed for community composition by denaturing gradient gel electrophoresis fingerprint. Community shift was observed between soybean and paddy fields and within each of them. The consistent occurrence of three bands 5, 6, and 7 in all samples showed their high adaptability for both arid cultivation and continuous rice paddy cultivation. Our data suggest that AOA and AOB are playing a more important role in nitrogen transformation in agricultural soils in oxic or anoxic environment and anammox bacteria may also contribute but in a less extent to N transformation in these agricultural soils

  8. Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria ▿

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    Furuya, Toshiki; Hirose, Satomi; Semba, Hisashi; Kino, Kuniki

    2011-01-01

    The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ54 factor, a gene encoding the σ54 factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria. PMID:21856847

  9. Downregulated kynurenine 3-monooxygenase gene expression and enzyme activity in schizophrenia and genetic association with schizophrenia endophenotypes.

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    Wonodi, Ikwunga; Stine, O Colin; Sathyasaikumar, Korrapati V; Roberts, Rosalinda C; Mitchell, Braxton D; Hong, L Elliot; Kajii, Yasushi; Thaker, Gunvant K; Schwarcz, Robert

    2011-07-01

    Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan degradation, is an antagonist at N-methyl-d-aspartate and α7 nicotinic acetylcholine receptors and modulates glutamate, dopamine, and acetylcholine signaling. Cortical kynurenic acid concentrations are elevated in the brain and cerebrospinal fluid of schizophrenia patients. The proximal cause may be an impairment of kynurenine 3-monooxygenase (KMO), a rate-limiting enzyme at the branching point of the kynurenine pathway. To examine KMO messenger RNA expression and KMO enzyme activity in postmortem tissue from the frontal eye field (FEF; Brodmann area 6) obtained from schizophrenia individuals compared with healthy control individuals and to explore the relationship between KMO single-nucleotide polymorphisms and schizophrenia oculomotor endophenotypes. Case-control postmortem and clinical study. Maryland Brain Collection, outpatient clinics. Postmortem specimens from schizophrenia patients (n = 32) and control donors (n = 32) and a clinical sample of schizophrenia patients (n = 248) and healthy controls (n = 228). Comparison of quantitative KMO messenger RNA expression and KMO enzyme activity in postmortem FEF tissue between schizophrenia patients and controls and association of KMO single-nucleotide polymorphisms with messenger RNA expression in postmortem FEF and schizophrenia and oculomotor endophenotypes (ie, smooth pursuit eye movements and oculomotor delayed response). In postmortem tissue, we found a significant and correlated reduction in KMO gene expression and KMO enzyme activity in the FEF in schizophrenia patients. In the clinical sample, KMO rs2275163 was not associated with a diagnosis of schizophrenia but showed modest effects on predictive pursuit and visuospatial working memory endophenotypes. Our results provide converging lines of evidence implicating reduced KMO activity in the etiopathophysiology of schizophrenia and related neurocognitive deficits.

  10. Diversity of alkane degrading bacteria associated with plants in a petroleum oil-contaminated environment and expression of alkane monooxygenase (alkB) genes

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    Andria, V.; Yousaf, S.; Reichenauer, T. G.; Smalla, K.; Sessitsch, A.

    2009-04-01

    Among twenty-six different plant species, Italian ryegrass (Lolium multiflorum var. Taurus), Birdsfoot trefoil (Lotus corniculatus var. Leo), and the combination of both plants performed well in a petroleum oil contaminated soil. Hydrocarbon degrading bacteria were isolated from the rhizosphere, root interior and shoot interior and subjected to the analysis of 16S rRNA, the 16S and 23S rRNA intergenic spacer region and alkane hydroxylase genes. Higher numbers of culturable, degrading bacteria were associated with Italian ryegrass, which were also characterized by a higher diversity, particularly in the plant interior. Only half of the isolated bacteria hosted known alkane hydroxylase genes (alkB and cytochrome P153-like). Our results indicated that alkB genes have spread through horizontal gene transfer, particularly in the Italian ryegrass rhizosphere, and suggested mobility of catabolic genes between Gram-negative and Gram-positive bacteria. We furthermore studied the colonization behaviour of selected hydrocarbon-degrading strains (comprising an endopyhte and a rhizosphere strain) as well as the expression of their alkane monooxygenase genes in association with Italian ryegrass. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior.

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

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    Maria Laura Mascotti

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

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

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

    2015-01-01

    The Baeyer—Villiger Monooxygenases (BVMOs) are enzymes belonging to the “Class B” of flavin monooxygenases and are capable of performing exquisite selective oxidations. These enzymes have been studied from a biotechnological perspective, but their physiological substrates and functional roles are widely unknown. Here, we investigated the origin, taxonomic distribution and evolutionary history of the BVMO genes. By using in silico approaches, 98 BVMO encoding genes were detected in the three domains of life: Archaea, Bacteria and Eukarya. We found evidence for the presence of these genes in Metazoa (Hydra vulgaris, Oikopleura dioica and Adineta vaga) and Haptophyta (Emiliania huxleyi) for the first time. Furthermore, a search for other “Class B” monooxygenases (flavoprotein monooxygenases –FMOs – and N-hydroxylating monooxygenases – NMOs) was conducted. These sequences were also found in the three domains of life. Phylogenetic analyses of all “Class B” monooxygenases revealed that NMOs and BVMOs are monophyletic, whereas FMOs form a paraphyletic group. Based on these results, we propose that BVMO genes were already present in the last universal common ancestor (LUCA) and their current taxonomic distribution is the result of differential duplication and loss of paralogous genes. PMID:26161776

  13. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

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    Jungmann, Volker; Molnár, István; Hammer, Philip E.; Hill, D. Steven; Zirkle, Ross; Buckel, Thomas G.; Buckel, Dagmar; Ligon, James M.; Pachlatko, J. Paul

    2005-01-01

    4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined. PMID:16269732

  14. MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental Samples

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    Pérez-de-Mora, Alfredo; Schulz, Stephan; Schloter, Michael

    Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

  15. A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants.

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    Rong Tan, Li; Chen Lu, Yi; Jing Zhang, Jing; Luo, Fang; Yang, Hong

    2015-09-01

    Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase.

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    Chen, Yin; Patel, Nisha A; Crombie, Andrew; Scrivens, James H; Murrell, J Colin

    2011-10-25

    Flavin-containing monooxygenases (FMOs) are one of the most important monooxygenase systems in Eukaryotes and have many important physiological functions. FMOs have also been found in bacteria; however, their physiological function is not known. Here, we report the identification and characterization of trimethylamine (TMA) monooxygenase, termed Tmm, from Methylocella silvestris, using a combination of proteomic, biochemical, and genetic approaches. This bacterial FMO contains the FMO sequence motif (FXGXXXHXXXF/Y) and typical flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-binding domains. The enzyme was highly expressed in TMA-grown M. silvestris and absent during growth on methanol. The gene, tmm, was expressed in Escherichia coli, and the purified recombinant protein had high Tmm activity. Mutagenesis of this gene abolished the ability of M. silvestris to grow on TMA as a sole carbon and energy source. Close homologs of tmm occur in many Alphaproteobacteria, in particular Rhodobacteraceae (marine Roseobacter clade, MRC) and the marine SAR11 clade (Pelagibacter ubique). We show that the ability of MRC to use TMA as a sole carbon and/or nitrogen source is directly linked to the presence of tmm in the genomes, and purified Tmm of MRC and SAR11 from recombinant E. coli showed Tmm activities. The tmm gene is highly abundant in the metagenomes of the Global Ocean Sampling expedition, and we estimate that 20% of the bacteria in the surface ocean contain tmm. Taken together, our results suggest that Tmm, a bacterial FMO, plays an important yet overlooked role in the global carbon and nitrogen cycles.

  17. Presence and Expression of Microbial Genes Regulating Soil Nitrogen Dynamics Along the Tanana River Successional Sequence

    Science.gov (United States)

    Boone, R. D.; Rogers, S. L.

    2004-12-01

    We report on work to assess the functional gene sequences for soil microbiota that control nitrogen cycle pathways along the successional sequence (willow, alder, poplar, white spruce, black spruce) on the Tanana River floodplain, Interior Alaska. Microbial DNA and mRNA were extracted from soils (0-10 cm depth) for amoA (ammonium monooxygenase), nifH (nitrogenase reductase), napA (nitrate reductase), and nirS and nirK (nitrite reductase) genes. Gene presence was determined by amplification of a conserved sequence of each gene employing sequence specific oligonucleotide primers and Polymerase Chain Reaction (PCR). Expression of the genes was measured via nested reverse transcriptase PCR amplification of the extracted mRNA. Amplified PCR products were visualized on agarose electrophoresis gels. All five successional stages show evidence for the presence and expression of microbial genes that regulate N fixation (free-living), nitrification, and nitrate reduction. We detected (1) nifH, napA, and nirK presence and amoA expression (mRNA production) for all five successional stages and (2) nirS and amoA presence and nifH, nirK, and napA expression for early successional stages (willow, alder, poplar). The results highlight that the existing body of previous process-level work has not sufficiently considered the microbial potential for a nitrate economy and free-living N fixation along the complete floodplain successional sequence.

  18. Physical map location of the multicopy genes coding for ammonia monooxygenase and hydroxylamine oxidoreductase in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11.

    Science.gov (United States)

    Hirota, R; Yamagata, A; Kato, J; Kuroda, A; Ikeda, T; Takiguchi, N; Ohtake, H

    2000-02-01

    Pulsed-field gel electrophoresis of PmeI digests of the Nitrosomonas sp. strain ENI-11 chromosome produced four bands ranging from 1,200 to 480 kb in size. Southern hybridizations suggested that a 487-kb PmeI fragment contained two copies of the amoCAB genes, coding for ammonia monooxygenase (designated amoCAB(1) and amoCAB(2)), and three copies of the hao gene, coding for hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)). In this DNA fragment, amoCAB(1) and amoCAB(2) were about 390 kb apart, while hao(1), hao(2), and hao(3) were separated by at least about 100 kb from each other. Interestingly, hao(1) and hao(2) were located relatively close to amoCAB(1) and amoCAB(2), respectively. DNA sequence analysis revealed that hao(1) and hao(2) shared 160 identical nucleotides immediately upstream of each translation initiation codon. However, hao(3) showed only 30% nucleotide identity in the 160-bp corresponding region.

  19. Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes

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    Martina eCappelletti

    2015-05-01

    Full Text Available Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from butane metabolism. Two gene clusters, prmABCD and smoABCD – coding for soluble di-iron monooxgenases (SDIMOs involved in gaseous n-alkanes oxidation – were detected in the BCP1 genome. By means of reverse transcriptase-quantitative PCR (RT-qPCR analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids or during the cell growth on rich medium (Luria Bertani broth. The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane and/or liquid (n-hexane short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step.

  20. Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes

    Science.gov (United States)

    Cappelletti, Martina; Presentato, Alessandro; Milazzo, Giorgio; Turner, Raymond J.; Fedi, Stefano; Frascari, Dario; Zannoni, Davide

    2015-01-01

    Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD—coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation—were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria–Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. PMID:26029173

  1. The peptidylglycine-α-amidating monooxygenase (PAM) gene rs13175330 A>G polymorphism is associated with hypertension in a Korean population.

    Science.gov (United States)

    Yoo, Hye Jin; Kim, Minjoo; Kim, Minkyung; Chae, Jey Sook; Lee, Sang-Hyun; Lee, Jong Ho

    2017-11-21

    Peptidylglycine-α-amidating monooxygenase (PAM) may play a role in the secretion of atrial natriuretic peptide (ANP), which is a hormone involved in the maintenance of blood pressure (BP). The objective of the present study was to determine whether PAM is a novel candidate gene for hypertension (HTN). A total of 2153 Korean participants with normotension and HTN were included. Genotype data were obtained using the Korean Chip. The rs13175330 polymorphism of the PAM gene was selected from the ten single nucleotide polymorphisms (SNPs) most strongly associated with BP. The presence of the G allele of the PAM rs13175330 A>G SNP was associated with a higher risk of HTN after adjustments for age, sex, BMI, smoking, and drinking [OR 1.607 (95% CI 1.220-2.116), p = 0.001]. The rs13175330 G allele carriers in the HTN group treated without antihypertensive therapy (HTN w/o therapy) had significantly higher systolic and diastolic BP than the AA carriers, whereas the G allele carriers in the HTN group treated with antihypertensive therapy (HTN w/ therapy) showed significantly higher diastolic BP. Furthermore, rs13175330 G allele carriers in the HTN w/o therapy group had significantly increased levels of insulin, insulin resistance, and oxidized low-density lipoprotein (LDL) and significantly decreased LDL-cholesterol levels and LDL particle sizes compared to the AA carriers. These results suggest that the PAM rs13175330 A>G SNP is a novel candidate gene for HTN in the Korean population. Additionally, the PAM rs13175330 G allele might be associated with insulin resistance and LDL atherogenicity in patients with HTN.

  2. Abundance and distribution of archaeal acetyl-CoA/propionyl-CoA carboxylase genes indicative for putatively chemoautotrophic Archaea in the tropical Atlantic's interior.

    Science.gov (United States)

    Bergauer, Kristin; Sintes, Eva; van Bleijswijk, Judith; Witte, Harry; Herndl, Gerhard J

    2013-06-01

    Recently, evidence suggests that dark CO2 fixation in the pelagic realm of the ocean does not only occur in the suboxic and anoxic water bodies but also in the oxygenated meso- and bathypelagic waters of the North Atlantic. To elucidate the significance and phylogeny of the key organisms mediating dark CO2 fixation in the tropical Atlantic, we quantified functional genes indicative for CO2 fixation. We used a Q-PCR-based assay targeting the bifunctional acetyl-CoA/propionyl-CoA carboxylase (accA subunit), a key enzyme powering inter alia the 3-hydroxypropionate/4-hydroxybutyrate cycle (HP/HB) and the archaeal ammonia monooxygenase (amoA). Quantification of accA-like genes revealed a consistent depth profile in the upper mesopelagial with increasing gene abundances from subsurface layers towards the oxygen minimum zone (OMZ), coinciding with an increase in archaeal amoA gene abundance. Gene abundance profiles of metabolic marker genes (accA, amoA) were correlated with thaumarchaeal 16S rRNA gene abundances as well as CO2 fixation rates to link the genetic potential to actual rate measurements. AccA gene abundances correlated with archaeal amoA gene abundance throughout the water column (r(2)  = 0.309, P < 0.0001). Overall, a substantial genetic predisposition of CO2 fixation was present in the dark realm of the tropical Atlantic in both Archaea and Bacteria. Hence, dark ocean CO2 fixation might be more widespread among prokaryotes inhabiting the oxygenated water column of the ocean's interior than hitherto assumed. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  3. Metagenomic identification of a novel salt tolerance gene from the human gut microbiome which encodes a membrane protein with homology to a brp/blh-family β-carotene 15,15'-monooxygenase.

    Directory of Open Access Journals (Sweden)

    Eamonn P Culligan

    Full Text Available The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane.

  4. Kynurenine 3-Monooxygenase Gene Associated With Nicotine Initiation and Addiction: Analysis of Novel Regulatory Features at 5′ and 3′-Regions

    Directory of Open Access Journals (Sweden)

    Hassan A. Aziz

    2018-06-01

    Full Text Available Tobacco smoking is widespread behavior in Qatar and worldwide and is considered one of the major preventable causes of ill health and death. Nicotine is part of tobacco smoke that causes numerous health risks and is incredibly addictive; it binds to the α7 nicotinic acetylcholine receptor (α7nAChR in the brain. Recent studies showed α7nAChR involvement in the initiation and addiction of smoking. Kynurenic acid (KA, a significant tryptophan metabolite, is an antagonist of α7nAChR. Inhibition of kynurenine 3-monooxygenase enzyme encoded by KMO enhances the KA levels. Modulating KMO gene expression could be a useful tactic for the treatment of tobacco initiation and dependence. Since KMO regulation is still poorly understood, we aimed to investigate the 5′ and 3′-regulatory factors of KMO gene to advance our knowledge to modulate KMO gene expression. In this study, bioinformatics methods were used to identify the regulatory sequences associated with expression of KMO. The displayed differential expression of KMO mRNA in the same tissue and different tissues suggested the specific usage of the KMO multiple alternative promoters. Eleven KMO alternative promoters identified at 5′-regulatory region contain TATA-Box, lack CpG Island (CGI and showed dinucleotide base-stacking energy values specific to transcription factor binding sites (TFBSs. The structural features of regulatory sequences can influence the transcription process and cell type-specific expression. The uncharacterized LOC105373233 locus coding for non-coding RNA (ncRNA located on the reverse strand in a convergent manner at the 3′-side of KMO locus. The two genes likely expressed by a promoter that lacks TATA-Box harbor CGI and two TFBSs linked to the bidirectional transcription, the NRF1, and ZNF14 motifs. We identified two types of microRNA (miR in the uncharacterized LOC105373233 ncRNA, which are like hsa-miR-5096 and hsa-miR-1285-3p and can target the miR recognition

  5. High-throughput analysis of ammonia oxidiser community composition via a novel, amoA-based functional gene array.

    Directory of Open Access Journals (Sweden)

    Guy C J Abell

    Full Text Available Advances in microbial ecology research are more often than not limited by the capabilities of available methodologies. Aerobic autotrophic nitrification is one of the most important and well studied microbiological processes in terrestrial and aquatic ecosystems. We have developed and validated a microbial diagnostic microarray based on the ammonia-monooxygenase subunit A (amoA gene, enabling the in-depth analysis of the community structure of bacterial and archaeal ammonia oxidisers. The amoA microarray has been successfully applied to analyse nitrifier diversity in marine, estuarine, soil and wastewater treatment plant environments. The microarray has moderate costs for labour and consumables and enables the analysis of hundreds of environmental DNA or RNA samples per week per person. The array has been thoroughly validated with a range of individual and complex targets (amoA clones and environmental samples, respectively, combined with parallel analysis using traditional sequencing methods. The moderate cost and high throughput of the microarray makes it possible to adequately address broader questions of the ecology of microbial ammonia oxidation requiring high sample numbers and high resolution of the community composition.

  6. Marine Microbial Gene Abundance and Community Composition in Response to Ocean Acidification and Elevated Temperature in Two Contrasting Coastal Marine Sediments

    Directory of Open Access Journals (Sweden)

    Ashleigh R. Currie

    2017-08-01

    Full Text Available Marine ecosystems are exposed to a range of human-induced climate stressors, in particular changing carbonate chemistry and elevated sea surface temperatures as a consequence of climate change. More research effort is needed to reduce uncertainties about the effects of global-scale warming and acidification for benthic microbial communities, which drive sedimentary biogeochemical cycles. In this research, mesocosm experiments were set up using muddy and sandy coastal sediments to investigate the independent and interactive effects of elevated carbon dioxide concentrations (750 ppm CO2 and elevated temperature (ambient +4°C on the abundance of taxonomic and functional microbial genes. Specific quantitative PCR primers were used to target archaeal, bacterial, and cyanobacterial/chloroplast 16S rRNA in both sediment types. Nitrogen cycling genes archaeal and bacterial ammonia monooxygenase (amoA and bacterial nitrite reductase (nirS were specifically targeted to identify changes in microbial gene abundance and potential impacts on nitrogen cycling. In muddy sediment, microbial gene abundance, including amoA and nirS genes, increased under elevated temperature and reduced under elevated CO2 after 28 days, accompanied by shifts in community composition. In contrast, the combined stressor treatment showed a non-additive effect with lower microbial gene abundance throughout the experiment. The response of microbial communities in the sandy sediment was less pronounced, with the most noticeable response seen in the archaeal gene abundances in response to environmental stressors over time. 16S rRNA genes (amoA and nirS were lower in abundance in the combined stressor treatments in sandy sediments. Our results indicated that marine benthic microorganisms, especially in muddy sediments, are susceptible to changes in ocean carbonate chemistry and seawater temperature, which ultimately may have an impact upon key benthic biogeochemical cycles.

  7. Lyophilization conditions for the storage of monooxygenases

    NARCIS (Netherlands)

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

    2015-01-01

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

  8. Expression of alkane monooxygenase (alkB) genes by plant-associated bacteria in the rhizosphere and endosphere of Italian ryegrass (Lolium multiflorum L.) grown in diesel contaminated soil

    International Nuclear Information System (INIS)

    Andria, Verania; Reichenauer, Thomas G.; Sessitsch, Angela

    2009-01-01

    For phytoremediation of organic contaminants, plants have to host an efficiently degrading microflora. To assess the role of endophytes in alkane degradation, Italian ryegrass was grown in sterile soil with 0, 1 or 2% diesel and inoculated either with an alkane degrading bacterial strain originally derived from the rhizosphere of Italian ryegrass or with an endophyte. We studied plant colonization of these strains as well as the abundance and expression of alkane monooxygenase (alkB) genes in the rhizosphere, shoot and root interior. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior. - Bacterial alkane degradation genes are expressed in the rhizosphere and in the plant interior.

  9. Expression of alkane monooxygenase (alkB) genes by plant-associated bacteria in the rhizosphere and endosphere of Italian ryegrass (Lolium multiflorum L.) grown in diesel contaminated soil

    Energy Technology Data Exchange (ETDEWEB)

    Andria, Verania [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria); Reichenauer, Thomas G. [AIT Austrian Institute of Technology GmbH, Unit of Environmental Resources and Technologies, A-2444 Seibersdorf (Austria); Sessitsch, Angela, E-mail: angela.sessitsch@ait.ac.a [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria)

    2009-12-15

    For phytoremediation of organic contaminants, plants have to host an efficiently degrading microflora. To assess the role of endophytes in alkane degradation, Italian ryegrass was grown in sterile soil with 0, 1 or 2% diesel and inoculated either with an alkane degrading bacterial strain originally derived from the rhizosphere of Italian ryegrass or with an endophyte. We studied plant colonization of these strains as well as the abundance and expression of alkane monooxygenase (alkB) genes in the rhizosphere, shoot and root interior. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior. - Bacterial alkane degradation genes are expressed in the rhizosphere and in the plant interior.

  10. Distribution of the octopamine receptor AmOA1 in the honey bee brain.

    Directory of Open Access Journals (Sweden)

    Irina Sinakevitch

    2011-01-01

    Full Text Available Octopamine plays an important role in many behaviors in invertebrates. It acts via binding to G protein coupled receptors located on the plasma membrane of responsive cells. Several distinct subtypes of octopamine receptors have been found in invertebrates, yet little is known about the expression pattern of these different receptor subtypes and how each subtype may contribute to different behaviors. One honey bee (Apis mellifera octopamine receptor, AmOA1, was recently cloned and characterized. Here we continue to characterize the AmOA1 receptor by investigating its distribution in the honey bee brain. We used two independent antibodies produced against two distinct peptides in the carboxyl-terminus to study the distribution of the AmOA1 receptor in the honey bee brain. We found that both anti-AmOA1 antibodies revealed labeling of cell body clusters throughout the brain and within the following brain neuropils: the antennal lobes; the calyces, pedunculus, vertical (alpha, gamma and medial (beta lobes of the mushroom body; the optic lobes; the subesophageal ganglion; and the central complex. Double immunofluorescence staining using anti-GABA and anti-AmOA1 receptor antibodies revealed that a population of inhibitory GABAergic local interneurons in the antennal lobes express the AmOA1 receptor in the cell bodies, axons and their endings in the glomeruli. In the mushroom bodies, AmOA1 receptors are expressed in a subpopulation of inhibitory GABAergic feedback neurons that ends in the visual (outer half of basal ring and collar regions and olfactory (lip and inner basal ring region calyx neuropils, as well as in the collar and lip zones of the vertical and medial lobes. The data suggest that one effect of octopamine via AmOA1 in the antennal lobe and mushroom body is to modulate inhibitory neurons.

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

    NARCIS (Netherlands)

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

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

  12. Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa zea larval development and pupation.

    Science.gov (United States)

    Jin, Shuangxia; Singh, Nameirakpam D; Li, Lebin; Zhang, Xianlong; Daniell, Henry

    2015-04-01

    In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/μg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  13. Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli.

    Science.gov (United States)

    Furuya, Toshiki; Hayashi, Mika; Kino, Kuniki

    2013-10-01

    Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

  14. Depth-related distribution of a key gene of the tetraether lipid biosynthetic pathway in marine Thaumarchaeota

    NARCIS (Netherlands)

    Villanueva, Laura; Schouten, Stefan; Sinninghe Damsté, Jaap S.

    2015-01-01

    The distribution of isoprenoid glycerol dialkyl glycerol tetraethers (GDGT) lipids synthesized by Thaumarchaeota has been shown to be temperature-dependent in world oceans. Depth-related differences in the ammonia monooxygenase (amoA) of Thaumarchaeota have led to the classification of 'shallow' and

  15. Sequencing and characterization of mixed function monooxygenase genes CYP1A1 and CYP1A2 of Mink (Mustela vison) to facilitate study of dioxin-like compounds

    International Nuclear Information System (INIS)

    Zhang Xiaowei; Moore, Jeremy N.; Newsted, John L.; Hecker, Markus; Zwiernik, Matthew J.; Jones, Paul D.; Bursian, Steven J.

    2009-01-01

    As part of an ongoing effort to understand aryl hydrocarbon receptor (AhR) mediated toxicity in mink, cDNAs encoding for CYP1A1 and the CYP1A2 mixed function monooxygenases were cloned and characterized. In addition, the effects of selected dibenzofurans on the expression of these genes and the presence of their respective proteins (P4501A) were investigated, and then correlated with the catalytic activities of these proteins as measured by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) activities. The predicted protein sequences for CYP1A1 and CYP1A2 comprise 517 and 512 amino acid residues, respectively. The phylogenetic analysis of the mink CYP1As with protein sequences of other mammals revealed high sequence homology with sea otter, seals and the dog, with amino acid identities ranging from 89 to 95% for CYP1A1 and 81 to 93% for CYP1A2. Since exposure to both 2,3,7,8-Tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) resulted in dose-dependent increases of CYP1A1 mRNA, CYP1A2 mRNA and CYP1A protein levels an underlying AhR-mediated mechanism is suggested. The up-regulation of CYP1A mRNA in liver was more consistent to the sum adipose TEQ concentration than to the liver TEQ concentration in minks treated with TCDF or PeCDF. The result suggested that the hepatic-sequestered fraction of PeCDF was biologically inactive to the induction of CYP1A1 and CYP1A2

  16. Archaea Dominate the Ammonia-Oxidizing Community in the Rhizosphere of the Freshwater Macrophyte Littorella uniflora

    DEFF Research Database (Denmark)

    Herrmann, Martina; Saunders, Aaron M.; Schramm, Andreas

    2008-01-01

    Archaeal and bacterial ammonia monooxygenase genes (amoA) had similar low relative abundances in freshwater sediment. In the rhizosphere of the submersed macrophyte Littorella uniflora, archaeal amoA was 500- to >8,000-fold enriched compared to bacterial amoA, suggesting that the enhanced nitrifi...

  17. New Dimensions in Microbial Ecology—Functional Genes in Studies to Unravel the Biodiversity and Role of Functional Microbial Groups in the Environment

    Science.gov (United States)

    Imhoff, Johannes F.

    2016-01-01

    During the past decades, tremendous advances have been made in the possibilities to study the diversity of microbial communities in the environment. The development of methods to study these communities on the basis of 16S rRNA gene sequences analysis was a first step into the molecular analysis of environmental communities and the study of biodiversity in natural habitats. A new dimension in this field was reached with the introduction of functional genes of ecological importance and the establishment of genetic tools to study the diversity of functional microbial groups and their responses to environmental factors. Functional gene approaches are excellent tools to study the diversity of a particular function and to demonstrate changes in the composition of prokaryote communities contributing to this function. The phylogeny of many functional genes largely correlates with that of the 16S rRNA gene, and microbial species may be identified on the basis of functional gene sequences. Functional genes are perfectly suited to link culture-based microbiological work with environmental molecular genetic studies. In this review, the development of functional gene studies in environmental microbiology is highlighted with examples of genes relevant for important ecophysiological functions. Examples are presented for bacterial photosynthesis and two types of anoxygenic phototrophic bacteria, with genes of the Fenna-Matthews-Olson-protein (fmoA) as target for the green sulfur bacteria and of two reaction center proteins (pufLM) for the phototrophic purple bacteria, with genes of adenosine-5′phosphosulfate (APS) reductase (aprA), sulfate thioesterase (soxB) and dissimilatory sulfite reductase (dsrAB) for sulfur oxidizing and sulfate reducing bacteria, with genes of ammonia monooxygenase (amoA) for nitrifying/ammonia-oxidizing bacteria, with genes of particulate nitrate reductase and nitrite reductases (narH/G, nirS, nirK) for denitrifying bacteria and with genes of methane

  18. New Dimensions in Microbial Ecology—Functional Genes in Studies to Unravel the Biodiversity and Role of Functional Microbial Groups in the Environment

    Directory of Open Access Journals (Sweden)

    Johannes F. Imhoff

    2016-05-01

    Full Text Available During the past decades, tremendous advances have been made in the possibilities to study the diversity of microbial communities in the environment. The development of methods to study these communities on the basis of 16S rRNA gene sequences analysis was a first step into the molecular analysis of environmental communities and the study of biodiversity in natural habitats. A new dimension in this field was reached with the introduction of functional genes of ecological importance and the establishment of genetic tools to study the diversity of functional microbial groups and their responses to environmental factors. Functional gene approaches are excellent tools to study the diversity of a particular function and to demonstrate changes in the composition of prokaryote communities contributing to this function. The phylogeny of many functional genes largely correlates with that of the 16S rRNA gene, and microbial species may be identified on the basis of functional gene sequences. Functional genes are perfectly suited to link culture-based microbiological work with environmental molecular genetic studies. In this review, the development of functional gene studies in environmental microbiology is highlighted with examples of genes relevant for important ecophysiological functions. Examples are presented for bacterial photosynthesis and two types of anoxygenic phototrophic bacteria, with genes of the Fenna-Matthews-Olson-protein (fmoA as target for the green sulfur bacteria and of two reaction center proteins (pufLM for the phototrophic purple bacteria, with genes of adenosine-5′phosphosulfate (APS reductase (aprA, sulfate thioesterase (soxB and dissimilatory sulfite reductase (dsrAB for sulfur oxidizing and sulfate reducing bacteria, with genes of ammonia monooxygenase (amoA for nitrifying/ammonia-oxidizing bacteria, with genes of particulate nitrate reductase and nitrite reductases (narH/G, nirS, nirK for denitrifying bacteria and with genes

  19. Abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China.

    Science.gov (United States)

    Song, Zhao-Qi; Wang, Li; Wang, Feng-Ping; Jiang, Hong-Chen; Chen, Jin-Quan; Zhou, En-Min; Liang, Feng; Xiao, Xiang; Li, Wen-Jun

    2013-09-01

    It has been suggested that archaea carrying the accA gene, encoding the alpha subunit of the acetyl CoA carboxylase, autotrophically fix CO2 using the 3-hydroxypropionate/4-hydroxybutyrate pathway in low-temperature environments (e.g., soils, oceans). However, little new information has come to light regarding the occurrence of archaeal accA genes in high-temperature ecosystems. In this study, we investigated the abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China, using DNA- and RNA-based phylogenetic analyses and quantitative polymerase chain reaction. The results showed that archaeal accA genes were present and expressed in the investigated Yunnan hot springs with a wide range of temperatures (66-96 °C) and pH (4.3-9.0). The majority of the amplified archaeal accA gene sequences were affiliated with the ThAOA/HWCG III [thermophilic ammonia-oxidizing archaea (AOA)/hot water crenarchaeotic group III]. The archaeal accA gene abundance was very close to that of AOA amoA gene, encoding the alpha subunit of ammonia monooxygenase. These data suggest that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.

  20. Terbinafine Resistance Mediated by Salicylate 1-Monooxygenase in Aspergillus nidulans

    Science.gov (United States)

    Graminha, Marcia A. S.; Rocha, Eleusa M. F.; Prade, Rolf A.; Martinez-Rossi, Nilce M.

    2004-01-01

    Resistance to antifungal agents is a recurring and growing problem among patients with systemic fungal infections. UV-induced Aspergillus nidulans mutants resistant to terbinafine have been identified, and we report here the characterization of one such gene. A sib-selected, 6.6-kb genomic DNA fragment encodes a salicylate 1-monooxygenase (salA), and a fatty acid synthase subunit (fasC) confers terbinafine resistance upon transformation of a sensitive strain. Subfragments carrying salA but not fasC confer terbinafine resistance. salA is present as a single-copy gene on chromosome VI and encodes a protein of 473 amino acids that is homologous to salicylate 1-monooxygenase, a well-characterized naphthalene-degrading enzyme in bacteria. salA transcript accumulation analysis showed terbinafine-dependent induction in the wild type and the UV-induced mutant Terb7, as well as overexpression in a strain containing the salA subgenomic DNA fragment, probably due to the multicopy effect caused by the transformation event. Additional naphthalene degradation enzyme-coding genes are present in fungal genomes, suggesting that resistance could follow degradation of the naphthalene ring contained in terbinafine. PMID:15328121

  1. Oxidative cyclization of prodigiosin by an alkylglycerol monooxygenase-like enzyme

    DEFF Research Database (Denmark)

    de Rond, Tristan; Stow, Parker; Eigl, Ian

    2017-01-01

    Prodiginines, which are tripyrrole alkaloids displaying a wide array of bioactivities, occur as linear and cyclic congeners. Identification of an unclustered biosynthetic gene led to the discovery of the enzyme responsible for catalyzing the regiospecific C–H activation and cyclization of prodigi...... of prodigiosin to cycloprodigiosin in Pseudoalteromonas rubra. This enzyme is related to alkylglycerol monooxygenase and unrelated to RedG, the Rieske oxygenase that produces cyclized prodiginines in Streptomyces, implying convergent evolution....

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

    NARCIS (Netherlands)

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

    2016-01-01

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

  3. Structural basis of kynurenine 3-monooxygenase inhibition.

    Science.gov (United States)

    Amaral, Marta; Levy, Colin; Heyes, Derren J; Lafite, Pierre; Outeiro, Tiago F; Giorgini, Flaviano; Leys, David; Scrutton, Nigel S

    2013-04-18

    Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington's-disease-relevant phenotypes in yeast, fruitfly and mouse models, as well as in a mouse model of Alzheimer's disease. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders, as well as cancer and several peripheral inflammatory conditions. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO-UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington's, Alzheimer's and Parkinson's diseases.

  4. Apis mellifera octopamine receptor 1 (AmOA1 expression in antennal lobe networks of the honey bee (Apis mellifera and fruit fly (Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Irina T Sinakevitch

    2013-10-01

    Full Text Available Octopamine (OA underlies reinforcement during appetitive conditioning in the honey bee and fruit fly, acting via different subtypes of receptors. Recently, antibodies raised against a peptide sequence of one honey bee OA receptor, AmOA1, were used to study the distribution of these receptors in the honey bee brain (Sinakevitch et al., 2011. These antibodies also recognize an isoform of the AmOA1 ortholog in the fruit fly (OAMB, mushroom body OA receptor. Here we describe in detail the distribution of AmOA1 receptors in different types of neurons in the honey bee and fruit fly antennal lobes. We integrate this information into a detailed anatomical analysis of olfactory receptor neurons (ORNs, uni- and multi-glomerular projection neurons (uPNs, and mPNs and local interneurons in glomeruli of the antennal lobe. These neurons were revealed by dye injection into the antennal nerve, antennal lobe, medial and lateral antenno-protocerbral tracts (m-APT and l-APT, and lateral protocerebral lobe by use of labeled cell lines in the fruit fly or by staining with anti-GABA. We found that ORN receptor terminals and uPNs largely do not show immunostaining for AmOA1. About seventeen GABAergic mPNs leave the antennal lobe through the ml-APT and branch into the lateral protocerebral lobe. Many, but not all, mPNs show staining for AmOA1. AmOA1 receptors are also in glomeruli on GABAergic processes associated with local interneurons. The data suggest that in both species one important action of OA in the antennal lobe involves modulation of different types of inhibitory neurons via AmOA1 receptors. We integrated this new information into a model of circuitry within glomeruli of the antennal lobes of these species.

  5. Apis mellifera octopamine receptor 1 (AmOA1) expression in antennal lobe networks of the honey bee (Apis mellifera) and fruit fly (Drosophila melanogaster)

    Science.gov (United States)

    Sinakevitch, Irina T.; Smith, Adrian N.; Locatelli, Fernando; Huerta, Ramon; Bazhenov, Maxim; Smith, Brian H.

    2013-01-01

    Octopamine (OA) underlies reinforcement during appetitive conditioning in the honey bee and fruit fly, acting via different subtypes of receptors. Recently, antibodies raised against a peptide sequence of one honey bee OA receptor, AmOA1, were used to study the distribution of these receptors in the honey bee brain (Sinakevitch et al., 2011). These antibodies also recognize an isoform of the AmOA1 ortholog in the fruit fly (OAMB, mushroom body OA receptor). Here we describe in detail the distribution of AmOA1 receptors in different types of neurons in the honey bee and fruit fly antennal lobes. We integrate this information into a detailed anatomical analysis of olfactory receptor neurons (ORNs), uni- and multi-glomerular projection neurons (uPNs, and mPNs) and local interneurons (LNs) in glomeruli of the antennal lobe. These neurons were revealed by dye injection into the antennal nerve, antennal lobe, medial and lateral antenno-protocerbral tracts (m-APT and l-APT), and lateral protocerebral lobe (LPL) by use of labeled cell lines in the fruit fly or by staining with anti-GABA. We found that ORN receptor terminals and uPNs largely do not show immunostaining for AmOA1. About seventeen GABAergic mPNs leave the antennal lobe through the ml-APT and branch into the LPL. Many, but not all, mPNs show staining for AmOA1. AmOA1 receptors are also in glomeruli on GABAergic processes associated with LNs. The data suggest that in both species one important action of OA in the antennal lobe involves modulation of different types of inhibitory neurons via AmOA1 receptors. We integrated this new information into a model of circuitry within glomeruli of the antennal lobes of these species. PMID:24187534

  6. Loss of Kynurenine 3-Mono-oxygenase Causes Proteinuria.

    Science.gov (United States)

    Korstanje, Ron; Deutsch, Konstantin; Bolanos-Palmieri, Patricia; Hanke, Nils; Schroder, Patricia; Staggs, Lynne; Bräsen, Jan H; Roberts, Ian S D; Sheehan, Susan; Savage, Holly; Haller, Hermann; Schiffer, Mario

    2016-11-01

    Changes in metabolite levels of the kynurenine pathway have been observed in patients with CKD, suggesting involvement of this pathway in disease pathogenesis. Our recent genetic analysis in the mouse identified the kynurenine 3-mono-oxygenase (KMO) gene (Kmo) as a candidate gene associated with albuminuria. This study investigated this association in more detail. We compared KMO abundance in the glomeruli of mice and humans under normal and diabetic conditions, observing a decrease in glomerular KMO expression with diabetes. Knockdown of kmo expression in zebrafish and genetic deletion of Kmo in mice each led to a proteinuria phenotype. We observed pronounced podocyte foot process effacement on long stretches of the filtration barrier in the zebrafish knockdown model and mild podocyte foot process effacement in the mouse model, whereas all other structures within the kidney remained unremarkable. These data establish the candidacy of KMO as a causal factor for changes in the kidney leading to proteinuria and indicate a functional role for KMO and metabolites of the tryptophan pathway in podocytes. Copyright © 2016 by the American Society of Nephrology.

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

    OpenAIRE

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

    2016-01-01

    Genetic variant T329S in flavin-containing monooxygenase 3 (FMO3) impairs trimethylamine (TMA) metabolism in birds. The TMA metabolism that under complex genetic and dietary regulation, closely linked to cardiovascular disease risk. We determined whether the genetic defects in TMA metabolism may change other metabolic traits in birds, determined whether the genetic effects depend on diets, and to identify genes or gene pathways that underlie the metabolic alteration induced by genetic and die...

  8. Effect of nutrient and selective inhibitor amendments on methane oxidation, nitrous oxide production, and key gene presence and expression in landfill cover soils: characterization of the role of methanotrophs, nitrifiers, and denitrifiers.

    Science.gov (United States)

    Lee, Sung-Woo; Im, Jeongdae; Dispirito, Alan A; Bodrossy, Levente; Barcelona, Michael J; Semrau, Jeremy D

    2009-11-01

    Methane and nitrous oxide are both potent greenhouse gasses, with global warming potentials approximately 25 and 298 times that of carbon dioxide. A matrix of soil microcosms was constructed with landfill cover soils collected from the King Highway Landfill in Kalamazoo, Michigan and exposed to geochemical parameters known to affect methane consumption by methanotrophs while also examining their impact on biogenic nitrous oxide production. It was found that relatively dry soils (5% moisture content) along with 15 mg NH (4) (+) (kg soil)(-1) and 0.1 mg phenylacetylene(kg soil)(-1) provided the greatest stimulation of methane oxidation while minimizing nitrous oxide production. Microarray analyses of pmoA showed that the methanotrophic community structure was dominated by Type II organisms, but Type I genera were more evident with the addition of ammonia. When phenylacetylene was added in conjunction with ammonia, the methanotrophic community structure was more similar to that observed in the presence of no amendments. PCR analyses showed the presence of amoA from both ammonia-oxidizing bacteria and archaea, and that the presence of key genes associated with these cells was reduced with the addition of phenylacetylene. Messenger RNA analyses found transcripts of pmoA, but not of mmoX, nirK, norB, or amoA from either ammonia-oxidizing bacteria or archaea. Pure culture analyses showed that methanotrophs could produce significant amounts of nitrous oxide, particularly when expressing the particulate methane monooxygenase (pMMO). Collectively, these data suggest that methanotrophs expressing pMMO played a role in nitrous oxide production in these microcosms.

  9. Cytochrome P450 monooxygenases and insecticide resistance in insects.

    OpenAIRE

    Bergé, J B; Feyereisen, R; Amichot, M

    1998-01-01

    Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the seque...

  10. Adaptive and Behavioral Changes in Kynurenine 3-Monooxygenase Knockout Mice: Relevance to Psychotic Disorders.

    Science.gov (United States)

    Erhardt, Sophie; Pocivavsek, Ana; Repici, Mariaelena; Liu, Xi-Cong; Imbeault, Sophie; Maddison, Daniel C; Thomas, Marian A R; Smalley, Joshua L; Larsson, Markus K; Muchowski, Paul J; Giorgini, Flaviano; Schwarcz, Robert

    2017-11-15

    Kynurenine 3-monooxygenase converts kynurenine to 3-hydroxykynurenine, and its inhibition shunts the kynurenine pathway-which is implicated as dysfunctional in various psychiatric disorders-toward enhanced synthesis of kynurenic acid, an antagonist of both α7 nicotinic acetylcholine and N-methyl-D-aspartate receptors. Possibly as a result of reduced kynurenine 3-monooxygenase activity, elevated central nervous system levels of kynurenic acid have been found in patients with psychotic disorders, including schizophrenia. In the present study, we investigated adaptive-and possibly regulatory-changes in mice with a targeted deletion of Kmo (Kmo -/- ) and characterized the kynurenine 3-monooxygenase-deficient mice using six behavioral assays relevant for the study of schizophrenia. Genome-wide differential gene expression analyses in the cerebral cortex and cerebellum of these mice identified a network of schizophrenia- and psychosis-related genes, with more pronounced alterations in cerebellar tissue. Kynurenic acid levels were also increased in these brain regions in Kmo -/- mice, with significantly higher levels in the cerebellum than in the cerebrum. Kmo -/- mice exhibited impairments in contextual memory and spent less time than did controls interacting with an unfamiliar mouse in a social interaction paradigm. The mutant animals displayed increased anxiety-like behavior in the elevated plus maze and in a light/dark box. After a D-amphetamine challenge (5 mg/kg, intraperitoneal), Kmo -/- mice showed potentiated horizontal activity in the open field paradigm. Taken together, these results demonstrate that the elimination of Kmo in mice is associated with multiple gene and functional alterations that appear to duplicate aspects of the psychopathology of several neuropsychiatric disorders. Copyright © 2016. Published by Elsevier Inc.

  11. Desaturation reactions catalyzed by soluble methane monooxygenase.

    Science.gov (United States)

    Jin, Y; Lipscomb, J D

    2001-09-01

    Soluble methane monooxygenase (MMO) is shown to be capable of catalyzing desaturation reactions in addition to the usual hydroxylation and epoxidation reactions. Dehydrogenated products are generated from MMO-catalyzed oxidation of certain substrates including ethylbenzene and cyclohexadienes. In the reaction of ethylbenzene, desaturation of ethyl C-H occurred along with the conventional hydroxvlations of ethyl and phenyl C-Hs. As a result, styrene is formed together with ethylphenols and phenylethanols. Similarly, when 1,3- and 1,4-cyclohexadienes were used as substrates, benzene was detected as a product in addition to the corresponding alcohols and epoxides. In all cases, reaction conditions were found to significantly affect the distribution among the different products. This new activity of MMO is postulated to be associated with the chemical properties of the substrates rather than fundamental changes in the nature of the oxygen and C-H activation chemistries. The formation of the desaturated products is rationalized by formation of a substrate cationic intermediate, possibly via a radical precursor. The cationic species is then proposed to partition between recombination (alcohol formation) and elimination (alkene production) pathways. This novel function of MMO indicates close mechanistic kinship between the hydroxylation and desaturation reactions catalyzed by the nonheme diiron clusters.

  12. Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida.

    Science.gov (United States)

    Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

    2008-04-01

    The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20 degrees C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.

  13. A novel methanotroph in the genus Methylomonas that contains a distinct clade of soluble methane monooxygenase.

    Science.gov (United States)

    Nguyen, Ngoc-Loi; Yu, Woon-Jong; Yang, Hye-Young; Kim, Jong-Geol; Jung, Man-Young; Park, Soo-Je; Roh, Seong-Woon; Rhee, Sung-Keun

    2017-10-01

    Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2-97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-01-29

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

  15. Transcriptional control of the isoeugenol monooxygenase of Pseudomonas nitroreducens Jin1 in Escherichia coli.

    Science.gov (United States)

    Ryu, Ji-Young; Seo, Jiyoung; Ahn, Joong-Hoon; Sadowsky, Michael J; Hur, Hor-Gil

    2012-01-01

    Vanillin is one of the most valuable compounds in the flavoring and fragrance industries, and many attempts to produce natural vanillin have been made in recent years. Isoeugenol monooxygenase (Iem) converts the phenylpropanoid compound isoeugenol to vanillin. In Pseudomonas nitroreducens Jin1, the positive regulatory protein IemR is divergently expressed from Iem, and the promoter region is located between the genes. In this study, we investigated the transcriptional regulation of iem in Escherichia coli. We focused on inducers and regulatory protein IemR. Transcription of iem was found to be dependent on the amounts of isoeugenol and IemR. Isoeugenol was found to be the best inducer of iem, followed by trans-anethole, which induced iem to 58% of the transcription level observed for isoeugenol. Overproduction of IemR in E. coli significantly increased the transcription of iem, up to 96-fold, even in the absence of isoeugenol, as compared to basally expressed IemR. Results of this study indicate that the transcription of iem iss dependent on the type of inducers and on IemR. They should contribute to the development of bioengineering strategies for increased production of vanillin through high-level expression of the isoeugenol monooxygenase gene in microorganisms.

  16. Identification of a Baeyer-Villiger monooxygenase sequence motif

    NARCIS (Netherlands)

    Fraaije, MW; Kamerbeek, NM; van Berkel, WJH; Janssen, DB; Kamerbeek, Nanne M.; Berkel, Willem J.H. van

    2002-01-01

    Baeyer-Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C-C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXRXXXW(P/D). Studies with

  17. Discovery of Baeyer-Villiger monooxygenases from photosynthetic eukaryotes

    NARCIS (Netherlands)

    Beneventi, Elisa; Niero, Mattia; Motterle, Riccardo; Fraaije, Marco; Bergantino, Elisabetta

    2013-01-01

    Baeyer-Villiger monooxygenases are attractive "green" catalysts able to produce chiral esters or lactones starting from ketones. They can act as natural equivalents of peroxyacids that are the catalysts classically used in the organic synthesis reactions, consisting in the cleavage of C-C bonds with

  18. Synthesis of methyl propanoate by Baeyer-Villiger monooxygenases

    NARCIS (Netherlands)

    van Beek, Hugo L.; Winter, Remko T.; Eastham, Graham R.; Fraaije, Marco W.

    2014-01-01

    Methyl propanoate is an important precursor for polymethyl methacrylates. The use of a Baeyer-Villiger monooxygenase (BVMO) to produce this compound was investigated. Several BVMOs were identified that produce the chemically non-preferred product methyl propanoate in addition to the normal product

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

    Directory of Open Access Journals (Sweden)

    Sven Sehlmeyer

    2010-05-01

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

  20. Diversity of Archaea and detection of crenarchaeotal amoA genes in the rivers Rhine and Têt

    NARCIS (Netherlands)

    Herfort, L.; Kim, J.H.; Coolen, M.J.L.; Abbas, B.; Schouten, S.; Herndl, G.J.; Sinninghe Damste, J.S.

    2009-01-01

    Pelagic archaeal phylogenetic diversity and the potential for crenarchaeotal nitrification of Group 1.1a were determined in the rivers Rhine and Têt by 16S rRNA sequencing, catalyzed reported deposition-fluorescence in situ hybridization (CARD–FISH) and quantification of 16S rRNA and functional

  1. Regulation of cytochrome P-450 monooxygenases in the mouse

    International Nuclear Information System (INIS)

    Kelley, M.F.

    1986-01-01

    Recently, the compound 1,4-bis[2-(3,4-dichloropyridyloxy)] benzene (TCPOBOP) has been identified as a highly potent phenobabital-like agonist in mice. This finding has led to the suggestion that a receptor-mediated process may govern the induction of cytochrome P-450 monooxygenases by phenobarbital and phenobarbital-like agonists. This dissertation examines: (1) the effects of structural alterations of the TCPOBOP molecule on enzyme induction activity, (2) the induction response to phenobarbital and TCPOBOP among inbred mouse strains, (3) the spectrum of monooxygenase activities induced by phenobarbital and TCPOBOP compared to 3-methylcholanthrene, isosafrole and pregnenolone 16α-carbonitrile (PCN) and (4) the binding of [ 3 H] TCPOBOP in hepatic cytosol. Changes in the structure of the pyridyloxy or benzene rings markedly affect enzyme induction activity and provide additional indirect evidence for a receptor-mediated response. An evaluation of monooxygenase induction by TCPOBOP for 27 inbred mouse strains and by phenobarbital for 15 inbred mouse strains failed to identify a strain which was completely nonresponsive to these compounds, although several strains exhibited decreased responsiveness for select monooxygenase reactions. TCPOBOP, PCN and phenobarbital were all found to significantly increase the rate of hydroxylation of testosterone at the 2α-, 6β- and 15β- positions but only TCPOBOP and phenobarbital dramatically increased the rate of pentoxyresorufin O-dealkylation. The results demonstrates that TCPOBOP most closely resembles phenobarbital in its mode of monooxygenase induction in mice. Sucrose density gradient analysis of [ 3 H] TCPOBOP-hepatic cytosol incubations failed to identify specific, saturable binding of [ 3 H] TCPOBOP to cytosolic marcomolecular elements

  2. Pam (Peptidylglycine α-amidating monooxygenase) heterozygosity alters brain copper handling with region specificity

    Science.gov (United States)

    Gaier, Eric D; Miller, Megan B; Ralle, Martina; Aryal, Dipendra; Wetsel, William C; Mains, Richard E; Eipper, Betty A

    2013-01-01

    Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in postsynaptic vesicular fractions. Cu followed a similar pattern, with ~20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) is selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not the hippocampus in PAM+/− mice, along with GABAB receptor mRNA levels. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. PMID:24032518

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

    DEFF Research Database (Denmark)

    Holtze, Maria; Saetre, Peter; Engberg, Göran

    2012-01-01

    on the activity of kynurenine 3-monooxygenase (KMO), the enzyme converting kynurenine to 3-hydroxykynurenine. Methods: We analyzed the association between KMO gene polymorphisms and CSF concentrations of KYNA in patients with schizophrenia and healthy controls. Fifteen single nucleotide polymorphisms (SNPs) were...... selected covering KMO and were analyzed in UNPHASED. Results: We included 17 patients with schizophrenia and 33 controls in our study. We found an association between a KMO SNP (rs1053230), encoding an amino acid change of potential importance for substrate interaction, and CSF concentrations of KYNA....... Limitations: Given the limited sample size, the results are tentative until replication. Conclusion: Our results suggest that the nonsynonymous KMO SNP rs1053230 influences CSF concentrations of KYNA....

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

    Science.gov (United States)

    Holtze, Maria; Saetre, Peter; Engberg, Göran; Schwieler, Lilly; Werge, Thomas; Andreassen, Ole A; Hall, Håkan; Terenius, Lars; Agartz, Ingrid; Jönsson, Erik G; Schalling, Martin; Erhardt, Sophie

    2012-01-01

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

  5. Source of Nitrous Oxide Emissions during the Cow Manure Composting Process as Revealed by Isotopomer Analysis of and amoA Abundance in Betaproteobacterial Ammonia-Oxidizing Bacteria▿ †

    Science.gov (United States)

    Maeda, Koki; Toyoda, Sakae; Shimojima, Ryosuke; Osada, Takashi; Hanajima, Dai; Morioka, Riki; Yoshida, Naohiro

    2010-01-01

    A molecular analysis of betaproteobacterial ammonia oxidizers and a N2O isotopomer analysis were conducted to study the sources of N2O emissions during the cow manure composting process. Much NO2−-N and NO3−-N and the Nitrosomonas europaea-like amoA gene were detected at the surface, especially at the top of the composting pile, suggesting that these ammonia-oxidizing bacteria (AOB) significantly contribute to the nitrification which occurs at the surface layer of compost piles. However, the 15N site preference within the asymmetric N2O molecule (SP = δ15Nα − δ15Nβ, where 15Nα and 15Nβ represent the 15N/14N ratios at the center and end sites of the nitrogen atoms, respectively) indicated that the source of N2O emissions just after the compost was turned originated mainly from the denitrification process. Based on these results, the reduction of accumulated NO2−-N or NO3−-N after turning was identified as the main source of N2O emissions. The site preference and bulk δ15N results also indicate that the rate of N2O reduction was relatively low, and an increased value for the site preference indicates that the nitrification which occurred mainly in the surface layer of the pile partially contributed to N2O emissions between the turnings. PMID:20048060

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

    Directory of Open Access Journals (Sweden)

    Linzhu Wang

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    DEFF Research Database (Denmark)

    Vendelboe, Trine Vammen

    catalytic domains called ascorbate dependent type IImonooxygenase domains and a C-terminal dimerization domain. DBM is related to peptidylglycine a-hydroxylating monooxygenase (PHM). They are 28 % identical over approximately 300 amino scids (AA) which corresponds to the catalytic domains. This is, among...... residue 47-596 in each chain, was hereafter manually built. The structure reveals the first structural insights into the DOMON domain and the C-terminal dimerization domain and it shows two different conformations of the catalytic domains. An open conformation, that resembles the structures known from PHM...

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

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    Langel, Dorothee; Ober, Dietrich

    2011-09-01

    Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Catalytic function of the mycobacterial binuclear iron monooxygenase in acetone metabolism.

    Science.gov (United States)

    Furuya, Toshiki; Nakao, Tomomi; Kino, Kuniki

    2015-10-01

    Mycobacteria such as Mycobacterium smegmatis strain mc(2)155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E. coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and ChiB) and a ......Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and Chi...... but different product profiles depending on the substrate. In LPMO-chitinase synergy experiments, CBP21 is able to boost the activity of both ChiA and ChiB more than LmLPMO10. Product analysis of the synergy assays revealed that the chitinases were unable to efficiently hydrolyse the LPMO products...... (chitooligosaccharide aldonic acids) with a degree of polymerization below four (ChiA and SmChiC) or three (ChiB). Gene transcription and protein expression analysis showed that LmLPMO10 is neither highly transcribed, nor abundantly secreted during the growth of L. monocytogenes in a chitin-containing medium...

  12. Flavin-containing monooxygenases in plants: looking beyond detox.

    Science.gov (United States)

    Schlaich, Nikolaus L

    2007-09-01

    Flavin-containing monooxygenases (FMOs) are known in bacteria, yeast and mammals where they catalyze the transfer of one atom of molecular O(2) to low molecular weight substrates. The predominant physiological function of animal FMOs appears to be detoxification of a vast spectrum of xenobiotics but until recently very little was known about the function of FMOs in plants. In the last two to three years, genetic and biochemical characterization has shown that plant FMOs can catalyze specific steps in the biosynthesis of auxin or in the metabolism of glucosinolates, and, furthermore, have a role in pathogen defence. Thus, plant FMOs hint that further FMO functions might be identified also in non-plant organisms and could stimulate novel research in this area.

  13. Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

    Science.gov (United States)

    Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

    2014-03-01

    Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification☆

    Science.gov (United States)

    Wilson, K.; Mole, D.J.; Binnie, M.; Homer, N.Z.M.; Zheng, X.; Yard, B.A.; Iredale, J.P.; Auer, M.; Webster, S.P.

    2014-01-01

    Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington’s disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

  15. Kynurenine-3-monooxygenase: a review of structure, mechanism, and inhibitors.

    Science.gov (United States)

    Smith, Jason R; Jamie, Joanne F; Guillemin, Gilles J

    2016-02-01

    Kynurenine monooxygenase (KMO) is an enzyme of the kynurenine (Kyn) pathway (KP), which is the major catabolic route of tryptophan. Kyn represents a branch point of the KP, being converted into the neurotoxin 3-hydroxykynurenine via KMO, neuroprotectant kynurenic acid, and anthranilic acid. As a result of this branch point, KMO is an attractive drug target for several neurodegenerative and/or neuroinflammatory diseases, especially Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) diseases. Although a neurological target, administration of KMO inhibitors in the periphery has demonstrated promising pharmacological results. In light of a recent crystal structure release and reports of preclinical candidates, here we provide a concise yet comprehensive update on the current state of research into the enzymology of KMO and related drug discovery efforts, highlighting areas where further work is required. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Catalytic diversity and homotropic allostery of two Cytochrome P450 monooxygenase like proteins from Trichoderma brevicompactum.

    Science.gov (United States)

    Hussain, Razak; Kumari, Indu; Sharma, Shikha; Ahmed, Mushtaq; Khan, Tabreiz Ahmad; Akhter, Yusuf

    2017-12-01

    Trichothecenes are the secondary metabolites produced by Trichoderma spp. Some of these molecules have been reported for their ability to stimulate plant growth by suppressing plant diseases and hence enabling Trichoderma spp. to be efficiently used as biocontrol agents in modern agriculture. Many of the proteins involved in the trichothecenes biosynthetic pathway in Trichoderma spp. are encoded by the genes present in the tri cluster. Tri4 protein catalyzes three consecutive oxygenation reaction steps during biosynthesis of isotrichodiol in the trichothecenes biosynthetic pathway, while tri11 protein catalyzes the C4 hydroxylation of 12, 13-epoxytrichothec-9-ene to produce trichodermol. In the present study, we have homology modelled the three-dimensional structures of tri4 and tri11 proteins. Furthermore, molecular dynamics simulations were carried out to elucidate the mechanism of their action. Both tri4 and tri11 encode for cytochrome P450 monooxygenase like proteins. These data also revealed effector-induced allosteric changes on substrate binding at an alternative binding site and showed potential homotropic negative cooperativity. These analyses also showed that their catalytic mechanism relies on protein-ligand and protein-heme interactions controlled by hydrophobic and hydrogen-bonding interactions which orient the complex in optimal conformation within the active sites.

  17. Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology.

    Science.gov (United States)

    Parrott, Jennifer M; O'Connor, Jason C

    2015-01-01

    Mounting evidence demonstrates that kynurenine metabolism may play an important pathogenic role in the development of multiple neurological and neuropsychiatric disorders. The kynurenine pathway consists of two functionally distinct branches that generate both neuroactive and oxidatively reactive metabolites. In the brain, the rate-limiting enzyme for one of these branches, kynurenine 3-monooxygenase (KMO), is predominantly expressed in microglia and has emerged as a pivotal point of metabolic regulation. KMO substrate and expression levels are upregulated by pro-inflammatory cytokines and altered by functional genetic mutations. Increased KMO metabolism results in the formation of metabolites that activate glutamate receptors and elevate oxidative stress, while recent evidence has revealed neurodevelopmental consequences of reduced KMO activity. Together, the evidence suggests that KMO is positioned at a critical metabolic junction to influence the development or trajectory of a myriad of neurological diseases. Understanding the mechanism(s) by which alterations in KMO activity are able to impair neuronal function, and viability will enhance our knowledge of related disease pathology and provide insight into novel therapeutic opportunities. This review will discuss the influence of KMO on brain kynurenine metabolism and the current understanding of molecular mechanisms by which altered KMO activity may contribute to neurodevelopment, neurodegenerative, and neuropsychiatric diseases.

  18. Kynurenine 3-monooxygenase: an influential mediator of neuropathology

    Directory of Open Access Journals (Sweden)

    Jennifer M Parrott

    2015-08-01

    Full Text Available Mounting evidence demonstrates that kynurenine metabolism may play an important pathogenic role in the development of multiple neurological and neuropsychiatric disorders. The kynurenine pathway consists of two functionally distinct branches that generate both neuroactive and oxidatively reactive metabolites. In the brain, the rate-limiting enzyme for one of these branches, kynurenine 3-monooxygenase (KMO, is predominantly expressed in microglia and has emerged as a pivotal point of metabolic regulation. KMO substrate and expression levels are up-regulated by pro-inflammatory cytokines and altered by functional genetic mutations. Increased KMO metabolism results in the formation of metabolites that activate glutamate receptors and elevate oxidative stress, while recent evidence has revealed neurodevelopmental consequences of reduced KMO activity. Together, the evidence suggests that KMO is positioned at a critical metabolic junction to influence the development or trajectory of a myriad of neurological diseases. Understanding the mechanism(s by which alterations in KMO activity are able to impair neuronal function and viability will enhance our knowledge of related disease pathology and provide insight into novel therapeutic opportunities. This review will discuss the influence of KMO on brain kynurenine metabolism and the current understanding of molecular mechanisms by which altered KMO activity may contribute to neurodevelopment, neurodegenerative and neuropsychiatric diseases.

  19. Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration

    Science.gov (United States)

    Zwilling, Daniel; Huang, Shao-Yi; Sathyasaikumar, Korrapati V.; Notarangelo, Francesca M.; Guidetti, Paolo; Wu, Hui-Qiu; Lee, Jason; Truong, Jennifer; Andrews-Zwilling, Yaisa; Hsieh, Eric W.; Louie, Jamie Y.; Wu, Tiffany; Scearce-Levie, Kimberly; Patrick, Christina; Adame, Anthony; Giorgini, Flaviano; Moussaoui, Saliha; Laue, Grit; Rassoulpour, Arash; Flik, Gunnar; Huang, Yadong; Muchowski, Joseph M.; Masliah, Eliezer; Schwarcz, Robert; Muchowski, Paul J.

    2011-01-01

    SUMMARY Metabolites in the kynurenine pathway of tryptophan degradation are thought to play an important role in neurodegenerative disorders such as Alzheimer’s disease and Huntington’s disease. Metabolites that cause glutamate receptor-mediated excitotoxicity and free radical formation are elevated in the blood and vulnerable brain regions in these diseases, while levels of the neuroprotective metabolite kynurenic acid are often decreased. Here we describe the synthesis and characterization of JM6, a novel small-molecule pro-drug inhibitor of kynurenine 3-monooxygenase (KMO). JM6 raises kynurenic acid and reduces extracellular glutamate in the brain after chronic oral administration by inhibiting KMO in blood. In a transgenic mouse model of Alzheimer’s disease, JM6 prevented spatial memory deficits, anxiety-related behavior, and synaptic loss. JM6 also extended life span, prevented synaptic loss, and decreased microglial activation in a mouse model of Huntington’s disease. These findings support a critical link between blood cells and neurodegeneration that is mediated by KMO and the kynurenine pathway. PMID:21640374

  20. Substrate and inhibitor specificity of kynurenine monooxygenase from Cytophaga hutchinsonii.

    Science.gov (United States)

    Phillips, Robert S; Anderson, Andrew D; Gentry, Harvey G; Güner, Osman F; Bowen, J Phillip

    2017-04-15

    Kynurenine monooxygenase (KMO) is a potential drug target for treatment of neurodegenerative disorders such as Huntington's and Alzheimer's diseases. We have evaluated substituted kynurenines as substrates or inhibitors of KMO from Cytophaga hutchinsonii. Kynurenines substituted with a halogen at the 5-position are excellent substrates, with values of k cat and k cat /K m comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with K I values lower than the K m for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a K I value of 1.5μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The K I for this compound was found to be 34μM, thus validating the pharmacophore model. We are using these results and our model to design more potent inhibitors of KMO. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Cloning, characterization and expression of OsFMO(t) in rice encoding a flavin monooxygenase.

    Science.gov (United States)

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

    2013-12-01

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

  2. Successional patterns of key genes and processes involved in the microbial nitrogen cycle in a salt marsh chronosequence

    NARCIS (Netherlands)

    Salles, Joana Falcao; Cassia Pereira e Silva , de Michele; Dini-Andreote, Francisco; Dias, Armando C. F.; Guillaumaud, Nadine; Poly, Franck; van Elsas, Jan Dirk

    Here, we investigated the patterns of microbial nitrogen cycling communities along a chronosequence of soil development in a salt marsh. The focus was on the abundance and structure of genes involved in N fixation (nifH), bacterial and archaeal ammonium oxidation (amoA; AOB and AOA), and the

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

    Science.gov (United States)

    Johansen, Katja S

    2016-02-01

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

  4. Targeted Deletion of Kynurenine 3-Monooxygenase in Mice

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Dyah Widiastuti

    2015-06-01

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

  6. Potential for drug interactions mediated by polymorphic flavin-containing monooxygenase 3 in human livers.

    Science.gov (United States)

    Shimizu, Makiko; Shiraishi, Arisa; Sato, Ayumi; Nagashima, Satomi; Yamazaki, Hiroshi

    2015-02-01

    Human flavin-containing monooxygenase 3 (FMO3) in the liver catalyzes a variety of oxygenations of nitrogen- and sulfur-containing medicines and xenobiotic substances. Because of growing interest in drug interactions mediated by polymorphic FMO3, benzydamine N-oxygenation by human FMO3 was investigated as a model reaction. Among the 41 compounds tested, trimethylamine, methimazole, itopride, and tozasertib (50 μM) suppressed benzydamine N-oxygenation at a substrate concentration of 50 μM by approximately 50% after co-incubation. Suppression of N-oxygenation of benzydamine, trimethylamine, itopride, and tozasertib and S-oxygenation of methimazole and sulindac sulfide after co-incubation with the other five of these six substrates was compared using FMO3 proteins recombinantly expressed in bacterial membranes. Apparent competitive inhibition by methimazole (0-50 μM) of sulindac sulfide S-oxygenation was observed with FMO3 proteins. Sulindac sulfide S-oxygenation activity of Arg205Cys variant FMO3 protein was likely to be suppressed more by methimazole than wild-type or Val257Met variant FMO3 protein was. These results suggest that genetic polymorphism in the human FMO3 gene may lead to changes of drug interactions for N- or S-oxygenations of xenobiotics and endogenous substances and that a probe battery system of benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities is recommended to clarify the drug interactions mediated by FMO3. Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  7. Peripheral kynurenine-3-monooxygenase deficiency as a potential risk factor for metabolic syndrome in schizophrenia patients.

    Science.gov (United States)

    Oxenkrug, Gregory; van der Hart, Marieke; Roeser, Julien; Summergrad, Paul

    2017-01-01

    Increased predisposition of schizophrenia patients (SP) to development of obesity and insulin resistance suggested common signaling pathway between metabolic syndrome (MetS) and schizophrenia. Deficiency of kynurenine-3-monooxygenase (KMO), enzyme catalyzing formation of 3-hydroxykynurenine (3-HK) from kynurenine (Kyn), a tryptophan (Trp) metabolite, might contribute to development of MetS as suggested by non-expression of KMO genes in human fat tissue and elevated serum concentrations of Kyn and its metabolites, kynurenic (KYNA) and anthranilic (ANA) acids, in diabetic patients and Zucker fatty rats (ZFR). Markers of KMO deficiency: decreased 3-HK and elevated Kyn, KYNA and ANA, were observed in brains and spinal fluids of SP, and in brains and serum of experimental animals with genetically- or pharmacologically-induced KMO deficiency. However, elevated concentrations of ANA and decreased 3-HK were reported in serum of SP without concurrent increase of Kyn and KYNA. Present study aimed to re-assess serum Kyn metabolites (HPLC-MS) in a sub-group of SP with elevated KYNA. We found increased Kyn concentrations (by 30%) and Kyn:Trp ratio (by 20%) in serum of SP with elevated KYNA concentrations (by 40%). Obtained results and our previous data suggest that peripheral KMO deficiency might be manifested by, at least, two different patterns: elevated ANA with decreased 3-HK; and elevated KYNA and KYN. The latter pattern was previously described in type 2 diabetes patients and might underline increased predisposition of SP to development of MetS. Assessment of peripheral KMO deficiency might identify SP predisposed to MetS. Attenuation of the consequences of peripheral KMO deficiency might be a new target for prevention/treatment of obesity and diabetes in SP.

  8. Flavin-containing monooxygenase 3 (FMO3) role in busulphan metabolic pathway

    Science.gov (United States)

    Terelius, Ylva; Abedi-Valugerdi, Manuchehr; Naughton, Seán; Saghafian, Maryam; Moshfegh, Ali; Mattsson, Jonas; Potácová, Zuzana; Hassan, Moustapha

    2017-01-01

    Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200μmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Simu eLiu

    2016-03-01

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

  11. Exploring the biocatalytic scope of a bacterial flavin-containing monooxygenase

    NARCIS (Netherlands)

    Rioz-Martinez, Ana; Kopacz, Malgorzata; de Gonzalo, Gonzalo; Pazmino, Daniel E. Torres; Gotor, Vicente; Fraaije, Marco W.

    2011-01-01

    A bacterial flavin-containing monooxygenase (FMO), fused to phosphite dehydrogenase, has been used to explore its biocatalytic potential. The bifunctional biocatalyst could be expressed in high amounts in Escherichia coli and was able to oxidize indole and indole derivatives into a variety of indigo

  12. Exploiting the enantioselectivity of Baeyer-Villiger monooxygenases via boron oxidation

    NARCIS (Netherlands)

    Brondani, Patricia B.; Dudek, Hanna; Reis, Joel S.; Fraaije, Marco W.; Andrade, Leandro H.

    2012-01-01

    The enantioselective carbon-boron bond oxidation of several chiral boron-containing compounds by Baeyer-Villiger monooxygenases was evaluated. PAMO and M446G PAMO conveniently oxidized 1-phenylethyl boronate into the corresponding 1-(phenyl)ethanol (ee = 82-91%). Cyclopropyl boronic esters were also

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

    Science.gov (United States)

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

    2015-02-11

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

  14. A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

    Science.gov (United States)

    Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

    2012-06-01

    N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Polycyclic Ketone Monooxygenase from the Thermophilic Fungus Thermothelomyces thermophila : A Structurally Distinct Biocatalyst for Bulky Substrates

    NARCIS (Netherlands)

    Fürst, Maximilian J L J; Savino, Simone; Dudek, Hanna M; Gómez Castellanos, J Rúben; Gutiérrez de Souza, Cora; Rovida, Stefano; Fraaije, Marco W; Mattevi, Andrea

    2017-01-01

    Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these

  16. Exploring the Substrate Scope of Baeyer–Villiger Monooxygenases with Branched Lactones as Entry towards Polyesters

    NARCIS (Netherlands)

    Delgove, Marie; Fürst, Maximilian; Fraaije, Marco; Bernaerts, Katrien; de Wildeman, Stefaan

    2018-01-01

    Baeyer–Villiger monooxygenases (BVMOs) are biocatalysts that are able to convert cyclic ketones into lactones by the insertion of oxygen. The aim of this study was to explore the substrate scope of several BVMOs with (biobased) cyclic ketones as precursors for the synthesis of branched polyesters.

  17. Exploring the substrate scope of Baeyer-Villiger monooxygenases with branched lactones as entry towards polyesters

    NARCIS (Netherlands)

    Delgove, Marie; Fürst, Maximilian; Fraaije, Marco; Bernaerts, Katrien; De Wildeman, Stefaan M A

    2018-01-01

    Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts able to convert cyclic ketones to lactones by the insertion of oxygen. The aim of this study was to explore the substrate scope of several BVMOs with (biobased) cyclic ketones as precursors for the synthesis of branched polyesters.The product

  18. Coupled reactions by coupled enzymes : alcohol to lactone cascade with alcohol dehydrogenase-cyclohexanone monooxygenase fusions

    NARCIS (Netherlands)

    Aalbers, Friso S; Fraaije, Marco W

    2017-01-01

    The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP(+) to oxidize cyclohexanol to form cyclohexanone and NADPH. Both

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

    DEFF Research Database (Denmark)

    Westereng, Bjørge; Arntzen, Magnus Ø.; Wittrup Agger, Jane

    2017-01-01

    Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the num...

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

    Directory of Open Access Journals (Sweden)

    Manivasagam Sindhu

    2011-04-01

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

  1. Advances in directed monooxygenase evolution : from diversity generation and flow cytometry screening to tailor-made monooxygenases

    OpenAIRE

    Ruff, Anna Joëlle

    2012-01-01

    Directed Evolution became a powerful tool for proteins engineers to generate tailor-made biocatalyst. Directed protein evolution consist of the following three consecutive main steps, which are performed in iterative cycles; Step 1 the gene diversity generation, Step 2 the screening for improved variants and Step 3 the isolation of gene encoding for improved proteins. In this thesis, methodological advancements in the two key steps of the directed evolution, the diversity generation (SeSaM me...

  2. Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates.

    Science.gov (United States)

    Carvalho, Alexandra T P; Dourado, Daniel F A R; Skvortsov, Timofey; de Abreu, Miguel; Ferguson, Lyndsey J; Quinn, Derek J; Moody, Thomas S; Huang, Meilan

    2017-10-11

    Phenylacetone monooxygenase (PAMO) is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenase family, and therefore it is an ideal candidate for the synthesis of industrially relevant compounds. However, its limited substrate scope has largely limited its industrial applications. In the present work, we provide, for the first time, the catalytic mechanism of PAMO for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations. We provide a theoretical basis for the preference of the enzyme for the native aromatic substrate over non-native linear substrates. Our study provides fundamental atomic-level insights that can be employed in the rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

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

    Science.gov (United States)

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

    2016-11-18

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

  4. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Heterologous Expression of the ema1 Cytochrome P450 Monooxygenase

    Science.gov (United States)

    Molnár, István; Hill, D. Steven; Zirkle, Ross; Hammer, Philip E.; Gross, Frank; Buckel, Thomas G.; Jungmann, Volker; Pachlatko, Johannes Paul; Ligon, James M.

    2005-01-01

    The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1. PMID:16269733

  5. Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback.

    Science.gov (United States)

    Wilson, K; Auer, M; Binnie, M; Zheng, X; Pham, N T; Iredale, J P; Webster, S P; Mole, D J

    2016-04-14

    Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.

  6. Conversion of chlorinated propanes by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

    OpenAIRE

    Bosma, T.; Janssen, D.B.

    1998-01-01

    Chlorinated propanes are important pollutants that may show persistent behaviour in the environment. The biotransformation of 1-chloropropane, 1,2-dichloropropane, 1,3-dichloropropane and 1,2,3-trichloropropane was studied using resting cell suspensions of Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. The transformation followed first-order kinetics. The rate constants were in the order 1-chloropropane > 1,3-dichloropropane > 1,2-dichloropropane > 1,2,3-trichloropr...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-31

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

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

    International Nuclear Information System (INIS)

    Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.; Beecher, Jean; Donadio, Giuliana; Saneei, Vahid; Dcunha, Stephlina A.; McGhie, Emma J.; Sayer, Christopher; Davenport, Colin F.; Lau, Peter C.; Hasegawa, Yoshie; Iwaki, Hiroaki; Kadow, Maria; Balke, Kathleen; Bornscheuer, Uwe T.; Bourenkov, Gleb; Littlechild, Jennifer A.

    2015-01-01

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

  9. EUI1, encoding a putative cytochrome P450 monooxygenase, regulates internode elongation by modulating gibberellin responses in rice.

    Science.gov (United States)

    Luo, Anding; Qian, Qian; Yin, Hengfu; Liu, Xiaoqiang; Yin, Changxi; Lan, Ying; Tang, Jiuyou; Tang, Zuoshun; Cao, Shouyun; Wang, Xiujie; Xia, Kai; Fu, Xiangdong; Luo, Da; Chu, Chengcai

    2006-02-01

    Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice.

  10. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Cirera, Susanna

    2007-01-01

    -microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase I (HPRT I), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB) and tyrosine 3-monooxygenase/tryptophan 5......-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ...

  11. Genetic variants of the kynurenine-3-monooxygenase and postpartum depressive symptoms after cesarean section in Chinese women.

    Science.gov (United States)

    Wang, Sai-Ying; Duan, Kai-Ming; Tan, Xiao-Fang; Yin, Ji-Ye; Mao, Xiao-Yuan; Zheng, Wei; Wang, Chun-Yan; Yang, Mi; Peng, Cheng; Zhou, Hong-Hao; Liu, Zhao-Qian

    2017-06-01

    New conceptualizations of depression have emphasized the role of the kynurenine pathway (KP) in the pathogenesis of postpartum depressive symptoms (PDS). Kynurenine 3-monooxygenase (KMO) is a rate-limiting enzyme of the KP, where it catalyzes the conversion of kynurenine (KYN) to 3-hydroxykynurenine (3-HK). Previous work indicates that KMO is closely linked to the pathophysiology of depressive disorders. The purpose of this study is to investigate whether variations in the KMO gene affect PDS development after cesarean section. A total of 710 Chinese women receiving cesarean section were enrolled in this study. PDS was determined by an Edinburgh Postnatal Depression Scale (EPDS) score ≥13. Subsequently, 24 women with PDS and 48 matched women without PDS were randomly selected for investigation of perinatal serum concentrations of KYN, 3-HK and the 3-HK/KYN ratio. The 3-HK/KYN ratio indicates the activity of KMO. In addition, 6 single nucleotide polymorphisms of the KMO gene were examined. Following this genotyping, 36 puerperant women carrying the KMO rs1053230 AG genotype and 72 matched puerperant women carrying the KMO rs1053230 GG genotype were selected for comparisons of KYN, 3-HK and 3-HK/KYN ratio levels. The results show the incidence of PDS in the Chinese population to be 7.3%, with PDS characterized by increased serum 3-HK concentration and 3-HK/KYN ratio, versus matched postpartum women without PDS (PKMO rs1053230 are significantly associated with the incidence of PDS (PKMO rs1053230 AG genotype are significantly higher than those in matched postpartum women carrying the KMO rs1053230 GG genotype. The presented data highlight the contribution of alterations in the KP to the pathogenesis of postpartum depression. Heightened KMO activity, including as arising from KMO rs1053230 G/A genetic variations, are indicated as one possible mechanism driving the biological underpinnings of PDS. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Lytic Polysaccharide Monooxygenases - Studies of Fungal Secretomes and Enzyme Properties

    DEFF Research Database (Denmark)

    Nekiunaite, Laura

    degradation, were also identified upstream the LPMO genes, providing evidence for a co-regulatory mechanism of LPMOs and amylolytic hydrolases. The second part of the PhD thesis is focused on understanding the binding properties of LPMOs to starch and starch mimic substrate. It was shown that LPMOs possessing...... to different substrates at the protein level. It could help to design better enzyme cocktails that increase efficiency of biomass degradation. The secretomes of A. nidulans revealed differences in growth and secretion of enzymes, depending on the type and properties of starches. A common characteristic...... conversion as they produce a wide diversity of degrading enzymes. In the first part of this PhD thesis, the secretomes of the well-known fungus Aspergillus nidulans grown on cereal and legume starches were analyzed. Secretomics is a powerful tool to unravel secretion patterns of fungi and their response...

  13. Genotyping of flavin-containing mono-oxygenase 3 (FMO3) gene by ...

    African Journals Online (AJOL)

    63.40%) of the 306 samples were genotyped using MAMA-PCR and 42 (13.72%) of the 306 samples were genotyped by both of PCR-RFLP and MAMA-PCR and genotyping data were validated by DNA sequencing. The results show that the ...

  14. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    International Nuclear Information System (INIS)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J.; Grognux, Johann; Reymond, Jean-Louis

    2004-01-01

    Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  15. Assessing and Modulating Kynurenine Pathway Dynamics in Huntington's Disease: Focus on Kynurenine 3-Monooxygenase.

    Science.gov (United States)

    Sathyasaikumar, Korrapati V; Breda, Carlo; Schwarcz, Robert; Giorgini, Flaviano

    2018-01-01

    The link between disturbances in kynurenine pathway (KP) metabolism and Huntington's disease (HD) pathogenesis has been explored for a number of years. Several novel genetic and pharmacological tools have recently been developed to modulate key regulatory steps in the KP such as the reaction catalyzed by the enzyme kynurenine 3-monooxygenase (KMO). This insight has offered new options for exploring the mechanistic link between this metabolic pathway and HD, and provided novel opportunities for the development of candidate drug-like compounds. Here, we present an overview of the field, focusing on some novel approaches for interrogating the pathway experimentally.

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

    Directory of Open Access Journals (Sweden)

    P.F. Zabrodsky

    2010-03-01

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

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

    Science.gov (United States)

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

  18. Change in gene abundance in the nitrogen biogeochemical cycle with temperature and nitrogen addition in Antarctic soils.

    Science.gov (United States)

    Jung, Jaejoon; Yeom, Jinki; Kim, Jisun; Han, Jiwon; Lim, Hyoun Soo; Park, Hyun; Hyun, Seunghun; Park, Woojun

    2011-12-01

    The microbial community (bacterial, archaeal, and fungi) and eight genes involved in the nitrogen biogeochemical cycle (nifH, nitrogen fixation; bacterial and archaeal amoA, ammonia oxidation; narG, nitrate reduction; nirS, nirK, nitrite reduction; norB, nitric oxide reduction; and nosZ, nitrous oxide reduction) were quantitatively assessed in this study, via real-time PCR with DNA extracted from three Antarctic soils. Interestingly, AOB amoA was found to be more abundant than AOA amoA in Antarctic soils. The results of microcosm studies revealed that the fungal and archaeal communities were diminished in response to warming temperatures (10 °C) and that the archaeal community was less sensitive to nitrogen addition, which suggests that those two communities are well-adapted to colder temperatures. AOA amoA and norB genes were reduced with warming temperatures. The abundance of only the nifH and nirK genes increased with both warming and the addition of nitrogen. NirS-type denitrifying bacteria outnumbered NirK-type denitrifiers regardless of the treatment used. Interestingly, dramatic increases in both NirS and NirK-types denitrifiers were observed with nitrogen addition. NirK types increase with warming, but NirS-type denitrifiers tend to be less sensitive to warming. Our findings indicated that the Antarctic microbial nitrogen cycle could be dramatically altered by temperature and nitrogen, and that warming may be detrimental to the ammonia-oxidizing archaeal community. To the best of our knowledge, this is the first report to investigate genes associated with each process of the nitrogen biogeochemical cycle in an Antarctic terrestrial soil environment. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  19. Methylocapsa aurea sp. nov., a facultative methanotroph possessing a particulate methane monooxygenase, and emended description of the genus Methylocapsa.

    Science.gov (United States)

    Dunfield, Peter F; Belova, Svetlana E; Vorob'ev, Alexey V; Cornish, Sabrina L; Dedysh, Svetlana N

    2010-11-01

    An aerobic, methanotrophic bacterium, designated KYG(T), was isolated from a forest soil in Germany. Cells of strain KYG(T) were Gram-negative, non-motile, slightly curved rods that multiplied by binary fission and produced yellow colonies. The cells contained intracellular granules of poly-β-hydroxybutyrate at each cell pole, a particulate methane monooxygenase (pMMO) and stacks of intracytoplasmic membranes (ICMs) packed in parallel along one side of the cell envelope. Strain KYG(T) grew at pH 5.2-7.2 and 2-33 °C and could fix atmospheric nitrogen under reduced oxygen tension. The major cellular fatty acid was C(18 : 1)ω7c (81.5 %) and the DNA G+C content was 61.4 mol%. Strain KYG(T) belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the obligate methanotroph Methylocapsa acidiphila B2(T) (98.1 % 16S rRNA gene sequence similarity and 84.7 % pmoA sequence similarity). Unlike Methylocapsa acidiphila B2(T), which grows only on methane and methanol, strain KYG(T) was able to grow facultatively on acetate. Facultative acetate utilization is a characteristic of the methanotrophs of the genus Methylocella, but the genus Methylocella does not produce pMMO or ICMs. Strain KYG(T) differed from Methylocapsa acidiphila B2(T) on the basis of substrate utilization pattern, pigmentation, pH range, cell ultrastructure and efficiency of dinitrogen fixation. Therefore, we propose a novel species, Methylocapsa aurea sp. nov., to accommodate this bacterium. The type strain is KYG(T) (=DSM 22158(T) =VKM B-2544(T)).

  20. Effects of submerged macrophytes on the abundance and community composition of ammonia-oxidizing prokaryotes in a eutrophic lake.

    Science.gov (United States)

    Zhao, Da-yong; Luo, Juan; Zeng, Jin; Wang, Meng; Yan, Wen-ming; Huang, Rui; Wu, Qinglong L

    2014-01-01

    Abundances and community compositions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in unvegetated sediment and the rhizosphere sediments of three submerged macrophytes (Ceratophyllum demersum, Vallisneria spinulosa, and Potamogeton crispus) were investigated in a large, eutrophic freshwater lake, Lake Taihu. Abundances of archaeal ammonia monooxygenase alpha-subunit (amoA) gene (from 6.56 × 10(6) copies to 1.06 × 10(7) copies per gram of dry sediment) were higher than those of bacterial amoA (from 6.13 × 10(5) to 3.21 × 10(6) copies per gram of dry sediment) in all samples. Submerged macrophytes exhibited no significant effect on the abundance and diversity of archaeal amoA gene. C. demersum and V. spinulosa increased the abundance and diversity of bacterial amoA gene in their rhizosphere sediment. However, the diversity of bacterial amoA gene in the rhizosphere sediments of P. crispus was decreased. The data obtained in this study would be helpful to elucidate the roles of submerged macrophytes involved in the nitrogen cycling of eutrophic lake ecosystems.

  1. Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Babić, Katarina Huić; Schauss, Kristina; Hai, Brigitte; Sikora, Sanja; Redzepović, Sulejman; Radl, Viviane; Schloter, Michael

    2008-11-01

    Inoculation of leguminous seeds with selected rhizobial strains is practised in agriculture to ameliorate the plant yield by enhanced root nodulation and nitrogen uptake of the plant. However, effective symbiosis between legumes and rhizobia does not only depend on the capacity of nitrogen fixation but also on the entire nitrogen turnover in the rhizosphere. We investigated the influence of seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting different efficiency concerning plant growth promotion on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within the nitrogen cycle was performed in rhizosphere samples before nodule formation, at bud development and at the late flowering stage. The results clearly demonstrated that effectiveness of rhizobial inocula is related to abundance of nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved in nitrogen turnover had been affected by the inocula, e.g. higher numbers of amoA copies were observed during flowering when the more effective strain had been inoculated. However, the respective gene abundances differed overall to a greater extent between the three plant development stages than between the inoculation variants.

  2. Draft Genome Sequence of Methyloferula stellata AR4, an Obligate Methanotroph Possessing Only a Soluble Methane Monooxygenase.

    Science.gov (United States)

    Dedysh, Svetlana N; Naumoff, Daniil G; Vorobev, Alexey V; Kyrpides, Nikos; Woyke, Tanja; Shapiro, Nicole; Crombie, Andrew T; Murrell, J Colin; Kalyuzhnaya, Marina G; Smirnova, Angela V; Dunfield, Peter F

    2015-03-05

    Methyloferula stellata AR4 is an aerobic acidophilic methanotroph, which, in contrast to most known methanotrophs but similar to Methylocella spp., possesses only a soluble methane monooxygenase. However, it differs from Methylocella spp. by its inability to grow on multicarbon substrates. Here, we report the draft genome sequence of this bacterium. Copyright © 2015 Dedysh et al.

  3. Draft Genome Sequence of Methyloferula stellata AR4, an Obligate Methanotroph Possessing Only a Soluble Methane Monooxygenase

    OpenAIRE

    Dedysh, Svetlana N.; Naumoff, Daniil G.; Vorobev, Alexey V.; Kyrpides, Nikos; Woyke, Tanja; Shapiro, Nicole; Crombie, Andrew T.; Murrell, J. Colin; Kalyuzhnaya, Marina G.; Smirnova, Angela V.; Dunfield, Peter F.

    2015-01-01

    Methyloferula stellata AR4 is an aerobic acidophilic methanotroph, which, in contrast to most known methanotrophs but similar to Methylocella spp., possesses only a soluble methane monooxygenase. However, it differs from Methylocella spp. by its inability to grow on multicarbon substrates. Here, we report the draft genome sequence of this bacterium.

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

    NARCIS (Netherlands)

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

    2011-01-01

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  7. A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase.

    Science.gov (United States)

    Bauman, Andrew T; Broers, Brenda A; Kline, Chelsey D; Blackburn, Ninian J

    2011-12-20

    The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase

  8. Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

    Science.gov (United States)

    Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

    2017-02-01

    Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC 50 ) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l -1 ) and acetamiprid (4.96 to 865 mg l -1 ). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

  9. ELONGATED UPPERMOST INTERNODE Encodes a Cytochrome P450 Monooxygenase That Epoxidizes Gibberellins in a Novel Deactivation Reaction in RiceW⃞

    Science.gov (United States)

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

    2006-01-01

    The recessive tall rice (Oryza sativa) mutant elongated uppermost internode (eui) is morphologically normal until its final internode elongates drastically at the heading stage. The stage-specific developmental effect of the eui mutation has been used in the breeding of hybrid rice to improve the performance of heading in male sterile cultivars. We found that the eui mutant accumulated exceptionally large amounts of biologically active gibberellins (GAs) in the uppermost internode. Map-based cloning revealed that the Eui gene encodes a previously uncharacterized cytochrome P450 monooxygenase, CYP714D1. Using heterologous expression in yeast, we found that EUI catalyzed 16α,17-epoxidation of non-13-hydroxylated GAs. Consistent with the tall and dwarfed phenotypes of the eui mutant and Eui-overexpressing transgenic plants, respectively, 16α,17-epoxidation reduced the biological activity of GA4 in rice, demonstrating that EUI functions as a GA-deactivating enzyme. Expression of Eui appeared tightly regulated during plant development, in agreement with the stage-specific eui phenotypes. These results indicate the existence of an unrecognized pathway for GA deactivation by EUI during the growth of wild-type internodes. The identification of Eui as a GA catabolism gene provides additional evidence that the GA metabolism pathway is a useful target for increasing the agronomic value of crops. PMID:16399803

  10. Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase

    Science.gov (United States)

    Hutchinson, Jonathan P.; Rowland, Paul; Taylor, Mark R. D.; Christodoulou, Erica M.; Haslam, Carl; Hobbs, Clare I.; Holmes, Duncan S.; Homes, Paul; Liddle, John; Mole, Damian J.; Uings, Iain; Walker, Ann L.; Webster, Scott P.; Mowat, Christopher G.; Chung, Chun-Wa

    2017-06-01

    Kynurenine-3-monooxygenase (KMO) is a key FAD-dependent enzyme of tryptophan metabolism. In animal models, KMO inhibition has shown benefit in neurodegenerative diseases such as Huntington's and Alzheimer's. Most recently it has been identified as a target for acute pancreatitis multiple organ dysfunction syndrome (AP-MODS); a devastating inflammatory condition with a mortality rate in excess of 20%. Here we report and dissect the molecular mechanism of action of three classes of KMO inhibitors with differentiated binding modes and kinetics. Two novel inhibitor classes trap the catalytic flavin in a previously unobserved tilting conformation. This correlates with picomolar affinities, increased residence times and an absence of the peroxide production seen with previous substrate site inhibitors. These structural and mechanistic insights culminated in GSK065(C1) and GSK366(C2), molecules suitable for preclinical evaluation. Moreover, revising the repertoire of flavin dynamics in this enzyme class offers exciting new opportunities for inhibitor design.

  11. Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

    DEFF Research Database (Denmark)

    Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K

    2013-01-01

    Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate......-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin...... ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together...

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

    International Nuclear Information System (INIS)

    Roccatano, Danilo

    2015-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

    StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst. PMID:24157359

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

    Science.gov (United States)

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

    2016-01-01

    Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Inactivation of Toluene 2-Monooxygenase in Burkholderia cepacia G4 by Alkynes

    Science.gov (United States)

    Yeager, Chris M.; Bottomley, Peter J.; Arp, Daniel J.; Hyman, Michael R.

    1999-01-01

    High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected. Toluene-dependent O2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min−1 to 2.45 min−1. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H2O2, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne—ethylene and propylene—were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent Ks and Vmax values of 39.7 μM and 112.3 nmol min−1 mg of protein−1 for ethylene and 32.3 μM and 89.2 nmol min−1 mg of protein−1 for propylene. PMID:9925593

  16. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

    Science.gov (United States)

    Gao, Jingjing; Yao, Licheng; Xia, Tingting; Liao, Xuebin; Zhu, Deyu; Xiang, Ye

    2018-04-01

    The human kynurenine 3-monooxygenase (hKMO) is a potential therapeutic target for neurodegenerative and neurologic disorders. Inhibition of KMO by Ro 61-8048, a potent, selective, and the most widely used inhibitor of KMO, was shown effective in various models of neurodegenerative or neurologic disorders. However, the molecular basis of hKMO inhibition by Ro 61-8048 is not clearly understood. Here, we report biochemistry studies on hKMO and crystal structures of an hKMO homolog, pfKMO from Pseudomonas fluorescens, in complex with the substrate l-kynurenine and Ro 61-8048. We found that the C-terminal ∼110 aa are essential for the enzymatic activity of hKMO and the homologous C-terminal region of pfKMO folds into a distinct, all-α-helical domain, which associates with the N-terminal catalytic domain to form a unique tunnel in proximity to the substrate-binding pocket. The tunnel binds the Ro 61-8048 molecule, which fills most of the tunnel, and Ro 61-8048 is hydrogen bonded with several completely conserved residues, including an essential catalytic residue. Modification of Ro 61-8048 and biochemical studies of the modified Ro 61-8048 derivatives suggested that Ro 61-8048 inhibits the enzyme in an allosteric manner by affecting the conformation of the essential catalytic residue and by blocking entry of the substrate or product release. The unique binding sites distinguish Ro 61-8048 as a noncompetitive and highly selective inhibitor from other competitive inhibitors, which should facilitate further optimization of Ro 61-8048 and the development of new inhibitory drugs to hKMO.-Gao, J., Yao, L., Xia, T., Liao, X., Zhu, D., Xiang, Y. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

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

    Science.gov (United States)

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

    2014-07-01

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

  18. Monooxygenase, a novel beta-cypermethrin degrading enzyme from Streptomyces sp.

    Directory of Open Access Journals (Sweden)

    Shaohua Chen

    Full Text Available The widely used insecticide beta-cypermethrin has become a public concern because of its environmental contamination and toxic effects on mammals. In this study, a novel beta-cypermethrin degrading enzyme designated as CMO was purified to apparent homogeneity from a Streptomyces sp. isolate capable of utilizing beta-cypermethrin as a growth substrate. The native enzyme showed a monomeric structure with a molecular mass of 41 kDa and pI of 5.4. The enzyme exhibited the maximal activity at pH 7.5 and 30°C. It was fairly stable in the pH range from 6.5-8.5 and at temperatures below 10°C. The enzyme activity was significantly stimulated by Fe(2+, but strongly inhibited by Ag(+, Al(3+, and Cu(2+. The enzyme catalyzed the degradation of beta-cypermethrin to form five products via hydroxylation and diaryl cleavage. A novel beta-cypermethrin detoxification pathway was proposed based on analysis of these products. The purified enzyme was identified as a monooxygenase by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis (MALDI-TOF-MS and N-terminal protein sequencing. Given that all the characterized pyrethroid-degrading enzymes are the members of hydrolase family, CMO represents the first pyrethroid-degrading monooxygenase identified from environmental microorganisms. Taken together, our findings depict a novel pyrethroid degradation mechanism and indicate that the purified enzyme may be a promising candidate for detoxification of beta-cypermethrin and environmental protection.

  19. Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic.

    Science.gov (United States)

    Yoshimoto, Naoko; Onuma, Misato; Mizuno, Shinya; Sugino, Yuka; Nakabayashi, Ryo; Imai, Shinsuke; Tsuneyoshi, Tadamitsu; Sumi, Shin-ichiro; Saito, Kazuki

    2015-09-01

    S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  20. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  1. Two Arabidopsis cytochrome P450 monooxygenases, CYP714A1 and CYP714A2, function redundantly in plant development through gibberellin deactivation.

    Science.gov (United States)

    Zhang, Yingying; Zhang, Baichen; Yan, Dawei; Dong, Weixin; Yang, Weibing; Li, Qun; Zeng, Longjun; Wang, Jianjun; Wang, Linyou; Hicks, Leslie M; He, Zuhua

    2011-07-01

    The rice gene ELONGATED UPPERMOST INTERNODE1 (EUI1) encodes a P450 monooxygenase that epoxidizes gibberellins (GAs) in a deactivation reaction. The Arabidopsis genome contains a tandemly duplicated gene pair ELA1 (CYP714A1) and ELA2 (CYP714A2) that encode EUI homologs. In this work, we dissected the functions of the two proteins. ELA1 and ELA2 exhibited overlapping yet distinct gene expression patterns. We showed that while single mutants of ELA1 or ELA2 exhibited no obvious morphological phenotype, simultaneous elimination of ELA1 and ELA2 expression in ELA1-RNAi/ela2 resulted in increased biomass and enlarged organs. By contrast, transgenic plants constitutively expressing either ELA1 or ELA2 were dwarfed, similar to those overexpressing the rice EUI gene. We also discovered that overexpression of ELA1 resulted in a severe dwarf phenotype, while overexpression of ELA2 gave rise to a breeding-favored semi-dwarf phenotype in rice. Consistent with the phenotypes, we found that the ELA1-RNAi/ela2 plants increased amounts of biologically active GAs that were decreased in the internodes of transgenic rice with ELA1 and ELA2 overexpression. In contrast, the precursor GA(12) slightly accumulated in the transgenic rice, and GA(19) highly accumulated in the ELA2 overexpression rice. Taken together, our study strongly suggests that the two Arabidopsis EUI homologs subtly regulate plant growth most likely through catalyzing deactivation of bioactive GAs similar to rice EUI. The two P450s may also function in early stages of the GA biosynthetic pathway. Our results also suggest that ELA2 could be an excellent tool for molecular breeding for high yield potential in cereal crops. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

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

    NARCIS (Netherlands)

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

    2014-01-01

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

  3. Unliganded and substrate bound structures of the cellooligosaccharide active lytic polysaccharide monooxygenase LsAA9A at low pH

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner; Poulsen, Jens-Christian Navarro; Tandrup, Tobias

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) have been found to be key components in microbial (bacterial and fungal) degradation of biomass. They are copper metalloenzymes that degrade polysaccharides oxidatively and act in synergy with glycoside hydrolases. Recently crystallographic studies...

  4. Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately methanotrophic bacterium that possesses only a soluble methane monooxygenase.

    Science.gov (United States)

    Vorobev, Alexey V; Baani, Mohamed; Doronina, Nina V; Brady, Allyson L; Liesack, Werner; Dunfield, Peter F; Dedysh, Svetlana N

    2011-10-01

    Two strains of aerobic methanotrophic bacteria, AR4(T) and SOP9, were isolated from acidic (pH 3.8-4.0) Sphagnum peat bogs in Russia. Another phenotypically similar isolate, strain LAY, was obtained from an acidic (pH 4.0) forest soil in Germany. Cells of these strains were Gram-negative, non-pigmented, non-motile, thin rods that multiplied by irregular cell division and formed rosettes or amorphous cell conglomerates. Similar to Methylocella species, strains AR4(T), SOP9 and LAY possessed only a soluble form of methane monooxygenase (sMMO) and lacked intracytoplasmic membranes. Growth occurred only on methane and methanol; the latter was the preferred growth substrate. mRNA transcripts of sMMO were detectable in cells when either methane or both methane and methanol were available. Carbon was assimilated via the serine and ribulose-bisphosphate (RuBP) pathways; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strains AR4(T), SOP9 and LAY were moderately acidophilic, mesophilic organisms capable of growth between pH 3.5 and 7.2 (optimum pH 4.8-5.2) and at 4-33 °C (optimum 20-23 °C). The major cellular fatty acid was 18 : 1ω7c and the quinone was Q-10. The DNA G+C content was 55.6-57.5 mol%. The isolates belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and were most closely related to the sMMO-possessing methanotrophs of the genus Methylocella (96.4-97.0 % 16S rRNA gene sequence similarity), particulate MMO (pMMO)-possessing methanotrophs of the genus Methylocapsa (96.1-97.0 %), facultative methylotrophs of the genus Methylovirgula (96.1-96.3 %) and non-methanotrophic organotrophs of the genus Beijerinckia (96.5-97.0 %). Phenotypically, strains AR4(T), SOP9 and LAY were most similar to Methylocella species, but differed from members of this genus by cell morphology, greater tolerance of low pH, detectable activities of RuBP pathway enzymes and inability to grow on multicarbon compounds. Therefore, we propose a novel

  5. Plant-Mediated Changes in Soil N-Cycling Genes during Revegetation of Copper Mine Tailings

    Directory of Open Access Journals (Sweden)

    Yang Li

    2017-11-01

    Full Text Available Nitrogen limitation represents a major bottleneck during the revegetation of mine tailings. However, controls over key nitrogen-cycling genes in rhizospheric soils under differential vegetation management remain poorly understood. The abundance and transcriptional activity of nitrogen-cycling genes and the enzymatic activity of nitrogen transformation are mediated differentially during revegetation of mine tailings by Imperata cylindrica and Chrysopogon zizanioides plants. Results showed that the highest total organic carbon (TOC, total nitrogen (TN, and NH4+-N contents were found in the rhizosphere of I. cylindrica. The nifH gene abundances differed between I. cylindrica and C. zizanioides, and were higher in I. cylindrica which demonstrated by 3.39-fold higher mRNA transcript abundance of the nifH gene and a 2.15-fold higher nitrogen fixation rate in the rhizosphere. In addition, C. zizanioides exhibited a 4.94-fold higher transcript abundance of the archaeal amoA gene and the highest nitrification rate (1.706 ± 0.293 μg N-NO2- g−1 h−1 in the rhizosphere. In conclusion, I. cylindrica and C. zizanioides stimulated the abundances and activities of nifH gene and archaeal amoA gene, respectively. In addition, I. cylindrica appears to be capable of enhancing nitrogen fixation and exhibited accelerated nitrogen accumulation, which may be particularly useful for the rehabilitation of mine tailings.

  6. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature

    DEFF Research Database (Denmark)

    Busk, Peter Kamp; Lange, Mette; Pilgaard, Bo

    2014-01-01

    The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence....... In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important...

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty......-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used...... as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes...

  8. Integrating cell-free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase.

    Science.gov (United States)

    Kwon, Yong-Chan; Oh, In-Seok; Lee, Nahum; Lee, Kyung-Ho; Yoon, Yeo Joon; Lee, Eun Yeol; Kim, Byung-Gee; Kim, Dong-Myung

    2013-04-01

    Harnessing the isolated protein synthesis machinery, cell-free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non-protein prosthetic groups for biological activity. Herein, we report the complete cell-free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real-time measurement of enzymatic activity during its synthesis. Copyright © 2012 Wiley Periodicals, Inc.

  9. Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

    Science.gov (United States)

    Bass, C; Carvalho, R A; Oliphant, L; Puinean, A M; Field, L M; Nauen, R; Williamson, M S; Moores, G; Gorman, K

    2011-12-01

    The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

  10. CYP79 P450 monooxygenases in gymnosperms: CYP79A118 is associated with the formation of taxiphyllin in Taxus baccata.

    Science.gov (United States)

    Luck, Katrin; Jia, Qidong; Huber, Meret; Handrick, Vinzenz; Wong, Gane Ka-Shu; Nelson, David R; Chen, Feng; Gershenzon, Jonathan; Köllner, Tobias G

    2017-09-01

    Conifers contain P450 enzymes from the CYP79 family that are involved in cyanogenic glycoside biosynthesis. Cyanogenic glycosides are secondary plant compounds that are widespread in the plant kingdom. Their biosynthesis starts with the conversion of aromatic or aliphatic amino acids into their respective aldoximes, catalysed by N-hydroxylating cytochrome P450 monooxygenases (CYP) of the CYP79 family. While CYP79s are well known in angiosperms, their occurrence in gymnosperms and other plant divisions containing cyanogenic glycoside-producing plants has not been reported so far. We screened the transcriptomes of 72 conifer species to identify putative CYP79 genes in this plant division. From the seven resulting full-length genes, CYP79A118 from European yew (Taxus baccata) was chosen for further characterization. Recombinant CYP79A118 produced in yeast was able to convert L-tyrosine, L-tryptophan, and L-phenylalanine into p-hydroxyphenylacetaldoxime, indole-3-acetaldoxime, and phenylacetaldoxime, respectively. However, the kinetic parameters of the enzyme and transient expression of CYP79A118 in Nicotiana benthamiana indicate that L-tyrosine is the preferred substrate in vivo. Consistent with these findings, taxiphyllin, which is derived from L-tyrosine, was the only cyanogenic glycoside found in the different organs of T. baccata. Taxiphyllin showed highest accumulation in leaves and twigs, moderate accumulation in roots, and only trace accumulation in seeds and the aril. Quantitative real-time PCR revealed that CYP79A118 was expressed in plant organs rich in taxiphyllin. Our data show that CYP79s represent an ancient family of plant P450s that evolved prior to the separation of gymnosperms and angiosperms. CYP79A118 from T. baccata has typical CYP79 properties and its substrate specificity and spatial gene expression pattern suggest that the enzyme contributes to the formation of taxiphyllin in this plant species.

  11. Effect of PCB 126 on aryl hydrocarbon receptor 1 (AHR1) and AHR1 nuclear translocator 1 (ARNT1) mRNA expression and CYP1 monooxygenase activity in chicken (Gallus domesticus) ovarian follicles.

    Science.gov (United States)

    Wójcik, Dagmara; Antos, Piotr A; Katarzyńska, Dorota; Hrabia, Anna; Sechman, Andrzej

    2015-12-03

    The aim of the experiment was to study the in vitro effect of 3,3',4,4',5-pentachlorobiphenyl (PCB 126; a coplanar PCB congener) on aryl hydrocarbon receptor (AHR1) and AHR1 nuclear translocator (ARNT1) mRNA expression and the activity of CYP1 family monooxygenases in chicken ovarian follicles. White (1-4 mm) and yellowish (4-8 mm) prehierarchical follicles as well as fragments of the theca and granulosa layers of the 3 largest preovulatory follicles (F3-F1) were incubated in a medium supplemented with 0 (control group), 1, 10 or 100 nM PCB 126. The incubation was carried out for 6 h or 24 h for determination of mRNA expression of AHR1 and ARNT1 genes (real-time qPCR) and CYP1 monooxygenase activity (EROD and MROD fluorometric assays), respectively. It was found that chicken ovarian follicles express mRNA of AHR1 and ARNT1 genes. A modulatory effect of PCB 126 on AHR1 and ARNT1 expression depended not only on the biphenyl concentration but also on the follicular layer and the maturational state of the follicle. EROD and MROD activities appeared predominantly in the granulosa layer of the yellow preovulatory follicles. PCB 126 induced these activities in a dose-dependent manner in all ovarian follicles. The obtained results suggest that ovarian follicles, especially the granulosa layer, are involved in the detoxification process of PCBs in the laying hen. Taking this finding into consideration it can be suggested that the granulosa layer of the yellow hierarchical follicles plays a key role in the protective mechanism which reduces the amount of transferred dioxin-like compounds into the yolk of the oocyte. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3-Monooxygenase.

    Science.gov (United States)

    Kim, Hyun Tae; Na, Byeong Kwan; Chung, Jiwoung; Kim, Sulhee; Kwon, Sool Ki; Cha, Hyunju; Son, Jonghyeon; Cho, Joong Myung; Hwang, Kwang Yeon

    2018-04-19

    Kynurenine 3-monooxygenase (KMO) inhibitors have been developed for the treatment of neurodegenerative disorders. The mechanisms of flavin reduction and hydrogen peroxide production by KMO inhibitors are unknown. Herein, we report the structure of human KMO and crystal structures of Saccharomyces cerevisiae (sc) and Pseudomonas fluorescens (pf) KMO with Ro 61-8048. Proton transfer in the hydrogen bond network triggers flavin reduction in p-hydroxybenzoate hydroxylase, but the mechanism triggering flavin reduction in KMO is different. Conformational changes via π-π interactions between the loop above the flavin and substrate or non-substrate effectors lead to disorder of the C-terminal α helix in scKMO and shifts of domain III in pfKMO, stimulating flavin reduction. Interestingly, Ro 61-8048 has two different binding modes. It acts as a competitive inhibitor in scKMO and as a non-substrate effector in pfKMO. These findings provide understanding of the catalytic cycle of KMO and insight for structure-based drug design of KMO inhibitors. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  14. A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

    Science.gov (United States)

    Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

    2015-02-01

    Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

  15. Absence of kynurenine 3-monooxygenase reduces mortality of acute viral myocarditis in mice.

    Science.gov (United States)

    Kubo, Hisako; Hoshi, Masato; Mouri, Akihiro; Tashita, Chieko; Yamamoto, Yasuko; Nabeshima, Toshitaka; Saito, Kuniaki

    2017-01-01

    Infection of the encephalomyocarditis virus (EMCV) in mice is an established model for viral myocarditis. Previously, we have demonstrated that indoleamine 2,3-dioxygenase (IDO), an L-tryptophan - kynurenine pathway (KP) enzyme, affects acute viral myocarditis. However, the roles of KP metabolites in EMCV infection remain unclear. Kynurenine 3-monooxygenase (KMO) is one of the key regulatory enzymes, which metabolizes kynurenine to 3-hydroxykynurenine in the KP. Therefore, we examined the role of KMO in acute viral infection by comparing between KMO -/- mice and KMO +/+ mice. KMO deficiency resulted in suppressed mortality after EMCV infection. The number of infiltrating cells and F4/80 + cells in KMO -/- mice was suppressed compared with those in KMO +/+ mice. KMO -/- mice showed significantly increased levels of serum KP metabolites, and induction of KMO expression upon EMCV infection was involved in its effect on mortality through EMCV suppression. Furthermore, KMO -/- mice showed significantly suppression of CCL2, CCL3 and CCL4 on day 2 and CXCL1 on day 4 after infection. These results suggest that increased KP metabolites reduced chemokine production, resulting in suppressed mortality upon KMO knockdown in EMCV infection. KP metabolites may thus provide an effective strategy for treating acute viral myocarditis. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  16. Kynurenine-3-monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis.

    Science.gov (United States)

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

    2016-02-01

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

  17. Targeting kynurenine 3-monooxygenase (KMO): implications for therapy in Huntington's disease.

    Science.gov (United States)

    Thevandavakkam, Mathuravani A; Schwarcz, Robert; Muchowski, Paul J; Giorgini, Flaviano

    2010-12-01

    Huntington's disease (HD) is an adult onset neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. Recent work has shown that perturbation of kynurenine pathway (KP) metabolism is a hallmark of HD pathology, and that changes in brain levels of KP metabolites may play a causative role in this disease. The KP contains three neuroactive metabolites, the neurotoxins 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN), and the neuroprotectant kynurenic acid (KYNA). In model systems in vitro and in vivo, 3-HK and QUIN have been shown to cause neurodegeneration via a combination of excitotoxic mechanisms and oxidative stress. Recent studies with HD patient samples and in HD model systems have supported the idea that a shift away from the synthesis of KYNA and towards the formation of 3-HK and QUIN may trigger the neuropathological features observed in HD. The enzyme kynurenine 3-monooxygenase (KMO) is located at a critical branching point in the KP such that inhibition of this enzyme by either pharmacological or genetic means shifts the flux in the pathway towards the formation of KYNA. This intervention ameliorates disease-relevant phenotypes in HD models. Here we review the work implicating the KP in HD pathology and discuss the potential of KMO as a therapeutic target for this disorder. As several neurodegenerative diseases exhibit alterations in KP metabolism, this concept has broader implications for the treatment of brain diseases.

  18. Reconstitution of β-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

    International Nuclear Information System (INIS)

    Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B.

    2006-01-01

    CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards β-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 o C. In this system, β-carotene was hydroxylated to β-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low β-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated β-carotene at 3- and also 3'-positions, resulting in β-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol β-cryptoxanthin produced per nmol P450 per min

  19. Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

    Science.gov (United States)

    Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K; Ejsing, Christer S; Carvalho, Pedro

    2013-01-01

    Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI: http://dx.doi.org/10.7554/eLife.00953.001 PMID:23898401

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

    Directory of Open Access Journals (Sweden)

    Nisha Hirani

    2016-05-01

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

  1. Linking Changes in Snow Cover with Nitrogen Cycling and Microbial Abundance and Functional Gene Expression in Agricultural Soils

    Science.gov (United States)

    Goyer, C.; Brin, L.; Zebarth, B.; Burton, D.; Wertz, S.; Chantigny, M.

    2016-12-01

    In eastern Canada, climate change-related warming and increased precipitation may alter winter snow cover, with potential consequences for soil conditions, microbes, and N2O fluxes. We conducted a two-year field study with snow removal, passive snow addition, and ambient treatments in a potato-barley crop system. We measured in situ greenhouse gas (N2O and CO2) fluxes and belowground gas accumulation, and quantified abundance and expression of denitrifier (nirS, nirK, nosZ) and nitrifier (ammonium oxidizing archaeal (AOA) and bacterial (AOB) amoA) genes. Soil gas accumulated throughout winter, and surface fluxes were greatest during spring thaw. Greatest mid-winter soil N2O accumulation and spring thaw N2O fluxes were associated with snow removal in winter 1 and ambient snow in winter 2. High N2O accumulation and fluxes may have been due to increased substrate availability with increased frost intensity in removal plots in winter 1, but with greatest water content in ambient plots in winter 2. In each winter, greatest abundances of nirS, nirK gene denitrifiers and/or amoA gene of AOA were observed in the treatments with the greatest N2O accumulation and fluxes. Gene expression did not vary with treatment, but highest expression of amoA gene of AOA and AOB, and nosZ gene was measured near 0ºC, indicating activity during periods of stable snow cover and spring thaw. Results suggest that the magnitude of fluxes during spring thaw were related to soil conditions and microbial communities present during the prior winter, and not solely those during thaw. Furthermore, the effects of changing snow cover on microbes and N2O fluxes were not a straightforward effect of snow depth, but were likely mediated by temperature and moisture.

  2. Community structure and function of planktonic Crenarchaeota: changes with depth in the South China Sea.

    Science.gov (United States)

    Hu, Anyi; Jiao, Nianzhi; Zhang, Chuanlun L

    2011-10-01

    Marine Crenarchaeota represent a widespread and abundant microbial group in marine ecosystems. Here, we investigated the abundance, diversity, and distribution of planktonic Crenarchaeota in the epi-, meso-, and bathypelagic zones at three stations in the South China Sea (SCS) by analysis of crenarchaeal 16S rRNA gene, ammonia monooxygenase gene amoA involved in ammonia oxidation, and biotin carboxylase gene accA putatively involved in archaeal CO(2) fixation. Quantitative PCR analyses indicated that crenarchaeal amoA and accA gene abundances varied similarly with archaeal and crenarchaeal 16S rRNA gene abundances at all stations, except that crenarchaeal accA genes were almost absent in the epipelagic zone. Ratios of the crenarchaeal amoA gene to 16S rRNA gene abundances decreased ~2.6 times from the epi- to bathypelagic zones, whereas the ratios of crenarchaeal accA gene to marine group I crenarchaeal 16S rRNA gene or to crenarchaeal amoA gene abundances increased with depth, suggesting that the metabolism of Crenarchaeota may change from the epi- to meso- or bathypelagic zones. Denaturing gradient gel electrophoresis profiling of the 16S rRNA genes revealed depth partitioning in archaeal community structures. Clone libraries of crenarchaeal amoA and accA genes showed two clusters: the "shallow" cluster was exclusively derived from epipelagic water and the "deep" cluster was from meso- and/or bathypelagic waters, suggesting that niche partitioning may take place between the shallow and deep marine Crenarchaeota. Overall, our results show strong depth partitioning of crenarchaeal populations in the SCS and suggest a shift in their community structure and ecological function with increasing depth.

  3. Hepatic Flavin-Containing Monooxygenase 3 Enzyme Suppressed by Type 1 Allergy-Produced Nitric Oxide.

    Science.gov (United States)

    Tanino, Tadatoshi; Bando, Toru; Komada, Akira; Nojiri, Yukie; Okada, Yuna; Ueda, Yukari; Sakurai, Eiichi

    2017-11-01

    Flavin-containing monooxygenases (FMOs) are major mammalian non-cytochrome P450 oxidative enzymes. T helper 2 cell-activated allergic diseases produce excess levels of nitric oxide (NO) that modify the functions of proteins. However, it remains unclear whether allergy-induced NO affects the pharmacokinetics of drugs metabolized by FMOs. This study investigated alterations of hepatic microsomal FMO1 and FMO3 activities in type 1 allergic mice and further examined the interaction of FMO1 and FMO3 with allergy-induced NO. Imipramine (IMP; FMO1 substrate) N- oxidation activity was not altered in allergic mice with high serum NO and immunoglobulin E levels. At 7 days after primary sensitization (PS7) or secondary sensitization (SS7), benzydamine (BDZ; FMO1 and FMO3 substrate) N- oxygenation was significantly decreased to 70% of individual controls. The expression levels of FMO1 and FMO3 proteins were not significantly changed in the sensitized mice. Hepatic inducible NO synthase (iNOS) mRNA level increased 5-fold and 15-fold in PS7 and SS7 mice, respectively, and hepatic tumor necrosis factor- α levels were greatly enhanced. When a selective iNOS inhibitor was injected into allergic mice, serum NO levels and BDZ N- oxygenation activity returned to control levels. NO directly suppressed BDZ N- oxygenation, which was probably related to FMO3-dependent metabolism in comparison with IMP N- oxidation. In hepatic microsomes from PS7 and SS7 mice, the suppression of BDZ N- oxygenation was restored by ascorbate. Therefore, type 1 allergic mice had differentially suppressed FMO3-dependent BDZ N- oxygenation. The suppression of FMO3 metabolism related to reversible S- nitrosyl modifications of iNOS-derived NO. NO is expected to alter FMO3-metabolic capacity-limited drug pharmacokinetics in humans. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

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

    International Nuclear Information System (INIS)

    Henderson, Marilyn C.; Siddens, Lisbeth K.; Krueger, Sharon K.; Stevens, J. Fred; Kedzie, Karen; Fang, Wenkui K.; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael; Gil, Daniel; Williams, David E.

    2014-01-01

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

  5. How pH Modulates the Reactivity and Selectivity of a Siderophore-Associated Flavin Monooxygenase

    Science.gov (United States)

    2015-01-01

    Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using O2, NADPH, and the flavin adenine dinucleotide (FAD) cofactor. The fungal FMO SidA initiates peptidic siderophore biosynthesis via the highly selective hydroxylation of l-ornithine, while the related amino acid l-lysine is a potent effector of reaction uncoupling to generate H2O2. We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H2O2 release, and therefore undertook a study of SidA’s pH-dependent reaction kinetics. Consistent with other FMOs that stabilize a C4a-OO(H) intermediate, SidA’s reductive half reaction is pH independent. The rate constant for the formation of the reactive C4a-OO(H) intermediate from reduced SidA and O2 is likewise independent of pH. However, the rate constants for C4a-OO(H) reactions, either to eliminate H2O2 or to hydroxylate l-Orn, were strongly pH-dependent and influenced by the nature of the bound amino acid. Solvent kinetic isotope effects of 6.6 ± 0.3 and 1.9 ± 0.2 were measured for the C4a-OOH/H2O2 conversion in the presence and absence of l-Lys, respectively. A model is proposed in which l-Lys accelerates H2O2 release via an acid–base mechanism and where side-chain position determines whether H2O2 or the hydroxylation product is observed. PMID:24490904

  6. Induction of liver monooxygenases by annatto and bixin in female rats

    Directory of Open Access Journals (Sweden)

    A.C.A.X. De-Oliveira

    2003-01-01

    Full Text Available Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'. Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and ß-Apo-8'-carotenal are known to be potent inducers of CYP1A1, a property not shared by others (ß-carotene, lycopene and lutein. Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin and bixin (95% pure on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight, bixin (250 mg/kg body weight or the vehicle only (corn oil, 3.75 g/kg body weight for 5 consecutive days, or were not treated (untreated control. The activities of aniline-4-hydroxylase (A4H, ethoxycoumarin-O-deethylase (ECOD, ethoxy- (EROD, methoxy- (MROD, pentoxy- (PROD and benzyloxy- (BROD resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg induced EROD (3.8x, MROD (4.2x, BROD (3.3x and PROD (2.4x. Bixin (250 mg/kg was a weaker inducer of EROD (2.7x, MROD (2.3x and BROD (1.9x and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.

  7. Kynurenine 3-monooxygenase is implicated in antidepressants-responsive depressive-like behaviors and monoaminergic dysfunctions.

    Science.gov (United States)

    Tashiro, Tomoyuki; Murakami, Yuki; Mouri, Akihiro; Imamura, Yukio; Nabeshima, Toshitaka; Yamamoto, Yasuko; Saito, Kuniaki

    2017-01-15

    l-Tryptophan (TRP) is metabolized via serotonin and kynurenine pathways (KP). Several studies have demonstrated that abnormality of both pathways is involved in the pathogenesis of major depressive disorder (MDD). Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the KP, has been suggested to play major roles in physiological and pathological events mediated by bioactive kynurenine metabolites. In this study, we investigated the role of KMO in the emotional and cognitive functions by using KMO knockout (KO) mice. We measured contents of TRP and monoamines and their metabolites in the serum and hippocampus of KMO KO mice. Further, we investigated whether antidepressants improved the depressive-like behaviors in KMO KO mice. KMO KO mice showed depressive-like behaviors such as decreased sucrose preference and increased immobility in the forced swimming test and high anxiety by decreased time spent in the center area of open field. But, there was no difference in spontaneous alternation in Y-maze test, counts of rearing or locomotor activity. Higher contents of TRP metabolites such as kynurenine (KYN), kynurenic acid (KA), anthranilic acid (AA), and 3-hydroxykynurenine (3-HK) in the serum and hippocampus and decreased serotonin turnover and higher content of normetanephrine (NM) in the hippocampus were observed in the KMO KO mice. Although both antidepressant attenuated increase of immobility, sertraline but not imipramine improved decrease of sucrose preference in the KMO KO mice. These findings suggested that KMO KO mice show antidepressants-responsive depressive-like behaviors and monoaminergic dysfunctions via abnormality of kynurenine metabolism with good validities as MDD model. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-03-01

    Recent studies have highlighted the involvement of the kynurenine pathway in the pathology of neurodegenerative diseases, but the role of this system in neuropathic pain requires further extensive research. Therefore, the aim of our study was to examine the role of kynurenine 3-monooxygenase (Kmo), an enzyme that is important in this pathway, in a rat model of neuropathy after chronic constriction injury (CCI) to the sciatic nerve. For the first time, we demonstrated that the injury-induced increase in the Kmo mRNA levels in the spinal cord and the dorsal root ganglia (DRG) was reduced by chronic administration of the microglial inhibitor minocycline and that this effect paralleled a decrease in the intensity of neuropathy. Further, minocycline administration alleviated the lipopolysaccharide (LPS)-induced upregulation of Kmo mRNA expression in microglial cell cultures. Moreover, we demonstrated that not only indirect inhibition of Kmo using minocycline but also direct inhibition using Kmo inhibitors (Ro61-6048 and JM6) decreased neuropathic pain intensity on the third and the seventh days after CCI. Chronic Ro61-6048 administration diminished the protein levels of IBA-1, IL-6, IL-1beta and NOS2 in the spinal cord and/or the DRG. Both Kmo inhibitors potentiated the analgesic properties of morphine. In summary, our data suggest that in neuropathic pain model, inhibiting Kmo function significantly reduces pain symptoms and enhances the effectiveness of morphine. The results of our studies show that the kynurenine pathway is an important mediator of neuropathic pain pathology and indicate that Kmo represents a novel pharmacological target for the treatment of neuropathy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Kynurenine pathway metabolic balance influences microglia activity: Targeting kynurenine monooxygenase to dampen neuroinflammation.

    Science.gov (United States)

    Garrison, Allison M; Parrott, Jennifer M; Tuñon, Arnulfo; Delgado, Jennifer; Redus, Laney; O'Connor, Jason C

    2018-08-01

    Chronic stress or inflammation increases tryptophan metabolism along the kynurenine pathway (KP), and the generation of neuroactive kynurenine metabolites contributes to subsequent depressive-like behaviors. Microglia regulate KP balance by preferentially producing oxidative metabolites, including quinolinic acid. Research has focused on the interplay between cytokines and HPA axis-derived corticosteroids in regulating microglial activity and effects of KP metabolites directly on neurons; however, the potential role that KP metabolites have directly on microglial activity is unknown. Here, murine microglia were stimulated with lipopolysaccharide(LPS). After 6 h, mRNA expression of interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α and inducible nitric oxide synthase(iNOS) was dose-dependently increased along with the rate-limiting enzymes for oxidative KP metabolism, indoleamine-2,3-dioxygenase(IDO)-1 and kynurenine 3-monooxygenase(KMO). By 24 h post-LPS, kynurenine and quinolinic acid in the media was elevated. Inhibiting KMO with Ro 61-8048 during LPS challenge attenuated extracellular nitrite accumulation and expression of KMO and TNF-α in response to LPS. Similarly, primary microglia isolated from KMO -/- mice exhibited a significantly reduced pro-inflammatory response to LPS compared to WT controls. To determine whether the substrate (kynurenine) or end product (quinolinic acid) of KMO-dependent metabolism modulates the LPS response, microglia were treated with increasing concentrations of L-kynurenine or quinolinic acid in combination with LPS or saline. Interestingly, quinolinic acid did not impact the microglial LPS response. However, L-kynurenine had dose-dependent inhibitory effect on the LPS response. These data are the first to show an anti-inflammatory effect of KMO inhibition on microglia during immune challenge and suggest that KP metabolic balance may play a direct role in regulating microglia activity. Published by Elsevier Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-15

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

  11. Characterization of alternate reductant binding and electron transfer in the dopamine β-monooxygenase reaction

    International Nuclear Information System (INIS)

    Stewart, L.C.; Klinman, J.P.

    1987-01-01

    The steady-state limiting kinetic parameters V/sub max/, V/K/sub DA/, and V/K/sub O 2 /, together with deuterium isotope effects on these parameters, have been determined for the dopamine β-monooxygenase (DβM) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascrobate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of V/sub max/ and V/K/sub DA/, and the authors conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/K/sub O 2 / have been measured with [2,2- 2 H 2 ]dopamine as substrate both in the presence and in the absence of added ascorbate. This key result unambiguously rules out an entry of electrons to enzyme forms leading from the enzyme-dopamine complex to enzyme-bound product and, hence, reaction mechanisms involving a reductive activation of the putative Cu(II)-OOH prior to substrate hydroxylation

  12. N-terminus determines activity and specificity of styrene monooxygenase reductases.

    Science.gov (United States)

    Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

    2017-12-01

    Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Ammonia and nitrite oxidation in the Eastern Tropical North Pacific

    Science.gov (United States)

    Peng, Xuefeng; Fuchsman, Clara A.; Jayakumar, Amal; Oleynik, Sergey; Martens-Habbena, Willm; Devol, Allan H.; Ward, Bess B.

    2015-12-01

    Nitrification plays a key role in the marine nitrogen (N) cycle, including in oceanic oxygen minimum zones (OMZs), which are hot spots for denitrification and anaerobic ammonia oxidation (anammox). Recent evidence suggests that nitrification links the source (remineralized organic matter) and sink (denitrification and anammox) of fixed N directly in the steep oxycline in the OMZs. We performed shipboard incubations with 15N tracers to characterize the depth distribution of nitrification in the Eastern Tropical North Pacific (ETNP). Additional experiments were conducted to investigate photoinhibition. Allylthiourea (ATU) was used to distinguish the contribution of archaeal and bacterial ammonia oxidation. The abundance of archaeal and β-proteobacterial ammonia monooxygenase gene subunit A (amoA) was determined by quantitative polymerase chain reaction. The rates of ammonia and nitrite oxidation showed distinct subsurface maxima, with the latter slightly deeper than the former. The ammonia oxidation maximum coincided with the primary nitrite concentration maximum, archaeal amoA gene maximum, and the subsurface nitrous oxide maximum. Negligible rates of ammonia oxidation were found at anoxic depths, where high rates of nitrite oxidation were measured. Archaeal amoA gene abundance was generally 1 to 2 orders of magnitude higher than bacterial amoA gene abundance, and inhibition of ammonia-oxidizing bacteria with 10 μM ATU did not affect ammonia oxidation rates, indicating the dominance of archaea in ammonia oxidation. These results depict highly dynamic activities of ammonia and nitrite oxidation in the oxycline of the ETNP OMZ.

  14. Microbial nitrification in throughfall of a Japanese cedar associated with archaea from the tree canopy.

    Science.gov (United States)

    Watanabe, Keiji; Kohzu, Ayato; Suda, Wataru; Yamamura, Shigeki; Takamatsu, Takejiro; Takenaka, Akio; Koshikawa, Masami Kanao; Hayashi, Seiji; Watanabe, Mirai

    2016-01-01

    To investigate the nitrification potential of phyllospheric microbes, we incubated throughfall samples collected under the canopies of Japanese cedar (Cryptomeria japonica) and analyzed the transformation of inorganic nitrogen in the samples. Nitrate concentration increased in the unfiltered throughfall after 4 weeks of incubation, but remained nearly constant in the filtered samples (pore size: 0.2 and 0.4 µm). In the unfiltered samples, δ(18)O and δ(15)N values of nitrate decreased during incubation. In addition, archaeal ammonia monooxygenase subunit A (amoA) genes, which participate in the oxidation of ammonia, were found in the throughfall samples, although betaproteobacterial amoA genes were not detected. The amoA genes recovered from the leaf surface of C. japonica were also from archaea. Conversely, nitrate production, decreased isotope ratios of nitrate, and the presence of amoA genes was not observed in rainfall samples collected from an open area. Thus, the microbial nitrification that occurred in the incubated throughfall is likely due to ammonia-oxidizing archaea that were washed off the tree canopy by precipitation.

  15. Linkage between N2O emission and functional gene abundance in an intensively managed calcareous fluvo-aquic soil

    Science.gov (United States)

    Yang, Liuqing; Zhang, Xiaojun; Ju, Xiaotang

    2017-02-01

    The linkage between N2O emissions and the abundance of nitrifier and denitrifier genes is unclear in the intensively managed calcareous fluvo-aquic soils of the North China Plain. We investigated the abundance of bacterial amoA for nitrification and narG, nirS, nirK, and nosZ for denitrification by in situ soil sampling to determine how the abundance of these genes changes instantly during N fertilization events and is related to high N2O emission peaks. We also investigated how long-term incorporated straw and/or manure affect(s) the abundance of these genes based on a seven-year field experiment. The overall results demonstrate that the long-term application of urea-based fertilizer and/or manure significantly enhanced the number of bacterial amoA gene copies leading to high N2O emission peaks after N fertilizer applications. These peaks contributed greatly to the annual N2O emissions in the crop rotation. A significant correlation between annual N2O emissions and narG, nirS, and nirK gene numbers indicates that the abundance of these genes is related to N2O emission under conditions for denitrification, thus partly contributing to the annual N2O emissions. These findings will help to draw up appropriate measures for mitigation of N2O emissions in this ‘hotspot’ region.

  16. Identification of the intermediates of in vivo oxidation of 1 ,4-dioxane by monooxygenase-containing bacteria.

    Science.gov (United States)

    Mahendra, Shaily; Petzold, Christopher J; Baidoo, Edward E; Keasling, Jay D; Alvarez-Cohen, Lisa

    2007-11-01

    1,4-dioxane is a probable human carcinogen and an emerging water contaminant. Monooxygenase-expressing bacteria have been shown to degrade dioxane via growth-supporting as well as cometabolic mechanisms. In this study, the intermediates of dioxane degradation by monooxygenase-expressing bacteria were determined by triple quadrupole-mass spectrometry and Fourier transform ion cyclotron resonance-mass spectrometry. The major intermediates were identified as 2-hydroxyethoxyacetic acid (HEAA), ethylene glycol, glycolate, and oxalate. Studies with uniformly labeled 14C dioxane showed that over 50% of the dioxane was mineralized to CO2 by CB1190, while 5% became biomass-associated after 48 h. Volatile organic acids and non-volatiles, respectively, accounted for 20 and 11% of the radiolabeled carbon. Although strains cometabolizing dioxane exhibited limited transformation capacities, nearly half of the initial dioxane was recovered as CO2. On the basis of these analytical results, we propose a pathway for dioxane oxidation by monooxygenase-expressing cells in which dioxane is first converted to 2-hydroxy-1,4-dioxane, which is spontaneously oxidized to HEAA. During a second monooxygenation step, HEAA is further hydroxylated, resulting in a mixture of dihydroxyethoxyacetic acids with a hydroxyl group at the ortho or para position. After cleavage of the second ether bond, small organic molecules such as ethylene glycol, glycolate, glyoxalate, and oxalate are progressively formed, which are then mineralized to CO2 via common cellular metabolic pathways. Bioremediation of dioxane via this pathway is not expected to cause an accumulation of toxic compounds in the environment.

  17. Ligand complex structures of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change.

    Science.gov (United States)

    Im, Dohyun; Matsui, Daisuke; Arakawa, Takatoshi; Isobe, Kimiyasu; Asano, Yasuhisa; Fushinobu, Shinya

    2018-03-01

    l-Amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 (l-AAO/MOG) catalyzes both the oxidative deamination and oxidative decarboxylation of the α-group of l-Lys to produce a keto acid and amide, respectively. l-AAO/MOG exhibits limited specificity for l-amino acid substrates with a basic side chain. We previously determined its ligand-free crystal structure and identified a key residue for maintaining the dual activities. Here, we determined the structures of l-AAO/MOG complexed with l-Lys, l-ornithine, and l-Arg and revealed its substrate recognition. Asp238 is located at the ceiling of a long hydrophobic pocket and forms a strong interaction with the terminal, positively charged group of the substrates. A mutational analysis on the D238A mutant indicated that the interaction is critical for substrate binding but not for catalytic control between the oxidase/monooxygenase activities. The catalytic activities of the D238E mutant unexpectedly increased, while the D238F mutant exhibited altered substrate specificity to long hydrophobic substrates. In the ligand-free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. In the l-Lys complex structure, a loop region is displaced to plug the channels. Moreover, the disordered region in the ligand-free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of l-AAO/MOG through this route. The atomic coordinates and structure factors (codes 5YB6, 5YB7, and 5YB8) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1.4.3.2 (l-amino acid oxidase), 1.13.12.2 (lysine 2-monooxygenase).

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

    Science.gov (United States)

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

    2017-04-27

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

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

    Science.gov (United States)

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

    2015-02-12

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

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

    OpenAIRE

    Cankar, K.; Houwelingen, van, A.M.M.L.; Bosch, H.J.; Sonke, Th.; Bouwmeester, H.J.; Beekwilder, M.J.

    2011-01-01

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-tr...

  1. LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.

    OpenAIRE

    Manns, M P; Griffin, K J; Sullivan, K F; Johnson, E F

    1991-01-01

    LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-31

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

  3. Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD–Transfer Reaction†

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2017-09-08

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

  5. Hypoxia inducible factor-1 (HIF-1)–flavin containing monooxygenase-2 (FMO-2) signaling acts in silver nanoparticles and silver ion toxicity in the nematode, Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Eom, Hyun-Jeong; Ahn, Jeong-Min; Kim, Younghun; Choi, Jinhee

    2013-01-01

    In the present study, nanotoxicity mechanism associated with silver nanoparticles (AgNPs) exposure was investigated on the nematode, Caenorhabditis elegans focusing on the hypoxia response pathway. In order to test whether AgNPs-induced hypoxia inducible factor-1 (HIF-1) activation was due to hypoxia or to oxidative stress, depletion of dissolved oxygen (DO) in the test media and a rescue effect using an antioxidant were investigated, respectively. The results suggested that oxidative stress was involved in activation of the HIF-1 pathway. We then investigated the toxicological implications of HIF-1 activation by examining the HIF-1 mediated transcriptional response. Of the genes tested, increased expression of the flavin containing monooxygenase-2 (FMO-2) gene was found to be the most significant as induced by AgNPs exposure. We found that AgNPs exposure induced FMO-2 activation in a HIF-1 and p38 MAPK PMK-1 dependent manner, and oxidative stress was involved in it. We conducted all experiments to include comparison of AgNPs and AgNO 3 in order to evaluate whether any observed toxicity was due to dissolution or particle specific. The AgNPs and AgNO 3 did not produce any qualitative differences in terms of exerting toxicity in the pathways observed in this study, however, considering equal amount of silver mass, in every endpoint tested the AgNPs were found to be more toxic than AgNO 3 . These results suggest that Ag nanotoxicity is dependent not only on dissolution of Ag ion but also on particle specific effects and HIF-1–FMO-2 pathway seems to be involved in it. - Highlights: • HIF-1 signaling was investigated in C. elegans exposed to AgNPs and AgNO 3 . • HIF-1 and PMK-1 were needed for AgNPs- and AgNO 3 -induced fmo-2 gene expression. • PMK-1–HIF-1–FMO-2 pathway was dependent on oxidative stress. • AgNPs and AgNO 3 did not produce any qualitative differences in HIF-1 signaling. • AgNPs were more toxic than an equal amount of silver mass contained

  6. Hypoxia inducible factor-1 (HIF-1)–flavin containing monooxygenase-2 (FMO-2) signaling acts in silver nanoparticles and silver ion toxicity in the nematode, Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Hyun-Jeong; Ahn, Jeong-Min [School of Environmental Engineering and Graduate School of Energy and Environmental System Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743 (Korea, Republic of); Kim, Younghun [Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-dong, Nowon-gu, Seoul 139-701 (Korea, Republic of); Choi, Jinhee, E-mail: jinhchoi@uos.ac.kr [School of Environmental Engineering and Graduate School of Energy and Environmental System Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743 (Korea, Republic of)

    2013-07-15

    In the present study, nanotoxicity mechanism associated with silver nanoparticles (AgNPs) exposure was investigated on the nematode, Caenorhabditis elegans focusing on the hypoxia response pathway. In order to test whether AgNPs-induced hypoxia inducible factor-1 (HIF-1) activation was due to hypoxia or to oxidative stress, depletion of dissolved oxygen (DO) in the test media and a rescue effect using an antioxidant were investigated, respectively. The results suggested that oxidative stress was involved in activation of the HIF-1 pathway. We then investigated the toxicological implications of HIF-1 activation by examining the HIF-1 mediated transcriptional response. Of the genes tested, increased expression of the flavin containing monooxygenase-2 (FMO-2) gene was found to be the most significant as induced by AgNPs exposure. We found that AgNPs exposure induced FMO-2 activation in a HIF-1 and p38 MAPK PMK-1 dependent manner, and oxidative stress was involved in it. We conducted all experiments to include comparison of AgNPs and AgNO{sub 3} in order to evaluate whether any observed toxicity was due to dissolution or particle specific. The AgNPs and AgNO{sub 3} did not produce any qualitative differences in terms of exerting toxicity in the pathways observed in this study, however, considering equal amount of silver mass, in every endpoint tested the AgNPs were found to be more toxic than AgNO{sub 3}. These results suggest that Ag nanotoxicity is dependent not only on dissolution of Ag ion but also on particle specific effects and HIF-1–FMO-2 pathway seems to be involved in it. - Highlights: • HIF-1 signaling was investigated in C. elegans exposed to AgNPs and AgNO{sub 3}. • HIF-1 and PMK-1 were needed for AgNPs- and AgNO{sub 3}-induced fmo-2 gene expression. • PMK-1–HIF-1–FMO-2 pathway was dependent on oxidative stress. • AgNPs and AgNO{sub 3} did not produce any qualitative differences in HIF-1 signaling. • AgNPs were more toxic than an equal

  7. Ammonia oxidation kinetics and temperature sensitivity of a natural marine community dominated by Archaea

    Science.gov (United States)

    Horak, Rachel E A; Qin, Wei; Schauer, Andy J; Armbrust, E Virginia; Ingalls, Anitra E; Moffett, James W; Stahl, David A; Devol, Allan H

    2013-01-01

    Archaeal ammonia oxidizers (AOAs) are increasingly recognized as prominent members of natural microbial assemblages. Evidence that links the presence of AOA with in situ ammonia oxidation activity is limited, and the abiotic factors that regulate the distribution of AOA natural assemblages are not well defined. We used quantitative PCR to enumerate amoA (encodes α-subunit of ammonia monooxygenase) abundances; AOA amoA gene copies greatly outnumbered ammonia-oxidizing bacteria and amoA transcripts were derived primarily from AOA throughout the water column of Hood Canal, Puget Sound, WA, USA. We generated a Michaelis–Menten kinetics curve for ammonia oxidation by the natural community and found that the measured Km of 98±14 nmol l−1 was close to that for cultivated AOA representative Nitrosopumilus maritimus SCM1. Temperature did not have a significant effect on ammonia oxidation rates for incubation temperatures ranging from 8 to 20 °C, which is within the temperature range for depths of measurable ammonia oxidation at the site. This study provides substantial evidence, through both amoA gene copies and transcript abundances and the kinetics response, that AOA are the dominant active ammonia oxidizers in this marine environment. We propose that future ammonia oxidation experiments use a Km for the natural community to better constrain ammonia oxidation rates determined with the commonly used 15NH4+ dilution technique. PMID:23657360

  8. EXPRESSION OF BRANCHIAL FLAVIN-CONTAINING MONOOXYGENASE IS DIRECTLY CORRELATED WITH SALINITY-INDUCED ALDICARB TOXICITY IN THE EURYHALINE FISH (ORYZIAS LATIPES). (R826109)

    Science.gov (United States)

    AbstractEarlier studies in our laboratory have demonstrated a reduction of flavin-containing monooxygenase (FMO) activity when salt-water adapted euryhaline fish were transferred to water of less salinity. Since FMOs have been shown to be responsible for the bioact...

  9. Daily fluctuation of hepatic P450 monooxygenase activities in male rats is controlled by the suprachiasmatic nucleus but remains unaffected by adrenal hormones.

    Science.gov (United States)

    Furukawa, T; Manabe, S; Watanabe, T; Sehata, S; Sharyo, S; Okada, T; Mori, Y

    1999-09-01

    Hepatic P450 monooxygenase activities, which strongly influence the efficacy and/or toxicity of drugs, are known to fluctuate daily. We also know that the P450 activities assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities fluctuate daily, with apparently high values during the dark period in male rats. However, there is little knowledge about the factors that regulate daily fluctuation of P450 monooxygenase activities. In the present study using rats, we induced lesions in the suprachiasmatic nucleus (SCN) of the brain, the known site of the body's internal clock, and examined the effects on the daily fluctuation of the ACD activities to clarify the relationship between the SCN and the daily fluctuation of P450 monooxygenase activities. In addition, adrenalectomy was performed to re-evaluate the influence of adrenal hormones on the P450 activities. Our results indicated that daily fluctuations of the hepatic ACD activities were completely eliminated in the SCN-lesioned rats. However, the ACD activities in the adrenalectomized rats showed apparent daily fluctuations with high values during the dark period and low values during the light period. Therefore, this study demonstrated that the daily fluctuation of the hepatic P450 monooxygenase activities in male rats is controlled by the SCN but remains unaffected by the adrenal hormones.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and β-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular...

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

    Science.gov (United States)

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

    2017-03-01

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

  12. Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

    Science.gov (United States)

    Jacobs, K R; Guillemin, G J; Lovejoy, D B

    2018-02-01

    Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC 50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

  13. Mammalian peptidylglycine alpha-amidating monooxygenase (PAM) mRNA expression can be modulated by the La autoantigen

    DEFF Research Database (Denmark)

    Brenet, Fabienne; Dussault, Nadège; Borch, Jonas

    2005-01-01

    Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated...... region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt...... downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most...

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

    Science.gov (United States)

    Cankar, Katarina; van Houwelingen, Adèle; Bosch, Dirk; Sonke, Theo; Bouwmeester, Harro; Beekwilder, Jules

    2011-01-03

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Rebecca C. Schugar

    2017-06-01

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

  16. Abundance and diversity of bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants revealed by high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Zhu Wang

    Full Text Available Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.

  17. Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus.

    Science.gov (United States)

    Ul-Hasan, Sabah; Burgess, Daniel M; Gajewiak, Joanna; Li, Qing; Hu, Hao; Yandell, Mark; Olivera, Baldomero M; Bandyopadhyay, Pradip K

    2013-11-01

    Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase

    Directory of Open Access Journals (Sweden)

    Dirk Tischler

    2018-04-01

    Full Text Available Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1 and a two domain protein (VpStyA2B harboring an epoxidase (A2 and a FAD-reductase (B domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s−1 were determined and resulted in a catalytic efficiency (kcat Km−1 of 0.64 s−1 μM−1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV was mutated. One mutant (AAAAA showed 18.7-fold higher affinity for FAD (kcat Km−1 of 5.21 s−1 μM−1 while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg−1 and 0.46 U mg−1, as well as on benzyl methyl sulfide of 1.62 U mg−1 and 3.11 U mg−1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h and produced dominantly (S-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.

  19. Molecular cloning of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

    DEFF Research Database (Denmark)

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

    1997-01-01

    conserved regions of PHM, we have now cloned a PHM from the sea anemone Calliactis parasitica showing 42% amino acid sequence identity with rat PHM. Among the conserved (identical) amino acid residues are five histidine and one methionine residue, which bind two Cu2+ atoms that are essential for PHM...... activity. No cDNA coding for PAL could be identified, suggesting that sea anemone PAL is coded for by a gene that is different from the sea anemone PHM gene, a situation similar to the one found in insects. This is the first report on the molecular cloning of a cnidarian PHM. Udgivelsesdato: 1997-Dec-18...

  20. Cytochrome P450 genes from the aquatic midge Chironomus tentans: Atrazine-induced up-regulation of CtCYP6EX3 contributing to oxidative activation of chlorpyrifos

    Science.gov (United States)

    The open reading frames of 19 cytochrome P450 monooxygenase (CYP) genes were sequenced from Chironomus tentans, a commonly used freshwater invertebrate model. Functional analysis of CtCYP6EX3 confirmed its atrazine-induced oxidative activation for chlorpyrifos by using a nanoparticle-based RNA inter...

  1. Monoterpenoid-based preparations in beehives affect learning, memory, and gene expression in the bee brain.

    Science.gov (United States)

    Bonnafé, Elsa; Alayrangues, Julie; Hotier, Lucie; Massou, Isabelle; Renom, Allan; Souesme, Guillaume; Marty, Pierre; Allaoua, Marion; Treilhou, Michel; Armengaud, Catherine

    2017-02-01

    Bees are exposed in their environment to contaminants that can weaken the colony and contribute to bee declines. Monoterpenoid-based preparations can be introduced into hives to control the parasitic mite Varroa destructor. The long-term effects of monoterpenoids are poorly investigated. Olfactory conditioning of the proboscis extension reflex (PER) has been used to evaluate the impact of stressors on cognitive functions of the honeybee such as learning and memory. The authors tested the PER to odorants on bees after exposure to monoterpenoids in hives. Octopamine receptors, transient receptor potential-like (TRPL), and γ-aminobutyric acid channels are thought to play a critical role in the memory of food experience. Gene expression levels of Amoa1, Rdl, and trpl were evaluated in parallel in the bee brain because these genes code for the cellular targets of monoterpenoids and some pesticides and neural circuits of memory require their expression. The miticide impaired the PER to odors in the 3 wk following treatment. Short-term and long-term olfactory memories were improved months after introduction of the monoterpenoids into the beehives. Chronic exposure to the miticide had significant effects on Amoa1, Rdl, and trpl gene expressions and modified seasonal changes in the expression of these genes in the brain. The decrease of expression of these genes in winter could partly explain the improvement of memory. The present study has led to new insights into alternative treatments, especially on their effects on memory and expression of selected genes involved in this cognitive function. Environ Toxicol Chem 2017;36:337-345. © 2016 SETAC. © 2016 SETAC.

  2. Niche specificity of ammonia-oxidizing archaeal and bacterial communities in a freshwater wetland receiving municipal wastewater in Daqing, Northeast China.

    Science.gov (United States)

    Lee, Kwok-Ho; Wang, Yong-Feng; Li, Hui; Gu, Ji-Dong

    2014-12-01

    Ecophysiological differences between ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) enable them to adapt to different niches in complex freshwater wetland ecosystems. The community characters of AOA and AOB in the different niches in a freshwater wetland receiving municipal wastewater, as well as the physicochemical parameters of sediment/soil samples, were investigated in this study. AOA community structures varied and separated from each other among four different niches. Wetland vegetation including aquatic macrophytes and terrestrial plants affected the AOA community composition but less for AOB, whereas sediment depths might contribute to the AOB community shift. The diversity of AOA communities was higher than that of AOB across all four niches. Archaeal and bacterial amoA genes (encoding for the alpha-subunit of ammonia monooxygenases) were most diverse in the dry-land niche, indicating O2 availability might favor ammonia oxidation. The majority of AOA amoA sequences belonged to the Soil/sediment Cluster B in the freshwater wetland ecosystems, while the dominant AOB amoA sequences were affiliated with Nitrosospira-like cluster. In the Nitrosospira-like cluster, AOB amoA gene sequences affiliated with the uncultured ammonia-oxidizing beta-proteobacteria constituted the largest portion (99%). Moreover, independent methods for phylogenetic tree analysis supported high parsimony bootstrap values. As a consequence, it is proposed that Nitrosospira-like amoA gene sequences recovered in this study represent a potentially novel cluster, grouping with the sequences from Gulf of Mexico deposited in the public databases.

  3. Niche partitioning of marine group I Crenarchaeota in the euphotic and upper mesopelagic zones of the East China Sea.

    Science.gov (United States)

    Hu, Anyi; Jiao, Nianzhi; Zhang, Rui; Yang, Zao

    2011-11-01

    Marine group I Crenarchaeota (MGI) represents a ubiquitous and numerically predominant microbial population in marine environments. An understanding of the spatial dynamics of MGI and its controlling mechanisms is essential for an understanding of the role of MGI in energy and element cycling in the ocean. In the present study, we investigated the diversity and abundance of MGI in the East China Sea (ECS) by analysis of crenarchaeal 16S rRNA gene, the ammonia monooxygenase gene amoA, and the biotin carboxylase gene accA. Quantitative PCR analyses revealed that these genes were higher in abundance in the mesopelagic than in the euphotic zone. In addition, the crenarchaeal amoA gene was positively correlated with the copy number of the MGI 16S rRNA gene, suggesting that most of the MGI in the ECS are nitrifiers. Furthermore, the ratios of crenarchaeal accA to amoA or to MGI 16S rRNA genes increased from the euphotic to the mesopelagic zone, suggesting that the role of MGI in carbon cycling may change from the epipelagic to the mesopelagic zones. Denaturing gradient gel electrophoretic profiling of the 16S rRNA genes revealed depth partitioning in MGI community structures. Clone libraries of the crenarchaeal amoA and accA genes showed both "shallow" and "deep" groups, and their relative abundances varied in the water column. Ecotype simulation analysis revealed that MGI in the upper ocean could diverge into special ecotypes associated with depth to adapt to the light gradient across the water column. Overall, our results showed niche partitioning of the MGI population and suggested a shift in their ecological functions between the euphotic and mesopelagic zones of the ECS.

  4. [Dynamic changes in functional genes for nitrogen bioremediation of petroleum-contaminated soil cycle during].

    Science.gov (United States)

    Wu, Bin-Bin; Lu, Dian-Nan; Liu, Zheng

    2012-06-01

    Microorganisms in nitrogen cycle serve as an important part of the ecological function of soil. The aim of this research was to monitor the abundance of nitrogen-fixing, denitrifying and nitrifying bacteria during bioaugmentation of petroleum-contaminated soil using real-time polymerase chain reaction (real-time PCR) of nifH, narG and amoA genes which encode the key enzymes in nitrogen fixation, nitrification and ammoniation respectively. Three different kinds of soils, which are petroleum-contaminated soil, normal soil, and remediated soil, were monitored. It was shown that the amounts of functional microorganisms in petroleum-contaminated soil were far less than those in normal soil, while the amounts in remediated soil and normal soil were comparable. Results of this experiment demonstrate that nitrogen circular functional bacteria are inhibited in petroleum-contaminated soil and can be recovered through bioremediation. Furthermore, copies of the three functional genes as well as total petroleum hydrocarbons (TPH) for soils with six different treatments were monitored. Among all treatments, the one, into which both E. cloacae as an inoculant and wheat straw as an additive were added, obtained the maximum copies of 2.68 x 10(6), 1.71 x 10(6) and 8.54 x 10(4) per gram dry soil for nifH, narG and amoA genes respectively, companying with the highest degradation rate (48% in 40 days) of TPH. The recovery of functional genes and removal of TPH were better in soil inoculated with E cloacae and C echinulata collectively than soil inoculated with E cloacae only. All above results suggest that the nitrogen circular functional genes could be applied to monitor and assess the bioremediation of petroleum-contaminated soil.

  5. Sparteine monooxygenase in brain and liver: Identified by the dopamine uptake blocker [3H]GBR-12935

    International Nuclear Information System (INIS)

    Kalow, W.; Tyndale, R.F.; Niznik, H.B.; Inaba, T.

    1990-01-01

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

  6. Monooxygenase system in Guerin’s carcinoma of rats under conditions of ω-3 polyunsaturated fatty acids administration

    Directory of Open Access Journals (Sweden)

    M. M. Marchenko

    2016-08-01

    Full Text Available The aim of the study was to determine the variations of function in components of monooxygenase system (MOS of rat Guerin’s carcinoma under ω-3 polyunsaturated fatty acids (PUFAs administration. The activity of Guerin’s carcinoma microsomal NADH-cytochrome b5 reductase, the content and the rate of cytochrome b5 oxidation-reduction, the content and the rate of cytochrome Р450 oxidation-reduction have been investigated in rats with tumor under conditions of ω-3 PUFAs administration. ω-3 PUFAs supplementation before and after transplantation of Guerin’s carcinoma resulted in the increase of NADH-cytochrome b5 reductase activity and decrease of cytochrome b5 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of carcinogenesis as compared to the tumor-bearing rats. Increased activity of NADH-cytochrome b5 reductase facilitates higher electron flow in redox-chain of MOS. Under decreased cytochrome b5 levels the electrons are transferred to oxygen, which leads to heightened generation of superoxide (O2•- in comparison to control. It was shown, that the decrease of cytochrome P450 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of oncogenesis under ω-3 PUFAs administration may be associated with its transition into an inactive form – cytochrome P420. This decrease in cytochrome P450 coincides with increased generation of superoxide by MOS oxygenase chain.

  7. Importance of kynurenine 3-monooxygenase for spontaneous firing and pharmacological responses of midbrain dopamine neurons: Relevance for schizophrenia.

    Science.gov (United States)

    Tufvesson-Alm, Maximilian; Schwieler, Lilly; Schwarcz, Robert; Goiny, Michel; Erhardt, Sophie; Engberg, Göran

    2018-06-05

    Kynurenine 3-monooxygenase (KMO) is an essential enzyme of the kynurenine pathway, converting kynurenine into 3-hydroxykynurenine. Inhibition of KMO increases kynurenine, resulting in elevated levels of kynurenic acid (KYNA), an endogenous N-methyl-d-aspartate and α*7-nicotinic receptor antagonist. The concentration of KYNA is elevated in the brain of patients with schizophrenia, possibly as a result of a reduced KMO activity. In the present study, using in vivo single cell recording techniques, we investigated the electrophysiological characteristics of ventral tegmental area dopamine (VTA DA) neurons and their response to antipsychotic drugs in a KMO knock-out (K/O) mouse model. KMO K/O mice exhibited a marked increase in spontaneous VTA DA neuron activity as compared to wild-type (WT) mice. Furthermore, VTA DA neurons showed clear-cut, yet qualitatively opposite, responses to the antipsychotic drugs haloperidol and clozapine in the two genotypes. The anti-inflammatory drug parecoxib successfully lowered the firing activity of VTA DA neurons in KMO K/O, but not in WT mice. Minocycline, an antibiotic and anti-inflammatory drug, produced no effect in this regard. Taken together, the present data further support the usefulness of KMO K/O mice for studying distinct aspects of the pathophysiology and pharmacological treatment of psychiatric disorders such as schizophrenia. Copyright © 2018. Published by Elsevier Ltd.

  8. Kynurenine 3-monooxygenase mediates inhibition of Th17 differentiation via catabolism of endogenous aryl hydrocarbon receptor ligands.

    Science.gov (United States)

    Stephens, Geoffrey L; Wang, Qun; Swerdlow, Bonnie; Bhat, Geetha; Kolbeck, Roland; Fung, Michael

    2013-07-01

    The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Targeted deletion of kynurenine 3-monooxygenase in mice: a new tool for studying kynurenine pathway metabolism in periphery and brain.

    Science.gov (United States)

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

    2013-12-20

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

  10. Prognostic significance of kynurenine 3-monooxygenase and effects on proliferation, migration, and invasion of human hepatocellular carcinoma.

    Science.gov (United States)

    Jin, Haojie; Zhang, Yurong; You, Haiyan; Tao, Xuemei; Wang, Cun; Jin, Guangzhi; Wang, Ning; Ruan, Haoyu; Gu, Dishui; Huo, Xisong; Cong, Wenming; Qin, Wenxin

    2015-06-23

    Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the kynurenine pathway of tryptophan degradation and plays a critical role in Huntington's and Alzheimer's diseases. This study aimed to examine the expression of KMO in human hepatocellular carcinoma (HCC) and investigate the relationship between its expression and prognosis of HCC patients. We first analyzed KMO expression in 120 paired HCC samples (HCC tissues vs matched adjacent non-cancerous liver tissues), and 205 clinical HCC specimens using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis showed that KMO expression was significantly higher in HCC tissues than that in normal liver tissues (all p KMO was an independent prognostic factor for overall survival (OS) and time to recurrence (TTR) (both pKMO positively regulated proliferation, migration, and invasion of HCC cells. These results suggest that KMO exhibits tumor-promoting effects towards HCC and it may serve as a novel prognostic marker in HCC.

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

    Directory of Open Access Journals (Sweden)

    Alexandre Ismail

    2016-01-01

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

  12. Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization.

    Science.gov (United States)

    Griffiths, Scott; Mesarich, Carl H; Saccomanno, Benedetta; Vaisberg, Abraham; De Wit, Pierre J G M; Cox, Russell; Collemare, Jérôme

    2016-06-21

    Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families.

  13. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

    Directory of Open Access Journals (Sweden)

    Peter K Busk

    Full Text Available The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.

  14. Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris

    Directory of Open Access Journals (Sweden)

    Harindra E. Amarasinghe

    2015-07-01

    Full Text Available Methylation has previously been associated with allele specific expression in ants. Recently, we found methylation is important in worker reproduction in the bumblebee Bombus terrestris. Here we searched for allele specific expression in twelve genes associated with worker reproduction in bees. We found allele specific expression in Ecdysone 20 monooxygenase and IMP-L2-like. Although we were unable to confirm a genetic or epigenetic cause for this allele specific expression, the expression patterns of the two genes match those predicted for imprinted genes.

  15. Insight into effects of electro-dewatering pretreatment on nitrous oxide emission involved in related functional genes in sewage sludge composting.

    Science.gov (United States)

    Wang, Ke; Wu, Yiqi; Wang, Zhe; Wang, Wei; Ren, Nanqi

    2018-05-26

    Electro-dewatering (ED) pretreatment could improve sludge dewatering performance and remove heavy metal, but the effect of ED pretreatment on nitrous oxide (N 2 O) emission and related functional genes in sludge composting process is still unknown, which was firstly investigated in this study. The results revealed that ED pretreatment changed the physicochemical characteristics of sludge and impacted N 2 O related functional genes, resulting in the reduction of cumulative N 2 O emission by 77.04% during 60 days composting. The higher pH and NH 4 + -N, but lower moisture, ORP and NO 2 - -N emerged in the composting of ED sludge compared to mechanical dewatering (MD) sludge. Furthermore, ED pretreatment reduced amoA, hao, narG, nirK and nosZ in ED sludge on Day-10 and Day-60 of composting. It was found that nirK reduction was the major factor impacting N 2 O generation in the initial composting of ED sludge, and the decline of amoA restricted N 2 O production in the curing period. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. The kynurenine pathway is activated in human obesity and shifted toward kynurenine monooxygenase activation.

    Science.gov (United States)

    Favennec, Marie; Hennart, Benjamin; Caiazzo, Robert; Leloire, Audrey; Yengo, Loïc; Verbanck, Marie; Arredouani, Abdelilah; Marre, Michel; Pigeyre, Marie; Bessede, Alban; Guillemin, Gilles J; Chinetti, Giulia; Staels, Bart; Pattou, François; Balkau, Beverley; Allorge, Delphine; Froguel, Philippe; Poulain-Godefroy, Odile

    2015-10-01

    This study characterized the kynurenine pathway (KP) in human obesity by evaluating circulating levels of kynurenines and the expression of KP enzymes in adipose tissue. Tryptophan and KP metabolite levels were measured in serum of individuals from the D.E.S.I.R. cohort (case-cohort study: 212 diabetic, 836 randomly sampled) and in women with obesity, diabetic or normoglycemic, from the ABOS cohort (n = 100). KP enzyme gene expressions were analyzed in omental and subcutaneous adipose tissue of women from the ABOS cohort, in human primary adipocytes and in monocyte-derived macrophages. In the D.E.S.I.R. cohort, kynurenine levels were positively associated with body mass index (BMI) (P = 4.68 × 10(-19) ) and with a higher HOMA2-IR insulin resistance index (P = 6.23 × 10(-4) ). The levels of kynurenine, kynurenic acid, and quinolinic acid were associated with higher BMI (P KMO], and kynurenine aminotransferase III [CCBL2]) was increased in the omental adipose tissue of women with obesity compared to lean (P KMO that is not expressed in these cells. The expressions of IDO1, KYNU, KMO, and CCBL2 were higher in proinflammatory than in anti-inflammatory macrophages (P KMO activation. © 2015 The Obesity Society.

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

    Science.gov (United States)

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

    2016-03-21

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

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

    Science.gov (United States)

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

    2014-04-01

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

  19. An Investigation into the Prediction of in Vivo Clearance for a Range of Flavin-containing Monooxygenase Substrates.

    Science.gov (United States)

    Jones, Barry C; Srivastava, Abhishek; Colclough, Nicola; Wilson, Joanne; Reddy, Venkatesh Pilla; Amberntsson, Sara; Li, Danxi

    2017-10-01

    Flavin-containing monooxygenases (FMO) are metabolic enzymes mediating the oxygenation of nucleophilic atoms such as nitrogen, sulfur, phosphorus, and selenium. These enzymes share similar properties to the cytochrome P450 system but can be differentiated through heat inactivation and selective substrate inhibition by methimazole. This study investigated 10 compounds with varying degrees of FMO involvement to determine the nature of the correlation between human in vitro and in vivo unbound intrinsic clearance. To confirm and quantify the extent of FMO involvement six of the compounds were investigated in human liver microsomal (HLM) in vitro assays using heat inactivation and methimazole substrate inhibition. Under these conditions FMO contribution varied from 21% (imipramine) to 96% (itopride). Human hepatocyte and HLM intrinsic clearance (CL int ) data were scaled using standard methods to determine the predicted unbound intrinsic clearance (predicted CL int u ) for each compound. This was compared with observed unbound intrinsic clearance (observed CL int u ) values back calculated from human pharmacokinetic studies. A good correlation was observed between the predicted and observed CL int u using hepatocytes ( R 2 = 0.69), with 8 of the 10 compounds investigated within or close to a factor of 2. For HLM the in vitro-in vivo correlation was maintained ( R 2 = 0.84) but the accuracy was reduced with only 3 out of 10 compounds falling within, or close to, twofold. This study demonstrates that human hepatocytes and HLM can be used with standard scaling approaches to predict the human in vivo clearance for FMO substrates. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  20. CD and MCD studies of the effects of component B variant binding on the biferrous active site of methane monooxygenase.

    Science.gov (United States)

    Mitić, Natasa; Schwartz, Jennifer K; Brazeau, Brian J; Lipscomb, John D; Solomon, Edward I

    2008-08-12

    The multicomponent soluble form of methane monooxygenase (sMMO) catalyzes the oxidation of methane through the activation of O 2 at a nonheme biferrous center in the hydroxylase component, MMOH. Reactivity is limited without binding of the sMMO effector protein, MMOB. Past studies show that mutations of specific MMOB surface residues cause large changes in the rates of individual steps in the MMOH reaction cycle. To define the structural and mechanistic bases for these observations, CD, MCD, and VTVH MCD spectroscopies coupled with ligand-field (LF) calculations are used to elucidate changes occurring near and at the MMOH biferrous cluster upon binding of MMOB and the MMOB variants. Perturbations to both the CD and MCD are observed upon binding wild-type MMOB and the MMOB variant that similarly increases O 2 reactivity. MMOB variants that do not greatly increase O 2 reactivity fail to cause one or both of these changes. LF calculations indicate that reorientation of the terminal glutamate on Fe2 reproduces the spectral perturbations in MCD. Although this structural change allows O 2 to bridge the diiron site and shifts the redox active orbitals for good overlap, it is not sufficient for enhanced O 2 reactivity of the enzyme. Binding of the T111Y-MMOB variant to MMOH induces the MCD, but not CD changes, and causes only a small increase in reactivity. Thus, both the geometric rearrangement at Fe2 (observed in MCD) coupled with a more global conformational change that may control O 2 access (probed by CD), induced by MMOB binding, are critical factors in the reactivity of sMMO.

  1. Biosynthesis of actinorhodin and related antibiotics: discovery of alternative routes for quinone formation encoded in the act gene cluster.

    Science.gov (United States)

    Okamoto, Susumu; Taguchi, Takaaki; Ochi, Kozo; Ichinose, Koji

    2009-02-27

    All known benzoisochromanequinone (BIQ) biosynthetic gene clusters carry a set of genes encoding a two-component monooxygenase homologous to the ActVA-ORF5/ActVB system for actinorhodin biosynthesis in Streptomyces coelicolor A3(2). Here, we conducted molecular genetic and biochemical studies of this enzyme system. Inactivation of actVA-ORF5 yielded a shunt product, actinoperylone (ACPL), apparently derived from 6-deoxy-dihydrokalafungin. Similarly, deletion of actVB resulted in accumulation of ACPL, indicating a critical role for the monooxygenase system in C-6 oxygenation, a biosynthetic step common to all BIQ biosyntheses. Furthermore, in vitro, we showed a quinone-forming activity of the ActVA-ORF5/ActVB system in addition to that of a known C-6 monooxygenase, ActVA-ORF6, by using emodinanthrone as a model substrate. Our results demonstrate that the act gene cluster encodes two alternative routes for quinone formation by C-6 oxygenation in BIQ biosynthesis.

  2. Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO).

    Science.gov (United States)

    Winkler, Dirk; Beconi, Maria; Toledo-Sherman, Leticia M; Prime, Michael; Ebneth, Andreas; Dominguez, Celia; Muñoz-Sanjuan, Ignacio

    2013-09-01

    Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells.

  3. First molecular modeling report on novel arylpyrimidine kynurenine monooxygenase inhibitors through multi-QSAR analysis against Huntington's disease: A proposal to chemists!

    Science.gov (United States)

    Amin, Sk Abdul; Adhikari, Nilanjan; Jha, Tarun; Gayen, Shovanlal

    2016-12-01

    Huntington's disease (HD) is caused by mutation of huntingtin protein (mHtt) leading to neuronal cell death. The mHtt induced toxicity can be rescued by inhibiting the kynurenine monooxygenase (KMO) enzyme. Therefore, KMO is a promising drug target to address the neurodegenerative disorders such as Huntington's diseases. Fiftysix arylpyrimidine KMO inhibitors are structurally explored through regression and classification based multi-QSAR modeling, pharmacophore mapping and molecular docking approaches. Moreover, ten new compounds are proposed and validated through the modeling that may be effective in accelerating Huntington's disease drug discovery efforts. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Nitrification inhibition by hexavalent chromium Cr(VI)--Microbial ecology, gene expression and off-gas emissions.

    Science.gov (United States)

    Kim, Young Mo; Park, Hongkeun; Chandran, Kartik

    2016-04-01

    The goal of this study was to investigate the responses in the physiology, microbial ecology and gene expression of nitrifying bacteria to imposition of and recovery from Cr(VI) loading in a lab-scale nitrification bioreactor. Exposure to Cr(VI) in the reactor strongly inhibited nitrification performance resulting in a parallel decrease in nitrate production and ammonia consumption. Cr(VI) exposure also led to an overall decrease in total bacterial concentrations in the reactor. However, the fraction of ammonia oxidizing bacteria (AOB) decreased to a greater extent than the fraction of nitrite oxidizing bacteria (NOB). In terms of functional gene expression, a rapid decrease in the transcript concentrations of amoA gene coding for ammonia oxidation in AOB was observed in response to the Cr(VI) shock. In contrast, transcript concentrations of the nxrA gene coding for nitrite oxidation in NOB were relatively unchanged compared to Cr(VI) pre-exposure levels. Therefore, Cr(VI) exposure selectively and directly inhibited activity of AOB, which indirectly resulted in substrate (nitrite) limitation to NOB. Significantly, trends in amoA expression preceded performance trends both during imposition of and recovery from inhibition. During recovery from the Cr(VI) shock, the high ammonia concentrations in the bioreactor resulted in an irreversible shift towards AOB populations, which are expected to be more competitive in high ammonia environments. An inadvertent impact during recovery was increased emission of nitrous oxide (N2O) and nitric oxide (NO), consistent with recent findings linking AOB activity and the production of these gases. Therefore, Cr(VI) exposure elicited multiple responses on the microbial ecology, gene expression and both aqueous and gaseous nitrogenous conversion in a nitrification process. A complementary interrogation of these multiple responses facilitated an understanding of both direct and indirect inhibitory impacts on nitrification. Copyright

  5. Induction of monooxygenases and incorporation of radioactivity from 2-14C-lysine into hepatic microsomes of phenobarbital-treated rats fed a diet deficient in lysine, methionine, threonine and vitamine A, C and E

    International Nuclear Information System (INIS)

    Nurmagambetov, T.Zh.; Amirov, B.B.; Kuanysheva, T.K.; Sharmanov, T.Sh.

    1991-01-01

    The effect of diet on induction of monooxygenases and distribution of label from 2- 14 C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital (80 mg/rg, three days) was studied. 2- 14 C-lysine was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monooxygenase induction, increase of relative liver weight and incorporation of label from 2- 14 C-lysine into fractions of liver homogenate in phenobarbital-treated rats were more pronounced as compared with the similarly trated rats that were fed a balanced diet. The possibility of mobilization of deficient essential components to liver from other organs and tissues for maintenance of monooxygenase induction is discussed

  6. Induction of monooxygenases and incorporation of radioactivity from 2-14C-lysine into hepatic microsomes of phenobarbital-treated rats fed a diet deficient in lysine, methionine, threonine and vitamines A, C, E

    International Nuclear Information System (INIS)

    Nurmagambetov, T.Zh.; Amirov, B.B.; Kuanysheva, T.G.; Sharmanov, T.Sh.

    1992-01-01

    The effect of diet on induction of monooxygenases and distribution of radioactivity from 2- 14 C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital was studied. 2- 14 C-lysin was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monooxygenase induction, increase of relative liver weight and incorporation of radioactivity from 2- 14 C-lysine into fractions of liver homogenate in phenobarbital-treated rats fed diet deficient in lysine, methionine, threonine and vitamins A, C, E were more pronounced as compared with the similarly treated rats which were fed a balanced diet. The possibility of mobilization of deficient essencial components to liver from other organs and tissues for maintenance of monooxygenase induction iis discussed

  7. cDNA-SRAP and Its Application in Differential Gene Expression Analysis: A Case Study in Erianthus arundinaceum

    Directory of Open Access Journals (Sweden)

    Youxiong Que

    2012-01-01

    Full Text Available Erianthus arundinaceum is a wild relative species of sugarcane. The aim of this research was to demonstrate the feasibility of cDNA-SRAP for differential gene expression and to explore the molecular mechanism of drought resistance in E. arundinaceum. cDNA-SRAP technique, for the first time, was applied in the analysis of differential gene expression in E. arundinaceum under drought stress. In total, eight differentially expressed genes with length of 185–427 bp were successfully isolated (GenBank Accession numbers: EU071770, EU071772, EU071774, EU071776, EU071777, EU071779, EU071780, and EU071781. Based on their homologies with genes in GenBank, these genes were assumed to encode ribonuclease III, vacuolar protein, ethylene insensitive protein, aerobactin biosynthesis protein, photosystem II protein, glucose transporter, leucine-rich repeat protein, and ammonia monooxygenase. Real-time PCR analysis on the expression profiling of gene (EU071774 encoding ethylene-insensitive protein and gene (EU071781 encoding ammonia monooxygenase revealed that the expression of these two genes was upregulated both by PEG and ABA treatments, suggesting that they may involve in the drought resistance of E. arundinaceum. This study constitutes the first report of genes activated in E. arundinaceum by drought stress and opens up the application of cDNA-SRAP in differential gene expression analysis in E. arundinaceum under certain stress conditions.

  8. Diversity of ammonia-oxidizing bacteria in relation to soil environment in Ebinur Lake Wetland

    Directory of Open Access Journals (Sweden)

    Wenge Hu

    2016-03-01

    Full Text Available Ammonia oxidation is the first and rate-limiting step of nitrification and is carried out by ammonia-oxidizing bacteria (AOB. Ebinur Lake Wetland, the most representative temperate arid zone wetland ecosystem in China, is the centre of oasis and desertification of the northern slope of Tianshan conjugate. Soil samples were collected from three sites (Tamarix ramosissima, Halocnemum strobilaceum and Phragmites australis and different soil layers (0–5, 5–15, 15–25 and 25–35 cm in this wetland in spring, summer and autumn and were used to characterize the diversity of AOB based on the ammonia monooxygenase (amoA gene. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE and bivariate correlation analysis were used to analyse the relationship between the diversity of AOB and soil environment factors. The PCR-DGGE indicated that the diversity of AOB was high in the entire sample and the Shannon diversity index varied from 1.369 to 2.471. The phylogenetic analysis showed that the amoA fragments were grouped into Nitrosospira sp. and Nitrosomonas sp. Most amoA gene sequences fell within the Nitrosospira sp. cluster, and only a few sequences were clustered with Nitrosomonas sp., indicating that Nitrosospira sp. may be more adaptable than Nitrosomonas sp. in this area. Bivariate correlation analysis showed that the diversity of AOB was significantly correlated with soil organic matter, conductivity, total phosphorus and nitrate in the Ebinur Lake Wetland in Xinjiang.

  9. Identification of active methanotrophs in a landfill cover soil through detection of expression of 16S rRNA and functional genes.

    Science.gov (United States)

    Chen, Yin; Dumont, Marc G; Cébron, Aurélie; Murrell, J Colin

    2007-11-01

    Active methanotrophs in a landfill soil were revealed by detecting the 16S rRNA of methanotrophs and the mRNA transcripts of key genes involved in methane oxidation. New 16S rRNA primers targeting type I and type II methanotrophs were designed and optimized for analysis by denaturing gradient gel electrophoresis. Direct extraction of RNA from soil enabled the analysis of the expression of the functional genes: mmoX, pmoA and mxaF, which encode subunits of soluble methane monooxygenase, particulate methane monooxygenase and methanol dehydrogenase respectively. The 16S rRNA polymerase chain reaction (PCR) primers for type I methanotrophs detected Methylomonas, Methylosarcina and Methylobacter sequences from both soil DNA and cDNA which was generated from RNA extracted directly from the landfill cover soil. The 16S rRNA primers for type II methanotrophs detected primarily Methylocella and some Methylocystis 16S rRNA genes. Phylogenetic analysis of mRNA recovered from the soil indicated that Methylobacter, Methylosarcina, Methylomonas, Methylocystis and Methylocella were actively expressing genes involved in methane and methanol oxidation. Transcripts of pmoA but not mmoX were readily detected by reverse transcription polymerase chain reaction (RT-PCR), indicating that particulate methane monooxygenase may be largely responsible for methane oxidation in situ.

  10. Magnetic resonance studies on the copper site of dopamine β-monooxygenase in the presence of cyanide and azide anions

    International Nuclear Information System (INIS)

    Obata, A.; Tanaka, H.; Kawazura, H.

    1987-01-01

    In order to elucidate the coordination state of water molecules in the Cu(II) site of dopamine [(3,4-dihydroxyphenyl)ethylamine] β-monooxygenase, measurements of the paramagnetic 1 H nuclear magnetic relaxation rate of solvent water in the enzyme solution containing cyanide or azide as an exogenous ligand were carried out to obtain the values of intrinsic paramagnetic relaxation rate decrements R/sub p/ 1 and R/sub p/ 2 for the ligand-enzyme 1:1 and 2:1 complexes, respectively. R/sub p/ 1 (percent) values were 53 (pH 5.5) and 52 (pH 7.0) for cyanide and 38 (pH 5.5) and 32 (pH 7.0) for azide, while R/sub p/ 2 (percent) values were 98 (pH 5.5) and 96 (pH 7.0) for azide. Although no R/sub p/ 2 values for cyanide were obtained because of its reducing power at the Cu(II) site, the R/sub p/ 1 and R/sub p/ 2 values obtained above prove that the Cu(II) center has two coordinated water molecules that are exchangeable for exogenous ligands at either pH. Supporting evidence was provided by electron paramagnetic resonance (EPR) titration, in which the enzyme solution containing cyanide-enzyme (1:1) complex in an equal proportion to uncomplexed enzyme gave an observed paramagnetic relaxation rate decrement, R/sub p/, of 23%. Another characteristic of the R/sub p/ 1 and R/sub p/ 2 values was their invariability with respect to pH, indicating that the three-dimensional structure of the Cu(II) site is pH-invariant within the range examined. Binding constants of ligand to enzyme K/sub b/ 1 and K/sub b/ 2 for 1:1 and 2:1 complex formation, respectively, were also determined through an analysis of the R/sub p/ values; it was found that K/sub b/ 1 was larger than K/sub b/ 2 irrespective of pH. On the basis of these results, together with the axial-symmetric EPR parameters of the 1:1 complexes, a possible coordination geometry of the two water molecules in the Cu(II) site of the enzyme is suggested

  11. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens.

    Directory of Open Access Journals (Sweden)

    Lehlohonolo Benedict Qhanya

    Full Text Available Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s, heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence. Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea, Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala, revealed the presence of numerous putative P450s ranging from 267 (A. mellea to 14 (M. osmundae. Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

  12. HHM motif at the CuH-site of peptidylglycine monooxygenase is a pH-dependent conformational switch.

    Science.gov (United States)

    Kline, Chelsey D; Mayfield, Mary; Blackburn, Ninian J

    2013-04-16

    Peptidylglycine monooxygenase is a copper-containing enzyme that catalyzes the amidation of neuropeptides hormones, the first step of which is the conversion of a glycine-extended pro-peptide to its α-hydroxyglcine intermediate. The enzyme contains two mononuclear Cu centers termed CuM (ligated to imidazole nitrogens of H242, H244 and the thioether S of M314) and CuH (ligated to imidazole nitrogens of H107, H108, and H172) with a Cu-Cu separation of 11 Å. During catalysis, the M site binds oxygen and substrate, and the H site donates the second electron required for hydroxylation. The WT enzyme shows maximum catalytic activity at pH 5.8 and undergoes loss of activity at lower pHs due to a protonation event with a pKA of 4.6. Low pH also causes a unique structural transition in which a new S ligand coordinates to copper with an identical pKA, manifest by a large increase in Cu-S intensity in the X- ray absorption spectroscopy. In previous work (Bauman, A. T., Broers, B. A., Kline, C. D., and Blackburn, N. J. (2011) Biochemistry 50, 10819-10828), we tentatively assigned the new Cu-S interaction to binding of M109 to the H-site (part of an HHM conserved motif common to all but one member of the family). Here we follow up on these findings via studies on the catalytic activity, pH-activity profiles, and spectroscopic (electron paramagnetic resonance, XAS, and Fourier transform infrared) properties of a number of H-site variants, including H107A, H108A, H172A, and M109I. Our results establish that M109 is indeed the coordinating ligand and confirm the prediction that the low pH structural transition with associated loss of activity is abrogated when the M109 thioether is absent. The histidine mutants show more complex behavior, but the almost complete lack of activity in all three variants coupled with only minor differences in their spectroscopic properties suggests that unique structural elements at H are critical for functionality. The data suggest a more general

  13. [Altered expressions of alkane monooxygenase and hypoxia inducible factor-1α expression in lung tissue of rat hypoxic pulmonary hypertension].

    Science.gov (United States)

    Deng, Hua-jun; Yuan, Ya-dong

    2013-10-29

    To explore the altered expressions of alkane monooxygenase (AlkB) and hypoxia-inducible factor-1α (HIF-1α) in a rat model of hypoxic pulmonary arterial hypertension. Twenty Wistar rats were divided randomly into normal control and hypoxia groups after 1-week adaptive feeding. Hypoxia group was raised in a homemade organic glass tank with a 24-h continuous supply of air and nitrogen atmospheric mixed gas. And the oxygen concentration of (10.0 ± 0.5)% was controlled by oxygen monitoring control system. The control group was maintained in room air. Both groups stayed in the same room with the same diet. After 8 weeks, the level of mean pulmonary pressure (mPAP) was measured by right-heart catheterization, right ventricular hypertrophy index (RVHI) calculated by the ratio of right ventricle to left ventricle plus septum and hypoxic pulmonary vascular remodeling (HPSR) observed under microscope. And the levels of AlkB and HIF-1α mRNA and protein in lungs were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. At 8 weeks post-hypoxia, compared with the control group [11.0 ± 0.7 mm Hg (1 mm Hg = 0.133 kPa), 0.210 ± 0.035], the levels of mPAP and RVHI in hypoxia group (33.3 ± 1.3 mm Hg, 0.448 ± 0.013) increased significantly (both P < 0.05), the expressions of AlkB mRNA and protein in pulmonary tissue decreased significantly (0.338 ± 0.085 vs 0.688 ± 0.020, P < 0.01) (0.483 ± 0.052 vs 0.204 ± 0.010, P < 0.01), and the expressions of HIF-1α mRNA and protein increased significantly (0.790 ± 0.161 vs 0.422 ± 0.096, P < 0.01) (0.893 ± 0.080 vs 0.346 ± 0.008, P < 0.01). The down-regulation of AlkB in lung tissue may increase the activity of HIF-1 to participate in the occurrence and development of pulmonary hypertension.

  14. Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

    Science.gov (United States)

    Eng, Heather; Sharma, Raman; Wolford, Angela; Di, Li; Ruggeri, Roger B; Buckbinder, Leonard; Conn, Edward L; Dalvie, Deepak K; Kalgutkar, Amit S

    2016-08-01

    N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more

  15. The lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Otoikhian, Adenike [Oregon Health & Sciences University; Barry, Amanda N. [Los Alamos National Laboratory; Mayfield, Mary [Oregon Health & Science University; Nilges, Mark [Illinois EPR Center; Huang, Yiping [Johns Hopkins University; Lutsenko, Svetlana [Johns Hopkins University; Blackburn, Ninian [Oregon Health & Science University

    2012-05-14

    Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper translocating ATPase (ATP7A or ATP7B) but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1, 15N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased towards 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met while at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.

  16. Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

    Science.gov (United States)

    Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

    2010-12-01

    l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

  17. Bidirectional gene sequences with similar homology to functional proteins of alkane degrading bacterium pseudomonas fredriksbergensis DNA

    International Nuclear Information System (INIS)

    Megeed, A.A.

    2011-01-01

    The potential for two overlapping fragments of DNA from a clone of newly isolated alkanes degrading bacterium Pseudomonas frederiksbergensis encoding sequences with similar homology to two parts of functional proteins is described. One strand contains a sequence with high homology to alkanes monooxygenase (alkB), a member of the alkanes hydroxylase family, and the other strand contains a sequence with some homology to alcohol dehydrogenase gene (alkJ). Overlapping of the genes on opposite strands has been reported in eukaryotic species, and is now reported in a bacterial species. The sequence comparisons and ORFS results revealed that the regulation and the genes organization involved in alkane oxidation represented in Pseudomonas frederiksberghensis varies among the different known alkane degrading bacteria. The alk gene cluster containing homologues to the known alkane monooxygenase (alkB), and rubredoxin (alkG) are oriented in the same direction, whereas alcohol dehydrogenase (alkJ) is oriented in the opposite direction. Such genomes encode messages on both strands of the DNA, or in an overlapping but different reading frames, of the same strand of DNA. The possibility of creating novel genes from pre-existing sequences, known as overprinting, which is a widespread phenomenon in small viruses. Here, the origin and evolution of the gene overlap to bacteriophages belonging to the family Microviridae have been investigated. Such a phenomenon is most widely described in extremely small genomes such as those of viruses or small plasmids, yet here is a unique phenomenon. (author)

  18. Characterization of abiotic stress genes from different species of eucalyptus

    International Nuclear Information System (INIS)

    Naz, S.; Kausar, H.; Saleem, F.; Zafarullah, A.

    2015-01-01

    The stresses causing dehydration damage to the plant cell like cold, drought, and high salinity are the most frequent environmental stresses that influence plant growth, development and restraining productivity in cultivated areas world-wide. Many drought, salinity and cold inducible genes causing tolerance to environmental stresses in many plants include Dehydrin1 (DHN1), Dehydrin2 (DHN2), Dehydrin10 (DHN10), putative phosphate transporter (Ecpt2), choline monooxygenase (CMO) and DREB/CBF1c genes. Gene specific primer pairs were designed for each gene using DNAStar software. These genes were amplified from different species of eucalyptus such as Eucalyptus camaldulensis, E. globulus, E. tereticornis and E. gunii through PCR. Dehydrin2 gene of E. camaldulensis and dehydrin10 gene of E. globulus were cloned using the TA Cloning Kit with pCR 2.1 vector and sequenced. The Dehydrin genes sequences were submitted to GeneBank: Eucalyptus globulus dehydrin10 gene (Accession No. HG915712) and E. camaldulensis dehydrin 2 gene (Accession No. HG813113). The amino acid sequence of Dehydrin10 from E. globulus showed 97% homology to E. globulus DHN10 (JN052210) and Dehydrin2 from E. camaldulensis presented 94% homology to E. globulus DHN2 (JN052209). These genes can be employed in generating drought resistant crop plants. (author)

  19. Ammonia-Oxidizing β-Proteobacteria from the Oxygen Minimum Zone off Northern Chile▿

    Science.gov (United States)

    Molina, Verónica; Ulloa, Osvaldo; Farías, Laura; Urrutia, Homero; Ramírez, Salvador; Junier, Pilar; Witzel, Karl-Paul

    2007-01-01

    The composition of ammonia-oxidizing bacteria from the β-Proteobacteria subclass (βAOB) was studied in the surface and upper-oxycline oxic waters (2- to 50-m depth, ∼200 to 44 μM O2) and within the oxygen minimum zone (OMZ) suboxic waters (50- to 400-m depth, ≤10 μM O2) of the eastern South Pacific off northern Chile. This study was carried out through cloning and sequencing of genes coding for 16S rRNA and the ammonia monooxygenase enzyme active subunit (amoA). Sequences affiliated with Nitrosospira-like cluster 1 dominated the 16S rRNA gene clone libraries constructed from both oxic and suboxic waters. Cluster 1 consists exclusively of yet-uncultivated βAOB from marine environments. However, a single clone, out of 224 obtained from the OMZ, was found to belong to Nitrosospira lineage cluster 0. To our knowledge, cluster 0 sequences have been derived from βAOB isolated only from sand, soil, and freshwater environments. Sequences in clone libraries of the amoA gene from the surface and upper oxycline could be grouped in a marine subcluster, also containing no cultured representatives. In contrast, all 74 amoA sequences originating from the OMZ were either closely affiliated with cultured Nitrosospira spp. from clusters 0 and 2 or with other yet-uncultured βAOB from soil and an aerated-anoxic Orbal process waste treatment plant. Our results reveal the presence of Nitrosospira-like βAOB in both oxic and suboxic waters associated with the OMZ but with a clear community shift at the functional level (amoA) along the strong oxygen gradient. PMID:17416686

  20. Genes and Gene Therapy

    Science.gov (United States)

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  1. Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Vermaas, Josh V.; Crowley, Michael F.; Beckham, Gregg T.; Payne, Christina M.

    2015-05-21

    In nature, polysaccharide glycosidic bonds are cleaved by hydrolytic enzymes for a vast array of biological functions. Recently, a new class of enzymes that utilize an oxidative mechanism to cleave glycosidic linkages was discovered; these enzymes are called lytic polysaccharide monooxygenases (LPMO). These oxidative enzymes are synergistic with cocktails of hydrolytic enzymes and are thought to act primarily on crystalline regions, in turn providing new sites of productive attachment and detachment for processive hydrolytic enzymes. In the case of cellulose, the homopolymer of ..beta..-1,4-d-glucose, enzymatic oxidation occurs at either the reducing end or the nonreducing end of glucose, depending on enzymatic specificity, and results in the generation of oxidized chemical substituents at polymer chain ends. LPMO oxidation of cellulose is thought to produce either a lactone at the reducing end of glucose that can spontaneously or enzymatically convert to aldonic acid or 4-keto-aldose at the nonreducing end that may further oxidize to a geminal diol. Here, we use molecular simulation to examine the effect of oxidation on the structure of crystalline cellulose. The simulations highlight variations in behaviors depending on the chemical identity of the oxidized species and its location within the cellulose fibril, as different oxidized species introduce steric effects that disrupt local crystallinity and in some cases reduce the work needed for polymer decrystallization. Reducing-end oxidations are easiest to decrystallize when located at the end of the fibril, whereas nonreducing end oxidations readily decrystallize from internal cleavage sites despite their lower solvent accessibility. The differential in decrystallization free energy suggests a molecular mechanism consistent with experimentally observed LPMO/cellobiohydrolase synergy. Additionally, the soluble oxidized cellobiose products released by hydrolytic cellulases may bind to the active sites of cellulases

  2. X-ray absorption spectroscopic studies of the dinuclear iron center in methane monooxygenase and the sulfure and chlorine centers in photographic materials

    Energy Technology Data Exchange (ETDEWEB)

    DeWitt, J.G.

    1992-12-01

    The dinuclear iron center of the hydroxylase component of soluble methane monooxygenase (MMO) from Methylococcus capsulatus and Methylosinus trichosporiwn has been studied by X-ray absorption spectroscopy. Analysis of the Fe K-edge EXAFS revealed that the first shell coordination of the Fe(HI)Fe(IH) oxidized state of the hydroxylase from M. capsulatus consists of approximately 6 N and 0 atoms at an average distance of 2.04 [Angstrom]. The Fe-Fe distance was determined to be 3.4 [Angstrom]. No evidence for the presence of a short oxo bridge in the iron center of the oxidized hydroxylase was found, suggesting that the active site of MMO is significantly different from the active sites of the dinuclear iron proteins hemery and ribonucleotide reductase. In addition, the results of the first shell fits suggest that there are more oxygen than nitrogen donor ligands.

  3. X-ray absorption spectroscopic studies of the dinuclear iron center in methane monooxygenase and the sulfure and chlorine centers in photographic materials

    Energy Technology Data Exchange (ETDEWEB)

    DeWitt, Jane G. [Stanford Univ., CA (United States)

    1992-12-01

    The dinuclear iron center of the hydroxylase component of soluble methane monooxygenase (MMO) from Methylococcus capsulatus and Methylosinus trichosporiwn has been studied by X-ray absorption spectroscopy. Analysis of the Fe K-edge EXAFS revealed that the first shell coordination of the Fe(HI)Fe(IH) oxidized state of the hydroxylase from M. capsulatus consists of approximately 6 N and 0 atoms at an average distance of 2.04 Å. The Fe-Fe distance was determined to be 3.4 Å. No evidence for the presence of a short oxo bridge in the iron center of the oxidized hydroxylase was found, suggesting that the active site of MMO is significantly different from the active sites of the dinuclear iron proteins hemery and ribonucleotide reductase. In addition, the results of the first shell fits suggest that there are more oxygen than nitrogen donor ligands.

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

    Science.gov (United States)

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

    1998-01-01

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

  5. Optimized formation of detergent micelles of beta-carotene and retinal production using recombinant human beta,beta-carotene 15,15'-monooxygenase.

    Science.gov (United States)

    Kim, Nam-Hee; Kim, Yeong-Su; Kim, Hye-Jung; Oh, Deok-Kun

    2008-01-01

    The formation of beta-carotene detergent micelles and their conversion into retinal by recombinant human beta,beta-carotene 15,15'-monooxygenase was optimized under aqueous conditions. Toluene was the most hydrophobic among the organic solvents tested; thus, it was used to dissolve beta-carotene, which is a hydrophobic compound. Tween 80 was selected as the detergent because it supported the highest level of retinal production among all of the detergents tested. The maximum production of retinal was achieved in detergent micelles containing 200 mg/L of beta-carotene and 2.4% (w/v) Tween 80. Under these conditions, the recombinant enzyme produced 97 mg/L of retinal after 16 h with a conversion yield of 48.5% (w/w). The amount of retinal produced, which is the highest ever reported, is a result of the ability of our system to dissolve large amounts of beta-carotene.

  6. Effects of Quinizarin and Five Synthesized Derivatives on Fifth Larval Instar Midgut Ecdysone 20-Monooxygenase Activity of the Tobacco Hornworm Manduca sexta

    Directory of Open Access Journals (Sweden)

    Christopher A. Drummond

    2014-01-01

    Full Text Available The plant allelochemical, quinizarin (1,4-dihydroxy-9,10-anthraquinone, and five anthraquinones that were synthesized from quinizarin, namely, 1,4-anthraquinone; 2-hydroxy-1,4-anthraquinone; 2-methoxy-1,4-anthraquinone; 9-hydroxy-1,4-anthraquinone; and 9-methoxy-1,4-anthraquinone, were assessed as to their effects on the essential, P450-dependent ecdysone 20-monooxygenase system of the insect model Manduca sexta (tobacco hornworm. This steroid hydroxylase converts the arthropod molting hormone, ecdysone, to the physiologically required 20-hydroxyecdysone form. M. sexta fifth larval instar midgut homogenates were incubated with increasing concentrations (10−8 to 10−3 M of each of the six anthraquinones followed by ecdysone 20-monooxygenase assessments using a radioenzymological assay. Four of the five anthraquinones exhibited I50’s of about 4×10-6 to 6×10-2 M. The most effective inhibitors were 2-methoxy-1,4-anthraquinone and 1,4-anthraquinone followed by 9-hydroxy-1,4 anthraquinone and 9-methoxy-1,4-anthraquinone. At lower concentrations the latter anthraquinone stimulated E20M activity. Quinizarin was less inhibitory and 2-hydroxy-1,4-anthraquinone was essentially without effect. Significantly, these studies make evident for the first time that anthraquinones can affect insect E20M activity, and thus insect endocrine regulation and development, and that a relationship between anthraquinone structure and effectiveness is apparent. These studies represent the first demonstrations of anthraquinones affecting any steroid hydroxylase system.

  7. Molecular dynamics analysis reveals structural insights into mechanism of nicotine N-demethylation catalyzed by tobacco cytochrome P450 mono-oxygenase.

    Directory of Open Access Journals (Sweden)

    Shan Wang

    Full Text Available CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys-trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase.

  8. Analysis of the functional gene structure and metabolic potential of microbial community in high arsenic groundwater.

    Science.gov (United States)

    Li, Ping; Jiang, Zhou; Wang, Yanhong; Deng, Ye; Van Nostrand, Joy D; Yuan, Tong; Liu, Han; Wei, Dazhun; Zhou, Jizhong

    2017-10-15

    Microbial functional potential in high arsenic (As) groundwater ecosystems remains largely unknown. In this study, the microbial community functional composition of nineteen groundwater samples was investigated using a functional gene array (GeoChip 5.0). Samples were divided into low and high As groups based on the clustering analysis of geochemical parameters and microbial functional structures. The results showed that As related genes (arsC, arrA), sulfate related genes (dsrA and dsrB), nitrogen cycling related genes (ureC, amoA, and hzo) and methanogen genes (mcrA, hdrB) in groundwater samples were correlated with As, SO 4 2- , NH 4 + or CH 4 concentrations, respectively. Canonical correspondence analysis (CCA) results indicated that some geochemical parameters including As, total organic content, SO 4 2- , NH 4 + , oxidation-reduction potential (ORP) and pH were important factors shaping the functional microbial community structures. Alkaline and reducing conditions with relatively low SO 4 2- , ORP, and high NH 4 + , as well as SO 4 2- and Fe reduction and ammonification involved in microbially-mediated geochemical processes could be associated with As enrichment in groundwater. This study provides an overall picture of functional microbial communities in high As groundwater aquifers, and also provides insights into the critical role of microorganisms in As biogeochemical cycling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Phylogenetic diversity of archaeal 16S rRNA and ammonia monooxygenase genes from tropical estuarine sediments on the central west coast of India

    Digital Repository Service at National Institute of Oceanography (India)

    Singh, S.K.; Verma, P.; Ramaiah, N.; Anil, A.C.; Shouche, Y.S.

    of Euryarchaeota. Our results indicate that archaeal diversity in tropical estuarine sediments is influenced by the mangrove vegetation bordering the lower stretches of both estuaries. Higher diversity may be related to elevated land drainage during the monsoon...

  10. Demonstration that menthofuran synthase of mint (Mentha) is a cytochrome P450 monooxygenase: cloning, functional expression, and characterization of the responsible gene

    NARCIS (Netherlands)

    Bertea, C.M.; Schalk, M.; Karp, F.; Maffei, M.; Croteau, R.

    2001-01-01

    ( )-Menthofuran is an undesirable monoterpenoid component of peppermint (Mentha x piperita) essential oil that is derived from the ,-unsaturated ketone ( )-pulegone. Microsomal preparations, from the oil gland secretory cells of a high ( )-menthofuran-producing chemotype of Mentha pulegium,

  11. The Arabidopsis YUCCA1 Flavin Monooxygenase Functions in the Indole-3-Pyruvic Acid Branch of Auxin Biosynthesis

    Czech Academy of Sciences Publication Activity Database

    Stepanova, A.N.; Yun, J.; Robles, L.M.; Novák, Ondřej; He, W.; Guo, H.W.; Ljung, K.; Alonso, J.M.

    2011-01-01

    Roč. 23, č. 11 (2011), s. 3961-3973 ISSN 1040-4651 R&D Projects: GA ČR GA301/08/1649 Keywords : PLANT DEVELOPMENT * GLUCOSINOLATE BIOSYNTHESIS * REPRODUCTIVE DEVELOPMENT * MASS-SPECTROMETRY * ALDEHYDE OXIDASE * THALIANA * GENE * METABOLISM * MUTANTS * PATHWAY Subject RIV: EF - Botanics Impact factor: 8.987, year: 2011

  12. FMNH2-dependent monooxygenases initiate catabolism of sulfonamides in Microbacterium sp strain BR1 subsisting on sulfonamide antibiotics

    Czech Academy of Sciences Publication Activity Database

    Ricken, B.; Kolvenbach, B.A.; Bergesch, C.; Benndorf, D.; Kroll, K.; Strnad, Hynek; Vlček, Čestmír; Adaixo, R.; Hammes, F.; Shahgaldian, P.; Schaeffer, A.; Kohler, H.P.E.; Corvini, P.F.X.

    2017-01-01

    Roč. 7, podzim (2017), č. článku 15783. ISSN 2045-2322 Institutional support: RVO:68378050 Keywords : resistance mechanism * clinical specimens * sulfamethoxazole * bacteria * degradation * benzylpenicillin * biodegradation * genes * sulfadiazine * arthrobacter Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Microbiology Impact factor: 4.259, year: 2016

  13. Genetic interrelations in the actinomycin biosynthetic gene clusters of Streptomyces antibioticus IMRU 3720 and Streptomyces chrysomallus ATCC11523, producers of actinomycin X and actinomycin C

    Science.gov (United States)

    Crnovčić, Ivana; Rückert, Christian; Semsary, Siamak; Lang, Manuel; Kalinowski, Jörn; Keller, Ullrich

    2017-01-01

    Sequencing the actinomycin (acm) biosynthetic gene cluster of Streptomyces antibioticus IMRU 3720, which produces actinomycin X (Acm X), revealed 20 genes organized into a highly similar framework as in the bi-armed acm C biosynthetic gene cluster of Streptomyces chrysomallus but without an attached additional extra arm of orthologues as in the latter. Curiously, the extra arm of the S. chrysomallus gene cluster turned out to perfectly match the single arm of the S. antibioticus gene cluster in the same order of orthologues including the the presence of two pseudogenes, scacmM and scacmN, encoding a cytochrome P450 and its ferredoxin, respectively. Orthologues of the latter genes were both missing in the principal arm of the S. chrysomallus acm C gene cluster. All orthologues of the extra arm showed a G +C-contents different from that of their counterparts in the principal arm. Moreover, the similarities of translation products from the extra arm were all higher to the corresponding translation products of orthologue genes from the S. antibioticus acm X gene cluster than to those encoded by the principal arm of their own gene cluster. This suggests that the duplicated structure of the S. chrysomallus acm C biosynthetic gene cluster evolved from previous fusion between two one-armed acm gene clusters each from a different genetic background. However, while scacmM and scacmN in the extra arm of the S. chrysomallus acm C gene cluster are mutated and therefore are non-functional, their orthologues saacmM and saacmN in the S. antibioticus acm C gene cluster show no defects seemingly encoding active enzymes with functions specific for Acm X biosynthesis. Both acm biosynthetic gene clusters lack a kynurenine-3-monooxygenase gene necessary for biosynthesis of 3-hydroxy-4-methylanthranilic acid, the building block of the Acm chromophore, which suggests participation of a genome-encoded relevant monooxygenase during Acm biosynthesis in both S. chrysomallus and S

  14. Determination of the human cytochrome P450 monooxygenase catalyzing the enantioselective oxidation of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183).

    Science.gov (United States)

    Nagayoshi, Haruna; Kakimoto, Kensaku; Konishi, Yoshimasa; Kajimura, Keiji; Nakano, Takeshi

    2017-10-17

    2,2',3,5',6-Pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183) possess axial chirality and form the aS and aR enantiomers. The enantiomers of these congeners have been reported to accumulate in the human body enantioselectively via unknown mechanisms. In this study, we determined the cytochrome P450 (CYP) monooxygenase responsible for the enantioselective oxidization of PCB 95 and PCB 183, using a recombinant human CYP monooxygenase. We evaluated 13 CYP monooxygenases, namely CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4F2, and aromatase (CYP19), and revealed that CYP2A6 preferably oxidizes aS-PCB 95 enantioselectively; however, it did not oxidize PCB 183. The enantiomer composition was elevated from 0.5 (racemate) to 0.54. In addition, following incubation with CYP2A6, the enantiomer fraction (EF) of PCB 95 demonstrated a time-dependent increase.

  15. The effects of oxygen on process rates and gene expression of anammox and denitrification in the Eastern South Pacific oxygen minimum zone

    DEFF Research Database (Denmark)

    Dalsgaard, Tage; Stewart, Frank; De Brabandere, Loreto

    Oxygen concentrations were consistently below our detection limit of 90 nM for a distance of > 2000 km in the oxygen minimum zone (OMZ) along the coasts of Chile and Peru. In most cases, anammox and denitrification were only detected when in situ oxygen concentrations were below detection...... differently to oxygen. When normalized to a housekeeping gene (rpoB), the expression of 4 out of 9 N-cycle-genes changed with increasing oxygen concentration: The expression of ammonium monooxygenase (amoC) was stimulated, whereas expression of nitrite reductase (nirS), nitric oxide reductase (nor...

  16. Temporal and Spatial Stability of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofilters

    KAUST Repository

    Bagchi, Samik

    2014-12-05

    Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4–5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for ≥81–86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium

  17. Temporal and Spatial Stability of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofilters

    KAUST Repository

    Bagchi, Samik; Vlaeminck, Siegfried E.; Sauder, Laura A.; Mosquera, Mariela; Neufeld, Josh D.; Boon, Nico; Poulain, Alexandre

    2014-01-01

    Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4–5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for ≥81–86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium

  18. Phylogenetic congruence and ecological coherence in terrestrial Thaumarchaeota.

    Science.gov (United States)

    Oton, Eduard Vico; Quince, Christopher; Nicol, Graeme W; Prosser, James I; Gubry-Rangin, Cécile

    2016-01-01

    Thaumarchaeota form a ubiquitously distributed archaeal phylum, comprising both the ammonia-oxidising archaea (AOA) and other archaeal groups in which ammonia oxidation has not been demonstrated (including Group 1.1c and Group 1.3). The ecology of AOA in terrestrial environments has been extensively studied using either a functional gene, encoding ammonia monooxygenase subunit A (amoA) or 16S ribosomal RNA (rRNA) genes, which show phylogenetic coherence with respect to soil pH. To test phylogenetic congruence between these two markers and to determine ecological coherence in all Thaumarchaeota, we performed high-throughput sequencing of 16S rRNA and amoA genes in 46 UK soils presenting 29 available contextual soil characteristics. Adaptation to pH and organic matter content reflected strong ecological coherence at various levels of taxonomic resolution for Thaumarchaeota (AOA and non-AOA), whereas nitrogen, total mineralisable nitrogen and zinc concentration were also important factors associated with AOA thaumarchaeotal community distribution. Other significant associations with environmental factors were also detected for amoA and 16S rRNA genes, reflecting different diversity characteristics between these two markers. Nonetheless, there was significant statistical congruence between the markers at fine phylogenetic resolution, supporting the hypothesis of low horizontal gene transfer between Thaumarchaeota. Group 1.1c Thaumarchaeota were also widely distributed, with two clusters predominating, particularly in environments with higher moisture content and organic matter, whereas a similar ecological pattern was observed for Group 1.3 Thaumarchaeota. The ecological and phylogenetic congruence identified is fundamental to understand better the life strategies, evolutionary history and ecosystem function of the Thaumarchaeota.

  19. Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

    Science.gov (United States)

    Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

    2015-05-01

    We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

  20. Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

    Science.gov (United States)

    Attar, Mayssa; Dong, Dahai; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

    2003-04-01

    Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.

  1. Co-immobilization of cyclohexanone monooxygenase and glucose-6-phosphate dehydrogenase onto polyethylenimine-porous agarose polymeric composite using γ irradiation to use in biotechnological processes

    International Nuclear Information System (INIS)

    Atia, K.S.

    2005-01-01

    The co-immobilization of cyclohexanone monooxygenase (CHMO) and glucose-6-phosphate dehydrogenase (G6PDH) was optimized by completely coating, via covalent immobilization, the surface aldehyde groups of porous agarose (glyoxyl-agarose) with amine groups of polyethylenimine (PEI). The highest immobilization efficiency (∼87%) (activity of enzyme per amount of immobilized enzyme) was obtained with a CHMO/G6PDH ratio 2:1. The effects of different ratios of the support to the amount of enzymes (CHMO:G6PDH=2:1), the optimum incubation pH and the incubation time on the enzymatic activity of the enzymes were determined and found to be 5:1, 8.5 and 30 min, respectively. Subjecting the co-immobilized enzymes to doses of γ-radiation (5-100 kGy) resulted in complete loss in the activity of the free enzymes at a dose of 40 kGy, while the co-immobilized ones showed relatively high resistance to γ-radiation up to a dose of 50 kGy

  2. Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

    Energy Technology Data Exchange (ETDEWEB)

    O; Dell, William B.; Swartz, Paul D.; Weiss, Kevin L.; Meilleur, Flora (ORNL); (NCSU)

    2017-01-19

    Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.

  3. The involvement of flavin-containing monooxygenase but not CYP3A4 in metabolism of itopride hydrochloride, a gastroprokinetic agent: comparison with cisapride and mosapride citrate.

    Science.gov (United States)

    Mushiroda, T; Douya, R; Takahara, E; Nagata, O

    2000-10-01

    The goals of the present study were to identify the enzyme responsible for metabolism of itopride hydrochloride (itopride) and to evaluate the likelihood of drug interaction involving itopride. In human liver microsomes, the involvement of flavin-containing monooxygenase in N-oxygenation, the major metabolic pathway of itopride, was indicated by the following results: inhibition by methimazole and thiourea, heat inactivation, and protection against heat inactivation by NADPH. When the effects of ketoconazole on the metabolism of itopride, cisapride, and mosapride citrate (mosapride) were examined using human liver microsomes, ketoconazole strongly inhibited the formation of the primary metabolites of cisapride and mosapride, but not itopride. Other cytochrome P450 (CYP) 3A4 inhibitors, cimetidine, erythromycin, and clarithromycin, also inhibited the metabolism of cisapride and mosapride. In an in vivo study, itopride (30 mg/kg), cisapride (1.5 mg/kg), or mosapride (3 mg/kg) was orally administered to male rats with or without oral pretreatment with ketoconazole (120 mg/kg) twice daily for 2 days. The ketoconazole pretreatment significantly increased the area under the serum concentration curve and the maximum serum concentration of cisapride and mosapride but had no significant effect on the pharmacokinetics of itopride. In addition, itopride did not inhibit five specific CYP-mediated reactions of human liver microsomes. These results suggest that itopride is unlikely to alter the pharmacokinetics of other concomitantly administered drugs.

  4. Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

    International Nuclear Information System (INIS)

    Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R.

    1985-01-01

    The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H 2 O 2 such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization

  5. Structures of the Apo and FAD-bound forms of 2-hydroxybiphenyl 3-monooxygenase (HbpA) locate activity hotspots identified by using directed evolution.

    Science.gov (United States)

    Jensen, Chantel N; Mielke, Tamara; Farrugia, Joseph E; Frank, Annika; Man, Henry; Hart, Sam; Turkenburg, Johan P; Grogan, Gideon

    2015-04-13

    The FAD-dependent monooxygenase HbpA from Pseudomonas azelaica HBP1 catalyses the hydroxylation of 2-hydroxybiphenyl (2HBP) to 2,3-dihydroxybiphenyl (23DHBP). HbpA has been used extensively as a model for studying flavoprotein hydroxylases under process conditions, and has also been subjected to directed-evolution experiments that altered its catalytic properties. The structure of HbpA has been determined in its apo and FAD-complex forms to resolutions of 2.76 and 2.03 Å, respectively. Comparisons of the HbpA structure with those of homologues, in conjunction with a model of the reaction product in the active site, reveal His48 as the most likely acid/base residue to be involved in the hydroxylation mechanism. Mutation of His48 to Ala resulted in an inactive enzyme. The structures of HbpA also provide evidence that mutants achieved by directed evolution that altered activity are comparatively remote from the substrate-binding site. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Metabolism of methoxychlor by the P450-monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after expression in cell cultures of Nicotiana tabacum.

    Science.gov (United States)

    Joussen, Nicole; Schuphan, Ingolf; Schmidt, Burkhard

    2010-03-01

    Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

  7. Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b.

    Science.gov (United States)

    Zhang, Tingting; Zhou, Jiti; Wang, Xiaowei; Zhang, Yu

    2017-02-01

    The coupled effects of nitrogen source and methane monooxygenase (MMO) on the growth and poly-β-hydroxybutyrate (PHB) accumulation capacity of methanotrophs were explored. The ammonia-supplied methanotrophs expressing soluble MMO (sMMO) grew at the highest rate, while N 2 -fixing bacteria expressing particulate MMO (pMMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing pMMO, which might cause their slightly lower growth rate. The highest PHB content (51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing pMMO. Ammonia-supplied bacteria also accumulated a higher content of PHB (45.2%) with the expression of pMMO, while N 2 -fixing bacteria containing pMMO only showed low PHB production capacity (32.1%). The maximal PHB contents of bacteria expressing sMMO were low, with no significant change under different nitrogen source conditions. The low MMO activity, low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N 2 with the expression of pMMO were greatly improved in the cyclic NO 3 - N 2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO. Copyright © 2016. Published by Elsevier B.V.

  8. Effect of a thymol application on olfactory memory and gene expression levels in the brain of the honeybee Apis mellifera.

    Science.gov (United States)

    Bonnafé, Elsa; Drouard, Florian; Hotier, Lucie; Carayon, Jean-Luc; Marty, Pierre; Treilhou, Michel; Armengaud, Catherine

    2015-06-01

    Essential oils are used by beekeepers to control the Varroa mites that infest honeybee colonies. So, bees can be exposed to thymol formulations in the hive. The effects of the monoterpenoid thymol were explored on olfactory memory and gene expression in the brain of the honeybee. In bees previously exposed to thymol (10 or 100 ng/bee), the specificity of the response to the conditioned stimulus (CS) was lost 24 h after learning. Besides, the octopamine receptor OA1 gene Amoa1 showed a significant decrease of expression 3 h after exposure with 10 or 100 ng/bee of thymol. With the same doses, expression of Rdl gene, coding for a GABA receptor subunit, was not significantly modified but the trpl gene was upregulated 1 and 24 h after exposure to thymol. These data indicated that the genes coding for the cellular targets of thymol could be rapidly regulated after exposure to this molecule. Memory and sensory processes should be investigated in bees after chronic exposure in the hive to thymol-based preparations.

  9. Do freshwater macrophytes influence the community structure of ammonia-oxidizing and denitrifying bacteria in the rhizospere?

    DEFF Research Database (Denmark)

    Herrmann, Martina; Schramm, Andreas

    2006-01-01

    to unvegetated sediment, especially with respect to the availability of oxygen, organic carbon, and inorganic nitrogen. We hypothesize that macrophyte species create specific niches for ammonia oxidizing and nitrate-reducing bacteria in their rhizosphere, leading to plant-dependant differences in abundance...... dortmanna have been shown to release oxygen from their roots and to stimulate nitrification and coupled nitrification-denitrification in the rhizosphere. Together with the excretion of root exudates, this effect leads to strongly modified microenvironments at the root surface and in the rhizosphere compared......-denitrification using the 15N isotope pairing technique. Ammonia-oxidizing and nitrate-reducing populations are analyzed based on the ammonia monooxygenase gene (amoA) and the nitrate reductase gene (narG) as functional markers. Preliminary data indicate that there in fact exist differences in the community composition...

  10. A comparative study of P450 gene expression in field and laboratory Musca domestica L. strains

    DEFF Research Database (Denmark)

    Højland, Dorte Heidi; Vagn Jensen, Karl-Martin; Kristensen, Michael

    2014-01-01

    BACKGROUND The housefly is a global pest that has developed resistance to most insecticides applied for its control. Resistance has been associated with cytochrome P450 monooxygenases (P450s). The authors compare the expression of six genes possibly associated with insecticide resistance in three...... unselected strains: a multiresistant strain (791a), a neonicotinoid-resistant strain (766b) and a new field strain (845b). RESULTS CYP4G2 was highly expressed throughout the range of strains and proved to be the one of the most interesting expression profiles of all P450s analysed. CYP6G4 was expressed up...... to 11-fold higher in 766b than in WHO-SRS. Significant differences between expression of P450 genes between F1 flies from 845b and established laboratory strains were shown. In general, P450 gene expression in 845b was 2–14-fold higher than in the reference strain (P

  11. The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

    International Nuclear Information System (INIS)

    Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

    2008-01-01

    Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO 3 and FMO 5 was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO 1 showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO 1 -catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation

  12. A histidine-rich linker region in peptidylglycine α-amidating monooxygenase has the properties of a pH sensor.

    Science.gov (United States)

    Vishwanatha, Kurutihalli; Bäck, Nils; Mains, Richard E; Eipper, Betty A

    2014-05-02

    Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

  13. Predicting the Metabolic Sites by Flavin-Containing Monooxygenase on Drug Molecules Using SVM Classification on Computed Quantum Mechanics and Circular Fingerprints Molecular Descriptors.

    Directory of Open Access Journals (Sweden)

    Chien-Wei Fu

    Full Text Available As an important enzyme in Phase I drug metabolism, the flavin-containing monooxygenase (FMO also metabolizes some xenobiotics with soft nucleophiles. The site of metabolism (SOM on a molecule is the site where the metabolic reaction is exerted by an enzyme. Accurate prediction of SOMs on drug molecules will assist the search for drug leads during the optimization process. Here, some quantum mechanics features such as the condensed Fukui function and attributes from circular fingerprints (called Molprint2D are computed and classified using the support vector machine (SVM for predicting some potential SOMs on a series of drugs that can be metabolized by FMO enzymes. The condensed Fukui function fA- representing the nucleophilicity of central atom A and the attributes from circular fingerprints accounting the influence of neighbors on the central atom. The total number of FMO substrates and non-substrates collected in the study is 85 and they are equally divided into the training and test sets with each carrying roughly the same number of potential SOMs. However, only N-oxidation and S-oxidation features were considered in the prediction since the available C-oxidation data was scarce. In the training process, the LibSVM package of WEKA package and the option of 10-fold cross validation are employed. The prediction performance on the test set evaluated by accuracy, Matthews correlation coefficient and area under ROC curve computed are 0.829, 0.659, and 0.877 respectively. This work reveals that the SVM model built can accurately predict the potential SOMs for drug molecules that are metabolizable by the FMO enzymes.

  14. A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.

    Science.gov (United States)

    Chua, Ngee Kiat; Howe, Vicky; Jatana, Nidhi; Thukral, Lipi; Brown, Andrew J

    2017-12-08

    Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Induction of indolamine 2,3-dioxygenase and kynurenine 3-monooxygenase in rat brain following a systemic inflammatory challenge: a role for IFN-gamma?

    Science.gov (United States)

    Connor, Thomas J; Starr, Neasa; O'Sullivan, Joan B; Harkin, Andrew

    2008-08-15

    Inflammation-mediated dysregulation of the kynurenine pathway has been implicated as a contributor to a number of major brain disorders. Consequently, we examined the impact of a systemic inflammatory challenge on kynurenine pathway enzyme expression in rat brain. Indoleamine 2,3-dioxygenase (IDO) expression was induced in cortex and hippocampus following systemic lipopolysaccharide (LPS) administration. Whilst IDO expression was paralleled by increased circulating interferon (IFN)-gamma concentrations, IFN-gamma expression in the brain was only modestly altered following LPS administration. In contrast, induction of IDO was associated with increased central tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 expression. Similarly, in cultured glial cells LPS-induced IDO expression was accompanied by increased TNF-alpha and IL-6 expression, whereas IFN-gamma was not detectable. These findings indicate that IFN-gamma is not required for LPS-induced IDO expression in brain. A robust increase in kynurenine-3-monooxygenase (KMO) expression was observed in rat brain 24h post LPS, without any change in kynurenine aminotransferase II (KAT II) expression. In addition, we report that constitutive expression of KAT II is approximately 8-fold higher than KMO in cortex and 20-fold higher in hippocampus. Similarly, in glial cells constitutive expression of KAT II was approximately 16-fold higher than KMO, and expression of KMO but not KAT II was induced by LPS. These data are the first to demonstrate that a systemic inflammatory challenge stimulates KMO expression in brain; a situation that is likely to favour kynurenine metabolism in a neurotoxic direction. However, our observation that expression of KAT II is much higher than KMO in rat brain is likely to counteract potential neurotoxicity that could arise from KMO induction following an acute inflammation.

  16. Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

    Science.gov (United States)

    Poda, Suresh B; Kobayashi, Masakazu; Nachane, Ruta; Menon, Veena; Gandhi, Adarsh S; Budac, David P; Li, Guiying; Campbell, Brian M; Tagmose, Lena

    2015-10-01

    Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-β-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

  17. Use of isotope effects to characterize intermediates in mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine

    International Nuclear Information System (INIS)

    Bossard, M.J.; Klinman, J.P.

    1990-01-01

    A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which bound alpha-aminoacetophenone is generated followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the inhibitory species. Based on the assumption that the ketone radical is the inhibitory intermediate, an analogous system was predicted and verified. In the present study, the role of alpha-aminoacetophenone as the proposed intermediate in the inactivation by beta-chlorophenethylamine was examined in greater detail. From the interdependence of tyramine and alpha-aminoacetophenone concentrations, ketone inactivation is concluded to occur at the substrate site as opposed to potential binding at the reductant-binding site. Using beta-[2-1H]- and beta-[2-2H]chlorophenethylamine, the magnitude of the deuterium isotope effect on inactivation under second-order conditions has been found to be identical to that observed under catalytic turnover, D(kappa inact/Ki) = D(kappa cat/Km) = 6-7. By contrast, the isotope effect on inactivation under conditions of substrate and oxygen saturation, D kappa inact = 2, is 3-fold smaller than that seen on catalytic turnover, D kappa cat = 6. This reduced isotope effect for inactivation is attributed to a normal isotope effect on substrate hydroxylation followed by an inverse isotope effect on the partitioning of the enol of alpha-aminoacetophenone between oxidation to a radical cation versus protonation to regenerate ketone. These findings are unusual in that two isotopically sensitive steps are present in the inactivation pathway whereas only one is observable in turnover

  18. Structural and molecular dynamics studies of a C1-oxidizing lytic polysaccharide monooxygenase from Heterobasidion irregulare reveal amino acids important for substrate recognition

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bing [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Kognole, Abhishek A. [Department of Chemical and Materials Engineering, University of Kentucky, Lexington KY USA; Wu, Miao [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Westereng, Bjørge [Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ås Norway; Crowley, Michael F. [Biosciences Center, National Renewable Energy Laboratory, Golden CO USA; Kim, Seonah [Biosciences Center, National Renewable Energy Laboratory, Golden CO USA; Dimarogona, Maria [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden; Department of Chemical Engineering, University of Patras, Greece; Payne, Christina M. [Department of Chemical and Materials Engineering, University of Kentucky, Lexington KY USA; Directorate of Engineering, Division of Chemical, Bioengineering, Environmental, and Transport Systems, National Science Foundation, Alexandria VA USA; Sandgren, Mats [Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala Sweden

    2018-04-24

    Lytic polysaccharide monooxygenases (LPMOs) are a group of recently discovered enzymes that play important roles in the decomposition of recalcitrant polysaccharides. Here, we report the biochemical, structural, and computational characterization of an LPMO from the white-rot fungus Heterobasidion irregulare (HiLPMO9B). This enzyme oxidizes cellulose at the C1 carbon of glycosidic linkages. The crystal structure of HiLPMO9B was determined at 2.1 A resolution using X-ray crystallography. Unlike the majority of the currently available C1-specific LPMO structures, the HiLPMO9B structure contains an extended L2 loop, connecting ..beta..-strands ..beta..2 and ..beta..3 of the ..beta..-sandwich structure. Molecular dynamics (MD) simulations suggest roles for both aromatic and acidic residues in the substrate binding of HiLPMO9B, with the main contribution from the residues located on the extended region of the L2 loop (Tyr20) and the LC loop (Asp205, Tyr207, and Glu210). Asp205 and Glu210 were found to be involved in the hydrogen bonding with the hydroxyl group of the C6 carbon of glucose moieties directly or via a water molecule. Two different binding orientations were observed over the course of the MD simulations. In each orientation, the active-site copper of this LPMO preferentially skewed toward the pyranose C1 of the glycosidic linkage over the targeted glycosidic bond. This study provides additional insight into cellulose binding by C1-specific LPMOs, giving a molecular-level picture of active site substrate interactions.

  19. Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

    Science.gov (United States)

    Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

    2018-02-01

    1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

  20. The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3.

    Science.gov (United States)

    Nikodinovic-Runic, Jasmina; Coulombel, Lydie; Francuski, Djordje; Sharma, Narain D; Boyd, Derek R; Ferrall, Rory Moore O; O'Connor, Kevin E

    2013-06-01

    Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min(-1) g cell dry weight(-1) resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite (S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[b]thiophene and 2-methylbenzo[b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group.

  1. Spatiotemporal Dynamics of Ammonia-Oxidizing Thaumarchaeota in Distinct Arctic Water Masses

    Directory of Open Access Journals (Sweden)

    Oliver Müller

    2018-01-01

    Full Text Available One of the most abundant archaeal groups on Earth is the Thaumarchaeota. They are recognized as major contributors to marine ammonia oxidation, a crucial step in the biogeochemical cycling of nitrogen. Their universal success is attributed to a high genomic flexibility and niche adaptability. Based on differences in the gene coding for ammonia monooxygenase subunit A (amoA, two different ecotypes with distinct distribution patterns in the water column have been identified. We used high-throughput sequencing of 16S rRNA genes combined with archaeal amoA functional gene clone libraries to investigate which environmental factors are driving the distribution of Thaumarchaeota ecotypes in the Atlantic gateway to the Arctic Ocean through an annual cycle in 2014. We observed the characteristic vertical pattern of Thaumarchaeota abundance with high values in the mesopelagic (>200 m water throughout the entire year, but also in the epipelagic (<200 m water during the dark winter months (January, March and November. The Thaumarchaeota community was dominated by three OTUs which on average comprised 76% ± 11 and varied in relative abundance according to water mass characteristics and not to depth or ammonium concentration, as suggested in previous studies. The ratios of the abundance of the different OTU types were similar to that of the functional amoA water cluster types. Together, this suggests a strong selection of ecotypes within different water masses, supporting the general idea of water mass characteristics as an important factor in defining microbial community structure. If indeed, as suggested in this study, Thaumarchaeota population dynamics are controlled by a set of factors, described here as water mass characteristics and not just depth alone, then changes in water mass flow will inevitably affect the distribution of the different ecotypes.

  2. Community Composition and Transcriptional Activity of Ammonia-Oxidizing Prokaryotes of Seagrass Thalassia hemprichii in Coral Reef Ecosystems

    Directory of Open Access Journals (Sweden)

    Juan Ling

    2018-01-01

    Full Text Available Seagrasses in coral reef ecosystems play important ecological roles by enhancing coral reef resilience under ocean acidification. However, seagrass primary productivity is typically constrained by limited nitrogen availability. Ammonia oxidation is an important process conducted by ammonia-oxidizing archaea (AOA and bacteria (AOB, yet little information is available concerning the community structure and potential activity of seagrass AOA and AOB. Therefore, this study investigated the variations in the abundance, diversity and transcriptional activity of AOA and AOB at the DNA and transcript level from four sample types: the leaf, root, rhizosphere sediment and bulk sediment of seagrass Thalassia hemprichii in three coral reef ecosystems. DNA and complementary DNA (cDNA were used to prepare clone libraries and DNA and cDNA quantitative PCR (qPCR assays, targeting the ammonia monooxygenase-subunit (amoA genes as biomarkers. Our results indicated that the closest relatives of the obtained archaeal and bacterial amoA gene sequences recovered from DNA and cDNA libraries mainly originated from the marine environment. Moreover, all the obtained AOB sequences belong to the Nitrosomonadales cluster. Nearly all the AOA communities exhibited higher diversity than the AOB communities at the DNA level, but the qPCR data demonstrated that the abundances of AOB communities were higher than that of AOA communities based on both DNA and RNA transcripts. Collectively, most of the samples shared greater community composition similarity with samples from the same location rather than sample type. Furthermore, the abundance of archaeal amoA gene in rhizosphere sediments showed significant relationships with the ammonium concentration of sediments and the nitrogen content of plant tissue (leaf and root at the DNA level (P < 0.05. Conversely, no such relationships were found for the AOB communities. This work provides new insight into the nitrogen cycle

  3. Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

    Directory of Open Access Journals (Sweden)

    Ravid Rivka

    2008-05-01

    Full Text Available Abstract Background Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD, Parkinson's disease (PD and dementia with Lewy bodies (DLB. Quantitative real-time PCR (RT qPCR is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. Results The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], β-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA] in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan® Gene Expression Assays. Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. Conclusion This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects.

  4. Regulation of the cytochrome P450 2A genes

    International Nuclear Information System (INIS)

    Su Ting; Ding Xinxin

    2004-01-01

    Cytochrome P450 monooxygenases of the CYP2A subfamily play important roles in xenobiotic disposition in the liver and in metabolic activation in extrahepatic tissues. Many of the CYP2A transcripts and enzymes are inducible by xenobiotic compounds, and the expression of at least some of the CYP2A genes is influenced by physiological status, such as circadian rhythm, and pathological conditions, such as inflammation, microbial infection, and tumorigenesis. Variability in the expression of the CYP2A genes, which differs by species, animal strain, gender, and organ, may alter the risks of chemical toxicity for numerous compounds that are CYP2A substrates. The mechanistic bases of these variabilities are generally not well understood. However, recent studies have yielded interesting findings in several areas, such as the role of nuclear factor 1 in the tissue-selective expression of CYP2A genes in the olfactory mucosa (OM); the roles of constitutive androstane receptor, pregnane X receptor (PXR), and possibly, peroxisome proliferator-activated receptors in transcriptional regulation of the Cyp2a5 gene; and the involvement of heterogeneous nuclear ribonucleoprotein A1 in pyrazole-induced stabilization of CYP2A5 mRNA. The aims of this minireview are to summarize current knowledge of the regulation of the CYP2A genes in rodents and humans, and to stimulate further mechanistic studies that will ultimately improve our ability to determine, and to understand, these variabilities in humans

  5. Nitrosospira sp. Govern Nitrous Oxide Emissions in a Tropical Soil Amended With Residues of Bioenergy Crop

    Directory of Open Access Journals (Sweden)

    Késia S. Lourenço

    2018-04-01

    Full Text Available Organic vinasse, a residue produced during bioethanol production, increases nitrous oxide (N2O emissions when applied with inorganic nitrogen (N fertilizer in soil. The present study investigated the role of the ammonia-oxidizing bacteria (AOB community on the N2O emissions in soils amended with organic vinasse (CV: concentrated and V: non-concentrated plus inorganic N fertilizer. Soil samples and N2O emissions were evaluated at 11, 19, and 45 days after fertilizer application, and the bacterial and archaea gene (amoA encoding the ammonia monooxygenase enzyme, bacterial denitrifier (nirK, nirS, and nosZ genes and total bacteria were quantified by real time PCR. We also employed a deep amoA amplicon sequencing approach to evaluate the effect of treatment on the community structure and diversity of the soil AOB community. Both vinasse types applied with inorganic N application increased the total N2O emissions and the abundance of AOB. Nitrosospira sp. was the dominant AOB in the soil and was correlated with N2O emissions. However, the diversity and the community structure of AOB did not change with vinasse and inorganic N fertilizer amendment. The results highlight the importance of residues and fertilizer management in sustainable agriculture and can be used as a reference and an input tool to determine good management practices for organic fertilization.

  6. Microbial community changes along the active seepage site of one cold seep in the Red Sea.

    KAUST Repository

    Cao, Huiluo

    2015-07-21

    The active seepage of the marine cold seeps could be a critical process for the exchange of energy between the submerged geosphere and the sea floor environment through organic-rich fluids, potentially even affecting surrounding microbial habitats. However, few studies have investigated the associated microbial community changes. In the present study, 16S rRNA genes were pyrosequenced to decipher changes in the microbial communities from the Thuwal seepage point in the Red Sea to nearby marine sediments in the brine pool, normal marine sediments and water, and benthic microbial mats. An unexpected number of reads from unclassified groups were detected in these habitats; however, the ecological functions of these groups remain unresolved. Furthermore, ammonia-oxidizing archaeal community structures were investigated using the ammonia monooxygenase subunit A (amoA) gene. Analysis of amoA showed that planktonic marine habitats, including seeps and marine water, hosted archaeal ammonia oxidizers that differed from those in microbial mats and marine sediments, suggesting modifications of the ammonia oxidizing archaeal (AOA) communities along the environmental gradient from active seepage sites to peripheral areas. Changes in the microbial community structure of AOA in different habitats (water vs. sediment) potentially correlated with changes in salinity and oxygen concentrations. Overall, the present results revealed for the first time unanticipated novel microbial groups and changes in the ammonia-oxidizing archaea in response to environmental gradients near the active seepages of a cold seep.

  7. Microbial community changes along the active seepage site of one cold seep in the Red Sea.

    KAUST Repository

    Cao, Huiluo; Zhang, Weipeng; Wang, Yong; Qian, Pei-Yuan

    2015-01-01

    The active seepage of the marine cold seeps could be a critical process for the exchange of energy between the submerged geosphere and the sea floor environment through organic-rich fluids, potentially even affecting surrounding microbial habitats. However, few studies have investigated the associated microbial community changes. In the present study, 16S rRNA genes were pyrosequenced to decipher changes in the microbial communities from the Thuwal seepage point in the Red Sea to nearby marine sediments in the brine pool, normal marine sediments and water, and benthic microbial mats. An unexpected number of reads from unclassified groups were detected in these habitats; however, the ecological functions of these groups remain unresolved. Furthermore, ammonia-oxidizing archaeal community structures were investigated using the ammonia monooxygenase subunit A (amoA) gene. Analysis of amoA showed that planktonic marine habitats, including seeps and marine water, hosted archaeal ammonia oxidizers that differed from those in microbial mats and marine sediments, suggesting modifications of the ammonia oxidizing archaeal (AOA) communities along the environmental gradient from active seepage sites to peripheral areas. Changes in the microbial community structure of AOA in different habitats (water vs. sediment) potentially correlated with changes in salinity and oxygen concentrations. Overall, the present results revealed for the first time unanticipated novel microbial groups and changes in the ammonia-oxidizing archaea in response to environmental gradients near the active seepages of a cold seep.

  8. Network analysis of ChIP-Seq data reveals key genes in prostate cancer.

    Science.gov (United States)

    Zhang, Yu; Huang, Zhen; Zhu, Zhiqiang; Liu, Jianwei; Zheng, Xin; Zhang, Yuhai

    2014-09-03

    Prostate cancer (PC) is the second most common cancer among men in the United States, and it imposes a considerable threat to human health. A deep understanding of its underlying molecular mechanisms is the premise for developing effective targeted therapies. Recently, deep transcriptional sequencing has been used as an effective genomic assay to obtain insights into diseases and may be helpful in the study of PC. In present study, ChIP-Seq data for PC and normal samples were compared, and differential peaks identified, based upon fold changes (with P-values calculated with t-tests). Annotations of these peaks were performed. Protein-protein interaction (PPI) network analysis was performed with BioGRID and constructed with Cytoscape, following which the highly connected genes were screened. We obtained a total of 5,570 differential peaks, including 3,726 differentially enriched peaks in tumor samples and 1,844 differentially enriched peaks in normal samples. There were eight significant regions of the peaks. The intergenic region possessed the highest score (51%), followed by intronic (31%) and exonic (11%) regions. The analysis revealed the top 35 highly connected genes, which comprised 33 differential genes (such as YWHAQ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein and θ polypeptide) from ChIP-Seq data and 2 differential genes retrieved from the PPI network: UBA52 (ubiquitin A-52 residue ribosomal protein fusion product (1) and SUMO2 (SMT3 suppressor of mif two 3 homolog (2) . Our findings regarding potential PC-related genes increase the understanding of PC and provides direction for future research.

  9. Gene Therapy

    Science.gov (United States)

    Gene therapy Overview Gene therapy involves altering the genes inside your body's cells in an effort to treat or stop disease. Genes contain your ... that don't work properly can cause disease. Gene therapy replaces a faulty gene or adds a new ...

  10. Organization of genes responsible for the stereospecific conversion of hydantoins to alpha-amino acids in Arthrobacter aurescens DSM 3747.

    Science.gov (United States)

    Wiese, A; Syldatk, C; Mattes, R; Altenbuchner, J

    2001-09-01

    Arthrobacter aurescens DSM 3747 hydrolyzes stereospecifically 5'-monosubstituted hydantoins to alpha-amino acids. The genes involved in hydantoin utilization (hyu) were isolated on an 8.7-kb DNA fragment, and by DNA sequence analysis eight ORFs were identified. The hyu gene cluster includes four genes: hyuP encoding a putative transport protein, the hydantoin racemase gene hyuA, the hydantoinase gene hyuH, and the carbamoylase gene hyuC. The four genes are transcribed in the same direction. Upstream of hyuP and in opposite orientation to the hyu genes, three ORFs were found showing similarities to cytochrome P450 monooxygenase (ORF1, incomplete), to membrane proteins (ORF2), and to ferredoxin (ORF3). ORF8 was found downstream of hyuC and again in opposite orientation to the hyu genes. The gene product of ORF8 displayed similarities to the LacI/GalR family of transcriptional regulators. Reverse transcriptase PCR experiments and Northern blot analysis revealed that the genes hyuPAHC are coexpressed in A. aurescens after induction with 3-N-CH3-IMH. The expression of the hyu operon was not regulated by the putative regulator ORF8 as shown by gene disruption and mobility-shift experiments.

  11. Response of ammonia oxidizing archaea and bacteria to decabromodiphenyl ether and copper contamination in river sediments.

    Science.gov (United States)

    Wang, Linqiong; Li, Yi; Niu, Lihua; Zhang, Wenlong; Zhang, Huanjun; Wang, Longfei; Wang, Peifang

    2018-01-01

    Ammonia oxidation plays a fundamental role in river nitrogen cycling ecosystems, which is normally governed by both ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB). Co-contamination of typical emerging pollutant Polybrominated diphenyl ethers (PBDEs) and heavy metal on AOA and AOB communities in river sediments remains unknown. In this study, multiple analytical tools, including high-throughput pyrosequencing and real-time quantitative PCR (qPCR), were used to reveal the ammonia monooxygenase (AMO) activity, subunit alpha (amoA) gene abundance, and community structures of AOA and AOB in river sediments. It was found that the inhibition of AMO activities was increased with the increase of decabromodiphenyl ether (BDE 209, 1-100 mg kg -1 ) and copper (Cu, 50-500 mg kg -1 ) concentrations. Moreover, the synergic effects of BDE 209 and Cu resulted in a higher AMO activity reduction than the individual pollutant BDE 209. The AOA amoA copy number declined by 75.9% and 83.2% and AOB amoA gene abundance declined 82.8% and 90.0% at 20 and 100 mg kg -1 BDE 209 with a 100 mg kg -1 Cu co-contamination, respectively. The pyrosequencing results showed that both AOB and AOA community structures were altered, with a higher change of AOB than that of AOA. The results demonstrated that the AOB microbial community may be better adapted to BDE 209 and Cu pollution, while AOA might possess a greater capacity for stress resistance. Our study provides a better understanding of the ecotoxicological effects of heavy metal and micropollutant combined exposure on AOA and AOB in river sediments. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Characterization of the expression of the thcB gene, coding for a pesticide-degrading cytochrome P-450 in Rhodococcus strains.

    OpenAIRE

    Shao, Z Q; Behki, R

    1996-01-01

    A cytochrome P-450 system in Rhodococcus strains, encoded by thcB, thcC, and thcD, participates in the degradation of thiocarbamates and several other pesticides. The regulation of the system was investigated by fusing a truncated lacZ in frame to thcB, the structural gene for the cytochrome P-450 monooxygenase. Analysis of the thcB-lacZ fusion showed that the expression of thcB was 10-fold higher in the presence of the herbicide EPTC (s-ethyl dipropylthiocarbamate). Similar enhancement of th...

  13. Detection of toxic effects of Cd{sup 2+} on different fish species via liver cytochrome P450-dependent monooxygenase activities and FTIR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Henczova, Maria; Deer, Aranka Kiss [University of Szeged, Department of Biochemistry, P.O. Box 533, Szeged (Hungary); Komlosi, Viktoria [Chemical Research Center of the Hungarian Academy of Sciences, Department of Molecular Spectroscopy, P.O. Box 17, Budapest (Hungary); Mink, Janos [Chemical Research Center of the Hungarian Academy of Sciences, Department of Molecular Spectroscopy, P.O. Box 17, Budapest (Hungary); University of Veszprem, Faculty of Information Technology, Research Institute of Chemistry and Process Engineering; Analytical Chemistry Research Group of the Hungarian Academy of Sciences, P.O. Box 158, Veszprem (Hungary)

    2006-06-15

    The in vivo and in vitro effects of Cd{sup 2+} and the CYP1A inductor {beta}-naphthoflavone({beta}-NF) on the hepatic cytochrome P450 (Cyt 450) monooxygenases were studied in silver carp (Hypophthalmichtys molitrix V.), wels (Silurus glanis L.), and carp (Cyprinus carpio). In vivo treatment of carp with a high dose of Cd{sup 2+} (10 mg kg{sup -1}, for 3 days) caused a strong inhibition of 7-ethoxyresorufin-O-deethylase (EROD) and a lower inhibition of 7-ethoxycoumarin-O-deethylase (ECOD) activity. The low-dose cadmium treatment (2 mg kg{sup -1} Cd{sup 2+}, for 6+3 days) resulted in 4-fold increase in EROD and a 3-fold increase in ECOD activity. The combined treatment with Cd{sup 2+} and {beta}-NF in both cases led to a loss of EROD inducibility. The silver carp and wels were treated with 10 mg L{sup -1} Cd{sup 2+} for 72 h in water. The Cyt P450 content in the wels liver microsomes was increased significantly after treatment for 48 h, whereas there was only a slight, not significant increase in Cyt P450 content in the silver carp microsomes. While the Cd{sup 2+} treatment resulted in inhibition of the CYP1A isoenzymes (EROD and ECOD), the APND (aminopyrene-N-demethylase, CYP2B or CYP3A isoenzyme) activity was increased 3- to 4-fold in both fish species. In vitro experiments of the effect of Cd{sup 2+} led to a concentration-dependent inhibition in all three investigated fish species. The ECOD isoenzyme of silver carp was the most sensitive to Cd{sup 2+}. The lowest concentration of Cd{sup 2+} resulted in 50% inhibition. The APND isoenzyme was similarly sensitive to Cd{sup 2+} in all three investigated fish species. The most sensitive species was the wels, and the least sensitive were the carp isoenzyme. FTIR spectroscopy confirmed that cadmium caused damage to the protein structure. These results support the enzyme activity measurements measured in vivo and in vitro. (orig.)

  14. Facile N-oxygenation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by the flavin-containing monooxygenase. A convenient synthesis of tritiated [methyl-3H]-4-phenyl-2,3-dihydropyridinium species

    International Nuclear Information System (INIS)

    Cashman, J.R.

    1988-01-01

    A rapid, efficient procedure useful for the radiosynthesis of [Me- 3 H]-MPDP+ ([methyl- 3 H]-4-phenyl-2,3-dihydropyridinium species) is described. Hog liver microsomes or the highly purified flavin-containing monooxygenase from hog liver quantitatively biotransforms [Me- 3 H]-MPTP to its corresponding radiolabeled N-oxide. For the small-scale synthesis required for radiolabeling procedures, this enzymatic process is superior to H 2 O 2 -mediated N-oxygenation of MPTP. In the presence of 0.5 mM NADPH, 4.5 mM n-octylamine, and 2 microCi [Me- 3 H]-MPTP, the only product detected in extracts from incubations performed with hog liver microsomes or purified hog liver flavin-containing monooxygenase is [Me- 3 H]-MPTP N-oxide. [Me- 3 H]-MPTP N-oxide is almost completely converted to [Me- 3 H]-MPDP+ by the action of trifluoroacetic anhydride. This procedure has the advantage of using a commercially available tritiated starting material, efficient transformations, and easily accomplished purification to afford a rapid synthesis of [Me- 3 H]-MPDP+

  15. The influence of different light quality and benzene on gene expression and benzene degradation of Chlorophytum comosum.

    Science.gov (United States)

    Setsungnern, Arnon; Treesubsuntorn, Chairat; Thiravetyan, Paitip

    2017-11-01

    Benzene, a carcinogenic compound, has been reported as a major indoor air pollutant. Chlorophytum comosum (C. comosum) was reported to be the highest efficient benzene removal plant among other screened plants. Our previous studies found that plants under light conditions could remove gaseous benzene higher than under dark conditions. Therefore, C. comosum exposure to airborne benzene was studied under different light quality at the same light intensity. C. comosum could remove 500 ppm gaseous benzene with the highest efficiency of 68.77% under Blue:Red = 1:1 LED treatments and the lowest one appeared 57.41% under white fluorescent treatment within 8 days. After benzene was uptaken by C. comosum, benzene was oxidized to be phenol in the plant cells by cytochrome P450 monooxygenase system. Then, phenol was catalyzed to be catechol that was confirmed by the up-regulation of phenol 2-monooxygenase (PMO) gene expression. After that, catechol was changed to cic, cis-muconic acid. Interestingly, cis,cis-muconic acid production was found in the plant tissues higher than phenol and catechol. The result confirmed that NADPH-cytochrome P450 reductase (CPR), cytochrome b5 (cyt b5), phenol 2-monooxygenase (PMO) and cytochrome P450 90B1 (CYP90B1) in plant cells were involved in benzene degradation or detoxification. In addition, phenol, catechol, and cis,cis-muconic acid production were found under the Blue-Red LED light conditions higher than under white fluorescent light conditions due to under LED light conditions gave higher NADPH contents. Hence, C. comosum under the Blue-Red LED light conditions had a high potential to remove benzene in a contaminated site. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Effect of benzene and ethylbenzene on the transcription of methyl-tert-butyl ether degradation genes of Methylibium petroleiphilum PM1.

    Science.gov (United States)

    Joshi, Geetika; Schmidt, Radomir; Scow, Kate M; Denison, Michael S; Hristova, Krassimira R

    2016-09-01

    Methyl-tert-butyl ether (MTBE) and its degradation by-product, tert-butyl alcohol (TBA), are widespread contaminants detected frequently in groundwater in California. Since MTBE was used as a fuel oxygenate for almost two decades, leaking underground fuel storage tanks are an important source of contamination. Gasoline components such as BTEX (benzene, toluene, ethylbenzene and xylenes) are often present in mixtures with MTBE and TBA. Investigations of interactions between BTEX and MTBE degradation have not yielded consistent trends, and the molecular mechanisms of BTEX compounds' impact on MTBE degradation are not well understood. We investigated trends in transcription of biodegradation genes in the MTBE-degrading bacterium, Methylibium petroleiphilum PM1 upon exposure to MTBE, TBA, ethylbenzene and benzene as individual compounds or in mixtures. We designed real-time quantitative PCR assays to target functional genes of strain PM1 and provide evidence for induction of genes mdpA (MTBE monooxygenase), mdpJ (TBA hydroxylase) and bmoA (benzene monooxygenase) in response to MTBE, TBA and benzene, respectively. Delayed induction of mdpA and mdpJ transcription occurred with mixtures of benzene and MTBE or TBA, respectively. bmoA transcription was similar in the presence of MTBE or TBA with benzene as in their absence. Our results also indicate that ethylbenzene, previously proposed as an inhibitor of MTBE degradation in some bacteria, inhibits transcription of mdpA, mdpJ and bmoAgenes in strain PM1.

  17. Selection of reference genes in different myocardial regions of an in vivo ischemia/reperfusion rat model for normalization of antioxidant gene expression

    Directory of Open Access Journals (Sweden)

    Vesentini Nicoletta

    2012-02-01

    Full Text Available Abstract Background Changes in cardiac gene expression due to myocardial injury are usually assessed in whole heart tissue. However, as the heart is a heterogeneous system, spatial and temporal heterogeneity is expected in gene expression. Results In an ischemia/reperfusion (I/R rat model we evaluated gene expression of mitochondrial and cytoplasmatic superoxide dismutase (MnSod, Cu-ZnSod and thioredoxin reductase (trxr1 upon short (4 h and long (72 h reperfusion times in the right ventricle (RV, and in the ischemic/reperfused (IRR and the remote region (RR of the left ventricle. Gene expression was assessed by Real-time reverse-transcription quantitative PCR (RT-qPCR. In order to select most stable reference genes suitable for normalization purposes, in each myocardial region we tested nine putative reference genes by geNorm analysis. The genes investigated were: Actin beta (actb, Glyceraldehyde-3-P-dehydrogenase (gapdh, Ribosomal protein L13A (rpl13a, Tyrosine 3-monooxygenase (ywhaz, Beta-glucuronidase (gusb, Hypoxanthine guanine Phosphoribosyltransferase 1 (hprt, TATA binding box protein (tbp, Hydroxymethylbilane synthase (hmbs, Polyadenylate-binding protein 1 (papbn1. According to our findings, most stable reference genes in the RV and RR were hmbs/hprt and hmbs/tbp/hprt respectively. In the IRR, six reference genes were recommended for normalization purposes; however, in view of experimental feasibility limitations, target gene expression could be normalized against the three most stable reference genes (ywhaz/pabp/hmbs without loss of sensitivity. In all cases MnSod and Cu-ZnSod expression decreased upon long reperfusion, the former in all myocardial regions and the latter in IRR alone. trxr1 expression did not vary. Conclusions This study provides a validation of reference genes in the RV and in the anterior and posterior wall of the LV of cardiac ischemia/reperfusion model and shows that gene expression should be assessed separately in

  18. Comammox in drinking water systems.

    Science.gov (United States)

    Wang, Yulin; Ma, Liping; Mao, Yanping; Jiang, Xiaotao; Xia, Yu; Yu, Ke; Li, Bing; Zhang, Tong

    2017-06-01

    The discovery of complete ammonia oxidizer (comammox) has fundamentally upended our perception of the global nitrogen cycle. Here, we reported four metagenome assembled genomes (MAGs) of comammox Nitrospira that were retrieved from metagenome datasets of tap water in Singapore (SG-bin1 and SG-bin2), Hainan province, China (HN-bin3) and Stanford, CA, USA (ST-bin4). Genes of phylogenetically distinct ammonia monooxygenase subunit A (amoA) and hydroxylamine dehydrogenase (hao) were identified in these four MAGs. Phylogenetic analysis based on ribosomal proteins, AmoA, hao and nitrite oxidoreductase (subunits nxrA and nxrB) sequences indicated their close relationships with published comammox Nitrospira. Canonical ammonia-oxidizing microbes (AOM) were also identified in the three tap water samples, ammonia-oxidizing bacteria (AOB) in Singapore's and Stanford's samples and ammonia-oxidizing archaea (AOA) in Hainan's sample. The comammox amoA-like sequences were also detected from some other drinking water systems, and even outnumbered the AOA and AOB amoA-like sequences. The findings of MAGs and the occurrences of AOM in different drinking water systems provided a significant clue that comammox are widely distributed in drinking water systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Community Composition of Nitrous Oxide-Related Genes in Salt Marsh Sediments Exposed to Nitrogen Enrichment.

    Science.gov (United States)

    Angell, John H; Peng, Xuefeng; Ji, Qixing; Craick, Ian; Jayakumar, Amal; Kearns, Patrick J; Ward, Bess B; Bowen, Jennifer L

    2018-01-01

    Salt marshes provide many key ecosystem services that have tremendous ecological and economic value. One critical service is the removal of fixed nitrogen from coastal waters, which limits the negative effects of eutrophication resulting from increased nutrient supply. Nutrient enrichment of salt marsh sediments results in higher rates of nitrogen cycling and, commonly, a concurrent increase in the flux of nitrous oxide, an important greenhouse gas. Little is known, however, regarding controls on the microbial communities that contribute to nitrous oxide fluxes in marsh sediments. To address this disconnect, we generated profiles of microbial communities and communities of micro-organisms containing specific nitrogen cycling genes that encode several enzymes ( amoA, norB, nosZ) related to nitrous oxide flux from salt marsh sediments. We hypothesized that communities of microbes responsible for nitrogen transformations will be structured by nitrogen availability. Taxa that respond positively to high nitrogen inputs may be responsible for the elevated rates of nitrogen cycling processes measured in fertilized sediments. Our data show that, with the exception of ammonia-oxidizing archaea, the community composition of organisms involved in the production and consumption of nitrous oxide was altered under nutrient enrichment. These results suggest that previously measured rates of nitrous oxide production and consumption are likely the result of changes in community structure, not simply changes in microbial activity.

  20. Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

    Science.gov (United States)

    Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

    2015-03-10

    Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification.

  1. Genetic interrelations in the actinomycin biosynthetic gene clusters of Streptomyces antibioticus IMRU 3720 and Streptomyces chrysomallus ATCC11523, producers of actinomycin X and actinomycin C

    Directory of Open Access Journals (Sweden)

    Crnovčić I

    2017-04-01

    biosynthetic gene clusters lack a kynurenine-3-monooxygenase gene necessary for biosynthesis of 3-hydroxy-4-methylanthranilic acid, the building block of the Acm chromophore, which suggests participation of a genome-encoded relevant monooxygenase during Acm biosynthesis in both S. chrysomallus and S. antibioticus. Keywords: actinomycin, actinomycin halves, biosynthesis, Streptomyces chrysomallus, ­Streptomyces anulatus Streptomyces antibioticus, genomes, 3-hydroxy-4-methylanthranilic acid (4-MHA, evolution of biosynthetic gene cluster, genetic transmission of biosynthetic gene cluster, actinomycin C, actinomycin X

  2. Specific microbial gene abundances and soil parameters contribute to C, N, and greenhouse gas process rates after land use change in Southern Amazonian Soils

    Directory of Open Access Journals (Sweden)

    Daniel Renato Lammel

    2015-10-01

    Full Text Available Ecological processes regulating soil carbon (C and nitrogen (N cycles are still poorly understood, especially in the world’s largest agricultural frontier in Southern Amazonia. We analyzed soil parameters in samples from pristine rainforest and after land use change to pasture and crop fields, and correlated them with abundance of functional and phylogenetic marker genes (amoA, nirK, nirS, norB, nosZ, nifH, mcrA, pmoA, and 16S/18S rRNA. Additionally, we integrated these parameters using path analysis and multiple regressions. Following forest removal, concentrations of soil C and N declined, and pH and nutrient levels increased, which influenced microbial abundances and biogeochemical processes. A seasonal trend was observed, suggesting that abundances of microbial groups were restored to near native levels after the dry winter fallow. Integration of the marker gene abundances with soil parameters using path analysis and multiple regressions provided good predictions of biogeochemical processes, such as the fluxes of NO3, N2O, CO2, and CH4. In the wet season, agricultural soil showed the highest abundance of nitrifiers (amoA and Archaea, however forest soils showed the highest abundances of denitrifiers (nirK, nosZ and high N, which correlated with increased N2O emissions. Methanogens (mcrA and methanotrophs (pmoA were more abundant in forest soil, but methane flux was highest in pasture sites, which was related to soil compaction. Rather than analyzing direct correlations, the data integration using multivariate tools provided a better overview of biogeochemical processes. Overall, in the wet season, land use change from forest to agriculture reduced the abundance of different functional microbial groups related to the soil C and N cycles; integrating the gene abundance data and soil parameters provided a comprehensive overview of these interactions. Path analysis and multiple regressions addressed the need for more comprehensive approaches

  3. Metagenomes reveal microbial structures, functional potentials, and biofouling-related genes in a membrane bioreactor.

    Science.gov (United States)

    Ma, Jinxing; Wang, Zhiwei; Li, Huan; Park, Hee-Deung; Wu, Zhichao

    2016-06-01

    Metagenomic sequencing was used to investigate the microbial structures, functional potentials, and biofouling-related genes in a membrane bioreactor (MBR). The results showed that the microbial community in the MBR was highly diverse. Notably, function analysis of the dominant genera indicated that common genes from different phylotypes were identified for important functional potentials with the observation of variation of abundances of genes in a certain taxon (e.g., Dechloromonas). Despite maintaining similar metabolic functional potentials with a parallel full-scale conventional activated sludge (CAS) system due to treating the identical wastewater, the MBR had more abundant nitrification-related bacteria and coding genes of ammonia monooxygenase, which could well explain its excellent ammonia removal in the low-temperature period. Furthermore, according to quantification of the genes involved in exopolysaccharide and extracellular polymeric substance (EPS) protein metabolism, the MBR did not show a much different potential in producing EPS compared to the CAS system, and bacteria from the membrane biofilm had lower abundances of genes associated with EPS biosynthesis and transport compared to the activated sludge in the MBR.

  4. Metagenomic survey of methanesulfonic acid (MSA catabolic genes in an Atlantic Ocean surface water sample and in a partial enrichment

    Directory of Open Access Journals (Sweden)

    Ana C. Henriques

    2016-10-01

    Full Text Available Methanesulfonic acid (MSA is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea.

  5. Salix purpurea Stimulates the Expression of Specific Bacterial Xenobiotic Degradation Genes in a Soil Contaminated with Hydrocarbons.

    Directory of Open Access Journals (Sweden)

    Antoine P Pagé

    Full Text Available The objectives of this study were to uncover Salix purpurea-microbe xenobiotic degradation systems that could be harnessed in rhizoremediation, and to identify microorganisms that are likely involved in these partnerships. To do so, we tested S. purpurea's ability to stimulate the expression of 10 marker microbial oxygenase genes in a soil contaminated with hydrocarbons. In what appeared to be a detoxification rhizosphere effect, transcripts encoding for alkane 1-monooxygenases, cytochrome P450 monooxygenases, laccase/polyphenol oxidases, and biphenyl 2,3-dioxygenase small subunits were significantly more abundant in the vicinity of the plant's roots than in bulk soil. This gene expression induction is consistent with willows' known rhizoremediation capabilities, and suggests the existence of S. purpurea-microbe systems that target many organic contaminants of interest (i.e. C4-C16 alkanes, fluoranthene, anthracene, benzo(apyrene, biphenyl, polychlorinated biphenyls. An enhanced expression of the 4 genes was also observed within the bacterial orders Actinomycetales, Rhodospirillales, Burkholderiales, Alteromonadales, Solirubrobacterales, Caulobacterales, and Rhizobiales, which suggest that members of these taxa are active participants in the exposed partnerships. Although the expression of the other 6 marker genes did not appear to be stimulated by the plant at the community level, signs of additional systems that rest on their expression by members of the orders Solirubrobacterales, Sphingomonadales, Actinomycetales, and Sphingobacteriales were observed. Our study presents the first transcriptomics-based identification of microbes whose xenobiotic degradation activity in soil appears stimulated by a plant. It paints a portrait that contrasts with the current views on these consortia's composition, and opens the door for the development of laboratory test models geared towards the identification of root exudate characteristics that limit the

  6. Clinical value of miR-452-5p expression in lung adenocarcinoma: A retrospective quantitative real-time polymerase chain reaction study and verification based on The Cancer Genome Atlas and Gene Expression Omnibus databases.

    Science.gov (United States)

    Gan, Xiao-Ning; Luo, Jie; Tang, Rui-Xue; Wang, Han-Lin; Zhou, Hong; Qin, Hui; Gan, Ting-Qing; Chen, Gang

    2017-05-01

    The role and mechanism of miR-452-5p in lung adenocarcinoma remain unclear. In this study, we performed a systematic study to investigate the clinical value of miR-452-5p expression in lung adenocarcinoma. The expression of miR-452-5p in 101 lung adenocarcinoma patients was detected by quantitative real-time polymerase chain reaction. The Cancer Genome Atlas and Gene Expression Omnibus databases were joined to verify the expression level of miR-452-5p in lung adenocarcinoma. Via several online prediction databases and bioinformatics software, pathway and network analyses of miR-452-5p target genes were performed to explore its prospective molecular mechanism. The expression of miR-452-5p in lung adenocarcinoma in house was significantly lower than that in adjacent tissues (p < 0.001). Additionally, the expression level of miR-452-5p was negatively correlated with several clinicopathological parameters including the tumor size (p = 0.014), lymph node metastasis (p = 0.032), and tumor-node-metastasis stage (p = 0.036). Data from The Cancer Genome Atlas also confirmed the low expression of miR-452 in lung adenocarcinoma (p < 0.001). Furthermore, reduced expression of miR-452-5p in lung adenocarcinoma (standard mean deviations = -0.393, 95% confidence interval: -0.774 to -0.011, p = 0.044) was validated by a meta-analysis. Five hub genes targeted by miR-452-5p, including SMAD family member 4, SMAD family member 2, cyclin-dependent kinase inhibitor 1B, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta, were significantly enriched in the cell-cycle pathway. In conclusion, low expression of miR-452-5p tends to play an essential role in lung adenocarcinoma. Bioinformatics analysis might be beneficial to reveal the potential mechanism of miR-452-5p in lung adenocarcinoma.

  7. Expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1) and CYP1 family monooxygenase mRNAs and their activity in chicken ovarian follicles following in vitro exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

    Science.gov (United States)

    Antos, Piotr A; Błachuta, Małgorzata; Hrabia, Anna; Grzegorzewska, Agnieszka K; Sechman, Andrzej

    2015-09-02

    The aim of this in vitro study was to determine the effect of TCDD and luteinizing hormone (LH) on mRNA expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1), and the CYP1 family monooxygenases (CYP1A4, CYP1A5, CYP1B1), and to assess the basal and TCDD-induced activity of these enzymes in chicken ovarian follicles. White (WF) and yellowish (YF) prehierarchical follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest preovulatory follicles (F3-F1) were exposed to TCDD (10nM), ovine LH (oLH; 10ng/mL) or a combination of TCDD (10nM) and oLH (10ng/mL), and increasing doses of TCDD (0.01-100nM). AHR1 and ARNT1 mRNA transcripts were found in all examined follicles. The effect of TCDD and oLH on AHR1 and ARNT1 mRNA expression depended on the maturational state of the follicle. CYP1A4 was predominantly expressed in the GL of the F3-F1 follicles; in comparison with the WF, a higher level of CYP1A5 mRNA was found both in the GL and TL of F3-F1 follicles. Alternatively, the highest level of CYP1B1 mRNA was noticed in the WF follicles. In different developmental stages of the follicle TCDD and oLH induced a different CYP1 isoform. TCDD increased EROD and MROD activities in all the investigated ovarian follicles. In conclusion, AHR1 and ARNT1 mRNA expression indicate that the chicken ovary is a target tissue for dioxin and dioxin-like compounds. The expression of CYP1-family genes and TCDD-inducible EROD and MROD activities in ovarian follicles suggest the possibility of xenobiotic detoxification in the chicken ovary. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Gene expression

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

    1983-01-01

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  9. Comparative expression profiling reveals gene functions in female meiosis and gametophyte development in Arabidopsis.

    Science.gov (United States)

    Zhao, Lihua; He, Jiangman; Cai, Hanyang; Lin, Haiyan; Li, Yanqiang; Liu, Renyi; Yang, Zhenbiao; Qin, Yuan

    2014-11-01

    Megasporogenesis is essential for female fertility, and requires the accomplishment of meiosis and the formation of functional megaspores. The inaccessibility and low abundance of female meiocytes make it particularly difficult to elucidate the molecular basis underlying megasporogenesis. We used high-throughput tag-sequencing analysis to identify genes expressed in female meiocytes (FMs) by comparing gene expression profiles from wild-type ovules undergoing megasporogenesis with those from the spl mutant ovules, which lack megasporogenesis. A total of 862 genes were identified as FMs, with levels that are consistently reduced in spl ovules in two biological replicates. Fluorescence-assisted cell sorting followed by RNA-seq analysis of DMC1:GFP-labeled female meiocytes confirmed that 90% of the FMs are indeed detected in the female meiocyte protoplast profiling. We performed reverse genetic analysis of 120 candidate genes and identified four FM genes with a function in female meiosis progression in Arabidopsis. We further revealed that KLU, a putative cytochrome P450 monooxygenase, is involved in chromosome pairing during female meiosis, most likely by affecting the normal expression pattern of DMC1 in ovules during female meiosis. Our studies provide valuable information for functional genomic analyses of plant germline development as well as insights into meiosis. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  10. Pathway and mechanism of nitrogen transformation during composting: Functional enzymes and genes under different concentrations of PVP-AgNPs.

    Science.gov (United States)

    Zeng, Guangming; Zhang, Lihua; Dong, Haoran; Chen, Yaoning; Zhang, Jiachao; Zhu, Yuan; Yuan, Yujie; Xie, Yankai; Fang, Wei

    2018-04-01

    Polyvinylpyrrolidone coated silver nanoparticles (PVP-AgNPs) were applied at different concentrations to reduce total nitrogen (TN) losses and the mechanisms of nitrogen bio-transformation were investigated in terms of the nitrogen functional enzymes and genes. Results showed that mineral N in pile 3 which was treated with AgNPs at a concentration of 10 mg/kg compost was the highest (6.58 g/kg dry weight (DW) compost) and the TN loss (47.07%) was the lowest at the end of composting. Correlation analysis indicated that TN loss was significantly correlated with amoA abundance. High throughput sequencing showed that the dominant family of ammonia-oxidizing bacteria (AOB) was Nitrosomonadaceae, and the number of Operational Taxonomic Units (OTUs) reduced after the beginning of composting when compared with day 1. In summary, treatment with AgNPs at a concentration of 10 mg/kg compost was considerable to reduce TN losses and reserve more mineral N during composting. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Nitrous oxide production and mRNA expression analysis of nitrifying and denitrifying bacterial genes under floodwater disappearance and fertilizer application.

    Science.gov (United States)

    Riya, Shohei; Takeuchi, Yuki; Zhou, Sheng; Terada, Akihiko; Hosomi, Masaaki

    2017-06-01

    A pulse of nitrous oxide (N 2 O) emission has been observed following the disappearance of floodwater by drainage. However, its mechanism is not well understood. We conducted a column study to clarify the mechanism for N 2 O production during floodwater disappearance by using a microsensor and determining the bacterial gene expression. An increase in N 2 O flux was observed following floodwater disappearance after the addition of NH 4 + , with a corresponding increase in the concentrations of NO 3 - and dissolved N 2 O in the oxic and anoxic soil layers, respectively. The transcription level of the bacterial amoA mRNA did not change, while that of nirK mRNA increased sharply after an hour of floodwater disappearance. An additional anoxic soil slurry experiment demonstrated that the addition of NO 3 - induced the expression of nirK gene and caused a concomitant increase in N 2 O production. These findings suggest that NO 3 - production in the oxic layers is important as it provides a substrate and induces the synthesis of denitrification enzymes in the anoxic layer during N 2 O production.

  12. Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection)

    International Nuclear Information System (INIS)

    O'Connor, Meeghan A.; Koza-Taylor, Petra; Campion, Sarah N.; Aleksunes, Lauren M.; Gu, Xinsheng; Enayetallah, Ahmed E.; Lawton, Michael P.; Manautou, José E.

    2014-01-01

    Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400 mg/kg) and then challenged 48 h later with 600 mg APAP/kg. Livers were obtained 4 or 24 h later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430 2 GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection. - Highlights: • Differential expression of genes in mice resistant to acetaminophen hepatotoxicity. • Increased gene expression of Flavin-containing monooxygenase 3 and Galectin-3. • Decrease in MAT1A expression and compensatory hepatocellular regeneration. • Two distinct gene expression

  13. Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection)

    Energy Technology Data Exchange (ETDEWEB)

    O' Connor, Meeghan A., E-mail: meeghan.oconnor@boehringer-ingelheim.com [Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269-3092 (United States); Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, CT 06877-0368 (United States); Koza-Taylor, Petra, E-mail: petra.h.koza-taylor@pfizer.com [Pfizer Inc., Groton, CT 06340 (United States); Campion, Sarah N., E-mail: sarah.campion@pfizer.com [Pfizer Inc., Groton, CT 06340 (United States); Aleksunes, Lauren M., E-mail: aleksunes@eohsi.rutgers.edu [Rutgers University, Department of Pharmacology and Toxicology, Environmental and Occupational Health Sciences Institute, Piscataway, NJ 08854 (United States); Gu, Xinsheng, E-mail: xinsheng.gu@uconn.edu [Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269-3092 (United States); Enayetallah, Ahmed E., E-mail: ahmed.enayetallah@pfizer.com [Pfizer Inc., Groton, CT 06340 (United States); Lawton, Michael P., E-mail: michael.lawton@pfizer.com [Pfizer Inc., Groton, CT 06340 (United States); Manautou, José E., E-mail: jose.manautou@uconn.edu [Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269-3092 (United States)

    2014-01-01

    Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400 mg/kg) and then challenged 48 h later with 600 mg APAP/kg. Livers were obtained 4 or 24 h later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430{sub 2} GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection. - Highlights: • Differential expression of genes in mice resistant to acetaminophen hepatotoxicity. • Increased gene expression of Flavin-containing monooxygenase 3 and Galectin-3. • Decrease in MAT1A expression and compensatory hepatocellular regeneration. • Two distinct gene

  14. Communities of archaea and bacteria in a subsurface radioactive thermal spring in the Austrian Central Alps, and evidence of ammonia-oxidizing Crenarchaeota.

    Science.gov (United States)

    Weidler, Gerhard W; Dornmayr-Pfaffenhuemer, Marion; Gerbl, Friedrich W; Heinen, Wolfgang; Stan-Lotter, Helga

    2007-01-01

    Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (alpha, beta, gamma, and delta), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (delta-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.

  15. Tyrosine hydroxylase (TH), its cofactor tetrahydrobiopterin (BH4), other catecholamine-related enzymes, and their human genes in relation to the drug and gene therapies of Parkinson's disease (PD): historical overview and future prospects.

    Science.gov (United States)

    Nagatsu, Toshiharu; Nagatsu, Ikuko

    2016-11-01

    Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic L-amino acid decarboxylase (AADC); dopamine β-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as L-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.

  16. Transcriptomic and phylogenetic analysis of Culex pipiens quinquefasciatus for three detoxification gene families

    Directory of Open Access Journals (Sweden)

    Yan Liangzhen

    2012-11-01

    Full Text Available Abstract Background The genomes of three major mosquito vectors of human diseases, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus, have been previously sequenced. C. p. quinquefasciatus has the largest number of predicted protein-coding genes, which partially results from the expansion of three detoxification gene families: cytochrome P450 monooxygenases (P450, glutathione S-transferases (GST, and carboxyl/cholinesterases (CCE. However, unlike An. gambiae and Ae. aegypti, which have large amounts of gene expression data, C. p. quinquefasciatus has limited transcriptomic resources. Knowledge of complete gene expression information is very important for the exploration of the functions of genes involved in specific biological processes. In the present study, the three detoxification gene families of C. p. quinquefasciatus were analyzed for phylogenetic classification and compared with those of three other dipteran insects. Gene expression during various developmental stages and the differential expression responsible for parathion resistance were profiled using the digital gene expression (DGE technique. Results A total of 302 detoxification genes were found in C. p. quinquefasciatus, including 71 CCE, 196 P450, and 35 cytosolic GST genes. Compared with three other dipteran species, gene expansion in Culex mainly occurred in the CCE and P450 families, where the genes of α-esterases, juvenile hormone esterases, and CYP325 of the CYP4 subfamily showed the most pronounced expansion on the genome. For the five DGE libraries, 3.5-3.8 million raw tags were generated and mapped to 13314 reference genes. Among 302 detoxification genes, 225 (75% were detected for expression in at least one DGE library. One fourth of the CCE and P450 genes were detected uniquely in one stage, indicating potential developmentally regulated expression. A total of 1511 genes showed different expression levels between a parathion-resistant and a

  17. Trichoderma genes

    Science.gov (United States)

    Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  18. The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells.

    Science.gov (United States)

    Nemoto, Keiichirou; Hara, Masamitsu; Goto, Shingo; Kasai, Kouji; Seki, Hikaru; Suzuki, Masashi; Oka, Atsuhiro; Muranaka, Toshiya; Mano, Yoshihiro

    2009-05-01

    Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.

  19. Identification of differentially expressed genes in brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae) responding to host plant resistance.

    Science.gov (United States)

    Yang, Zhifan; Zhang, Futie; Zhu, Lili; He, Guangcun

    2006-02-01

    The brown planthopper Nilaparvata lugens Stål is one of the major insect pests of rice Oryza sativa L. The host resistance exhibits profound effects on growth, development and propagation of N. lugens. To investigate the molecular response of N. lugens to host resistance, a cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique was employed to identify the differentially expressed genes in the nymphs feeding on three rice varieties. Of the 2,800 cDNA bands analysed, 54 were up-regulated and seven down-regulated qualitatively in N. lugens when the ingestion sources were changed from susceptible rice plants to resistant ones. Sequence analysis of the differential transcript-derived fragments showed that the genes involved in signalling, stress response, gene expression regulation, detoxification and metabolism were regulated by host resistance. Four of the transcript-derived fragments corresponding to genes encoding for a putative B subunit of phosphatase PP2A, a nemo kinase, a cytochrome P450 monooxygenase and a prolyl endopeptidase were further characterized in detail. Northern blot analysis confirmed that the expression of the four genes was enhanced in N. lugens feeding on resistant rice plants. The roles of these genes in the defensive response of N. lugens to host plant resistance were discussed.

  20. Identification of genes and pathways associated with aluminum stress and tolerance using transcriptome profiling of wheat near-isogenic lines.

    Science.gov (United States)

    Houde, Mario; Diallo, Amadou Oury

    2008-08-27

    Aluminum is considered the most limiting factor for plant productivity in acidic soils, which cover large areas of the world's potential arable lands. The inhibition of root growth is recognized as the primary effect of Al toxicity. To identify genes associated with Al stress and tolerance, transcriptome analyses of four different wheat lines (2 Al-tolerant and 2 Al sensitive) that differ in their response to Al were performed. Microarray expression profiling revealed that 83 candidate genes are associated with Al stress and 25 are associated with tolerance. The stress-associated genes include important enzymes such as pyruvate dehydrogenase, alternative oxidase, and galactonolactone oxidase, ABC transporter and ascorbate oxido-reducatase. The Al tolerance-associated genes include the ALMT-1 malate transporter, glutathione S-transferase, germin/oxalate oxidase, fructose 1,6-bisphosphatase, cysteine-rich proteins, cytochrome P450 monooxygenase, cellulose synthase, zinc finger transcription factor, disease resistance response protein and F-box containing domain protein. In this survey, we identified stress- and tolerance-associated genes that may be involved in the detoxification of Al and reactive oxygen species. Alternative pathways could help maintain the supply of important metabolites (H2O2, ascorbate, NADH, and phosphate) needed for Al tolerance and root growth. The Al tolerance-associated genes may be key factors that regulate these pathways.

  1. Identification of genes and pathways associated with aluminum stress and tolerance using transcriptome profiling of wheat near-isogenic lines

    Directory of Open Access Journals (Sweden)

    Diallo Amadou

    2008-08-01

    Full Text Available Abstract Background Aluminum is considered the most limiting factor for plant productivity in acidic soils, which cover large areas of the world's potential arable lands. The inhibition of root growth is recognized as the primary effect of Al toxicity. To identify genes associated with Al stress and tolerance, transcriptome analyses of four different wheat lines (2 Al-tolerant and 2 Al sensitive that differ in their response to Al were performed. Results Microarray expression profiling revealed that 83 candidate genes are associated with Al stress and 25 are associated with tolerance. The stress-associated genes include important enzymes such as pyruvate dehydrogenase, alternative oxidase, and galactonolactone oxidase, ABC transporter and ascorbate oxido-reducatase. The Al tolerance-associated genes include the ALMT-1 malate transporter, glutathione S-transferase, germin/oxalate oxidase, fructose 1,6-bisphosphatase, cysteine-rich proteins, cytochrome P450 monooxygenase, cellulose synthase, zinc finger transcription factor, disease resistance response protein and F-box containing domain protein. Conclusion In this survey, we identified stress- and tolerance-associated genes that may be involved in the detoxification of Al and reactive oxygen species. Alternative pathways could help maintain the supply of important metabolites (H2O2, ascorbate, NADH, and phosphate needed for Al tolerance and root growth. The Al tolerance-associated genes may be key factors that regulate these pathways.

  2. Isolation and characterization of styrene metabolism genes from styrene-assimilating soil bacteria Rhodococcus sp. ST-5 and ST-10.

    Science.gov (United States)

    Toda, Hiroshi; Itoh, Nobuya

    2012-01-01

    Styrene metabolism genes were isolated from styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10. Strain ST-5 had a gene cluster containing four open reading frames which encoded styrene degradation enzymes. The genes showed high similarity to styABCD of Pseudomonas sp. Y2. On the other hand, strain ST-10 had only two genes which encoded styrene monooxygenase and flavin oxidoreductase (styAB). Escherichia coli transformants possessing the sty genes of strains ST-5 and ST-10 produced (S)-styrene oxide from styrene, indicating that these genes function as styrene degradation enzymes. Metabolite analysis by resting-cell reaction with gas chromatography-mass spectrometry revealed that strain ST-5 converts styrene to phenylacetaldehyde via styrene oxide by styrene oxide isomerase (styC) reaction. On the other hand, strain ST-10 lacked this enzyme, and thus accumulated styrene oxide as an intermediate. HPLC analysis showed that styrene oxide was spontaneously isomerized to phenylacetaldehyde by chemical reaction. The produced phenylacetaldehyde was converted to phenylacetic acid (PAA) in strain ST-10 as well as in strain ST-5. Furthermore, phenylacetic acid was converted to phenylacetyl-CoA by the catalysis of phenylacetate-CoA ligase in strains ST-5 and ST-10. This study proposes possible styrene metabolism pathways in Rhodococcus sp. strains ST-5 and ST-10. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

    Science.gov (United States)

    Oba, Yuichi; Ojika, Makoto; Inouye, Satoshi

    2004-03-31

    This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

  4. Functional gene diversity of soil microbial communities from five oil-contaminated fields in China.

    Science.gov (United States)

    Liang, Yuting; Van Nostrand, Joy D; Deng, Ye; He, Zhili; Wu, Liyou; Zhang, Xu; Li, Guanghe; Zhou, Jizhong

    2011-03-01

    To compare microbial functional diversity in different oil-contaminated fields and to know the effects of oil contaminant and environmental factors, soil samples were taken from typical oil-contaminated fields located in five geographic regions of China. GeoChip, a high-throughput functional gene array, was used to evaluate the microbial functional genes involved in contaminant degradation and in other major biogeochemical/metabolic processes. Our results indicated that the overall microbial community structures were distinct in each oil-contaminated field, and samples were clustered by geographic locations. The organic contaminant degradation genes were most abundant in all samples and presented a similar pattern under oil contaminant stress among the five fields. In addition, alkane and aromatic hydrocarbon degradation genes such as monooxygenase and dioxygenase were detected in high abundance in the oil-contaminated fields. Canonical correspondence analysis indicated that the microbial functional patterns were highly correlated to the local environmental variables, such as oil contaminant concentration, nitrogen and phosphorus contents, salt and pH. Finally, a total of 59% of microbial community variation from GeoChip data can be explained by oil contamination, geographic location and soil geochemical parameters. This study provided insights into the in situ microbial functional structures in oil-contaminated fields and discerned the linkages between microbial communities and environmental variables, which is important to the application of bioremediation in oil-contaminated sites.

  5. A comparative study of P450 gene expression in field and laboratory Musca domestica L. strains.

    Science.gov (United States)

    Højland, Dorte H; Vagn Jensen, Karl-Martin; Kristensen, Michael

    2014-08-01

    The housefly is a global pest that has developed resistance to most insecticides applied for its control. Resistance has been associated with cytochrome P450 monooxygenases (P450s). The authors compare the expression of six genes possibly associated with insecticide resistance in three unselected strains: a multiresistant strain (791a), a neonicotinoid-resistant strain (766b) and a new field strain (845b). CYP4G2 was highly expressed throughout the range of strains and proved to be the one of the most interesting expression profiles of all P450s analysed. CYP6G4 was expressed up to 11-fold higher in 766b than in WHO-SRS. Significant differences between expression of P450 genes between F1 flies from 845b and established laboratory strains were shown. In general, P450 gene expression in 845b was 2-14-fold higher than in the reference strain (P resistance. There is a strong indication that CYP6G4 is a major insecticide resistance gene involved in neonicotinoid resistance. © 2013 Society of Chemical Industry.

  6. Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA.

    Science.gov (United States)

    Shirey, Carolyn; Badieyan, Somayesadat; Sobrado, Pablo

    2013-11-08

    SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

  7. Report on a survey in fiscal 1999. Direct oxidation of hydrocarbons by manifestation of functions of methane mono-oxygenase (MMO); 1999 nendo metamonookishinaze (MMO) no kino hatsugen ni yoru tanka suiso no chokusetsu sanka seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    The metallic enzyme, methane mono-oxygenase (MMO) collected from methanotrophic bacteria, may perform a reaction that has a possibility to proceed direct conversion from methane to methanol under normal temperatures and pressures. However, its utilization of biological bacteria makes massive cultivation and handling difficult, not having realized its practical use. Therefore, research and development has been carried out on a process that can convert directly and selectively hydrocarbons including methane under normal temperatures and pressures, mimicking the excellent functions of MMO. To achieve the development, surveys and discussions were given on the following elementary researches: elucidation of the reaction mechanism in the activation point in microorganism enzymes; analysis of structures in microorganism MMO; creation of a technology to develop a bio-mimetic catalyst; improvement in selectivity of the bio-mimetic catalyst; and international joint research (basic analysis of the catalyst mechanism). As a result, technological problems in developing the mimetic catalyst were put into order, and guidelines and measures for specific catalyst designing are being proposed. Furthermore, a way was opened for international joint research with the complex synthesis research group in CNRS in France, and progress into the step of demonstrating and discussing the feasibility thereof is now ready. (NEDO)

  8. Land-use systems affect Archaeal community structure and functional diversity in western Amazon soils

    Directory of Open Access Journals (Sweden)

    Acácio Aparecido Navarrete

    2011-10-01

    Full Text Available The study of the ecology of soil microbial communities at relevant spatial scales is primordial in the wide Amazon region due to the current land use changes. In this study, the diversity of the Archaea domain (community structure and ammonia-oxidizing Archaea (richness and community composition were investigated using molecular biology-based techniques in different land-use systems in western Amazonia, Brazil. Soil samples were collected in two periods with high precipitation (March 2008 and January 2009 from Inceptisols under primary tropical rainforest, secondary forest (5-20 year old, agricultural systems of indigenous people and cattle pasture. Denaturing gradient gel electrophoresis of polymerase chain reaction-amplified DNA (PCR-DGGE using the 16S rRNA gene as a biomarker showed that archaeal community structures in crops and pasture soils are different from those in primary forest soil, which is more similar to the community structure in secondary forest soil. Sequence analysis of excised DGGE bands indicated the presence of crenarchaeal and euryarchaeal organisms. Based on clone library analysis of the gene coding the subunit of the enzyme ammonia monooxygenase (amoA of Archaea (306 sequences, the Shannon-Wiener function and Simpson's index showed a greater ammonia-oxidizing archaeal diversity in primary forest soils (H' = 2.1486; D = 0.1366, followed by a lower diversity in soils under pasture (H' = 1.9629; D = 0.1715, crops (H' = 1.4613; D = 0.3309 and secondary forest (H' = 0.8633; D = 0.5405. All cloned inserts were similar to the Crenarchaeota amoA gene clones (identity > 95 % previously found in soils and sediments and distributed primarily in three major phylogenetic clusters. The findings indicate that agricultural systems of indigenous people and cattle pasture affect the archaeal community structure and diversity of ammonia-oxidizing Archaea in western Amazon soils.

  9. Microbial community structure of relict niter-beds previously used for saltpeter production.

    Directory of Open Access Journals (Sweden)

    Takashi Narihiro

    Full Text Available From the 16th to the 18th centuries in Japan, saltpeter was produced using a biological niter-bed process and was formed under the floor of gassho-style houses in the historic villages of Shirakawa-go and Gokayama, which are classified as United Nations Educational, Scientific and Cultural Organization (UNESCO World Heritage Sites. The relict niter-beds are now conserved in the underfloor space of gassho-style houses, where they are isolated from destabilizing environmental factors and retain the ability to produce nitrate. However, little is known about the nitrifying microbes in such relict niter-bed ecosystems. In this study, the microbial community structures within nine relict niter-bed soils were investigated using 454 pyrotag analysis targeting the 16S rRNA gene and the bacterial and archaeal ammonia monooxygenase gene (amoA. The 16S rRNA gene pyrotag analysis showed that members of the phyla Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes, and Planctomycetes were major microbial constituents, and principal coordinate analysis showed that the NO3-, Cl-, K+, and Na+ contents were potential determinants of the structures of entire microbial communities in relict niter-bed soils. The bacterial and archaeal amoA libraries indicated that members of the Nitrosospira-type ammonia-oxidizing bacteria (AOB and "Ca. Nitrososphaera"-type ammonia-oxidizing archaea (AOA, respectively, predominated in relict niter-bed soils. In addition, soil pH and organic carbon content were important factors for the ecological niche of AOB and AOA in relict niter-bed soil ecosystems.

  10. Use of gene probes to assess the impact and effectiveness of aerobic in situ bioremediation of TCE

    Energy Technology Data Exchange (ETDEWEB)

    Hazen, Terry C.; Chakraborty, Romy; Fleming, James M.; Gregory, Ingrid R.; Bowman, John P.; Jimenez, Luis; Zhang, Dai; Pfiffner, Susan M.; Brockman, Fred J.; Sayler, Gary S.

    2009-03-15

    Gene probe hybridization was used to determine distribution and expression of co-metabolic genes at a contaminated site as it underwent in situ methanotrophic bioremediation of trichloroethylene (TCE). The bioremediation strategies tested included a series of air, air:methane, and air:methane:nutrient pulses of the test plot using horizontal injection wells. During the test period, the levels of TCE reduced drastically in almost all test samples. Sediment core samples (n = 367) taken from 0 m (surface)-43 m depth were probed for gene coding for methanotrophic soluble methane monooxygenase (sMMO) and heterotrophic toluene dioxygenase (TOD), which are known to co-metabolize TCE. The same sediment samples were also probed for genes coding for methanol dehydrogenase (MDH) (catalyzing the oxidation of methanol to formaldehyde) to assess specifically changes in methylotrophic bacterial populations in the site. Gene hybridization results showed that the frequency of detection of sMMO genes were stimulated approximately 250% following 1% methane:air (v/v) injection. Subsequent injection of 4% methane:air (v/v) resulted in an 85% decline probably due to nutrient limitations, since addition of nutrients (gaseous nitrogen and phosphorus) thereafter caused an increase in the frequency of detection of sMMO genes. Detection of TOD genes declined during the process, and eventually they were non-detectable by the final treatment, suggesting that methanotrophs displaced the TOD gene containing heterotrophs. Active transcription of sMMO and TOD was evidenced by hybridization to mRNA. These analyses combined with results showing the concomitant decline in TCE concentrations, increases in chloride concentration and increases in methanotroph viable counts, provide multiple lines of evidence that TCE remediation was caused specifically by methanotrophs. Our results suggest that sMMO genes are responsible for most, if not all, of the observed biodegradation of TCE. This study

  11. Ageing genes

    DEFF Research Database (Denmark)

    Rattan, Suresh

    2018-01-01

    The idea of gerontogenes is in line with the evolutionary explanation of ageing as being an emergent phenomenon as a result of the imperfect maintenance and repair systems. Although evolutionary processes did not select for any specific ageing genes that restrict and determine the lifespan...... of an individual, the term ‘gerontogenes’ primarily refers to any genes that may seem to influence ageing and longevity, without being specifically selected for that role. Such genes can also be called ‘virtual gerontogenes’ by virtue of their indirect influence on the rate and process of ageing. More than 1000...... virtual gerontogenes have been associated with ageing and longevity in model organisms and humans. The ‘real’ genes, which do influence the essential lifespan of a species, and have been selected for in accordance with the evolutionary life history of the species, are known as the longevity assurance...

  12. Molecular dynamics of detoxification and toxin-tolerance genes in brown planthopper (Nilaparvata lugens Stål., Homoptera: Delphacidae) feeding on resistant rice plants.

    Science.gov (United States)

    Yang, Zhifan; Zhang, Futie; He, Qing; He, Guangcun

    2005-06-01

    To investigate the molecular response of brown planthopper, Nilaparvata lugens (BPH) to BPH-resistant rice plants, we isolated cDNA fragments of the genes encoding for carboxylesterase (CAR), trypsin (TRY), cytochrome P450 monooxygenase (P450), NADH-quinone oxidoreductase (NQO), acetylcholinesterase (ACE), and Glutathione S-transferase (GST). Expression profiles of the genes were monitored on fourth instar nymphs feeding on rice varieties with different resistance levels. Northern blot hybridization showed that, compared with BPH reared on susceptible rice TN1, expression of the genes for P450 and CAR was apparently up-regulated and TRY mRNA decreased in BPH feeding on a highly resistant rice line B5 and a moderately resistant rice variety MH63, respectively. Two transcripts of GST increased in BPH feeding on B5; but in BPH feeding on MH63, this gene was inducible and its expression reached a maximum level at 24 h, and then decreased slightly. The expression of NQO gene was enhanced in BPH on B5 plants but showed a constant expression in BPH on MH63 plants. No difference in ACE gene expression among BPH on different rice plants was detected by the RT-PCR method. The results suggest these genes may play important roles in the defense response of BPH to resistant rice.

  13. Network analysis of inflammatory genes and their transcriptional regulators in coronary artery disease.

    Directory of Open Access Journals (Sweden)

    Jiny Nair

    Full Text Available Network analysis is a novel method to understand the complex pathogenesis of inflammation-driven atherosclerosis. Using this approach, we attempted to identify key inflammatory genes and their core transcriptional regulators in coronary artery disease (CAD. Initially, we obtained 124 candidate genes associated with inflammation and CAD using Polysearch and CADgene database for which protein-protein interaction network was generated using STRING 9.0 (Search Tool for the Retrieval of Interacting Genes and visualized using Cytoscape v 2.8.3. Based on betweenness centrality (BC and node degree as key topological parameters, we identified interleukin-6 (IL-6, vascular endothelial growth factor A (VEGFA, interleukin-1 beta (IL-1B, tumor necrosis factor (TNF and prostaglandin-endoperoxide synthase 2 (PTGS2 as hub nodes. The backbone network constructed with these five hub genes showed 111 nodes connected via 348 edges, with IL-6 having the largest degree and highest BC. Nuclear factor kappa B1 (NFKB1, signal transducer and activator of transcription 3 (STAT3 and JUN were identified as the three core transcription factors from the regulatory network derived using MatInspector. For the purpose of validation of the hub genes, 97 test networks were constructed, which revealed the accuracy of the backbone network to be 0.7763 while the frequency of the hub nodes remained largely unaltered. Pathway enrichment analysis with ClueGO, KEGG and REACTOME showed significant enrichment of six validated CAD pathways - smooth muscle cell proliferation, acute-phase response, calcidiol 1-monooxygenase activity, toll-like receptor signaling, NOD-like receptor signaling and adipocytokine signaling pathways. Experimental verification of the above findings in 64 cases and 64 controls showed increased expression of the five candidate genes and the three transcription factors in the cases relative to the controls (p<0.05. Thus, analysis of complex networks aid in the

  14. MOXD2, a Gene Possibly Associated with Olfaction, Is Frequently Inactivated in Birds.

    Directory of Open Access Journals (Sweden)

    Chul Jun Goh

    Full Text Available Vertebrate MOXD2 encodes a monooxygenase DBH-like 2 protein that could be involved in neurotransmitter metabolism, potentially during olfactory transduction. Loss of MOXD2 in apes and whales has been proposed to be associated with evolution of olfaction in these clades. We analyzed 57 bird genomes to identify MOXD2 sequences and found frequent loss of MOXD2 in 38 birds. Among the 57 birds, 19 species appeared to have an intact MOXD2 that encoded a full-length protein; 32 birds had a gene with open reading frame-disrupting point mutations and/or exon deletions; and the remaining 6 species did not show any MOXD2 sequence, suggesting a whole-gene deletion. Notably, among 10 passerine birds examined, 9 species shared a common genomic deletion that spanned several exons, implying the gene loss occurred in a common ancestor of these birds. However, 2 closely related penguin species, each of which had an inactive MOXD2, did not share any mutation, suggesting an independent loss after their divergence. Distribution of the 38 birds without an intact MOXD2 in the bird phylogenetic tree clearly indicates that MOXD2 loss is widespread and independent in bird lineages. We propose that widespread MOXD2 loss in some bird lineages may be implicated in the evolution of olfactory perception in these birds.

  15. MOXD2, a Gene Possibly Associated with Olfaction, Is Frequently Inactivated in Birds.

    Science.gov (United States)

    Goh, Chul Jun; Choi, Dongjin; Park, Dong-Bin; Kim, Hyein; Hahn, Yoonsoo

    2016-01-01

    Vertebrate MOXD2 encodes a monooxygenase DBH-like 2 protein that could be involved in neurotransmitter metabolism, potentially during olfactory transduction. Loss of MOXD2 in apes and whales has been proposed to be associated with evolution of olfaction in these clades. We analyzed 57 bird genomes to identify MOXD2 sequences and found frequent loss of MOXD2 in 38 birds. Among the 57 birds, 19 species appeared to have an intact MOXD2 that encoded a full-length protein; 32 birds had a gene with open reading frame-disrupting point mutations and/or exon deletions; and the remaining 6 species did not show any MOXD2 sequence, suggesting a whole-gene deletion. Notably, among 10 passerine birds examined, 9 species shared a common genomic deletion that spanned several exons, implying the gene loss occurred in a common ancestor of these birds. However, 2 closely related penguin species, each of which had an inactive MOXD2, did not share any mutation, suggesting an independent loss after their divergence. Distribution of the 38 birds without an intact MOXD2 in the bird phylogenetic tree clearly indicates that MOXD2 loss is widespread and independent in bird lineages. We propose that widespread MOXD2 loss in some bird lineages may be implicated in the evolution of olfactory perception in these birds.

  16. Gene doping.

    Science.gov (United States)

    Haisma, H J; de Hon, O

    2006-04-01

    Together with the rapidly increasing knowledge on genetic therapies as a promising new branch of regular medicine, the issue has arisen whether these techniques might be abused in the field of sports. Previous experiences have shown that drugs that are still in the experimental phases of research may find their way into the athletic world. Both the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) have expressed concerns about this possibility. As a result, the method of gene doping has been included in the list of prohibited classes of substances and prohibited methods. This review addresses the possible ways in which knowledge gained in the field of genetic therapies may be misused in elite sports. Many genes are readily available which may potentially have an effect on athletic performance. The sporting world will eventually be faced with the phenomena of gene doping to improve athletic performance. A combination of developing detection methods based on gene arrays or proteomics and a clear education program on the associated risks seems to be the most promising preventive method to counteract the possible application of gene doping.

  17. Revisiting nitrification in the Eastern Tropical South Pacific: A focus on controls

    Science.gov (United States)

    Peng, Xuefeng; Fuchsman, Clara A.; Jayakumar, Amal; Warner, Mark J.; Devol, Allan H.; Ward, Bess B.

    2016-03-01

    Nitrification, the oxidation of ammonium (NH4+) to nitrite (NO2-) and to nitrate (NO3-), is a component of the nitrogen (N) cycle internal to the fixed N pool. In oxygen minimum zones (OMZs), which are hotspots for oceanic fixed N loss, nitrification plays a key role because it directly supplies substrates for denitrification and anaerobic ammonia oxidation (anammox), and may compete for substrates with these same processes. However, the control of oxygen and substrate concentrations on nitrification are not well understood. We performed onboard incubations with 15N-labeled substrates to measure rates of NH4+ and NO2- oxidation in the eastern tropical South Pacific (ETSP). The spatial and depth distributions of NH4+ and NO2- oxidation rates were primarily controlled by NH4+ and NO2- availability, oxygen concentration, and light. In the euphotic zone, nitrification was partially photoinhibited. In the anoxic layer, NH4+ oxidation was negligible or below detection, but high rates of NO2- oxidation were observed. NH4+ oxidation displayed extremely high affinity for both NH4+ and oxygen. The positive linear correlations between NH4+ oxidation rates and in situ NH4+ concentrations and ammonia monooxygenase subunit A (amoA) gene abundances in the upper oxycline indicate that the natural assemblage of ammonia oxidizers responds to in situNH4+ concentrations or supply by adjusting their population size, which determines the NH4+ oxidation potential. The depth distribution of archaeal and bacterial amoA gene abundances and N2O concentration, along with independently reported simultaneous direct N2O production rate measurements, suggests that AOA were predominantly responsible for NH4+ oxidation, which was a major source of N2O production at oxygen concentrations > 5 µM.

  18. Coupling of diversification and pH adaptation during the evolution of terrestrial Thaumarchaeota

    Science.gov (United States)

    Gubry-Rangin, Cécile; Kratsch, Christina; Williams, Tom A.; McHardy, Alice C.; Embley, T. Martin; Prosser, James I.; Macqueen, Daniel J.

    2015-01-01

    The Thaumarchaeota is an abundant and ubiquitous phylum of archaea that plays a major role in the global nitrogen cycle. Previous analyses of the ammonia monooxygenase gene amoA suggest that pH is an important driver of niche specialization in these organisms. Although the ecological distribution and ecophysiology of extant Thaumarchaeota have been studied extensively, the evolutionary rise of these prokaryotes to ecological dominance in many habitats remains poorly understood. To characterize processes leading to their diversification, we investigated coevolutionary relationships between amoA, a conserved marker gene for Thaumarchaeota, and soil characteristics, by using deep sequencing and comprehensive environmental data in Bayesian comparative phylogenetics. These analyses reveal a large and rapid increase in diversification rates during early thaumarchaeotal evolution; this finding was verified by independent analyses of 16S rRNA. Our findings suggest that the entire Thaumarchaeota diversification regime was strikingly coupled to pH adaptation but less clearly correlated with several other tested environmental factors. Interestingly, the early radiation event coincided with a period of pH adaptation that enabled the terrestrial Thaumarchaeota ancestor to initially move from neutral to more acidic and alkaline conditions. In contrast to classic evolutionary models, whereby niches become rapidly filled after adaptive radiation, global diversification rates have remained stably high in Thaumarchaeota during the past 400–700 million years, suggesting an ongoing high rate of niche formation or switching for these microbes. Our study highlights the enduring importance of environmental adaptation during thaumarchaeotal evolution and, to our knowledge, is the first to link evolutionary diversification to environmental adaptation in a prokaryotic phylum. PMID:26170282

  19. Gene Locater

    DEFF Research Database (Denmark)

    Anwar, Muhammad Zohaib; Sehar, Anoosha; Rehman, Inayat-Ur

    2012-01-01

    software's for calculating recombination frequency is mostly limited to the range and flexibility of this type of analysis. GENE LOCATER is a fully customizable program for calculating recombination frequency, written in JAVA. Through an easy-to-use interface, GENE LOCATOR allows users a high degree...... of flexibility in calculating genetic linkage and displaying linkage group. Among other features, this software enables user to identify linkage groups with output visualized graphically. The program calculates interference and coefficient of coincidence with elevated accuracy in sample datasets. AVAILABILITY...

  20. Kynurenine 3-monooxygenase from Pseudomonas fluorescens: substrate-like inhibitors both stimulate flavin reduction and stabilize the flavin-peroxo intermediate yet result in the production of hydrogen peroxide.

    Science.gov (United States)

    Crozier-Reabe, Karen R; Phillips, Robert S; Moran, Graham R

    2008-11-25

    Kynurenine 3-monooxygenase (KMO) is a flavin-dependent hydroxylase that catalyzes the conversion of l-kynurenine (l-Kyn) to 3-hydroxykynurenine (3OHKyn) in the pathway for tryptophan catabolism. KMO inhibition has been widely suggested as an early treatment for stroke and other neurological disorders that involve ischemia. We have investigated the reductive and the oxidative half-reactions of a stable form of KMO from Pseudomonas fluorescens (KMO). The binding of l-Kyn by the enzyme is relatively slow and involves at least two reversible steps. The rate constant for reduction of the flavin cofactor by NADPH increases by a factor of approximately 2.5 x 10(3) when l-Kyn is bound. The rate of reduction of the KMO.l-Kyn complex is 160 s(-1), and the K(d) for the NADPH complex is 200 microM with charge-transfer absorption bands for the KMO(RED).l-Kyn.NADP(+) complex accumulating after reduction. The reduction potential of KMO is -188 mV and is unresponsive to the addition of l-Kyn or other inhibitory ligands. KMO inhibitors whose structures are reminiscent of l-Kyn such as m-nitrobenzoylalanine and benzoylalanine also stimulate reduction of flavin by NADPH and, in the presence of dioxygen, result in the stoichiometric liberation of hydrogen peroxide, diminishing the perceived therapeutic potential of inhibitors of this type. In the presence of the native substrate, the oxidative half-reaction exhibits triphasic absorbance data. A spectrum consistent with that of a peroxyflavin species accumulates and then decays to yield the oxidized enzyme. This species then undergoes minor spectral changes that, based on flavin difference spectra defined in the presence of 3OHKyn, can be correlated with product release. The oxidative half-reaction observed in the presence of saturating benzoylalanine or m-nitrobenzoylalanine also shows the accumulation of a peroxyflavin species that then decays to yield hydrogen peroxide without hydroxylation.

  1. Pyrethroid Resistance in Malaysian Populations of Dengue Vector Aedes aegypti Is Mediated by CYP9 Family of Cytochrome P450 Genes.

    Science.gov (United States)

    Ishak, Intan H; Kamgang, Basile; Ibrahim, Sulaiman S; Riveron, Jacob M; Irving, Helen; Wondji, Charles S

    2017-01-01

    Dengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance. Genome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise pyrethroids, DDT and bendiocarb. The predominant

  2. Gene mdpC plays a regulatory role in the methyl-tert-butyl ether degradation pathway of Methylibium petroleiphilum strain PM1.

    Science.gov (United States)

    Joshi, Geetika; Schmidt, Radomir; Scow, Kate M; Denison, Michael S; Hristova, Krassimira R

    2015-04-01

    Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC(-). We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris.

    Science.gov (United States)

    Wang, Sanhong; Yao, Quanhong; Tao, Jianmin; Qiao, Yushan; Zhang, Zhen

    2007-12-01

    The genes encoding the two enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) of glycine betaine synthesis in Suaeda salsa were cloned and fused with the 2A region of foot-and-mouth disease virus in a single open reading frame. The fused genes were placed under the control of the alcohol oxidase (AOX1) promoter in pPIC3B and transformed into P. pastoris GS115. The expression of the fused genes in P. pastoris and the ability of recombinant yeasts to tolerate environmental stresses were studied. The results showed that induced with 0.5% methanol for 96 h, the maximal activities of CMO and BADH in the tested recombinant yeasts were 45- and 44-fold higher than those in the control yeast transformed empty vector only, respectively; the content of glycine betaine in the recombinant yeasts was 28- to 35-fold higher than that in the control. The fused genes linked by 2A region of foot-and-mouth disease virus were expressed in P. pastoris successfully and the polyprotein was 'cleaved' to each functional protein. The yeasts transformed the fused genes, which were more resistant to salt, methanol, and high temperature stresses than the control as result of glycine betaine synthesis genes introduced.

  4. Archaeal enrichment in the hypoxic zone in the northern Gulf of Mexico.

    Science.gov (United States)

    Gillies, Lauren E; Thrash, J Cameron; deRada, Sergio; Rabalais, Nancy N; Mason, Olivia U

    2015-10-01

    Areas of low oxygen have spread exponentially over the past 40 years, and are cited as a key stressor on coastal ecosystems. The world's second largest coastal hypoxic (≤ 2 mg of O2 l(-1)) zone occurs annually in the northern Gulf of Mexico. The net effect of hypoxia is the diversion of energy flow away from higher trophic levels to microorganisms. This energy shunt is consequential to the overall productivity of hypoxic water masses and the ecosystem as a whole. In this study, water column samples were collected at 39 sites in the nGOM, 21 of which were hypoxic. Analysis of the microbial community along a hypoxic to oxic dissolved oxygen gradient revealed that the relative abundance (iTag) of Thaumarchaeota species 16S rRNA genes (> 40% of the microbial community in some hypoxic samples), the absolute abundance (quantitative polymerase chain reaction; qPCR) of Thaumarchaeota 16S rRNA genes and archaeal ammonia-monooxygenase gene copy number (qPCR) were significantly higher in hypoxic samples. Spatial interpolation of the microbial and chemical data revealed a continuous, shelfwide band of low dissolved oxygen waters that were dominated by Thaumarchaeota (and Euryarchaeota), amoA genes and high concentrations of phosphate in the nGOM, thus implicating physicochemical forcing on microbial abundance. © 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  5. The tryptophan hydroxylase 1 (TPH1) gene, schizophrenia susceptibility, and suicidal behavior: a multi-centre case-control study and meta-analysis

    DEFF Research Database (Denmark)

    Saetre, Peter; Lundmark, Per; Wang, August

    2010-01-01

    Serotonin (5-hydroxytryptamin; 5-HT) alternations has since long been suspected in the pathophysiology of schizophrenia. Tryptophan hydroxylase (tryptophan 5-monooxygenase; TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT, and sequence variation in intron 6 of the TPH1 gene has been...... affected individuals having attempted suicide at least once and patients with no history of suicide attempts (P = 0.84). A systematic literature review and meta-analysis support the A218C polymorphism as a susceptibility locus for schizophrenia (odds ratio 1.17, 95% confidence interval 1.......07-1.29). Association studies on suicide attempts are however conflicting (heterogeneity index I(2) = 0.54) and do not support the A218C/A779C polymorphisms being a susceptibility locus for suicidal behavior among individuals diagnosed with a psychiatric disorder (OR = 0.96 [0.80-1.16]). We conclude that the TPH1 A218...

  6. The tryptophan hydroxylase 1 (TPH1) gene, schizophrenia susceptibility, and suicidal behavior: a multi-centre case-control study and meta-analysis

    DEFF Research Database (Denmark)

    Saetre, Peter; Lundmark, Per; Wang, August

    2010-01-01

    Serotonin (5-hydroxytryptamin; 5-HT) alternations has since long been suspected in the pathophysiology of schizophrenia. Tryptophan hydroxylase (tryptophan 5-monooxygenase; TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT, and sequence variation in intron 6 of the TPH1 gene has been...... associated with schizophrenia. The minor allele (A) of this polymorphism (A218C) is also more frequent in patients who have attempted suicide and individuals who died by suicide, than in healthy control individuals. In an attempt to replicate previous findings, five single nucleotide polymorphisms (SNPs......) were genotyped in 837 Scandinavian schizophrenia patients and 1,473 controls. Three SNPs spanning intron 6 and 7, including the A218C and A779C polymorphisms, were associated with schizophrenia susceptibility (P = 0.019). However there were no differences in allele frequencies of these loci between...

  7. Gene Ontology

    Directory of Open Access Journals (Sweden)

    Gaston K. Mazandu

    2012-01-01

    Full Text Available The wide coverage and biological relevance of the Gene Ontology (GO, confirmed through its successful use in protein function prediction, have led to the growth in its popularity. In order to exploit the extent of biological knowledge that GO offers in describing genes or groups of genes, there is a need for an efficient, scalable similarity measure for GO terms and GO-annotated proteins. While several GO similarity measures exist, none adequately addresses all issues surrounding the design and usage of the ontology. We introduce a new metric for measuring the distance between two GO terms using the intrinsic topology of the GO-DAG, thus enabling the measurement of functional similarities between proteins based on their GO annotations. We assess the performance of this metric using a ROC analysis on human protein-protein interaction datasets and correlation coefficient analysis on the selected set of protein pairs from the CESSM online tool. This metric achieves good performance compared to the existing annotation-based GO measures. We used this new metric to assess functional similarity between orthologues, and show that it is effective at determining whether orthologues are annotated with similar functions and identifying cases where annotation is inconsistent between orthologues.

  8. Expression induction of P450 genes by imidacloprid in Nilaparvata lugens: A genome-scale analysis.

    Science.gov (United States)

    Zhang, Jianhua; Zhang, Yixi; Wang, Yunchao; Yang, Yuanxue; Cang, Xinzhu; Liu, Zewen

    2016-09-01

    The overexpression of P450 monooxygenase genes is a main mechanism for the resistance to imidacloprid, a representative neonicotinoid insecticide, in Nilaparvata lugens (brown planthopper, BPH). However, only two P450 genes (CYP6AY1 and CYP6ER1), among fifty-four P450 genes identified from BPH genome database, have been reported to play important roles in imidacloprid resistance until now. In this study, after the confirmation of important roles of P450s in imidacloprid resistance by the synergism analysis, the expression induction by imidacloprid was determined for all P450 genes. In the susceptible (Sus) strain, eight P450 genes in Clade4, eight in Clade3 and two in Clade2 were up-regulated by imidacloprid, among which three genes (CYP6CS1, CYP6CW1 and CYP6ER1, all in Clade3) were increased to above 4.0-fold and eight genes to above 2.0-fold. In contrast, no P450 genes were induced in Mito clade. Eight genes induced to above 2.0-fold were selected to determine their expression and induced levels in Huzhou population, in which piperonyl butoxide showed the biggest effects on imidacloprid toxicity among eight field populations. The expression levels of seven P450 genes were higher in Huzhou population than that in Sus strain, with the biggest differences for CYP6CS1 (9.8-fold), CYP6ER1 (7.7-fold) and CYP6AY1 (5.1-fold). The induction levels for all tested genes were bigger in Sus strain than that in Huzhou population except CYP425B1. Screening the induction of P450 genes by imidacloprid in the genome-scale will provide an overall view on the possible metabolic factors in the resistance to neonicotinoid insecticides. The further work, such as the functional study of recombinant proteins, will be performed to validate the roles of these P450s in imidacloprid resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Gene doping: gene delivery for olympic victory

    OpenAIRE

    Gould, David

    2012-01-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted...

  10. Comparison of partial and full nitrification processes applied for treating high-strength nitrogen wastewaters: microbial ecology through nitrous oxide production.

    Science.gov (United States)

    Ahn, Joon Ho; Kwan, Tiffany; Chandran, Kartik

    2011-04-01

    The goal of this study was to compare the microbial ecology, gene expression, biokinetics, and N2O emissions from a lab-scale bioreactor operated sequentially in full-nitrification and partial-nitrification modes. Based on sequencing of 16S rRNA and ammonia monooxygenase subunit A (amoA) genes, ammonia oxidizing bacteria (AOB) populations during full- and partial-nitrification modes were distinct from one another. The concentrations of AOB (XAOB) and their respiration rates during full- and partial-nitrification modes were statistically similar, whereas the concentrations of nitrite oxidizing bacteria (XNOB) and their respiration rates declined significantly after the switch from full- to partial-nitrification. The transition from full-nitrification to partial nitrification resulted in a protracted transient spike of nitrous oxide (N2O) and nitric oxide (NO) emissions, which later stabilized. The trends in N2O and NO emissions correlated well with trends in the expression of nirK and norB genes that code for the production of these gases in AOB. Both the transient and stabilized N2O and NO emissions during partial nitrification were statistically higher than those during steady-state full-nitrification. Based on these results, partial nitrification strategies for biological nitrogen removal, although attractive for their reduced operating costs and energy demand, may need to be optimized against the higher carbon foot-print attributed to their N2O emissions.

  11. Metagenomic potential for and diversity of N-cycle driving microorganisms in the Bothnian Sea sediment.

    Science.gov (United States)

    Rasigraf, Olivia; Schmitt, Julia; Jetten, Mike S M; Lüke, Claudia

    2017-08-01

    The biological nitrogen cycle is driven by a plethora of reactions transforming nitrogen compounds between various redox states. Here, we investigated the metagenomic potential for nitrogen cycle of the in situ microbial community in an oligotrophic, brackish environment of the Bothnian Sea sediment. Total DNA from three sediment depths was isolated and sequenced. The characterization of the total community was performed based on 16S rRNA gene inventory using SILVA database as reference. The diversity of diagnostic functional genes coding for nitrate reductases (napA;narG), nitrite:nitrate oxidoreductase (nxrA), nitrite reductases (nirK;nirS;nrfA), nitric oxide reductase (nor), nitrous oxide reductase (nosZ), hydrazine synthase (hzsA), ammonia monooxygenase (amoA), hydroxylamine oxidoreductase (hao), and nitrogenase (nifH) was analyzed by blastx against curated reference databases. In addition, Polymerase chain reaction (PCR)-based amplification was performed on the hzsA gene of anammox bacteria. Our results reveal high genomic potential for full denitrification to N 2 , but minor importance of anaerobic ammonium oxidation and dissimilatory nitrite reduction to ammonium. Genomic potential for aerobic ammonia oxidation was dominated by Thaumarchaeota. A higher diversity of anammox bacteria was detected in metagenomes than with PCR-based technique. The results reveal the importance of various N-cycle driving processes and highlight the advantage of metagenomics in detection of novel microbial key players. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  12. Genes and Hearing Loss

    Science.gov (United States)

    ... ENTCareers Marketplace Find an ENT Doctor Near You Genes and Hearing Loss Genes and Hearing Loss Patient ... mutation may only have dystopia canthorum. How Do Genes Work? Genes are a road map for the ...

  13. β-Carotene-9′,10′-Oxygenase Status Modulates the Impact of Dietary Tomato and Lycopene on Hepatic Nuclear Receptor–, Stress-, and Metabolism-Related Gene Expression in Mice123

    Science.gov (United States)

    Tan, Hsueh-Li; Moran, Nancy E.; Cichon, Morgan J.; Riedl, Ken M.; Schwartz, Steven J.; Erdman, John W.; Pearl, Dennis K.; Thomas-Ahner, Jennifer M.; Clinton, Steven K.

    2014-01-01

    Tomato and lycopene (ψ, ψ-carotene) consumption is hypothesized to protect against nonalcoholic steatohepatitis and hepatocarcinogenesis, processes that may depend upon diet and gene interactions. To investigate the interaction of tomato or lycopene feeding with β-carotene-9′,10′-monooxygenase (Bco2) on hepatic metabolic and signaling pathways, male wild-type (WT) and Bco2−/− mice (3-wk-old; n = 36) were fed semi-purified control, 10% tomato powder–containing, or 0.25% lycopene beadlet–containing diets for 3 wk. Serum lycopene concentrations were higher in lycopene- and tomato-fed Bco2−/− mice compared with WT (P = 0.03). Tomato- and lycopene-fed mice had detectable hepatic apolipoprotein (apo)-6′-, apo-8′-, and apo-12′-lycopenal concentrations. Hepatic expression of β-carotene-15,15’-monooxygenase was increased in Bco2−/− mice compared with WT (P = 0.02), but not affected by diet. Evaluation of hepatic gene expression by focused quantitative reverse transcriptase-polymerase chain reaction arrays for nuclear receptors and coregulators (84 genes) and stress and metabolism (82 genes) genes indicates that tomato feeding affected 31 genes (≥1.5-fold, P lycopene feeding affected 19 genes, 16 of which were affected by both diets. Lycopene down-regulation of 7 nuclear receptors and coregulators, estrogen-related receptor-α, histone deacetylase 3, nuclear receptor coactivator 4, RevErbA-β, glucocorticoid receptor, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ, coactivator 1 β was dependent upon interaction with Bco2 status. Lycopene and tomato feeding induced gene expression patterns consistent with decreased lipid uptake, decreased cell proliferation and mitosis, down-regulated aryl hydrocarbon receptor signaling, and decreased expression of genes involved in retinoid X receptor heterodimer activation. Tomato feeding also caused expression changes consistent with down-regulation of DNA synthesis and terpenoid

  14. Linking isoprenoidal GDGT membrane lipid distributions with gene abundances of ammonia-oxidizing Thaumarchaeota and uncultured crenarchaeotal groups in the water column of a tropical lake (Lake Challa, East Africa).

    Science.gov (United States)

    Buckles, Laura K; Villanueva, Laura; Weijers, Johan W H; Verschuren, Dirk; Damsté, Jaap S Sinninghe

    2013-09-01

    Stratified lakes are important reservoirs of microbial diversity and provide habitats for niche differentiation of Archaea. In this study, we used a lipid biomarker/DNA-based approach to reveal the diversity and abundance of Archaea in the water column of Lake Challa (East Africa). Concentrations of intact polar lipid (IPL) crenarchaeol, a specific biomarker of Thaumarchaeota, were enhanced (1 ng l(-1) ) at the oxycline/nitrocline. The predominance of the more labile IPL hexose-phosphohexose crenarchaeol indicated the presence of an actively living community of Thaumarchaeota. Archaeal 16S rRNA clone libraries revealed the presence of thaumarchaeotal groups 1.1a and 1.1b at and above the oxycline. In the anoxic deep water, amoA gene abundance was an order of magnitude lower than at the oxycline and high abundance (∼90 ng l(-1) ) of an IPL with the acyclic glycerol dialkyl glycerol tetraether (GDGT-0) was evident. The predominance of archaeal 16S rRNA sequences affiliated to the uncultured crenarchaeota groups 1.2 and miscellaneous crenarchaeotic group (MCG) points to an origin of GDGT-0 from uncultured crenarchaeota. This study demonstrates the importance of thermal stratification and nutrient availability in the distribution of archaeal groups in lakes, which is relevant to constrain and validate temperature proxies based on archaeal GDGTs (i.e. TEX86 ). © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

  15. P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia

    International Nuclear Information System (INIS)

    Matteson, K.J.; Phillips, J.A. III; Miller, W.L.; Chung, B.C.; Orlando, P.J.; Frisch, H.; Ferrandez, A.; Burr, I.M.

    1987-01-01

    Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P450XXIA2 gene encodes cytochrome P450c21 [steroid 21-monooxygenase (steroid 21-hydroxylase)], which mediates 21-hydroxylation. The P450XXIA2 gene may be distinguished from the duplicated P450XXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P450XXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. The authors have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAH. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, they conclude that the P450XXIA2 gene deletions widely reported in CAH patients probably represent gene conversions, unequal crossovers,or polymorphisms rather than simple gene deletions

  16. Gene expression and gene therapy imaging

    International Nuclear Information System (INIS)

    Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

    2007-01-01

    The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

  17. Imaging reporter gene for monitoring gene therapy

    International Nuclear Information System (INIS)

    Beco, V. de; Baillet, G.; Tamgac, F.; Tofighi, M.; Weinmann, P.; Vergote, J.; Moretti, J.L.; Tamgac, G.

    2002-01-01

    Scintigraphic images can be obtained to document gene function at cellular level. This approach is presented here and the use of a reporter gene to monitor gene therapy is described. Two main ways are presented: either the use of a reporter gene coding for an enzyme the action of which will be monitored by radiolabeled pro-drug, or a cellular receptor gene, the action of which is documented by a radio labeled cognate receptor ligand. (author)

  18. Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties.

    Science.gov (United States)

    Hoang, Van L T; Innes, David J; Shaw, P Nicholas; Monteith, Gregory R; Gidley, Michael J; Dietzgen, Ralf G

    2015-07-30

    Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3'-hydroxylase (F3'H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and

  19. Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba skin biopsies

    Directory of Open Access Journals (Sweden)

    Casini Silvia

    2006-09-01

    Full Text Available Abstract Background Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. Results Ten commonly used housekeeping genes (HKGs were partially sequenced in the striped dolphin (Stenella coeruleoalba and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH and tyrosine 3-monooxygenase (YWHAZ always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4 and S18 (RPS18 also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC, phosphoglycerate kinase 1 (PGK1, hypoxanthine ribosyltransferase (HPRT1 and β-2-microglobin (B2M show variable expression

  20. Genes involved in degradation of para-nitrophenol are differentially arranged in form of non-contiguous gene clusters in Burkholderia sp. strain SJ98.

    Directory of Open Access Journals (Sweden)

    Surendra Vikram

    Full Text Available Biodegradation of para-Nitrophenol (PNP proceeds via two distinct pathways, having 1,2,3-benzenetriol (BT and hydroquinone (HQ as their respective terminal aromatic intermediates. Genes involved in these pathways have already been studied in different PNP degrading bacteria. Burkholderia sp. strain SJ98 degrades PNP via both the pathways. Earlier, we have sequenced and analyzed a ~41 kb fragment from the genomic library of strain SJ98. This DNA fragment was found to harbor all the lower pathway genes; however, genes responsible for the initial transformation of PNP could not be identified within this fragment. Now, we have sequenced and annotated the whole genome of strain SJ98 and found two ORFs (viz., pnpA and pnpB showing maximum identity at amino acid level with p-nitrophenol 4-monooxygenase (PnpM and p-benzoquinone reductase (BqR. Unlike the other PNP gene clusters reported earlier in different bacteria, these two ORFs in SJ98 genome are physically separated from the other genes of PNP degradation pathway. In order to ascertain the identity of ORFs pnpA and pnpB, we have performed in-vitro assays using recombinant proteins heterologously expressed and purified to homogeneity. Purified PnpA was found to be a functional PnpM and transformed PNP into benzoquinone (BQ, while PnpB was found to be a functional BqR which catalyzed the transformation of BQ into hydroquinone (HQ. Noticeably, PnpM from strain SJ98 could also transform a number of PNP analogues. Based on the above observations, we propose that the genes for PNP degradation in strain SJ98 are arranged differentially in form of non-contiguous gene clusters. This is the first report for such arrangement for gene clusters involved in PNP degradation. Therefore, we propose that PNP degradation in strain SJ98 could be an important model system for further studies on differential evolution of PNP degradation functions.

  1. Inhibition of bacterial ammonia oxidation by organohydrazines in soil microcosms

    Directory of Open Access Journals (Sweden)

    Yucheng eWu

    2012-01-01

    Full Text Available Hydroxylamine oxidation by hydroxylamine oxidoreductase (HAO is a key step for energy-yielding in support of the growth of ammonia-oxidizing bacteria (AOB. Organohydrazines have been shown to inactivate HAO from Nitrosomonas europaea, and may serve as selective inhibitors to differentiate bacterial from archaeal ammonia oxidation due to the absence of bacterial HAO gene homologue in known ammonia-oxidizing archaea (AOA. In this study, the effects of three organohydrazines on activity, abundance and composition of AOB and AOA were evaluated in soil microcosms. The results indicate that phenylhydrazine and methylhydrazine at the concentration of 100 mol per gram dry weight soil completely suppressed the activity of soil nitrification. DGGE fingerprinting and sequencing analysis of bacterial ammonia monooxygenase subunit A gene (amoA clearly demonstrated that nitrification activity change is well paralleled with the growth of Nitrosomonas europaea-like AOB in soil microcosms. No significant correlation between AOA community structure and nitrification activity was observed among all treatments during the incubation period, although incomplete inhibition of nitrification activity occurred in 2-hydroxyethylhydrazine-amended soil microcosms. These findings show that the HAO-targeted organohydrazines can effectively inhibit bacterial nitrification in soil, and the mechanism of organohydrazine affecting AOA remains unclear.

  2. Identification of two combined genes responsible for dechlorination of 3,5,6-trichloro-2-pyridinol (TCP) in Cupriavidus pauculus P2.

    Science.gov (United States)

    Cao, Li; Xu, Jianhong; Wu, Guang; Li, Mingxing; Jiang, Jiandong; He, Jian; Li, Shunpeng; Hong, Qing

    2013-09-15

    Dehalogenation is an important mechanism for degrading and detoxifying halogenated aromatics in microbes. However, the biochemical and molecular mechanisms of dehalogenation of 3,5,6-trichloro-2-pyridinol (TCP) are still unknown. In this study, a novel 6012 bp gene cluster was cloned from TCP-degrading strain P2, which was responsible for the dehalogenation of TCP. The cluster included a monooxygenase gene (tcpA1), a flavin reductase gene (tcpB1), tcpR1, orf1 and orf2. TcpA1 and TcpB1 were indispensable for the dehalogenation of TCP. They worked together to catalyze the dehalogenation of three chlorine of TCP, and generated a more readily biodegradable product of 3,6-dihydroxypyridine-2,5-dione. TcpA1 displayed the highest activity against TCP at 40°C and at pH 8.0. Cu(2+), Zn(2+), and Hg(2+) significantly inhibited enzyme activity. To the best of our knowledge, this is the first report on a gene cluster responsible for TCP degradation. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Differential regulation by heat stress of novel cytochrome P450 genes from the dinoflagellate symbionts of reef-building corals.

    Science.gov (United States)

    Rosic, Nedeljka N; Pernice, Mathieu; Dunn, Simon; Dove, Sophie; Hoegh-Guldberg, Ove

    2010-05-01

    Exposure to heat stress has been recognized as one of the major factors leading to the breakdown of the coral-alga symbiosis and coral bleaching. Here, we describe the presence of three new cytochrome P450 (CYP) genes from the reef-building coral endosymbiont Symbiodinium (type C3) and changes in their expression during exposure to severe and moderate heat stress conditions. Sequence analysis of the CYP C-terminal region and two conserved domains, the "PERF" and "heme-binding" domains, confirmed the separate identities of the CYP genes analyzed. In order to explore the effects of different heat stress scenarios, samples of the scleractinian coral Acropora millepora were exposed to elevated temperatures incrementally over an 18-h period (rapid thermal stress) and over a 120-h period (gradual thermal stress). After 18 h of gradual heating and incubation at 26 degrees C, the Symbiodinium CYP mRNA pool was approximately 30% larger, while a further 6 degrees C increase to a temperature above the average sea temperature (29 degrees C after 72 h) resulted in a 2- to 4-fold increase in CYP expression. Both rapid heat stress and gradual heat stress at 32 degrees C resulted in 50% to 90% decreases in CYP gene transcript abundance. Consequently, the initial upregulation of expression of CYP genes at moderately elevated temperatures (26 degrees C and 29 degrees C) was followed by a decrease in expression under the greater thermal stress conditions at 32 degrees C. These findings indicate that in the coral-alga symbiosis under heat stress conditions there is production of chemical stressors and/or transcriptional factors that regulate the expression of genes, such as the genes encoding cytochrome P450 monooxygenases, that are involved in the first line of an organism's chemical defense.

  4. Transcriptome Profiling to Identify Genes Involved in Mesosulfuron-Methyl Resistance in Alopecurus aequalis

    Directory of Open Access Journals (Sweden)

    Ning Zhao

    2017-08-01

    Full Text Available Non-target-site resistance (NTSR to herbicides is a worldwide concern for weed control. However, as the dominant NTSR mechanism in weeds, metabolic resistance is not yet well-characterized at the genetic level. For this study, we have identified a shortawn foxtail (Alopecurus aequalis Sobol. population displaying both TSR and NTSR to mesosulfuron-methyl and fenoxaprop-P-ethyl, yet the molecular basis for this NTSR remains unclear. To investigate the mechanisms of metabolic resistance, an RNA-Seq transcriptome analysis was used to find candidate genes that may confer metabolic resistance to the herbicide mesosulfuron-methyl in this plant population. The RNA-Seq libraries generated 831,846,736 clean reads. The de novo transcriptome assembly yielded 95,479 unigenes (averaging 944 bp in length that were assigned putative annotations. Among these, a total of 29,889 unigenes were assigned to 67 GO terms that contained three main categories, and 14,246 unigenes assigned to 32 predicted KEGG metabolic pathways. Global gene expression was measured using the reads generated from the untreated control (CK, water-only control (WCK, and mesosulfuron-methyl treatment (T of R and susceptible (S. Contigs that showed expression differences between mesosulfuron-methyl-treated R and S biotypes, and between mesosulfuron-methyl-treated, water-treated and untreated R plants were selected for further quantitative real-time PCR (qRT-PCR validation analyses. Seventeen contigs were consistently highly expressed in the resistant A. aequalis plants, including four cytochrome P450 monooxygenase (CytP450 genes, two glutathione S-transferase (GST genes, two glucosyltransferase (GT genes, two ATP-binding cassette (ABC transporter genes, and seven additional contigs with functional annotations related to oxidation, hydrolysis, and plant stress physiology. These 17 contigs could serve as major candidate genes for contributing to metabolic mesosulfuron-methyl resistance; hence

  5. Molecular Characterization of Ferulate 5-Hydroxylase Gene from Kenaf (Hibiscus cannabinus L.

    Directory of Open Access Journals (Sweden)

    Jonggeun Kim

    2013-01-01

    Full Text Available The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.. Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84, is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF encoding 518 amino acids (GenBank Accession number JX524278. The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78% with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h, wounding (24 h, cold (24 h, abscisic acid (24 h, and methyl jasmonate (24 h.

  6. RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster.

    Science.gov (United States)

    Gellatly, Kyle J; Yoon, Kyong Sup; Doherty, Jeffery J; Sun, Weilin; Pittendrigh, Barry R; Clark, J Marshall

    2015-06-01

    4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Imaging gene expression in gene therapy

    International Nuclear Information System (INIS)

    Wiebe, Leonard I.

    1997-01-01

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on 'suicide gene therapy' of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k + ) has been use for 'suicide' in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k + gene expression where the H S V-1 t k + gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([ 18 F]F H P G; [ 18 F]-A C V), and pyrimidine- ([ 123 / 131 I]I V R F U; [ 124 / 131I ]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [ 123 / 131I ]I V R F U imaging with the H S V-1 t k + reporter gene will be presented

  8. Imaging gene expression in gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Wiebe, Leonard I. [Alberta Univ., Edmonton (Canada). Noujaim Institute for Pharmaceutical Oncology Research

    1997-12-31

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on `suicide gene therapy` of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k{sup +}) has been use for `suicide` in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k{sup +} gene expression where the H S V-1 t k{sup +} gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([{sup 18} F]F H P G; [{sup 18} F]-A C V), and pyrimidine- ([{sup 123}/{sup 131} I]I V R F U; [{sup 124}/{sup 131I}]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [{sup 123}/{sup 131I}]I V R F U imaging with the H S V-1 t k{sup +} reporter gene will be presented

  9. Monitoring Methanotrophic Bacteria in Hybrid Anaerobic-Aerobic Reactors with PCR and a Catabolic Gene Probe

    Science.gov (United States)

    Miguez, Carlos B.; Shen, Chun F.; Bourque, Denis; Guiot, Serge R.; Groleau, Denis

    1999-01-01

    We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreactors the metabolic association found in nature between methanogens and methanotrophs. UASB bioreactors were inoculated with pure cultures of methanotrophs, and the bioreactors were operated by using continuous low-level oxygenation in order to favor growth and/or survival of methanotrophs. Unlike the reactors in other similar studies, the hybrid anaerobic-aerobic bioreactors which we used were operated synchronously, not sequentially. Here, emphasis was placed on monitoring various methanotrophic populations by using classical methods and also a PCR amplification assay based on the mmoX gene fragment of the soluble methane monooxygenase (sMMO). The following results were obtained: (i) under the conditions used, Methylosinus sporium appeared to survive better than Methylosinus trichosporium; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotrophs per g (wet weight) of granular sludge; (iii) inoculation of the bioreactors with pure cultures of methanotrophs contributed greatly to increases in the sMMO-containing population (although the sMMO-containing population decreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (iv) in general, there was a good correlation between populations with the sMMO gene and populations that exhibited sMMO activity; and (v) inoculation with sMMO-positive cultures helped increase significantly the proportion of sMMO-positive methanotrophs in reactors, even after several weeks of operation under various regimes. At some point, anaerobic-aerobic bioreactors like those described here might be used for biodegradation of various chlorinated pollutants. PMID:9925557

  10. Gene doping: gene delivery for olympic victory.

    Science.gov (United States)

    Gould, David

    2013-08-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. © 2012 The Author. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

  11. Discriminating activated sludge flocs from biofilm microbial communities in a novel pilot-scale reciprocation MBR using high-throughput 16S rRNA gene sequencing.

    Science.gov (United States)

    De Sotto, Ryan; Ho, Jaeho; Lee, Woonyoung; Bae, Sungwoo

    2018-03-29

    Membrane bioreactors (MBRs) are a well-established filtration technology that has become a popular solution for treating wastewater. One of the drawbacks of MBRs, however, is the formation of biofilm on the surface of membrane modules. The occurrence of biofilms leads to biofouling, which eventually compromises water quality and damages the membranes. To prevent this, it is vital to understand the mechanism of biofilm formation on membrane surfaces. In this pilot-scale study, a novel reciprocation membrane bioreactor was operated for a period of 8 months and fed with domestic wastewater from an aerobic tank of a local WWTP. Water quality parameters were monitored and the microbial composition of the attached biofilm and suspended aggregates was evaluated in this reciprocating MBR configuration. The abundance of nitrifiers and composition of microbial communities from biofilm and suspended solids samples were investigated using qPCR and high throughput 16S amplicon sequencing. Removal efficiencies of 29%, 16%, and 15% of chemical oxygen demand, total phosphorus and total nitrogen from the influent were observed after the MBR process with average effluent concentrations of 16 mg/L, 4.6 mg/L, and 5.8 mg/L respectively. This suggests that the energy-efficient MBR, apart from reducing the total energy consumption, was able to maintain effluent concentrations that are within regulatory standards for discharge. Molecular analysis showed the presence of amoA Bacteria and 16S Nitrospira genes with the occurrence of nitrification. Candidatus Accumulibacter, a genus with organisms that can accumulate phosphorus, was found to be present in both groups which explains why phosphorus removal was observed in the system. High-throughput 16S rRNA amplicon sequencing revealed the genus Saprospira to be the most abundant species from the total OTUs of both the membrane tank and biofilm samples. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

    Science.gov (United States)

    Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

    2016-07-01

    Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

  13. Evolution of homeobox genes.

    Science.gov (United States)

    Holland, Peter W H

    2013-01-01

    Many homeobox genes encode transcription factors with regulatory roles in animal and plant development. Homeobox genes are found in almost all eukaryotes, and have diversified into 11 gene classes and over 100 gene families in animal evolution, and 10 to 14 gene classes in plants. The largest group in animals is the ANTP class which includes the well-known Hox genes, plus other genes implicated in development including ParaHox (Cdx, Xlox, Gsx), Evx, Dlx, En, NK4, NK3, Msx, and Nanog. Genomic data suggest that the ANTP class diversified by extensive tandem duplication to generate a large array of genes, including an NK gene cluster and a hypothetical ProtoHox gene cluster that duplicated to generate Hox and ParaHox genes. Expression and functional data suggest that NK, Hox, and ParaHox gene clusters acquired distinct roles in patterning the mesoderm, nervous system, and gut. The PRD class is also diverse and includes Pax2/5/8, Pax3/7, Pax4/6, Gsc, Hesx, Otx, Otp, and Pitx genes. PRD genes are not generally arranged in ancient genomic clusters, although the Dux, Obox, and Rhox gene clusters arose in mammalian evolution as did several non-clustered PRD genes. Tandem duplication and genome duplication expanded the number of homeobox genes, possibly contributing to the evolution of developmental complexity, but homeobox gene loss must not be ignored. Evolutionary changes to homeobox gene expression have also been documented, including Hox gene expression patterns shifting in concert with segmental diversification in vertebrates and crustaceans, and deletion of a Pitx1 gene enhancer in pelvic-reduced sticklebacks. WIREs Dev Biol 2013, 2:31-45. doi: 10.1002/wdev.78 For further resources related to this article, please visit the WIREs website. The author declares that he has no conflicts of interest. Copyright © 2012 Wiley Periodicals, Inc.

  14. Gene cluster statistics with gene families.

    Science.gov (United States)

    Raghupathy, Narayanan; Durand, Dannie

    2009-05-01

    Identifying genomic regions that descended from a common ancestor is important for understanding the function and evolution of genomes. In distantly related genomes, clusters of homologous gene pairs are evidence of candidate homologous regions. Demonstrating the statistical significance of such "gene clusters" is an essential component of comparative genomic analyses. However, currently there are no practical statistical tests for gene clusters that model the influence of the number of homologs in each gene family on cluster significance. In this work, we demonstrate empirically that failure to incorporate gene family size in gene cluster statistics results in overestimation of significance, leading to incorrect conclusions. We further present novel analytical methods for estimating gene cluster significance that take gene family size into account. Our methods do not require complete genome data and are suitable for testing individual clusters found in local regions, such as contigs in an unfinished assembly. We consider pairs of regions drawn from the same genome (paralogous clusters), as well as regions drawn from two different genomes (orthologous clusters). Determining cluster significance under general models of gene family size is computationally intractable. By assuming that all gene families are of equal size, we obtain analytical expressions that allow fast approximation of cluster probabilities. We evaluate the accuracy of this approximation by comparing the resulting gene cluster probabilities with cluster probabilities obtained by simulating a realistic, power-law distributed model of gene family size, with parameters inferred from genomic data. Surprisingly, despite the simplicity of the underlying assumption, our method accurately approximates the true cluster probabilities. It slightly overestimates these probabilities, yielding a conservative test. We present additional simulation results indicating the best choice of parameter values for data

  15. Carboxylesterase 1 genes

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Madsen, Majbritt Busk

    2018-01-01

    The carboxylesterase 1 gene (CES1) encodes a hydrolase that metabolizes commonly used drugs. The CES1-related pseudogene, carboxylesterase 1 pseudogene 1 (CES1P1), has been implicated in gene exchange with CES1 and in the formation of hybrid genes including the carboxylesterase 1A2 gene (CES1A2...

  16. Detection by denaturing gradient gel electrophoresis of ammonia-oxidizing bacteria in microcosms of crude oil-contaminated mangrove sediments.

    Science.gov (United States)

    dos Santos, A C F; Marques, E L S; Gross, E; Souza, S S; Dias, J C T; Brendel, M; Rezende, R P

    2012-01-27

    Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay.

  17. Molecular characterization and geological microenvironment of a microbial community inhabiting weathered receding shale cliffs.

    Science.gov (United States)

    Cockell, Charles S; Pybus, David; Olsson-Francis, Karen; Kelly, Laura; Petley, David; Rosser, Nick; Howard, Kieren; Mosselmans, Fred

    2011-01-01

    Shales play an important role in many earth system processes including coastal erosion, and they form the foundations of many engineering structures. The geobiology of the interior of pyrite-containing receding shale cliffs on the coast of northeast England was examined. The surface of the weathered shales was characterised by a thin layer of disordered authigenic iron oxyhydroxides and localised acicular, platy and aggregated gypsum, which was characterised by Raman spectroscopy, XAS and SEM. These chemical changes are likely to play an important role in causing rock weakening along fractures at the micron scale, which ultimately lead to coastal retreat at the larger scale. The surface of the shale hosts a novel, low-diversity microbial community. The bacterial community was dominated by Proteobacteria, with phylotypes closely associating with Methylocella and other members of the γ-subdivision. The second largest phylogenetic group corresponded to Nitrospira. The archaeal 16S rRNA phylotypes were dominated by a single group of sequences that matched phylotypes reported from South African gold mines and possessed ammonia monooxygenase (amoA) genes. Both the phylogenetic and the mineral data show that acidic microenvironments play an important role in shale weathering, but the shale has a higher microbial diversity than previously described pyritic acid mine drainage sites. The presence of a potentially biogeochemically active microbial population on the rock surface suggests that microorganisms may contribute to early events of shale degradation and coastal erosion.

  18. High dark inorganic carbon fixation rates by specific microbial groups in the Atlantic off the Galician coast (NW Iberian margin).

    Science.gov (United States)

    Guerrero-Feijóo, Elisa; Sintes, Eva; Herndl, Gerhard J; Varela, Marta M

    2018-02-01

    Bulk dark dissolved inorganic carbon (DIC) fixation rates were determined and compared to microbial heterotrophic production in subsurface, meso- and bathypelagic Atlantic waters off the Galician coast (NW Iberian margin). DIC fixation rates were slightly higher than heterotrophic production throughout the water column, however, more prominently in the bathypelagic waters. Microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization (MICRO-CARD-FISH) allowed us to identify several microbial groups involved in dark DIC uptake. The contribution of SAR406 (Marinimicrobia), SAR324 (Deltaproteobacteria) and Alteromonas (Gammaproteobacteria) to the dark DIC fixation was significantly higher than that of SAR202 (Chloroflexi) and Thaumarchaeota, in agreement with their contribution to microbial abundance. Q-PCR on the gene encoding for the ammonia monooxygenase subunit A (amoA) from the putatively high versus low ammonia concentration ecotypes revealed their depth-stratified distribution pattern. Taken together, our results indicate that chemoautotrophy is widespread among microbes in the dark ocean, particularly in bathypelagic waters. This chemolithoautotrophic biomass production in the dark ocean, depleted in bio-available organic matter, might play a substantial role in sustaining the dark ocean's food web. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. High abundances of potentially active ammonia-oxidizing bacteria and archaea in oligotrophic, high-altitude lakes of the Sierra Nevada, California, USA.

    Directory of Open Access Journals (Sweden)

    Curtis J Hayden

    Full Text Available Nitrification plays a central role in the nitrogen cycle by determining the oxidation state of nitrogen and its subsequent bioavailability and cycling. However, relatively little is known about the underlying ecology of the microbial communities that carry out nitrification in freshwater ecosystems--and particularly within high-altitude oligotrophic lakes, where nitrogen is frequently a limiting nutrient. We quantified ammonia-oxidizing archaea (AOA and bacteria (AOB in 9 high-altitude lakes (2289-3160 m in the Sierra Nevada, California, USA, in relation to spatial and biogeochemical data. Based on their ammonia monooxygenase (amoA genes, AOB and AOA were frequently detected. AOB were present in 88% of samples and were more abundant than AOA in all samples. Both groups showed >100 fold variation in abundance between different lakes, and were also variable through time within individual lakes. Nutrient concentrations (ammonium, nitrite, nitrate, and phosphate were generally low but also varied across and within lakes, suggestive of active internal nutrient cycling; AOB abundance was significantly correlated with phosphate (r(2 = 0.32, p<0.1, whereas AOA abundance was inversely correlated with lake elevation (r(2 = 0.43, p<0.05. We also measured low rates of ammonia oxidation--indicating that AOB, AOA, or both, may be biogeochemically active in these oligotrophic ecosystems. Our data indicate that dynamic populations of AOB and AOA are found in oligotrophic, high-altitude, freshwater lakes.

  20. The Influence of Ecological and Conventional Plant Production Systems on Soil Microbial Quality under Hops (Humulus lupulus)

    Science.gov (United States)

    Oszust, Karolina; Frąc, Magdalena; Gryta, Agata; Bilińska, Nina

    2014-01-01

    The knowledge about microorganisms—activity and diversity under hop production is still limited. We assumed that, different systems of hop production (within the same soil and climatic conditions) significantly influence on the composition of soil microbial populations and its functional activity (metabolic potential). Therefore, we compared a set of soil microbial properties in the field experiment of two hop production systems (a) ecological based on the use of probiotic preparations and organic fertilization (b) conventional—with the use of chemical pesticides and mineral fertilizers. Soil analyses included following microbial properties: The total number microorganisms, a bunch of soil enzyme activities, the catabolic potential was also assessed following Biolog EcoPlates®. Moreover, the abundance of ammonia-oxidizing archaea (AOA) was characterized by terminal restriction fragment length polymorphism analysis (T-RFLP) of PCR ammonia monooxygenase α-subunit (amoA) gene products. Conventional and ecological systems of hop production were able to affect soil microbial state in different seasonal manner. Favorable effect on soil microbial activity met under ecological, was more probably due to livestock-based manure and fermented plant extracts application. No negative influence on conventional hopyard soil was revealed. Both type of production fulfilled fertilizing demands. Under ecological production it was due to livestock-based manure fertilizers and fermented plant extracts application. PMID:24897025

  1. The Influence of Ecological and Conventional Plant Production Systems on Soil Microbial Quality under Hops (Humulus lupulus

    Directory of Open Access Journals (Sweden)

    Karolina Oszust

    2014-06-01

    Full Text Available The knowledge about microorganisms—activity and diversity under hop production is still limited. We assumed that, different systems of hop production (within the same soil and climatic conditions significantly influence on the composition of soil microbial populations and its functional activity (metabolic potential. Therefore, we compared a set of soil microbial properties in the field experiment of two hop production systems (a ecological based on the use of probiotic preparations and organic fertilization (b conventional—with the use of chemical pesticides and mineral fertilizers. Soil analyses included following microbial properties: The total number microorganisms, a bunch of soil enzyme activities, the catabolic potential was also assessed following Biolog EcoPlates®. Moreover, the abundance of ammonia-oxidizing archaea (AOA was characterized by terminal restriction fragment length polymorphism analysis (T-RFLP of PCR ammonia monooxygenase α-subunit (amoA gene products. Conventional and ecological systems of hop production were able to affect soil microbial state in different seasonal manner. Favorable effect on soil microbial activity met under ecological, was more probably due to livestock-based manure and fermented plant extracts application. No negative influence on conventional hopyard soil was revealed. Both type of production fulfilled fertilizing demands. Under ecological production it was due to livestock-based manure fertilizers and fermented plant extracts application.

  2. Inhibition effect of zinc in wastewater on the N2O emission from coastal loam soils.

    Science.gov (United States)

    Huang, Yan; Ou, Danyun; Chen, Shunyang; Chen, Bin; Liu, Wenhua; Bai, Renao; Chen, Guangcheng

    2017-03-15

    The effects of zinc (Zn) on nitrous oxide (N 2 O) fluxes from coastal loam soil and the abundances of soil nitrifier and denitrifier were studied in a tidal microcosm receiving livestock wastewater with different Zn levels. Soil N 2 O emission significantly increased due to discharge of wastewater rich in ammonia (NH 4 + -N) while the continuous measurements of gas flux showed a durative reduction in N 2 O flux by high Zn input (40mgL -1 ) during the low tide period. Soil inorganic nitrogen concentrations increased at the end of the experiment and even more soil NH 4 + -N was measured in the high-Zn-level treatment, indicating an inhibition of ammonia oxidation by Zn input. Quantitative PCR of soil amoA, narG and nirK genes encoding ammonia monooxygenase, nitrate reductase and nitrite reductase, respectively, showed that the microbial abundances involved in these metabolisms were neither affected by wastewater discharge nor Zn contamination. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Gene doping in sports.

    Science.gov (United States)

    Unal, Mehmet; Ozer Unal, Durisehvar

    2004-01-01

    Gene or cell doping is defined by the World Anti-Doping Agency (WADA) as "the non-therapeutic use of genes, genetic elements and/or cells that have the capacity to enhance athletic performance". New research in genetics and genomics will be used not only to diagnose and treat disease, but also to attempt to enhance human performance. In recent years, gene therapy has shown progress and positive results that have highlighted the potential misuse of this technology and the debate of 'gene doping'. Gene therapies developed for the treatment of diseases such as anaemia (the gene for erythropoietin), muscular dystrophy (the gene for insulin-like growth factor-1) and peripheral vascular diseases (the gene for vascular endothelial growth factor) are potential doping methods. With progress in gene technology, many other genes with this potential will be discovered. For this reason, it is important to develop timely legal regulations and to research the field of gene doping in order to develop methods of detection. To protect the health of athletes and to ensure equal competitive conditions, the International Olympic Committee, WADA and International Sports Federations have accepted performance-enhancing substances and methods as being doping, and have forbidden them. Nevertheless, the desire to win causes athletes to misuse these drugs and methods. This paper reviews the current status of gene doping and candidate performance enhancement genes, and also the use of gene therapy in sports medicine and ethics of genetic enhancement. Copyright 2004 Adis Data Information BV

  4. UPLC/Q-TOF MS-based metabolomics and qRT-PCR in enzyme gene screening with key role in triterpenoid saponin biosynthesis of Polygala tenuifolia.

    Science.gov (United States)

    Zhang, Fusheng; Li, Xiaowei; Li, Zhenyu; Xu, Xiaoshuang; Peng, Bing; Qin, Xuemei; Du, Guanhua

    2014-01-01

    The dried root of Polygala tenuifolia, named Radix Polygalae, is a well-known traditional Chinese medicine. Triterpenoid saponins are some of the most important components of Radix Polygalae extracts and are widely studied because of their valuable pharmacological properties. However, the relationship between gene expression and triterpenoid saponin biosynthesis in P. tenuifolia is unclear. In this study, ultra-performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS)-based metabolomic analysis was performed to identify and quantify the different chemical constituents of the roots, stems, leaves, and seeds of P. tenuifolia. A total of 22 marker compounds (VIP>1) were explored, and significant differences in all 7 triterpenoid saponins among the different tissues were found. We also observed an efficient reference gene GAPDH for different tissues in this plant and determined the expression level of some genes in the triterpenoid saponin biosynthetic pathway. Results showed that MVA pathway has more important functions in the triterpenoid saponin biosynthesis of P. tenuifolia. The expression levels of squalene synthase (SQS), squalene monooxygenase (SQE), and beta-amyrin synthase (β-AS) were highly correlated with the peak area intensity of triterpenoid saponins compared with data from UPLC/Q-TOF MS-based metabolomic analysis. This finding suggested that a combination of UPLC/Q-TOF MS-based metabolomics and gene expression analysis can effectively elucidate the mechanism of triterpenoid saponin biosynthesis and can provide useful information on gene discovery. These findings can serve as a reference for using the overexpression of genes encoding for SQS, SQE, and/or β-AS to increase the triterpenoid saponin production of P. tenuifolia.

  5. UPLC/Q-TOF MS-based metabolomics and qRT-PCR in enzyme gene screening with key role in triterpenoid saponin biosynthesis of Polygala tenuifolia.

    Directory of Open Access Journals (Sweden)

    Fusheng Zhang

    Full Text Available The dried root of Polygala tenuifolia, named Radix Polygalae, is a well-known traditional Chinese medicine. Triterpenoid saponins are some of the most important components of Radix Polygalae extracts and are widely studied because of their valuable pharmacological properties. However, the relationship between gene expression and triterpenoid saponin biosynthesis in P. tenuifolia is unclear.In this study, ultra-performance liquid chromatography (UPLC coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS-based metabolomic analysis was performed to identify and quantify the different chemical constituents of the roots, stems, leaves, and seeds of P. tenuifolia. A total of 22 marker compounds (VIP>1 were explored, and significant differences in all 7 triterpenoid saponins among the different tissues were found. We also observed an efficient reference gene GAPDH for different tissues in this plant and determined the expression level of some genes in the triterpenoid saponin biosynthetic pathway. Results showed that MVA pathway has more important functions in the triterpenoid saponin biosynthesis of P. tenuifolia. The expression levels of squalene synthase (SQS, squalene monooxygenase (SQE, and beta-amyrin synthase (β-AS were highly correlated with the peak area intensity of triterpenoid saponins compared with data from UPLC/Q-TOF MS-based metabolomic analysis.This finding suggested that a combination of UPLC/Q-TOF MS-based metabolomics and gene expression analysis can effectively elucidate the mechanism of triterpenoid saponin biosynthesis and can provide useful information on gene discovery. These findings can serve as a reference for using the overexpression of genes encoding for SQS, SQE, and/or β-AS to increase the triterpenoid saponin production of P. tenuifolia.

  6. Human Gene Therapy: Genes without Frontiers?

    Science.gov (United States)

    Simon, Eric J.

    2002-01-01

    Describes the latest advancements and setbacks in human gene therapy to provide reference material for biology teachers to use in their science classes. Focuses on basic concepts such as recombinant DNA technology, and provides examples of human gene therapy such as severe combined immunodeficiency syndrome, familial hypercholesterolemia, and…

  7. [Distribution Characteristics of Nitrifiers and Denitrifiers in the River Sediments of Tongling City].

    Science.gov (United States)

    Cheng, Jian-hua; Dou, Zhi-yong; Sun, Qing-ye

    2016-04-15

    Rivers in mining areas were influenced by contaminants such as nitrogen, phosphorus and organic matter due to domestic and agricultural wastewater discharge in addition to pollutants caused by mining activities. In this study, surface sediment samples of rivers in Tongling city were collected to address the effect of season and pollution type on the abundance of nitrifiers and denitrifiers using quantitative polymerase chain reaction (QPCR) technique targeting at the ammonia monooxygenase (amoA) and nitrite reductase (nir) genes. The results showed that the average ahundance of ammonia oxidizing archaea (AGA) (ranging from 1.74 x 10⁵ to 1.45 x 10⁸ copies · g⁻¹) was 4.39 times that of ammonia oxidizing hacteria (AGH) (ranging from 1.39 x 10⁵ to 3.39 x 10⁷ copies · g⁻¹); and the average abundance of nirK gene (ranging from 4.45 x 10⁶ to 1.51 x 10⁸ copies · g) was almost a thirtieth part of nirS gene (ranging from 1.69 x 10⁷ to 8.55 x 10⁹ copies · g⁻¹). The abundance of AOA was higher in spring and autumn, and lower in summer and winter. And sediment AOB abundance was higher in spring and winter than in summer and autumn. Meanwhile, the abundance of nir genes was in the order of spring (nirS )/autumn (nirK) > summer > winter > autumn (nirS )/spring (nirK). Moreover, the abundance of bacterial and archaeal arnoA and nirS genes in sediments influenced by mine pollution was generally higher than that in sediments influenced by agricultural non-point pollution, whereas the abundance of nirK gene showed an opposite trend.

  8. Differentiation in the microbial ecology and activity of suspended and attached bacteria in a nitritation-anammox process.

    Science.gov (United States)

    Park, Hongkeun; Sundar, Suneethi; Ma, Yiwei; Chandran, Kartik

    2015-02-01

    A directed differentiation between the biofilm and suspension was observed in the molecular microbial ecology and gene expression of different bacteria in a biofilm nitritation-anammox process operated at varying hydraulic residence times (HRT) and nitrogen loading rates (NLR). The highest degree of enrichment observed in the biofilm was of anaerobic ammonia-oxidizing bacteria (AMX) followed by that of Nitrospira spp. related nitrite-oxidizing bacteria (NOB). For AMX, a major shift from Candidatus "Brocadia fulgida" to Candidatus "Kuenenia stuttgartiensis" in both suspension and biofilm was observed with progressively shorter HRT, using discriminatory biomarkers targeting the hydrazine synthase (hzsA) gene. In parallel, expression of the hydrazine oxidoreductase gene (hzo), a functional biomarker for AMX energy metabolism, became progressively prominent in the biofilm. A marginal but statistically significant enrichment in the biofilm was observed for Nitrosomonas europaea related ammonia-oxidizing bacteria (AOB). In direct contrast to AMX, the gene expression of ammonia monooxygenase subunit A (amoA), a functional biomarker for AOB energy metabolism, progressively increased in suspension. Using gene expression and biomass concentration measures in conjunction, it was determined that signatures of AOB metabolism were primarily present in the biofilm throughout the study. On the other hand, AMX metabolism gradually shifted from being uniformly distributed in both the biofilm and suspension to primarily the biofilm at shorter HRTs and higher NLRs. These results therefore highlight the complexity and key differences in the microbial ecology, gene expression and activity between the biofilm and suspension of a nitritation-anammox process and the biokinetic and metabolic drivers for such niche segregation. © 2014 Wiley Periodicals, Inc.

  9. Tumor targeted gene therapy

    International Nuclear Information System (INIS)

    Kang, Joo Hyun

    2006-01-01

    Knowledge of molecular mechanisms governing malignant transformation brings new opportunities for therapeutic intervention against cancer using novel approaches. One of them is gene therapy based on the transfer of genetic material to an organism with the aim of correcting a disease. The application of gene therapy to the cancer treatment had led to the development of new experimental approaches such as suicidal gene therapy, inhibition of oncogenes and restoration of tumor-suppressor genes. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a prodrug into a toxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1-tk) and cytosine deaminase (CD). Especially, physicians and scientists of nuclear medicine field take an interest in suicidal gene therapy because they can monitor the location and magnitude, and duration of expression of HSV1-tk and CD by PET scanner

  10. Essential Bacillus subtilis genes

    DEFF Research Database (Denmark)

    Kobayashi, K.; Ehrlich, S.D.; Albertini, A.

    2003-01-01

    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximate to4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were...... predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related...... to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from...

  11. Potential for Nitrogen Fixation and Nitrification in the Granite-Hosted Subsurface at Henderson Mine, CO

    Science.gov (United States)

    Swanner, Elizabeth D.; Templeton, Alexis S.

    2011-01-01

    The existence of life in the deep terrestrial subsurface is established, yet few studies have investigated the origin of nitrogen that supports deep life. Previously, 16S rRNA gene surveys cataloged a diverse microbial community in subsurface fluids draining from boreholes 3000 feet deep at Henderson Mine, CO, USA (Sahl et al., 2008). The prior characterization of the fluid chemistry and microbial community forms the basis for the further investigation here of the source of NH4+. The reported fluid chemistry included N2, NH4+ (5–112 μM), NO2− (27–48 μM), and NO3− (17–72 μM). In this study, the correlation between low NH4+ concentrations in dominantly meteoric fluids and higher NH4+ in rock-reacted fluids is used to hypothesize that NH4+ is sourced from NH4+-bearing biotite. However, biotite samples from the host rocks and ore-body minerals were analyzed by Fourier transform infrared (FTIR) microscopy and none-contained NH4+. However, the nitrogenase-encoding gene nifH was successfully amplified from DNA of the fluid sample with high NH4+, suggesting that subsurface microbes have the capability to fix N2. If so, unregulated nitrogen fixation may account for the relatively high NH4+ concentrations in the fluids. Additionally, the amoA and nxrB genes for archaeal ammonium monooxygenase and nitrite oxidoreductase, respectively, were amplified from the high NH4+ fluid DNA, while bacterial amoA genes were not. Putative nitrifying organisms are closely related to ammonium-oxidizing Crenarchaeota and nitrite-oxidizing Nitrospira detected in other subsurface sites based upon 16S rRNA sequence analysis. Thermodynamic calculations underscore the importance of NH4+ as an energy source in a subsurface nitrification pathway. These results suggest that the subsurface microbial community at Henderson is adapted to the low nutrient and energy environment by their capability of fixing nitrogen, and that fixed nitrogen may support subsurface biomass via

  12. Radiotechnologies and gene therapy

    International Nuclear Information System (INIS)

    Xia Jinsong

    2001-01-01

    Gene therapy is an exciting frontier in medicine today. Radiologist will make an uniquely contribution to these exciting new technologies at every level by choosing sites for targeting therapy, perfecting and establishing routes of delivery, developing imaging strategies to monitor therapy and assess gene expression, developing radiotherapeutic used of gene therapy

  13. Discovering genes underlying QTL

    Energy Technology Data Exchange (ETDEWEB)

    Vanavichit, Apichart [Kasetsart University, Kamphaengsaen, Nakorn Pathom (Thailand)

    2002-02-01

    A map-based approach has allowed scientists to discover few genes at a time. In addition, the reproductive barrier between cultivated rice and wild relatives has prevented us from utilizing the germ plasm by a map-based approach. Most genetic traits important to agriculture or human diseases are manifested as observable, quantitative phenotypes called Quantitative Trait Loci (QTL). In many instances, the complexity of the phenotype/genotype interaction and the general lack of clearly identifiable gene products render the direct molecular cloning approach ineffective, thus additional strategies like genome mapping are required to identify the QTL in question. Genome mapping requires no prior knowledge of the gene function, but utilizes statistical methods to identify the most likely gene location. To completely characterize genes of interest, the initially mapped region of a gene location will have to be narrowed down to a size that is suitable for cloning and sequencing. Strategies for gene identification within the critical region have to be applied after the sequencing of a potentially large clone or set of clones that contains this gene(s). Tremendous success of positional cloning has been shown for cloning many genes responsible for human diseases, including cystic fibrosis and muscular dystrophy as well as plant disease resistance genes. Genome and QTL mapping, positional cloning: the pre-genomics era, comparative approaches to gene identification, and positional cloning: the genomics era are discussed in the report. (M. Suetake)

  14. Effect of two-linked mutations of the FMO3 gene on itopride metabolism in Chinese healthy volunteers.

    Science.gov (United States)

    Zhou, Li-Ping; Tan, Zhi-Rong; Chen, Hao; Guo, Dong; Chen, Yao; Huang, Wei-Hua; Wang, Lian-Sheng; Zhang, Guo-Gang

    2014-11-01

    Itopride is an effective gastroprokinetic agent mainly used for the treatment of functional dyspepsia. Flavin-containing monooxygenase 3 (FMO3) has been confirmed to be the key enzyme involved in the main itopride metabolic pathway. We investigated whether the FMO3 genotypes can affect itopride metabolism in Chinese healthy volunteers. Twelve healthy volunteers who had been genotyped for FMO3 gene were selected to participate in our study. Volunteers were given 50 mg itopride orally and then blood samples were collected from 0 to 24 h. The plasma concentrations of itopride and itopride N-oxide were determined by HPLC-MS/MS method. Itopride and itopride N-oxide both exhibit FMO3 genotype-dependent pharmacokinetic profiles. The area under the plasma concentration-time curve (AUC) of itopride increased by 127.82 ± 41.99 % (P itopride N-oxide decreased by 30.30 ± 25.70 % (P itopride and itopride N-oxide were observed between these two genotypes. The FMO3 allele can significantly affect the metabolism of itopride. The pharmacokinetic parameters of both itopride and itopride N-oxide were significantly different between these two genotypes.

  15. Gene therapy: An overview

    Directory of Open Access Journals (Sweden)

    Sudip Indu

    2013-01-01

    Full Text Available Gene therapy "the use of genes as medicine" involves the transfer of a therapeutic or working copy of a gene into specific cells of an individual in order to repair a faulty gene copy. The technique may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favorably modify the clinical course of a condition. The objective of gene therapy is to introduce new genetic material into target cells while causing no damage to the surrounding healthy cells and tissues, hence the treatment related morbidity is decreased. The delivery system includes a vector that delivers a therapeutic gene into the patient′s target cell. Functional proteins are created from the therapeutic gene causing the cell to return to a normal stage. The vectors used in gene therapy can be viral and non-viral. Gene therapy, an emerging field of biomedicine, is still at infancy and much research remains to be done before this approach to the treatment of condition will realize its full potential.

  16. Gene therapy in periodontics.

    Science.gov (United States)

    Chatterjee, Anirban; Singh, Nidhi; Saluja, Mini

    2013-03-01

    GENES are made of DNA - the code of life. They are made up of two types of base pair from different number of hydrogen bonds AT, GC which can be turned into instruction. Everyone inherits genes from their parents and passes them on in turn to their children. Every person's genes are different, and the changes in sequence determine the inherited differences between each of us. Some changes, usually in a single gene, may cause serious diseases. Gene therapy is 'the use of genes as medicine'. It involves the transfer of a therapeutic or working gene copy into specific cells of an individual in order to repair a faulty gene copy. Thus it may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favorably modify the clinical course of a condition. It has a promising era in the field of periodontics. Gene therapy has been used as a mode of tissue engineering in periodontics. The tissue engineering approach reconstructs the natural target tissue by combining four elements namely: Scaffold, signaling molecules, cells and blood supply and thus can help in the reconstruction of damaged periodontium including cementum, gingival, periodontal ligament and bone.

  17. Gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle-associated pathogen Grosmannia clavigera.

    Science.gov (United States)

    Wang, Ye; Lim, Lynette; Madilao, Lina; Lah, Ljerka; Bohlmann, Joerg; Breuil, Colette

    2014-08-01

    To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.

  18. Primetime for Learning Genes.

    Science.gov (United States)

    Keifer, Joyce

    2017-02-11

    Learning genes in mature neurons are uniquely suited to respond rapidly to specific environmental stimuli. Expression of individual learning genes, therefore, requires regulatory mechanisms that have the flexibility to respond with transcriptional activation or repression to select appropriate physiological and behavioral responses. Among the mechanisms that equip genes to respond adaptively are bivalent domains. These are specific histone modifications localized to gene promoters that are characteristic of both gene activation and repression, and have been studied primarily for developmental genes in embryonic stem cells. In this review, studies of the epigenetic regulation of learning genes in neurons, particularly the brain-derived neurotrophic factor gene ( BDNF ), by methylation/demethylation and chromatin modifications in the context of learning and memory will be highlighted. Because of the unique function of learning genes in the mature brain, it is proposed that bivalent domains are a characteristic feature of the chromatin landscape surrounding their promoters. This allows them to be "poised" for rapid response to activate or repress gene expression depending on environmental stimuli.

  19. GmCYP82A3, a Soybean Cytochrome P450 Family Gene Involved in the Jasmonic Acid and Ethylene Signaling Pathway, Enhances Plant Resistance to Biotic and Abiotic Stresses.

    Directory of Open Access Journals (Sweden)

    Qiang Yan

    Full Text Available The cytochrome P450 monooxygenases (P450s represent a large and important enzyme superfamily in plants. They catalyze numerous monooxygenation/hydroxylation reactions in biochemical pathways, P450s are involved in a variety of metabolic pathways and participate in the homeostasis of phytohormones. The CYP82 family genes specifically reside in dicots and are usually induced by distinct environmental stresses. However, their functions are largely unknown, especially in soybean (Glycine max L.. Here, we report the function of GmCYP82A3, a gene from soybean CYP82 family. Its expression was induced by Phytophthora sojae infection, salinity and drought stresses, and treatment with methyl jasmonate (MeJA or ethephon (ETH. Its expression levels were consistently high in resistant cultivars. Transgenic Nicotiana benthamiana plants overexpressing GmCYP82A3 exhibited strong resistance to Botrytis cinerea and Phytophthora parasitica, and enhanced tolerance to salinity and drought stresses. Furthermore, transgenic plants were less sensitive to jasmonic acid (JA, and the enhanced resistance was accompanied with increased expression of the JA/ET signaling pathway-related genes.

  20. Genes and Social Behavior

    OpenAIRE

    Robinson, Gene E.; Fernald, Russell D.; Clayton, David F.

    2008-01-01

    What specific genes and regulatory sequences contribute to the organization and functioning of brain circuits that support social behavior? How does social experience interact with information in the genome to modulate these brain circuits? Here we address these questions by highlighting progress that has been made in identifying and understanding two key “vectors of influence” that link genes, brain, and social behavior: 1) social information alters gene readout in the brain to influence beh...

  1. History of gene therapy.

    Science.gov (United States)

    Wirth, Thomas; Parker, Nigel; Ylä-Herttuala, Seppo

    2013-08-10

    Two decades after the initial gene therapy trials and more than 1700 approved clinical trials worldwide we not only have gained much new information and knowledge regarding gene therapy in general, but also learned to understand the concern that has persisted in society. Despite the setbacks gene therapy has faced, success stories have increasingly emerged. Examples for these are the positive recommendation for a gene therapy product (Glybera) by the EMA for approval in the European Union and the positive trials for the treatment of ADA deficiency, SCID-X1 and adrenoleukodystrophy. Nevertheless, our knowledge continues to grow and during the course of time more safety data has become available that helps us to develop better gene therapy approaches. Also, with the increased understanding of molecular medicine, we have been able to develop more specific and efficient gene transfer vectors which are now producing clinical results. In this review, we will take a historical view and highlight some of the milestones that had an important impact on the development of gene therapy. We will also discuss briefly the safety and ethical aspects of gene therapy and address some concerns that have been connected with gene therapy as an important therapeutic modality. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Refining discordant gene trees.

    Science.gov (United States)

    Górecki, Pawel; Eulenstein, Oliver

    2014-01-01

    Evolutionary studies are complicated by discordance between gene trees and the species tree in which they evolved. Dealing with discordant trees often relies on comparison costs between gene and species trees, including the well-established Robinson-Foulds, gene duplication, and deep coalescence costs. While these costs have provided credible results for binary rooted gene trees, corresponding cost definitions for non-binary unrooted gene trees, which are frequently occurring in practice, are challenged by biological realism. We propose a natural extension of the well-established costs for comparing unrooted and non-binary gene trees with rooted binary species trees using a binary refinement model. For the duplication cost we describe an efficient algorithm that is based on a linear time reduction and also computes an optimal rooted binary refinement of the given gene tree. Finally, we show that similar reductions lead to solutions for computing the deep coalescence and the Robinson-Foulds costs. Our binary refinement of Robinson-Foulds, gene duplication, and deep coalescence costs for unrooted and non-binary gene trees together with the linear time reductions provided here for computing these costs significantly extends the range of trees that can be incorporated into approaches dealing with discordance.

  3. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop.

    Science.gov (United States)

    Kyndt, Tina; Quispe, Dora; Zhai, Hong; Jarret, Robert; Ghislain, Marc; Liu, Qingchang; Gheysen, Godelieve; Kreuze, Jan F

    2015-05-05

    Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world's arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.

  4. Wastewater and Saltwater: Studying the Biogeochemistry and Microbial Activity Associated with Wastewater Inputs to San Francisco Bay

    Science.gov (United States)

    Challenor, T.; Menendez, A. D.; Damashek, J.; Francis, C. A.; Casciotti, K. L.

    2014-12-01

    Nitrification is the process of converting ammonium (NH­­4+) into nitrate (NO3-), and is a crucial step in removing nitrogen (N) from aquatic ecosystems. This process is governed by ammonia-oxidizing bacteria (AOB) and archaea (AOA) that utilize the ammonia monooxygenase gene (amoA). Studying the rates of nitrification and the abundances of ammonia-oxidizing microorganisms in south San Francisco Bay's Artesian Slough, which receives treated effluent from the massive San Jose-Santa Clara Regional Wastewater Facility, are important for understanding the cycling of nutrients in this small but complex estuary. Wastewater inputs can have negative environmental impacts, such as the release of nitrous oxide, a byproduct of nitrification and a powerful greenhouse gas. Nutrient inputs can also increase productivity and sometimes lead to oxygen depletion. Assessing the relative abundance and diversity of AOA and AOB, along with measuring nitrification rates gives vital information about the biology and biogeochemistry of this important N-cycling process. To calculate nitrification rates, water samples were spiked with 15N-labeled ammonium and incubated in triplicate for 24 hours. Four time-points were extracted across the incubation and the "denitrifier" method was used to measure the isotopic ratio of nitrate in the samples over time. In order to determine relative ratios of AOB to AOA, DNA was extracted from water samples and used in clade-specific amoA PCR assays. Nitrification rates were detectable in all locations sampled and were higher than in other regions of the bay, as were concentrations of nitrate and ammonium. Rates were highest in the regions of Artesian Slough most directly affected by wastewater effluent. AOB vastly outnumbered AOA, which is consistent with other studies showing that AOB prefer high nutrient environments. AOB diversity includes clades of Nitrosospira and Nitrosomonas prevalent in estuarine settings. Many of the sequenced genes are related

  5. Chromatin loops, gene positioning, and gene expression

    NARCIS (Netherlands)

    Holwerda, S.; de Laat, W.

    2012-01-01

    Technological developments and intense research over the last years have led to a better understanding of the 3D structure of the genome and its influence on genome function inside the cell nucleus. We will summarize topological studies performed on four model gene loci: the alpha- and beta-globin

  6. Your Genes, Your Choices

    Science.gov (United States)

    Table of Contents Your Genes, Your Choices describes the Human Genome Project, the science behind it, and the ethical, legal, and social issues that are ... Nothing could be further from the truth. Your Genes, Your Choices points out how the progress of ...

  7. DNA repair genes

    International Nuclear Information System (INIS)

    Morimyo, Mitsuoki

    1995-01-01

    Fission yeast S. pombe is assumed to be a good model for cloning of human DNA repair genes, because human gene is normally expressed in S. pombe and has a very similar protein sequence to yeast protein. We have tried to elucidate the DNA repair mechanisms of S. pombe as a model system for those of mammals. (J.P.N.)

  8. Antisense gene silencing

    DEFF Research Database (Denmark)

    Nielsen, Troels T; Nielsen, Jørgen E

    2013-01-01

    Since the first reports that double-stranded RNAs can efficiently silence gene expression in C. elegans, the technology of RNA interference (RNAi) has been intensively exploited as an experimental tool to study gene function. With the subsequent discovery that RNAi could also be applied...

  9. Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance.

    Science.gov (United States)

    Hu, Z; Lin, Q; Chen, H; Li, Z; Yin, F; Feng, X

    2014-12-01

    Insect cytochrome P450 monooxygenases (P450s) play an important role in catalysis of many reactions leading to insecticides resistance. Our previous studies on transcriptome analysis of chlorantraniliprole-resistant development in the diamondback moth, Plutella xylostella revealed that up-regulation of cytochrome P450s are one of the main factors leading to the development of chlorantraniliprole resistance. Here, we report for the first time a novel cytochrome P450 gene CYP321E1, which belongs to the cytochrome P450 gene family CYP321. Real-time quantitative PCR (RT-qPCR) analyses indicated that CYP321E1 was expressed at all developmental stages of P. xylostella but was highest in the fourth-instar larvae; furthermore, the relatively high expression was observed in the midgut of the fourth-instar larvae, followed by fat bodies and epidermis. The expression of CYP321E1 in P. xylostella was differentially affected by three representative insecticides, including alphamethrin, abamectin and chlorantraniliprole. Among them, the exposure to chlorantraniliprole resulted in the largest transcript level of this cytochrome P450 gene. The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of P. xylostella. To assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNA interference (RNAi)-mediated gene silencing by double stranded RNA (dsRNA) injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24 h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321E1 dsRNA was observed after 24 h of exposure to chlorantraniliprole. These results strongly support our notion that this novel cytochrome P450 gene plays an important role in chlorantraniliprole detoxification in the diamondback moth and is partly responsible for its resistance.

  10. Radionuclide reporter gene imaging

    International Nuclear Information System (INIS)

    Min, Jung Joon

    2004-01-01

    Recent progress in the development of non-invasive imaging technologies continues to strengthen the role of molecular imaging biological research. These tools have been validated recently in variety of research models, and have been shown to provide continuous quantitative monitoring of the location(s), magnitude, and time-variation of gene expression. This article reviews the principles, characteristics, categories and the use of radionuclide reporter gene imaging technologies as they have been used in imaging cell trafficking, imaging gene therapy, imaging endogenous gene expression and imaging molecular interactions. The studies published to date demonstrate that reporter gene imaging technologies will help to accelerate model validation as well as allow for clinical monitoring of human diseases

  11. Radionuclide reporter gene imaging

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon [School of Medicine, Chonnam National Univ., Gwangju (Korea, Republic of)

    2004-04-01

    Recent progress in the development of non-invasive imaging technologies continues to strengthen the role of molecular imaging biological research. These tools have been validated recently in variety of research models, and have been shown to provide continuous quantitative monitoring of the location(s), magnitude, and time-variation of gene expression. This article reviews the principles, characteristics, categories and the use of radionuclide reporter gene imaging technologies as they have been used in imaging cell trafficking, imaging gene therapy, imaging endogenous gene expression and imaging molecular interactions. The studies published to date demonstrate that reporter gene imaging technologies will help to accelerate model validation as well as allow for clinical monitoring of human diseases.

  12. Identification and expression analysis of cytokinin metabolic genes IPTs, CYP735A and CKXs in the biofuel plant Jatropha curcas

    Science.gov (United States)

    Cai, Li; Zhang, Lu

    2018-01-01

    The seed oil of Jatropha curcas is considered a potential bioenergy source that could replace fossil fuels. However, the seed yield of Jatropha is low and has yet to be improved. We previously reported that exogenous cytokinin treatment increased the seed yield of Jatropha. Cytokinin levels are directly regulated by isopentenyl transferase (IPT), cytochrome P450 monooxygenase, family 735, subfamily A (CYP735A), and cytokinin oxidase/dehydrogenase (CKX). In this study, we cloned six IPT genes, one JcCYP735A gene, and seven JcCKX genes. The expression patterns of these 14 genes in various organs were determined using real-time quantitative PCR. JcIPT1 was primarily expressed in roots and seeds, JcIPT2 was expressed in roots, apical meristems, and mature leaves, JcIPT3 was expressed in stems and mature leaves, JcIPT5 was expressed in roots and mature leaves, JcIPT6 was expressed in seeds at 10 days after pollination, and JcIPT9 was expressed in mature leaves. JcCYP735A was mainly expressed in roots, flower buds, and seeds. The seven JcCKX genes also showed different expression patterns in different organs of Jatropha. In addition, CK levels were detected in flower buds and seeds at different stages of development. The concentration of N6-(Δ2-isopentenyl)-adenine (iP), iP-riboside, and trans-zeatin (tZ) increased with flower development, and the concentration of iP decreased with seed development, while that of tZ increased. We further analyzed the function of JcCYP735A using the CRISPR-Cas9 system, and found that the concentrations of tZ and tZ-riboside decreased significantly in the Jccyp735a mutants, which showed severely retarded growth. These findings will be helpful for further studies of the functions of cytokinin metabolic genes and understanding the roles of cytokinins in Jatropha growth and development. PMID:29785355

  13. Identification and expression analysis of cytokinin metabolic genes IPTs, CYP735A and CKXs in the biofuel plant Jatropha curcas

    Directory of Open Access Journals (Sweden)

    Li Cai

    2018-05-01

    Full Text Available The seed oil of Jatropha curcas is considered a potential bioenergy source that could replace fossil fuels. However, the seed yield of Jatropha is low and has yet to be improved. We previously reported that exogenous cytokinin treatment increased the seed yield of Jatropha. Cytokinin levels are directly regulated by isopentenyl transferase (IPT, cytochrome P450 monooxygenase, family 735, subfamily A (CYP735A, and cytokinin oxidase/dehydrogenase (CKX. In this study, we cloned six IPT genes, one JcCYP735A gene, and seven JcCKX genes. The expression patterns of these 14 genes in various organs were determined using real-time quantitative PCR. JcIPT1 was primarily expressed in roots and seeds, JcIPT2 was expressed in roots, apical meristems, and mature leaves, JcIPT3 was expressed in stems and mature leaves, JcIPT5 was expressed in roots and mature leaves, JcIPT6 was expressed in seeds at 10 days after pollination, and JcIPT9 was expressed in mature leaves. JcCYP735A was mainly expressed in roots, flower buds, and seeds. The seven JcCKX genes also showed different expression patterns in different organs of Jatropha. In addition, CK levels were detected in flower buds and seeds at different stages of development. The concentration of N6-(Δ2-isopentenyl-adenine (iP, iP-riboside, and trans-zeatin (tZ increased with flower development, and the concentration of iP decreased with seed development, while that of tZ increased. We further analyzed the function of JcCYP735A using the CRISPR-Cas9 system, and found that the concentrations of tZ and tZ-riboside decreased significantly in the Jccyp735a mutants, which showed severely retarded growth. These findings will be helpful for further studies of the functions of cytokinin metabolic genes and understanding the roles of cytokinins in Jatropha growth and development.

  14. Quail FMO3 gene cloning, tissue expression profiling, polymorphism detection and association analysis with fishy taint in eggs.

    Directory of Open Access Journals (Sweden)

    Fengtao Mo

    Full Text Available Quail eggs comprise a significant and favourable part of table eggs in certain countries. Some quail eggs, however, present fishy off-flavor which directly influences their quality. It is reported that flavin-containing monooxygenase 3 (FMO3 is associated with fish-odour trait in human and animal products. FMO3 is responsible for the degradation of trimethylamine (TMA in vivo. Loss-of-function mutations in FMO3 gene can result in defective TMA N-oxygenation, giving rise to disorder known as "fish-odour syndrome" in human, as well as the fishy off-flavor in cow milk and chicken eggs. In order to reveal the genetic factor of fishy taint in quail eggs, we cloned the cDNA sequence of quail FMO3 gene, investigated FMO3 mRNA expression level in various tissues, detected SNPs in the coding region of the gene and conducted association analysis between a mutation and the TMA content in quail egg yolks. The 1888 bp cDNA sequence of quail FMO3 gene encoding 532 amino acids was obtained and characterized. The phylogenetic analysis revealed quail FMO3 had a closer relationship with chicken FMO3. The FMO3 mRNA was highly expressed in liver and kidney of quail. Nine SNPs were detected in the coding sequence of quail FMO3 gene, including a nonsense mutation (Q319X which was significantly associated with the elevated TMA content in quail egg yolks. Genotype TT at Q319X mutation loci was sensitive to choline. With addition of choline in the feed, the quails with homozygote TT at the Q319X mutation loci laid fish-odour eggs, indicating an interaction between genotype and diet. The results indicated that Q319X mutation was associated with the fishy off-flavor in quail eggs. Identification of the unfavorable allele T of quail FMO3 gene can be applied in future quail breeding to eliminate fishy off-flavor trait in quail eggs.

  15. Thaumarchaeal ammonium oxidation and evidence for a nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps

    Directory of Open Access Journals (Sweden)

    Friedrich Wolfgang Gerbl

    2014-05-01

    Full Text Available Previous studies had suggested the presence of ammonium oxidizing Thaumarchaeota as well as nitrite oxidizing Bacteria in the subsurface spring called Franz Josef Quelle (FJQ, a slightly radioactive thermal mineral spring with a temperature of 43.6 - 47oC near the alpine village of Bad Gastein, Austria. The microbiological consortium of the FJQ was investigated for its utilization of nitrogen compounds and the putative presence of a subsurface nitrogen cycle. Microcosm experiments made with samples from the spring water, containing planktonic microorganisms, or from biofilms, were used in this study. Three slightly different media, enriched with vitamins and trace elements, and two incubation temperatures (30 and 40oC, respectively were employed. Under aerobic conditions, high rates of conversion of ammonium to nitrite, as well as nitrite to nitrate were measured. Under oxygen-limited conditions nitrate was converted to gaseous compounds. Stable isotope probing with 15NH4Cl or (15NH42SO4 as sole energy sources revealed incorporation of 15N into community DNA. Genomic DNA as well as RNA were extracted from all microcosms. The following genes or fragments of genes were successfully amplified, cloned and sequenced by standard PCR from DNA extracts: Ammonia monooxygenase subunit A (amoA, nitrite oxidoreductase subunits A and B (nxrA and nxrB, nitrate reductase (narG, nitrite reductase (nirS, nitric oxide reductases (cnorB and qnorB, nitrous oxide reductase (nosZ. Reverse transcription of extracted total RNA and real-time PCR suggested the expression of each of those genes. Nitrogen fixation (as probed with nifH and nifD was not detected. However, a geological origin of NH4+ in the water of the FJQ cannot be excluded, considering the silicate, granite and gneiss containing environment. The data suggested the operation of a nitrogen cycle in the subsurface environment of the FJQ.

  16. Thaumarchaeal ammonium oxidation and evidence for a nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps.

    Science.gov (United States)

    Gerbl, Friedrich W; Weidler, Gerhard W; Wanek, Wolfgang; Erhardt, Angelika; Stan-Lotter, Helga

    2014-01-01

    Previous studies had suggested the presence of ammonium oxidizing Thaumarchaeota as well as nitrite oxidizing Bacteria in the subsurface spring called Franz Josef Quelle (FJQ), a slightly radioactive thermal mineral spring with a temperature of 43.6-47°C near the alpine village of Bad Gastein, Austria. The microbiological consortium of the FJQ was investigated for its utilization of nitrogen compounds and the putative presence of a subsurface nitrogen cycle. Microcosm experiments made with samples from the spring water, containing planktonic microorganisms, or from biofilms, were used in this study. Three slightly different media, enriched with vitamins and trace elements, and two incubation temperatures (30 and 40°C, respectively) were employed. Under aerobic conditions, high rates of conversion of ammonium to nitrite, as well as nitrite to nitrate were measured. Under oxygen-limited conditions nitrate was converted to gaseous compounds. Stable isotope probing with (15)NH4Cl or ((15)NH4)2SO4as sole energy sources revealed incorporation of (15)N into community DNA. Genomic DNA as well as RNA were extracted from all microcosms. The following genes or fragments of genes were successfully amplified, cloned and sequenced by standard PCR from DNA extracts: Ammonia monooxygenase subunit A (amoA), nitrite oxidoreductase subunits A and B (nxrA and nxrB), nitrate reductase (narG), nitrite reductase (nirS), nitric oxide reductases (cnorB and qnorB), nitrous oxide reductase (nosZ). Reverse transcription of extracted total RNA and real-time PCR suggested the expression of each of those genes. Nitrogen fixation (as probed with nifH and nifD) was not detected. However, a geological origin of NH(+) 4 in the water of the FJQ cannot be excluded, considering the silicate, granite and gneiss containing environment. The data suggested the operation of a nitrogen cycle in the subsurface environment of the FJQ.

  17. Gene amplification in carcinogenesis

    Directory of Open Access Journals (Sweden)

    Lucimari Bizari

    2006-01-01

    Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

  18. Methanogenesis and methane genes

    International Nuclear Information System (INIS)

    Reeve, J.N.; Shref, B.A.

    1991-01-01

    An overview of the pathways leading to methane biosynthesis is presented. The steps investigated to date by gene cloning and DNA sequencing procedures are identified and discussed. The primary structures of component C of methyl coenzyme M reductase encoded by mcr operons in different methanogens are compared. Experiments to detect the primary structure of the genes encoding F420 reducing hydrogenase (frhABG) and methyl hydrogen reducing hydrogenase (mvhDGA) in methanobacterium thermoautotrophicum strain H are compared with each other and with eubacterial hydrogenase encoding genes. A biotechnological use for hydrogenases from hypermorphillic archaebacteria is suggested. (author)

  19. Identification and Characterization of CYP9A40 from the Tobacco Cutworm Moth (Spodoptera litura), a Cytochrome P450 Gene Induced by Plant Allelochemicals and Insecticides

    Science.gov (United States)

    Wang, Rui-Long; Staehelin, Christian; Xia, Qing-Qing; Su, Yi-Juan; Zeng, Ren-Sen

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) of insects play crucial roles in the metabolism of endogenous and dietary compounds. Tobacco cutworm moth (Spodoptera litura), an important agricultural pest, causes severe yield losses in many crops. In this study, we identified CYP9A40, a novel P450 gene of S. litura, and investigated its expression profile and potential role in detoxification of plant allelochemicals and insecticides. The cDNA contains an open reading frame encoding 529 amino acid residues. CYP9A40 transcripts were found to be accumulated during various development stages of S. litura and were highest in fifth and sixth instar larvae. CYP9A40 was mainly expressed in the midgut and fat body. Larval consumption of xenobiotics, namely plant allelochemicals (quercetin and cinnamic acid) and insecticides (deltamethrin and methoxyfenozide) induced accumulation of CYP9A40 transcripts in the midgut and fat body. Injection of dsCYP9A40 (silencing of CYP9A40 by RNA interference) significantly increased the susceptibility of S. litura larvae to the tested plant allelochemicals and insecticides. These results indicate that CYP9A40 expression in S. litura is related to consumption of xenobiotics and suggest that CYP9A40 is involved in detoxification of these compounds. PMID:26393579

  20. Potent Nematicidal Activity and New Hybrid Metabolite Production by Disruption of a Cytochrome P450 Gene Involved in the Biosynthesis of Morphological Regulatory Arthrosporols in Nematode-Trapping Fungus Arthrobotrys oligospora.

    Science.gov (United States)

    Song, Tian-Yang; Xu, Zi-Fei; Chen, Yong-Hong; Ding, Qiu-Yan; Sun, Yu-Rong; Miao, Yang; Zhang, Ke-Qin; Niu, Xue-Mei

    2017-05-24

    Types of polyketide synthase-terpenoid synthase (PKS-TPS) hybrid metabolites, including arthrosporols with significant morphological regulatory activity, have been elucidated from nematode-trapping fungus Arthrobotrys oligospora. A previous study suggested that the gene cluster AOL_s00215 in A. oligospora was involved in the production of arthrosporols. Here, we report that disruption of one cytochrome P450 monooxygenase gene AOL_s00215g280 in the cluster resulted in significant phenotypic difference and much aerial hyphae. A further bioassay indicated that the mutant showed a dramatic decrease in the conidial formation but developed numerous traps and killed 85% nematodes within 6 h in contact with prey, in sharp contrast to the wild-type strain with no obvious response. Chemical investigation revealed huge accumulation of three new PKS-TPS epoxycyclohexone derivatives with different oxygenated patterns around the epoxycyclohexone moiety and the absence of arthrosporols in the cultural broth of the mutant ΔAOL_s00215g280. These findings suggested that a study on the biosynthetic pathway for morphological regulatory metabolites in nematode-trapping fungus would provide an efficient way to develop new fungal biocontrol agents.

  1. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  2. Finding Genes for Schizophrenia

    OpenAIRE

    Åberg, Karolina

    2005-01-01

    Schizophrenia is one of our most common psychiatric diseases. It severely affects all aspects of psychological functions and results in loss of contact with reality. No cure exists and the treatments available today produce only partial relief for disease symptoms. The aim of this work is to better understand the etiology of schizophrenia by identification of candidate genes and gene pathways involved in the development of the disease. In a preliminarily study, the effects of medication and g...

  3. Epigenetics: beyond genes

    CSIR Research Space (South Africa)

    Fossey, A

    2009-06-01

    Full Text Available in forestry breeding. Keywords Gene regulation; chromatin; histone code hyporthesis; RNA silencing; post transcriptional gene silencing; forestry. Introduction to epigenetic phenomena Most living organisms share a vast amount of genetic information... (Rapp and Wendel, 2005). Epigenetic phenomena pervade all aspects of cell proliferation and plant development and are often in conflict with Mendelian models of genetics (Grant-Downton and Dickinson, 2005). A key element in many epigenetic effects...

  4. Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response

    Directory of Open Access Journals (Sweden)

    Ryan James C

    2010-08-01

    Full Text Available Abstract Background Ciguatoxins (CTXs are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO and molecular pathway enrichment of the gene expression data. Results A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p Conclusions Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against

  5. Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response.

    Science.gov (United States)

    Ryan, James C; Morey, Jeanine S; Bottein, Marie-Yasmine Dechraoui; Ramsdell, John S; Van Dolah, Frances M

    2010-08-26

    Ciguatoxins (CTXs) are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO) and molecular pathway enrichment of the gene expression data. A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p < 0.0001) with microarray results. Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against neuroinflammation. Pathologic

  6. Gene-Gene and Gene-Environment Interactions in the Etiology of Breast Cancer

    National Research Council Canada - National Science Library

    Adegoke, Olufemi

    2003-01-01

    The objective of this CDA is to evaluate the gene-gene and gene-environment interactions in the etiology of breast cancer in two ongoing case-control studies, the Shanghai Breast Cancer Study (SBCS...

  7. Radiosensitivity and genes

    Energy Technology Data Exchange (ETDEWEB)

    Qiyue, Hu; Mingyue, Lun [Suzhou Medical Coll., JS (China)

    1995-07-01

    Reported effects of some oncogenes, tumour suppressor genes and DNA repair genes on sensitivity of cells to ionizing radiation are reviewed. The role of oncogenes in cellular response to irradiation is discussed, especially the extensively studied oncogenes such as the ras gene family. For tumour suppressor genes, mainly the p53, which is increasingly implicated as a gene affecting radiosensitivity, is reviewed. It is considered that there is a cell cycle checkpoint determinant which is postulated to be able to arrest the irradiated cells in G{sub 1} phase to allow them to repair damage before they undergo DNA synthesis. So far there are six DNA repair genes which have been cloned in mammalian cells, but only one, XRCC1, appears to be involved in repair of human X-ray damage. XRCC1 can correct high sisterchromatid exchange levels when transferred into EM{sub 9} cells, but its expression seems to have no correlation with radiosensitivity of human neck and head tumour cells. Radiosensitivity is an intricate issue which may involve many factors. A scheme of cellular reactions after exposure to irradiation is proposed to indicate a possible sequence of events initiated by ionizing radiation.

  8. Radiosensitivity and genes

    International Nuclear Information System (INIS)

    Hu Qiyue; Lun Mingyue

    1995-07-01

    Reported effects of some oncogenes, tumour suppressor genes and DNA repair genes on sensitivity of cells to ionizing radiation are reviewed. The role of oncogenes in cellular response to irradiation is discussed, especially the extensively studied oncogenes such as the ras gene family. For tumour suppressor genes, mainly the p53, which is increasingly implicated as a gene affecting radiosensitivity, is reviewed. It is considered that there is a cell cycle checkpoint determinant which is postulated to be able to arrest the irradiated cells in G 1 phase to allow them to repair damage before they undergo DNA synthesis. So far there are six DNA repair genes which have been cloned in mammalian cells, but only one, XRCC1, appears to be involved in repair of human X-ray damage. XRCC1 can correct high sisterchromatid exchange levels when transferred into EM 9 cells, but its expression seems to have no correlation with radiosensitivity of human neck and head tumour cells. Radiosensitivity is an intricate issue which may involve many factors. A scheme of cellular reactions after exposure to irradiation is proposed to indicate a possible sequence of events initiated by ionizing radiation

  9. Evidence for homosexuality gene

    Energy Technology Data Exchange (ETDEWEB)

    Pool, R.

    1993-07-16

    A genetic analysis of 40 pairs of homosexual brothers has uncovered a region on the X chromosome that appears to contain a gene or genes for homosexuality. When analyzing the pedigrees of homosexual males, the researcheres found evidence that the trait has a higher likelihood of being passed through maternal genes. This led them to search the X chromosome for genes predisposing to homosexuality. The researchers examined the X chromosomes of pairs of homosexual brothers for regions of DNA that most or all had in common. Of the 40 sets of brothers, 33 shared a set of five markers in the q28 region of the long arm of the X chromosome. The linkage has a LOD score of 4.0, which translates into a 99.5% certainty that there is a gene or genes in this area that predispose males to homosexuality. The chief researcher warns, however, that this one site cannot explain all instances of homosexuality, since there were some cases where the trait seemed to be passed paternally. And even among those brothers where there was no evidence that the trait was passed paternally, seven sets of brothers did not share the Xq28 markers. It seems likely that homosexuality arises from a variety of causes.

  10. A nonsense mutation in the beta-carotene oxygenase 2 (BCO2 gene is tightly associated with accumulation of carotenoids in adipose tissue in sheep (Ovis aries

    Directory of Open Access Journals (Sweden)

    Boman Inger A

    2010-02-01

    Full Text Available Abstract Background Sheep carcasses with yellow fat are sporadically observed at Norwegian slaughter houses. This phenomenon is known to be inherited as a recessive trait, and is caused by accumulation of carotenoids in adipose tissue. Two enzymes are known to be important in carotenoid degradation in mammals, and are therefore potential candidate genes for this trait. These are beta-carotene 15,15'-monooxygenase 1 (BCMO1 and the beta-carotene oxygenase 2 (BCO2. Results In the present study the coding region of the BCMO1 and the BCO2 gene were sequenced in yellow fat individuals and compared to the corresponding sequences from control animals with white fat. In the yellow fat individuals a nonsense mutation was found in BCO2 nucleotide position 196 (c.196C>T, introducing a stop codon in amino acid position 66. The full length protein consists of 575 amino acids. In spite of a very low frequency of this mutation in the Norwegian AI-ram population, 16 out of 18 yellow fat lambs were found to be homozygous for this mutation. Conclusion In the present study a nonsense mutation (c.196C>T in the beta-carotene oxygenase 2 (BCO2 gene is found to strongly associate with the yellow fat phenotype in sheep. The existence of individuals lacking this mutation, but still demonstrating yellow fat, suggests that additional mutations may cause a similar phenotype in this population. The results demonstrate a quantitatively important role for BCO2 in carotenoid degradation, which might indicate a broad enzyme specificity for carotenoids. Animals homozygous for the mutation are not reported to suffer from any negative health or development traits, pointing towards a minor role of BCO2 in vitamin A formation. Genotyping AI rams for c.196C>T can now be actively used in selection against the yellow fat trait.

  11. Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene

    Directory of Open Access Journals (Sweden)

    Makino Takuya

    2012-07-01

    Full Text Available Abstract Background Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds. Results Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2, were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl flavanones were respectively converted to apigenin and (hydroxyl flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms, and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds. Conclusion We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity for various small molecules, allowing the biosynthesis of not only flavones (from flavanones but also a variety of

  12. Subsurface Nitrogen-Cycling Microbial Communities at Uranium Contaminated Sites in the Colorado River Basin

    Science.gov (United States)

    Cardarelli, E.; Bargar, J.; Williams, K. H.; Dam, W. L.; Francis, C.

    2015-12-01

    Throughout the Colorado River Basin (CRB), uranium (U) persists as a relic contaminant of former ore processing activities. Elevated solid-phase U levels exist in fine-grained, naturally-reduced zone (NRZ) sediments intermittently found within the subsurface floodplain alluvium of the following Department of Energy-Legacy Management sites: Rifle, CO; Naturita, CO; and Grand Junction, CO. Coupled with groundwater fluctuations that alter the subsurface redox conditions, previous evidence from Rifle, CO suggests this resupply of U may be controlled by microbially-produced nitrite and nitrate. Nitrification, the two-step process of archaeal and bacterial ammonia-oxidation followed by bacterial nitrite oxidation, generates nitrate under oxic conditions. Our hypothesis is that when elevated groundwater levels recede and the subsurface system becomes anoxic, the nitrate diffuses into the reduced interiors of the NRZ and stimulates denitrification, the stepwise anaerobic reduction of nitrate/nitrite to dinitrogen gas. Denitrification may then be coupled to the oxidation of sediment-bound U(IV) forming mobile U(VI), allowing it to resupply U into local groundwater supplies. A key step in substantiating this hypothesis is to demonstrate the presence of nitrogen-cycling organisms in U-contaminated, NRZ sediments from the upper CRB. Here we investigate how the diversity and abundances of nitrifying and denitrifying microbial populations change throughout the NRZs of the subsurface by using functional gene markers for ammonia-oxidation (amoA, encoding the α-subunit of ammonia monooxygenase) and denitrification (nirK, nirS, encoding nitrite reductase). Microbial diversity has been assessed via clone libraries, while abundances have been determined through quantitative polymerase chain reaction (qPCR), elucidating how relative numbers of nitrifiers (amoA) and denitrifiers (nirK, nirS) vary with depth, vary with location, and relate to uranium release within NRZs in sediment

  13. The Mycoplasma hominis vaa gene displays a mosaic gene structure

    DEFF Research Database (Denmark)

    Boesen, Thomas; Emmersen, Jeppe M. G.; Jensen, Lise T.

    1998-01-01

    Mycoplasma hominis contains a variable adherence-associated (vaa) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolated by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular...

  14. Relative contributions of archaea and bacteria to microbial ammonia oxidation differ under different conditions during agricultural waste composting.

    Science.gov (United States)

    Zeng, Guangming; Zhang, Jiachao; Chen, Yaoning; Yu, Zhen; Yu, Man; Li, Hui; Liu, Zhifeng; Chen, Ming; Lu, Lunhui; Hu, Chunxiao

    2011-10-01

    The aim of this study was to compare the relative contribution of ammonia-oxidizing archaea (AOA) and bacteria (AOB) to nitrification during agricultural waste composting. The AOA and AOB amoA gene abundance and composition were determined by quantitative PCR and denaturing gradient gel electrophoresis (DGGE), respectively. The results showed that the archaeal amoA gene was abundant throughout the composting process, while the bacterial amoA gene abundance decreased to undetectable level during the thermophilic and cooling stages. DGGE showed more diverse archaeal amoA gene composition when the potential ammonia oxidation (PAO) rate reached peak values. A significant positive relationship was observed between the PAO rate and the archaeal amoA gene abundance (R²=0.554; Parchaea dominated ammonia oxidation during the thermophilic and cooling stages. Bacteria were also related to ammonia oxidation activity (R²=0.503; P=0.03) especially during the mesophilic and maturation stages. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Benthic Ammonia Oxidizers Differ in Community Structure and Biogeochemical Potential Across a Riverine Delta

    Directory of Open Access Journals (Sweden)

    Julian eDamashek

    2015-01-01

    Full Text Available Nitrogen pollution in coastal zones is a widespread issue, particularly in ecosystems with urban or agricultural watersheds. California’s Sacramento-San Joaquin Delta, at the landward reaches of San Francisco Bay, is highly impacted by both agricultural runoff and sewage effluent, leading to chronically high nutrient loadings. In particular, the massive discharge of ammonium into the Sacramento River has altered this ecosystem by increasing ammonium concentrations and thus changing the stoichiometry of inorganic nitrogen stocks, with potential effects throughout the food web. To date, however, there has been little research examining N biogeochemistry or N-cycling microbial communities in this system. We report the first data on benthic ammonia-oxidizing microbial communities and potential nitrification rates for the Sacramento-San Joaquin Delta, focusing on the functional gene amoA (encoding the α-subunit of ammonia monooxygenase. There were stark regional differences in ammonia-oxidizing communities, with ammonia-oxidizing bacteria (AOB outnumbering ammonia-oxidizing archaea (AOA only in the ammonium-rich Sacramento River. High potential nitrification rates in the Sacramento River suggested these communities may be capable of oxidizing significant amounts of ammonium, compared to the San Joaquin River and the upper reaches of San Francisco Bay. Gene diversity also showed regional patterns, as well as phylogenetically unique ammonia oxidizers in the Sacramento River. The community structure and biogeochemical function of benthic ammonia oxidizers appears related to nutrient loadings. Unraveling the microbial ecology and biogeochemistry of N cycling pathways is a critical step toward understanding how such ecosystems respond to the changing environmental conditions wrought by human development and climate change.

  16. A review of the microbiology of the Rehai geothermal field in Tengchong, Yunnan Province, China

    Directory of Open Access Journals (Sweden)

    Brian P. Hedlund

    2012-05-01

    Full Text Available The Rehai Geothermal Field, located in Tengchong County, in central-western Yunnan Province, is the largest and most intensively studied geothermal field in China. A wide physicochemical diversity of springs (ambient to ∼97 °C; pH from ≤1.8 to ≥9.3 provides a multitude of niches for extremophilic microorganisms. A variety of studies have focused on the cultivation, identification, basic physiology, taxonomy, and biotechnological potential of thermophilic microorganisms from Rehai. Thermophilic bacteria isolated from Rehai belong to the phyla Firmicutes and Deinococcus-Thermus. Firmicutes include neutrophilic or alkaliphilic Anoxybacillus, Bacillus, Caldalkalibacillus, Caldanaerobacter, Laceyella, and Geobacillus, as well as thermoacidophilic Alicyclobacillus and Sulfobacillus. Isolates from the Deinococcus-Thermus phylum include several Meiothermus and Thermus species. Many of these bacteria synthesize thermostable polymer-degrading enzymes that may be useful for biotechnology. The thermoacidophilic archaea Acidianus, Metallosphaera, and Sulfolobus have also been isolated and studied. A few studies have reported the isolation of thermophilic viruses belonging to Siphoviridae (TTSP4 and TTSP10 and Fuselloviridae (STSV1 infecting Thermus spp. and Sulfolobus spp., respectively. More recently, cultivation-independent studies using 16S rRNA gene sequences, shotgun metagenomics, or “functional gene” sequences have revealed a much broader diversity of microorganisms than represented in culture. Studies of the gene and mRNA encoding the large subunit of the ammonia monooxygenase (amoA of ammonia-oxidizing Archaea (AOA and the tetraether lipid crenarchaeol, a potential biomarker for AOA, suggest a wide diversity, but possibly low abundance, of thermophilic AOA in Rehai. Finally, we introduce the Tengchong Partnerships in International Research and Education (PIRE project, an international collaboration between Chinese and U.S. scientists with

  17. Evolutionary history and functional divergence of the cytochrome P450 gene superfamily between Arabidopsis thaliana and Brassica species uncover effects of whole genome and tandem duplications.

    Science.gov (United States)

    Yu, Jingyin; Tehrim, Sadia; Wang, Linhai; Dossa, Komivi; Zhang, Xiurong; Ke, Tao; Liao, Boshou

    2017-09-18

    The cytochrome P450 monooxygenase (P450) superfamily is involved in the biosynthesis of various primary and secondary metabolites. However, little is known about the effects of whole genome duplication (WGD) and tandem duplication (TD) events on the evolutionary history and functional divergence of P450s in Brassica after splitting from a common ancestor with Arabidopsis thaliana. Using Hidden Markov Model search and manual curation, we detected that Brassica species have nearly 1.4-fold as many P450 members as A. thaliana. Most P450s in A. thaliana and Brassica species were located on pseudo-chromosomes. The inferred phylogeny indicated that all P450s were clustered into two different subgroups. Analysis of WGD event revealed that different P450 gene families had appeared after evolutionary events of species. For the TD event analyses, the P450s from TD events in Brassica species can be divided into ancient and recent parts. Our comparison of influence of WGD and TD events on the P450 gene superfamily between A. thaliana and Brassica species indicated that the family-specific evolution in the Brassica lineage can be attributed to both WGD and TD, whereas WGD was recognized as the major mechanism for the recent evolution of the P450 super gene family. Expression analysis of P450s from A. thaliana and Brassica species indicated that WGD-type P450s showed the same expression pattern but completely different expression with TD-type P450s across different tissues in Brassica species. Selection force analysis suggested that P450 orthologous gene pairs between A. thaliana and Brassica species underwent negative selection, but no significant differences were found between P450 orthologous gene pairs in A. thaliana-B. rapa and A. thaliana-B. oleracea lineages, as well as in different subgenomes in B. rapa or B. oleracea compared with A. thaliana. This study is the first to investigate the effects of WGD and TD on the evolutionary history and functional divergence of P450

  18. FunGene: the functional gene pipeline and repository.

    Science.gov (United States)

    Fish, Jordan A; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C Titus; Tiedje, James M; Cole, James R

    2013-01-01

    Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

  19. FunGene: the Functional Gene Pipeline and Repository

    Directory of Open Access Journals (Sweden)

    Jordan A. Fish

    2013-10-01

    Full Text Available Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer.While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/ offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

  20. Gene therapy prospects--intranasal delivery of therapeutic genes.

    Science.gov (United States)

    Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

    2012-01-01

    Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

  1. Radionuclide reporter gene imaging for cardiac gene therapy

    International Nuclear Information System (INIS)

    Inubushi, Masayuki; Tamaki, Nagara

    2007-01-01

    In the field of cardiac gene therapy, angiogenic gene therapy has been most extensively investigated. The first clinical trial of cardiac angiogenic gene therapy was reported in 1998, and at the peak, more than 20 clinical trial protocols were under evaluation. However, most trials have ceased owing to the lack of decisive proof of therapeutic effects and the potential risks of viral vectors. In order to further advance cardiac angiogenic gene therapy, remaining open issues need to be resolved: there needs to be improvement of gene transfer methods, regulation of gene expression, development of much safer vectors and optimisation of therapeutic genes. For these purposes, imaging of gene expression in living organisms is of great importance. In radionuclide reporter gene imaging, ''reporter genes'' transferred into cell nuclei encode for a protein that retains a complementary ''reporter probe'' of a positron or single-photon emitter; thus expression of the reporter genes can be imaged with positron emission tomography or single-photon emission computed tomography. Accordingly, in the setting of gene therapy, the location, magnitude and duration of the therapeutic gene co-expression with the reporter genes can be monitored non-invasively. In the near future, gene therapy may evolve into combination therapy with stem/progenitor cell transplantation, so-called cell-based gene therapy or gene-modified cell therapy. Radionuclide reporter gene imaging is now expected to contribute in providing evidence on the usefulness of this novel therapeutic approach, as well as in investigating the molecular mechanisms underlying neovascularisation and safety issues relevant to further progress in conventional gene therapy. (orig.)

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